Genetic diversity of late Neanderthals in northwestern Europe

Abstract: Archaeological, osteological and genetic evidence suggests that Neanderthals lived in small groups1,2; however, less is known about whether these groups were part of isolated communities or belonged to larger, well-connected populations3. The dense concentration of broadly contemporaneous Neanderthal sites in the Meuse Basin, Belgium4, provides a rare opportunity to study regional populations at high resolution. Here we generated genetic data from 27 Neanderthals who lived less than approximately 52,500 years ago from ten archaeological sites in Belgium and France, including a high-coverage genome from a 45,000-year-old individual from Goyet, Belgium. We show that most of these individuals are more closely related to one another than to other contemporaneous late Neanderthals in Europe. Further, some of these individuals carry DNA from a Neanderthal lineage predating the split of late Neanderthals. Although these Neanderthals overlapped temporally with early modern humans in northwestern Europe from around 47,000 years ago, we find no evidence of recent gene flow from modern humans. They also do not show the genetic signatures of mating among close relatives found in Altai Neanderthals, suggesting that they lived in larger or better-connected groups. Moreover, genetic load did not accumulate over time, arguing against progressive genetic deterioration as a driver of Neanderthal extinction.

Bossoms Mesa A
Essel E
Peyrégne S
Sümer AP
Iasi LNM
Heide C
Popli D
de Filippo C
Gansauge MT
Gerullat L
Lippik L
Nagel S
Nickel B
Schellbach B
Schmidt A
Visagie J
Weihmann A
Zeberg H
Zorn J
Rougier H
Crevecoeur I
Semal P
Abrams G
Devièse T
Pirson S
Di Modica K
Cattelain P
Draily C
Toussaint M
De Groote I
Welker F
Posth C
Soressi M
Hublin JJ
Krause J
Pääbo S
Meyer M
Kelso J
Peter BM
Hajdinjak M

Animals
Female
Humans
Male
Belgium
France
Gene Flow
Genetic Variation
History, Ancient
Neanderthals
Phylogeny


Regulation of NAT1 activity in modern humans by a novel phosphorylation site

Abstract: N-acetyltransferase 1 (NAT1) is an enzyme that acetylates certain drugs and carcinogens and is involved in folate metabolism. Some NAT1 variants are associated with susceptibility to cancer and birth defects. We show that while nearly all present-day humans carry a valine residue at position 149 and a serine residue at position 214, some carry the ancestral amino acids isoleucine and alanine at these positions because of gene flow from Neanderthals. Neither substitution affects enzymatic activity. However, replacing the serine residue, a known phosphorylation site, with a phosphomimetic aspartate reduces NAT1 activity by ~60%. Phosphorylation at this site may allow NAT1 to be down-regulated during pregnancy to reduce birth defect risk, while maintaining high activity in adults for xenobiotic clearance. This regulatory site is a molecular feature that differs between modern humans and Neanderthals.

Fast L
Alberro ML
Rakava E
Ågren R
Riesenberg S
Huttner WB
Kelso J
Pääbo S
Zeberg H

Humans
Phosphorylation
Animals
Arylamine N-Acetyltransferase
Isoenzymes
Neanderthals
Female


A high-coverage Neandertal genome from the Altai Mountains reveals population structure among Neandertals

Abstract: We present a genome sequenced to ~37-fold genomic coverage from an approximately 110,000-y-old male Neandertal from Denisova Cave in the Altai Mountains and analyze it together with previously published Neandertal genomes of high quality. We show that he belonged to a population more closely related to a ~120,000-y-old Neandertal from Denisova Cave than to Neandertals in Europe or to a ~80,000-y-old Neandertal from Chagyrskaya Cave in the Altai Mountains. Both Neandertals from Denisova Cave show evidence of gene flow from Denisovans, a pattern not seen in later Neandertals from the Altai region or from Western Europe. The extent of chromosomal regions of homozygosity in Neandertals from the Altai region between 120,000 and 80,000 y ago indicates that they lived in smaller and more isolated groups than later Neandertals in Europe (54,000 to 40,000 y ago). We estimate the extent of allele frequency differentiation among Neandertal populations and find that the older Eastern Neandertals in the Altai region and younger Western Neandertals in Europe were as differentiated as the most differentiated present-day human populations worldwide.

Proceedings of the National Academy of Sciences of the United States of America

Massilani D
Peyrégne S
Iasi LNM
de Filippo C
Mafessoni F
Bossoms Mesa A
Sümer AP
Swiel Y
Popli D
Silverman S
Boyle MJ
Kozlikin MB
Shunkov MV
Derevianko AP
Higham T
Douka K
Meyer M
Zeberg H
Kelso J
Pääbo S

Animals
Neanderthals
Male
Gene Flow
Genome
Humans
Gene Frequency
Genetics, Population


The evolution of gene regulation in mammalian cerebellum development

Abstract: Gene regulatory changes are considered major drivers of evolutionary innovations, including the cerebellum's expansion during human evolution, yet they remain largely unexplored. In this study, we combined single-nucleus measurements of gene expression and chromatin accessibility from six mammals (human, bonobo, macaque, marmoset, mouse, and opossum) to uncover conserved and diverged regulatory networks in cerebellum development. We identified core regulators of cell identity and developed sequence-based models that revealed conserved regulatory codes. By predicting chromatin accessibility across 240 mammalian species, we reconstructed the evolutionary histories of human cis-regulatory elements, identifying sets associated with positive selection and gene expression changes, including the recent gain of THRB expression in cerebellar progenitor cells. Collectively, our work reveals the shared and mammalian lineage-specific regulatory programs governing cerebellum development.

Sarropoulos I
Sepp M
Yamada T
Schäfer PSL
Trost N
Schmidt J
Schneider C
Drummer C
Mißbach S
Taskiran II
Hecker N
Bravo González-Blas C
Frömel R
Joshi P
Leushkin E
Arnskötter F
Leiss K
Okonechnikov K
Lisgo S
Palkovits M
Pääbo S
Cardoso-Moreira M
Kutscher LM
Behr R
Pfister SM
Aerts S
Kaessmann H

Gene Expression Regulation, Developmental


Search-and-remove genome editing allows selection of cells by DNA sequence

Abstract: The selection of cells that have acquired a desired gene edit is often done by the introduction of additional genes that confer drug resistance or encode fluorophores. However, such marker genes can have unintended physiological effects and are not compatible with editing of single nucleotides. Here, we present SNIPE, a method that allows the marker-free selection of edited cells based on single nucleotide differences to unedited cells. SNIPE drastically enriches for cells, which have been precisely edited (median 7-fold). We validate the approach for 42 different edits using Cas9 or Cas12a in different cell types and species. We use it to enrich for combinations of substitutions that change missense mutations carried by all people today back to the ancestral state seen in Neandertals and Denisovans. We also show that it can be used to kill cultured tumor cells with aberrant genotypes and to repair heterozygous tumorigenic mutations.

Fast L
Omar M
Kanis P
Schaffer T
Chowdhury D
Rakava E
Pääbo S
Riesenberg S

Gene Editing
Humans
Animals
CRISPR-Cas Systems
Neanderthals
Mutation, Missense
CRISPR-Associated Protein 9
Base Sequence
Cell Line, Tumor


Denisovan mitochondrial DNA from dental calculus of the >146,000-year-old Harbin cranium

Abstract: Denisovans have yet to be directly associated with a hominin cranium, limiting our understanding of their morphology and geographical distribution. We have attempted to retrieve DNA from a nearly complete Middle Pleistocene cranium from Harbin (>146 ka), northeastern China. Although no DNA could be retrieved from a tooth or the petrous bone, mitochondrial DNA (mtDNA) could be isolated from dental calculus. The mtDNA falls within Denisovan mtDNA variation and is related to an mtDNA branch carried by early Denisovan individuals in southern Siberia, previously observed in Denisova Cave. This suggests that Denisovans inhabited a large geographical range in Asia in the Middle Pleistocene. The association of Denisovan mtDNA with the Harbin cranium allows a better understanding of the morphological relationships between Denisovans and other East Asian Middle Pleistocene fossils. Furthermore, the retrieval of host DNA from dental calculus opens new possibilities for genetic research on Middle Pleistocene hominins.

Fu Q
Cao P
Dai Q
Bennett EA
Feng X
Yang MA
Ping W
Pääbo S
Ji Q

DNA, Mitochondrial
Dental Calculus
Animals
Fossils
Hominidae
Skull
China
Phylogeny
DNA, Ancient
Humans


The activity and expression of adenylosuccinate lyase were reduced during modern human evolution, affecting brain and behavior

Abstract: Adenylosuccinate lyase (ADSL), an enzyme that is crucial for purine biosynthesis, carries an amino acid substitution that is present in almost all humans today but absent in Neandertals and Denisovans. This substitution reduces the stability of the enzyme, but what functional consequences it has are unknown. Here, we show that when introduced into mice, this substitution causes substrates of the enzyme to accumulate in amounts that correlate negatively with ADSL expression levels. In the brain, where the expression of the enzyme is low, the substitution results in particularly high substrate levels. When the behavior of the mice is analyzed, female mice expressing the modern human-like version of ADSL access water more efficiently for drinking than their wild-type littermates. In addition to the amino acid substitution, a haplotype in the ADSL gene occurs at a carrier frequency of >97% in present-day humans and exhibits evidence of positive selection. It is associated with less ADSL expression as well as with increased concentrations of succinyladenosine, one of the substrates of the enzyme, in cerebrospinal fluid. Thus, two genetic changes have reduced ADSL activity in human tissues since modern and archaic humans separated, affecting purine biosynthesis, particularly in the brain.

Ju XC
Lee SY
Ågren R
Machado LC
Xing J
Azama C
Roy MC
Endo T
Huttner W
Siepel A
Fukunaga I
Zeberg H
Pääbo S

Adenylosuccinate Lyase
Animals
Humans
Brain
Mice
Female
Evolution, Molecular
Amino Acid Substitution
Biological Evolution
Behavior, Animal


Muscle AMP deaminase activity was lower in Neandertals than in modern humans

Abstract: The enzyme AMPD1 is expressed in skeletal muscle and is involved in ATP production. All available Neandertal genomes carry a lysine-to-isoleucine substitution at position 287 in AMPD1. This variant, which occurs at an allele frequency of 0-8% outside Africa, was introduced to modern humans by gene flow from Neandertals. Here, we show that the catalytic activity of the purified Neandertal AMPD1 is ~25% lower than the ancestral enzyme, and when introduced in mice, it reduces AMPD activity in muscle extracts by ~80%. Among present-day Europeans, another AMPD1 variant encoding a stop codon occurs at an allele frequency of 9-14%. Individuals heterozygous for this variant are less likely to be top-performing athletes in various sports, but otherwise reduced AMPD1 activity is well tolerated in present-day humans. While being conserved among vertebrates, AMPD1 seems to have become less functionally important among Neandertals and modern humans.

Macak D
Lee SY
Nyman T
Ampah-Korsah H
Strandback E
Pääbo S
Zeberg H

Animals
Neanderthals
Humans
AMP Deaminase
Muscle, Skeletal
Mice
Gene Frequency


Pleistocene chronology and history of hominins and fauna at Denisova Cave

Abstract: Denisova Cave in southern Siberia is the only site known to have been occupied by Denisovans, Neanderthals and modern humans. The cave consists of three chambers (Main, East and South), with the archaeological assemblages and remains of hominins, fauna and flora recovered from Main and East Chambers being the most thoroughly investigated to date. Here we report the results of analyses of the Palaeolithic artefacts, faunal remains and hominin and mammalian mitochondrial (mt) DNA recovered from renewed excavations in South Chamber. We construct a calendar-year time scale for the stratified Pleistocene deposits from optical dating of the sediments. The timing of hominin occupation and major turnovers in the mtDNA of Denisovans and large mammals largely accords with the patterns detected in Main and East Chambers. Time gaps in those sequences are partly filled by the South Chamber data and the sediment DNA record of Denisovans after 80,000 years ago is more than doubled in size. We combine the sediment dating and DNA records for all three chambers to reveal the whole-of-cave history of this unique site and the climatic conditions experienced by hominins and fauna over the past 300,000 years, including potential changes in habitat suitability for Denisovans and Neanderthals.

Jacobs Z
Zavala EI
Li B
O'Gorman K
Shunkov MV
Kozlikin MB
Derevianko AP
Uliyanov VA
Goldberg P
Agadjanian AK
Vasiliev SK
Brink F
Peyrégne S
Slon V
Pääbo S
Kelso J
Meyer M
Roberts RG

Animals
Caves
Siberia
Hominidae
DNA, Mitochondrial
Fossils
Humans
Neanderthals
Geologic Sediments
Archaeology
DNA, Ancient
Mammals
History, Ancient


The modern human aryl hydrocarbon receptor is more active when ancestralized by genome editing

Abstract: The aryl hydrocarbon receptor (AHR) is a transcription factor that has many functions in mammals. Its best known function is that it binds aromatic hydrocarbons and induces the expression of cytochrome P450 genes, which encode enzymes that metabolize aromatic hydrocarbons and other substrates. All present-day humans carry an amino acid substitution at position 381 in the AHR that occurred after the divergence of modern humans from Neandertals and Denisovans. Previous studies that have expressed the ancestral and modern versions of AHR from expression vectors have yielded conflicting results with regard to their activities. Here, we use genome editing to modify the endogenous AHR gene so that it encodes to the ancestral, Neandertal-like AHR protein in human cells. In the absence of exogenous ligands, the expression of AHR target genes is higher in cells expressing the ancestral AHR than in cells expressing the modern AHR, and similar to the expression in chimpanzee cells. Furthermore, the modern human AHR needs higher doses of three ligands than the ancestral AHR to induce the expression of target genes. Thus, the ability of AHR to induce the expression of many of its target genes is reduced in modern humans.

Helmbrecht N
Lackner M
Maricic T
Pääbo S

Receptors, Aryl Hydrocarbon
Humans
Gene Editing
Animals
Basic Helix-Loop-Helix Proteins
Evolution, Molecular
Pan troglodytes
Neanderthals
Ligands


Functional synergy of a human-specific and an ape-specific metabolic regulator in human neocortex development

Abstract: Metabolism has recently emerged as a major target of genes implicated in the evolutionary expansion of human neocortex. One such gene is the human-specific gene ARHGAP11B. During human neocortex development, ARHGAP11B increases the abundance of basal radial glia, key progenitors for neocortex expansion, by stimulating glutaminolysis (glutamine-to-glutamate-to-alpha-ketoglutarate) in mitochondria. Here we show that the ape-specific protein GLUD2 (glutamate dehydrogenase 2), which also operates in mitochondria and converts glutamate-to-αKG, enhances ARHGAP11B's ability to increase basal radial glia abundance. ARHGAP11B + GLUD2 double-transgenic bRG show increased production of aspartate, a metabolite essential for cell proliferation, from glutamate via alpha-ketoglutarate and the TCA cycle. Hence, during human evolution, a human-specific gene exploited the existence of another gene that emerged during ape evolution, to increase, via concerted changes in metabolism, progenitor abundance and neocortex size.

Xing L
Gkini V
Nieminen AI
Zhou HC
Aquilino M
Naumann R
Reppe K
Tanaka K
Carmeliet P
Heikinheimo O
Pääbo S
Huttner WB
Namba T

Neocortex
Humans
Animals
Glutamate Dehydrogenase
GTPase-Activating Proteins
Ketoglutaric Acids
Neuroglia
Glutamic Acid
Mitochondria
Mice
Citric Acid Cycle
Female

Journal Article
Research Support, Non-U.S. Gov't


The genetic changes that shaped Neandertals, Denisovans, and modern humans

Abstract: Modern human ancestors diverged from the ancestors of Neandertals and Denisovans about 600,000 years ago. Until about 40,000 years ago, these three groups existed in parallel, occasionally met, and exchanged genes. A critical question is why modern humans, and not the other two groups, survived, became numerous, and developed complex cultures. Here, we discuss genetic differences among the groups and some of their functional consequences. As more present-day genome sequences become available from diverse groups, we predict that very few, if any, differences will distinguish all modern humans from all Neandertals and Denisovans. We propose that the genetic basis of what constitutes a modern human is best thought of as a combination of genetic features, where perhaps none of them is present in each and every present-day individual.

Animals
Humans
Neanderthals
Research
Hominidae
Human Genetics


Efficient high-precision homology-directed repair-dependent genome editing by HDRobust

Abstract: Homology-directed repair (HDR), a method for repair of DNA double-stranded breaks can be leveraged for the precise introduction of mutations supplied by synthetic DNA donors, but remains limited by low efficiency and off-target effects. In this study, we report HDRobust, a high-precision method that, via the combined transient inhibition of nonhomologous end joining and microhomology-mediated end joining, resulted in the induction of point mutations by HDR in up to 93% (median 60%, s.e.m. 3) of chromosomes in populations of cells. We found that, using this method, insertions, deletions and rearrangements at the target site, as well as unintended changes at other genomic sites, were largely abolished. We validated this approach for 58 different target sites and showed that it allows efficient correction of pathogenic mutations in cells derived from patients suffering from anemia, sickle cell disease and thrombophilia.

Riesenberg S
Kanis P
Macak D
Wollny D
Düsterhöft D
Kowalewski J
Helmbrecht N
Maricic T
Pääbo S

Humans
Gene Editing
CRISPR-Cas Systems
Recombinational DNA Repair
DNA Breaks, Double-Stranded
DNA End-Joining Repair
DNA


Major Genetic Risk Factors for Dupuytren's Disease Are Inherited From Neandertals

Abstract: Dupuytren's disease is characterized by fingers becoming permanently bent in a flexed position. Whereas people of African ancestry are rarely afflicted by Dupuytren's disease, up to ∼30% of men over 60 years suffer from this condition in northern Europe. Here, we meta-analyze 3 biobanks comprising 7,871 cases and 645,880 controls and find 61 genome-wide significant variants associated with Dupuytren's disease. We show that 3 of the 61 loci harbor alleles of Neandertal origin, including the second and third most strongly associated ones (P = 6.4 × 10-132 and P = 9.2 × 10-69, respectively). For the most strongly associated Neandertal variant, we identify EPDR1 as the causal gene. Dupuytren's disease is an example of how admixture with Neandertals has shaped regional differences in disease prevalence.

Ågren R
Patil S
Zhou X
FinnGen.
Sahlholm K
Pääbo S
Zeberg H

Animals
Humans
Male
Alleles
Dupuytren Contracture
Neanderthals
Risk Factors


Ancient human DNA recovered from a Palaeolithic pendant

Abstract: Artefacts made from stones, bones and teeth are fundamental to our understanding of human subsistence strategies, behaviour and culture in the Pleistocene. Although these resources are plentiful, it is impossible to associate artefacts to specific human individuals1 who can be morphologically or genetically characterized, unless they are found within burials, which are rare in this time period. Thus, our ability to discern the societal roles of Pleistocene individuals based on their biological sex or genetic ancestry is limited2-5. Here we report the development of a non-destructive method for the gradual release of DNA trapped in ancient bone and tooth artefacts. Application of the method to an Upper Palaeolithic deer tooth pendant from Denisova Cave, Russia, resulted in the recovery of ancient human and deer mitochondrial genomes, which allowed us to estimate the age of the pendant at approximately 19,000-25,000 years. Nuclear DNA analysis identifies the presumed maker or wearer of the pendant as a female individual with strong genetic affinities to a group of Ancient North Eurasian individuals who lived around the same time but were previously found only further east in Siberia. Our work redefines how cultural and genetic records can be linked in prehistoric archaeology.

Essel E
Zavala EI
Schulz-Kornas E
Kozlikin MB
Fewlass H
Vernot B
Shunkov MV
Derevianko AP
Douka K
Barnes I
Soulier MC
Schmidt A
Szymanski M
Tsanova T
Sirakov N
Endarova E
McPherron SP
Hublin JJ
Kelso J
Pääbo S
Hajdinjak M
Soressi M
Meyer M

Animals
Female
Humans
Archaeology
Bone and Bones
Deer
DNA, Ancient
DNA, Mitochondrial
History, Ancient
Siberia
Tooth
Caves
Russia

Historical Article
Journal Article
Research Support, Non-U.S. Gov't


Response to Comment on "Human TKTL1 implies greater neurogenesis in frontal neocortex of modern humans than Neanderthals"

Abstract: Herai et al. discuss the known fact that a low percentage of modern humans who lack any overt phenotypes carry the ancestral TKTL1 allele. Our paper demonstrates that the amino acid substitution in TKTL1 increases neural progenitor cells and neurogenesis in the developing brain. It is another question if, and to what extent, this has consequences for the adult brain.

Pinson A
Maricic T
Zeberg H
Pääbo S
Huttner WB

Animals
Humans
Neanderthals
Neocortex
Neural Stem Cells
Neurogenesis
Transketolase


Functional dissection of two amino acid substitutions unique to the human FOXP2 protein

Abstract: The transcription factor forkhead box P2 (FOXP2) is involved in the development of language and speech in humans. Two amino acid substitutions (T303N, N325S) occurred in the human FOXP2 after the divergence from the chimpanzee lineage. It has previously been shown that when they are introduced into the FOXP2 protein of mice they alter striatal synaptic plasticity by increasing long-term depression in medium spiny neurons. Here we introduce each of these amino acid substitutions individually into mice and analyze their effects in the striatum. We find that long-term depression in medium spiny neurons is increased in mice carrying only the T303N substitution to the same extent as in mice carrying both amino acid substitutions. In contrast, the N325S substitution has no discernable effects.

Bornschein U
Zeberg H
Enard W
Hevers W
Pääbo S

Humans
Animals
Mice
Amino Acid Substitution
Blood Group Antigens
Corpus Striatum
Dissection
Extremities
Pan troglodytes
Forkhead Transcription Factors
Repressor Proteins


Detection of unintended on-target effects in CRISPR genome editing by DNA donors carrying diagnostic substitutions

Abstract: CRISPR nucleases can introduce double-stranded DNA breaks in genomes at positions specified by guide RNAs. When repaired by the cell, this may result in the introduction of insertions and deletions or nucleotide substitutions provided by exogenous DNA donors. However, cellular repair can also result in unintended on-target effects, primarily larger deletions and loss of heterozygosity due to gene conversion. Here we present a strategy that allows easy and reliable detection of unintended on-target effects as well as the generation of control cells that carry wild-type alleles but have demonstratively undergone genome editing at the target site. Our 'sequence-ascertained favorable editing' (SAFE) donor approach relies on the use of DNA donor mixtures containing the desired nucleotide substitutions or the wild-type alleles together with combinations of additional 'diagnostic' substitutions unlikely to have any effects. Sequencing of the target sites then results in that two different sequences are seen when both chromosomes are edited with 'SAFE' donors containing different sets of substitutions, while a single sequence indicates unintended effects such as deletions or gene conversion. We analyzed more than 850 human embryonic stem cell clones edited with 'SAFE' donors and detect all copy number changes and almost all clones with gene conversion.

Lackner M
Helmbrecht N
Pääbo S
Riesenberg S

Clustered Regularly Interspaced Short Palindromic Repeats


The molecular evolution of spermatogenesis across mammals

Abstract: The testis produces gametes through spermatogenesis and evolves rapidly at both the morphological and molecular level in mammals1-6, probably owing to the evolutionary pressure on males to be reproductively successful7. However, the molecular evolution of individual spermatogenic cell types across mammals remains largely uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes from 11 species that cover the three main mammalian lineages (eutherians, marsupials and monotremes) and birds (the evolutionary outgroup), and include seven primates. We find that the rapid evolution of the testis was driven by accelerated fixation rates of gene expression changes, amino acid substitutions and new genes in late spermatogenic stages, probably facilitated by reduced pleiotropic constraints, haploid selection and transcriptionally permissive chromatin. We identify temporal expression changes of individual genes across species and conserved expression programs controlling ancestral spermatogenic processes. Genes predominantly expressed in spermatogonia (germ cells fuelling spermatogenesis) and Sertoli (somatic support) cells accumulated on X chromosomes during evolution, presumably owing to male-beneficial selective forces. Further work identified transcriptomal differences between X- and Y-bearing spermatids and uncovered that meiotic sex-chromosome inactivation (MSCI) also occurs in monotremes and hence is common to mammalian sex-chromosome systems. Thus, the mechanism of meiotic silencing of unsynapsed chromatin, which underlies MSCI, is an ancestral mammalian feature. Our study illuminates the molecular evolution of spermatogenesis and associated selective forces, and provides a resource for investigating the biology of the testis across mammals.

Murat F
Mbengue N
Winge SB
Trefzer T
Leushkin E
Sepp M
Cardoso-Moreira M
Schmidt J
Schneider C
Mößinger K
Brüning T
Lamanna F
Belles MR
Conrad C
Kondova I
Bontrop R
Behr R
Khaitovich P
Pääbo S
Marques-Bonet T
Grützner F
Almstrup K
Schierup MH
Kaessmann H

Animals
Male
Chromatin
Evolution, Molecular
Mammals
Meiosis
Spermatogenesis
Testis
Transcriptome
Single-Cell Analysis
Birds
Primates
Gene Expression Regulation
Spermatogonia
Sertoli Cells
X Chromosome
Y Chromosome
Dosage Compensation, Genetic
Gene Silencing

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't


Genetic insights into the social organization of Neanderthals

Abstract: Genomic analyses of Neanderthals have previously provided insights into their population history and relationship to modern humans1-8, but the social organization of Neanderthal communities remains poorly understood. Here we present genetic data for 13 Neanderthals from two Middle Palaeolithic sites in the Altai Mountains of southern Siberia: 11 from Chagyrskaya Cave9,10 and 2 from Okladnikov Cave11-making this one of the largest genetic studies of a Neanderthal population to date. We used hybridization capture to obtain genome-wide nuclear data, as well as mitochondrial and Y-chromosome sequences. Some Chagyrskaya individuals were closely related, including a father-daughter pair and a pair of second-degree relatives, indicating that at least some of the individuals lived at the same time. Up to one-third of these individuals' genomes had long segments of homozygosity, suggesting that the Chagyrskaya Neanderthals were part of a small community. In addition, the Y-chromosome diversity is an order of magnitude lower than the mitochondrial diversity, a pattern that we found is best explained by female migration between communities. Thus, the genetic data presented here provide a detailed documentation of the social organization of an isolated Neanderthal community at the easternmost extent of their known range.

Skov L
Peyrégne S
Popli D
Iasi LNM
Devièse T
Slon V
Zavala EI
Hajdinjak M
Sümer AP
Grote S
Bossoms Mesa A
López Herráez D
Nickel B
Nagel S
Richter J
Essel E
Gansauge M
Schmidt A
Korlević P
Comeskey D
Derevianko AP
Kharevich A
Markin SV
Talamo S
Douka K
Krajcarz MT
Roberts RG
Higham T
Viola B
Krivoshapkin AI
Kolobova KA
Kelso J
Meyer M
Pääbo S
Peter BM

Animals
Female
Humans
Caves
Genome
Hybridization, Genetic
Neanderthals
Siberia
DNA, Mitochondrial
Y Chromosome
Male
Family
Homozygote


Human TKTL1 implies greater neurogenesis in frontal neocortex of modern humans than Neanderthals

Abstract: Neanderthal brains were similar in size to those of modern humans. We sought to investigate potential differences in neurogenesis during neocortex development. Modern human transketolase-like 1 (TKTL1) differs from Neanderthal TKTL1 by a lysine-to-arginine amino acid substitution. Using overexpression in developing mouse and ferret neocortex, knockout in fetal human neocortical tissue, and genome-edited cerebral organoids, we found that the modern human variant, hTKTL1, but not the Neanderthal variant, increases the abundance of basal radial glia (bRG) but not that of intermediate progenitors (bIPs). bRG generate more neocortical neurons than bIPs. The hTKTL1 effect requires the pentose phosphate pathway and fatty acid synthesis. Inhibition of these metabolic pathways reduces bRG abundance in fetal human neocortical tissue. Our data suggest that neocortical neurogenesis in modern humans differs from that in Neanderthals.

Pinson A
Xing L
Namba T
Kalebic N
Peters J
Oegema CE
Traikov S
Reppe K
Riesenberg S
Maricic T
Derihaci R
Wimberger P
Pääbo S
Huttner WB

Animals
Ependymoglial Cells
Ferrets
Humans
Mice
Neanderthals
Neocortex
Neurogenesis
Transketolase


Longer metaphase and fewer chromosome segregation errors in modern human than Neanderthal brain development

Abstract: Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals.

Mora-Bermúdez F
Kanis P
Macak D
Peters J
Naumann R
Xing L
Sarov M
Winkler S
Oegema CE
Haffner C
Wimberger P
Riesenberg S
Maricic T
Huttner WB
Pääbo S

Animals
Brain
Chromosome Segregation
Hominidae
Humans
Kinesins
Metaphase
Mice
Neanderthals


The evolutionary history of human spindle genes includes back-and-forth gene flow with Neandertals

Abstract: Proteins associated with the spindle apparatus, a cytoskeletal structure that ensures the proper segregation of chromosomes during cell division, experienced an unusual number of amino acid substitutions in modern humans after the split from the ancestors of Neandertals and Denisovans. Here, we analyze the history of these substitutions and show that some of the genes in which they occur may have been targets of positive selection. We also find that the two changes in the kinetochore scaffold 1 (KNL1) protein, previously believed to be specific to modern humans, were present in some Neandertals. We show that the KNL1 gene of these Neandertals shared a common ancestor with present-day Africans about 200,000 years ago due to gene flow from the ancestors (or relatives) of modern humans into Neandertals. Subsequently, some non-Africans inherited this modern human-like gene variant from Neandertals, but none inherited the ancestral gene variants. These results add to the growing evidence of early contacts between modern humans and archaic groups in Eurasia and illustrate the intricate relationships among these groups.

Animals
Biological Evolution
Black People
Fossils
Gene Flow
Hominidae
Humans
Neanderthals


The clinically relevant CYP2C8*3 and CYP2C9*2 haplotype is inherited from Neandertals

Abstract: Genetic variation in genes encoding cytochrome P450 enzymes influences the metabolism of drugs and endogenous compounds. The locus containing the cytochrome genes CYP2C8 and CYP2C9 on chromosome 10 exhibits linkage disequilibrium between the CYP2C8*3 and CYP2C9*2 alleles, forming a haplotype of ~300 kilobases. This haplotype is associated with altered metabolism of several drugs, most notably reduced metabolism of warfarin and phenytoin, leading to toxicity at otherwise therapeutic doses. Here we show that this haplotype is inherited from Neandertals.

Haeggström S
Ingelman-Sundberg M
Pääbo S
Zeberg H

Animals
Aryl Hydrocarbon Hydroxylases
Cytochrome P-450 CYP2C8
Cytochrome P-450 CYP2C9
Gene Frequency
Haplotypes
Humans
Neanderthals


High-throughput muscle fiber typing from RNA sequencing data

Abstract: Skeletal muscle fiber type distribution has implications for human health, muscle function, and performance. This knowledge has been gathered using labor-intensive and costly methodology that limited these studies. Here, we present a method based on muscle tissue RNA sequencing data (totRNAseq) to estimate the distribution of skeletal muscle fiber types from frozen human samples, allowing for a larger number of individuals to be tested. By using single-nuclei RNA sequencing (snRNAseq) data as a reference, cluster expression signatures were produced by averaging gene expression of cluster gene markers and then applying these to totRNAseq data and inferring muscle fiber nuclei type via linear matrix decomposition. This estimate was then compared with fiber type distribution measured by ATPase staining or myosin heavy chain protein isoform distribution of 62 muscle samples in two independent cohorts (n = 39 and 22). The correlation between the sequencing-based method and the other two were rATPas = 0.44 [0.13-0.67], [95% CI], and rmyosin = 0.83 [0.61-0.93], with p = 5.70 × 10-3 and 2.00 × 10-6, respectively. The deconvolution inference of fiber type composition was accurate even for very low totRNAseq sequencing depths, i.e., down to an average of ~ 10,000 paired-end reads. This new method ( https://github.com/OlaHanssonLab/PredictFiberType ) consequently allows for measurement of fiber type distribution of a larger number of samples using totRNAseq in a cost and labor-efficient way. It is now feasible to study the association between fiber type distribution and e.g. health outcomes in large well-powered studies.

Oskolkov N
Santel M
Parikh HM
Ekström O
Camp GJ
Miyamoto-Mikami E
Ström K
Mir BA
Kryvokhyzha D
Lehtovirta M
Kobayashi H
Kakigi R
Naito H
Eriksson KF
Nystedt B
Fuku N
Treutlein B
Pääbo S
Hansson O

Base Sequence
Humans
Muscle Fibers, Skeletal
RNA
Sequence Analysis, RNA
Exome Sequencing


Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage

Abstract: The first step in CRISPR-Cas9-mediated genome editing is the cleavage of target DNA sequences that are complementary to so-called spacer sequences in CRISPR guide RNAs (gRNAs). However, some DNA sequences are refractory to CRISPR-Cas9 cleavage, which is at least in part due to gRNA misfolding. To overcome this problem, we have engineered gRNAs with highly stable hairpins in their constant parts and further enhanced their stability by chemical modifications. The 'Genome-editing Optimized Locked Design' (GOLD)-gRNA increases genome editing efficiency up to around 1000-fold (from 0.08 to 80.5%) with a mean increase across different other targets of 7.4-fold. We anticipate that this improved gRNA will allow efficient editing regardless of spacer sequence composition and will be especially useful if a desired genomic site is difficult to edit.

Riesenberg S
Helmbrecht N
Kanis P
Maricic T
Pääbo S

CRISPR-Cas Systems
Cell Line
Gene Editing
Genome
Humans
Nucleic Acid Conformation
Oligonucleotides
RNA, Guide, CRISPR-Cas Systems


A substitution in the glutathione reductase lowers electron leakage and inflammation in modern humans

Abstract: Glutathione reductase is a critical enzyme for preventing oxidative stress and maintaining a reduced intracellular environment. Almost all present-day humans carry an amino acid substitution (S232G) in this enzyme relative to apes and Neanderthals. We express the modern human and the ancestral enzymes and show that whereas the activity and stability are unaffected by the amino acid substitution, the ancestral enzyme produces more reactive oxygen species and increases cellular levels of transcripts encoding cytokines. We furthermore show that the ancestral enzyme has been reintroduced into the modern human gene pool by gene flow from Neanderthals and is associated with multiple traits in present-day people, including increased susceptibility for inflammatory-associated disorders and vascular disease.

Coppo L
Mishra P
Siefert N
Holmgren A
Pääbo S
Zeberg H


Microstratigraphic preservation of ancient faunal and hominin DNA in Pleistocene cave sediments

Abstract: Ancient DNA recovered from Pleistocene sediments represents a rich resource for the study of past hominin and environmental diversity. However, little is known about how DNA is preserved in sediments and the extent to which it may be translocated between archaeological strata. Here, we investigate DNA preservation in 47 blocks of resin-impregnated archaeological sediment collected over the last four decades for micromorphological analyses at 13 prehistoric sites in Europe, Asia, Africa, and North America and show that such blocks can preserve DNA of hominins and other mammals. Extensive microsampling of sediment blocks from Denisova Cave in the Altai Mountains reveals that the taxonomic composition of mammalian DNA differs drastically at the millimeter-scale and that DNA is concentrated in small particles, especially in fragments of bone and feces (coprolites), suggesting that these are substantial sources of DNA in sediments. Three microsamples taken in close proximity in one of the blocks yielded Neanderthal DNA from at least two male individuals closely related to Denisova 5, a Neanderthal toe bone previously recovered from the same layer. Our work indicates that DNA can remain stably localized in sediments over time and provides a means of linking genetic information to the archaeological and ecological records on a microstratigraphic scale.

Massilani D
Morley MW
Mentzer SM
Aldeias V
Vernot B
Miller C
Stahlschmidt M
Kozlikin MB
Shunkov MV
Derevianko AP
Conard NJ
Wurz S
Henshilwood CS
Vasquez J
Essel E
Nagel S
Richter J
Nickel B
Roberts RG
Pääbo S
Slon V
Goldberg P
Meyer M

Animals
Caves
DNA, Ancient
Fossils
Hominidae
Neanderthals


The earliest Denisovans and their cultural adaptation

Abstract: Since the initial identification of the Denisovans a decade ago, only a handful of their physical remains have been discovered. Here we analysed ~3,800 non-diagnostic bone fragments using collagen peptide mass fingerprinting to locate new hominin remains from Denisova Cave (Siberia, Russia). We identified five new hominin bones, four of which contained sufficient DNA for mitochondrial analysis. Three carry mitochondrial DNA of the Denisovan type and one was found to carry mtDNA of the Neanderthal type. The former come from the same archaeological layer near the base of the cave's sequence and are the oldest securely dated evidence of Denisovans at 200 ka (thousand years ago) (205-192 ka at 68.2% or 217-187 ka at 95% probability). The stratigraphic context in which they were located contains a wealth of archaeological material in the form of lithics and faunal remains, allowing us to determine the material culture associated with these early hominins and explore their behavioural and environmental adaptations. The combination of bone collagen fingerprinting and genetic analyses has so far more-than-doubled the number of hominin bones at Denisova Cave and has expanded our understanding of Denisovan and Neanderthal interactions, as well as their archaeological signatures.

Brown S
Massilani D
Kozlikin MB
Shunkov MV
Derevianko AP
Stoessel A
Jope-Street B
Meyer M
Kelso J
Pääbo S
Higham T
Douka K

Animals
Archaeology
Caves
DNA, Mitochondrial
Hominidae
Neanderthals


Comment on "Reintroduction of the archaic variant of NOVA1 in cortical organoids alters neurodevelopment"

Abstract: Trujillo et al. (Research Articles, 12 February 2021, eaax2537) conclude that the reintroduction of an ancestral amino acid substitution in the protein NOVA1 drastically alters the development of brain organoids. We show that cell lines used by the authors carry heterozygous deletions of the target DNA sequence, providing another plausible explanation for the effects observed.

Maricic T
Helmbrecht N
Riesenberg S
Macak D
Kanis P
Lackner M
Pugach-Matveeva AD
Pääbo S


Pleistocene sediment DNA reveals hominin and faunal turnovers at Denisova Cave

Abstract: Denisova Cave in southern Siberia is the type locality of the Denisovans, an archaic hominin group who were related to Neanderthals1-4. The dozen hominin remains recovered from the deposits also include Neanderthals5,6 and the child of a Neanderthal and a Denisovan7, which suggests that Denisova Cave was a contact zone between these archaic hominins. However, uncertainties persist about the order in which these groups appeared at the site, the timing and environmental context of hominin occupation, and the association of particular hominin groups with archaeological assemblages5,8-11. Here we report the analysis of DNA from 728 sediment samples that were collected in a grid-like manner from layers dating to the Pleistocene epoch. We retrieved ancient faunal and hominin mitochondrial (mt)DNA from 685 and 175 samples, respectively. The earliest evidence for hominin mtDNA is of Denisovans, and is associated with early Middle Palaeolithic stone tools that were deposited approximately 250,000 to 170,000 years ago; Neanderthal mtDNA first appears towards the end of this period. We detect a turnover in the mtDNA of Denisovans that coincides with changes in the composition of faunal mtDNA, and evidence that Denisovans and Neanderthals occupied the site repeatedly-possibly until, or after, the onset of the Initial Upper Palaeolithic at least 45,000 years ago, when modern human mtDNA is first recorded in the sediments.

Zavala EI
Jacobs Z
Vernot B
Shunkov MV
Kozlikin MB
Derevianko AP
Essel E
de Fillipo C
Nagel S
Richter J
Romagné F
Schmidt A
Li B
O'Gorman K
Slon V
Kelso J
Pääbo S
Roberts RG
Meyer M

Animals
Archaeology
Caves
DNA, Ancient
DNA, Mitochondrial
Fossils
Geologic Sediments
History, Ancient
Hominidae
Neanderthals
Siberia


Reduced purine biosynthesis in humans after their divergence from Neandertals

Abstract: We analyze the metabolomes of humans, chimpanzees, and macaques in muscle, kidney and three different regions of the brain. Although several compounds in amino acid metabolism occur at either higher or lower concentrations in humans than in the other primates, metabolites downstream of adenylosuccinate lyase, which catalyzes two reactions in purine synthesis, occur at lower concentrations in humans. This enzyme carries an amino acid substitution that is present in all humans today but absent in Neandertals. By introducing the modern human substitution into the genomes of mice, as well as the ancestral, Neandertal-like substitution into the genomes of human cells, we show that this amino acid substitution contributes to much or all of the reduction of de novo synthesis of purines in humans.

Stepanova V
Moczulska KE
Vacano GN
Kurochkin I
Ju X
Riesenberg S
Macak D
Maricic T
Dombrowski L
Schörnig M
Anastassiadis K
Baker O
Naumann R
Khrameeva E
Vanushkina A
Stekolshchikova E
Egorova A
Tkachev A
Mazzarino R
Duval N
Zubkov D
Giavalisco P
Wilkinson TG
Patterson D
Khaitovich P
Pääbo S

Animals
Biosynthetic Pathways
Female
Gene Editing
Humans
Macaca
Male
Metabolome
Mice
Mice, Transgenic
Mutation, Missense
Neanderthals
Pan troglodytes
Purines


Initial Upper Palaeolithic humans in Europe had recent Neanderthal ancestry

Abstract: Modern humans appeared in Europe by at least 45,000 years ago1-5, but the extent of their interactions with Neanderthals, who disappeared by about 40,000 years ago6, and their relationship to the broader expansion of modern humans outside Africa are poorly understood. Here we present genome-wide data from three individuals dated to between 45,930 and 42,580 years ago from Bacho Kiro Cave, Bulgaria1,2. They are the earliest Late Pleistocene modern humans known to have been recovered in Europe so far, and were found in association with an Initial Upper Palaeolithic artefact assemblage. Unlike two previously studied individuals of similar ages from Romania7 and Siberia8 who did not contribute detectably to later populations, these individuals are more closely related to present-day and ancient populations in East Asia and the Americas than to later west Eurasian populations. This indicates that they belonged to a modern human migration into Europe that was not previously known from the genetic record, and provides evidence that there was at least some continuity between the earliest modern humans in Europe and later people in Eurasia. Moreover, we find that all three individuals had Neanderthal ancestors a few generations back in their family history, confirming that the first European modern humans mixed with Neanderthals and suggesting that such mixing could have been common.

Hajdinjak M
Mafessoni F
Skov L
Vernot B
Hübner A
Fu Q
Essel E
Nagel S
Nickel B
Richter J
Moldovan OT
Constantin S
Endarova E
Zahariev N
Spasov R
Welker F
Smith GM
Sinet-Mathiot V
Paskulin L
Fewlass H
Talamo S
Rezek Z
Sirakova S
Sirakov N
McPherron SP
Tsanova T
Hublin JJ
Peter BM
Meyer M
Skoglund P
Kelso J
Pääbo S

Alleles
Americas
Animals
Archaeology
Bulgaria
Caves
DNA, Ancient
Asia, Eastern
Female
Genome, Human
History, Ancient
Humans
Male
Neanderthals
Phylogeny


Point-of-care bulk testing for SARS-CoV-2 by combining hybridization capture with improved colorimetric LAMP

Abstract: Efforts to contain the spread of SARS-CoV-2 have spurred the need for reliable, rapid, and cost-effective diagnostic methods which can be applied to large numbers of people. However, current standard protocols for the detection of viral nucleic acids while sensitive, require a high level of automation and sophisticated laboratory equipment to achieve throughputs that allow whole communities to be tested on a regular basis. Here we present Cap-iLAMP (capture and improved loop-mediated isothermal amplification) which combines a hybridization capture-based RNA extraction of gargle lavage samples with an improved colorimetric RT-LAMP assay and smartphone-based color scoring. Cap-iLAMP is compatible with point-of-care testing and enables the detection of SARS-CoV-2 positive samples in less than one hour. In contrast to direct addition of the sample to improved LAMP (iLAMP), Cap-iLAMP prevents false positives and allows single positive samples to be detected in pools of 25 negative samples, reducing the reagent cost per test to ~1 Euro per individual.

Bokelmann L
Nickel O
Maricic T
Pääbo S
Meyer M
Borte S
Riesenberg S

COVID-19
COVID-19 Testing
Colorimetry
Coronavirus Nucleocapsid Proteins
Humans
Molecular Diagnostic Techniques
Nucleic Acid Amplification Techniques
Nucleic Acid Hybridization
Phosphoproteins
Point-of-Care Testing
RNA, Viral
SARS-CoV-2
Sensitivity and Specificity


A genomic region associated with protection against severe COVID-19 is inherited from Neandertals

Abstract: It was recently shown that the major genetic risk factor associated with becoming severely ill with COVID-19 when infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is inherited from Neandertals. New, larger genetic association studies now allow additional genetic risk factors to be discovered. Using data from the Genetics of Mortality in Critical Care (GenOMICC) consortium, we show that a haplotype at a region on chromosome 12 associated with requiring intensive care when infected with the virus is inherited from Neandertals. This region encodes proteins that activate enzymes that are important during infections with RNA viruses. In contrast to the previously described Neandertal haplotype that increases the risk for severe COVID-19, this Neandertal haplotype is protective against severe disease. It also differs from the risk haplotype in that it has a more moderate effect and occurs at substantial frequencies in all regions of the world outside Africa. Among ancient human genomes in western Eurasia, the frequency of the protective Neandertal haplotype may have increased between 20,000 and 10,000 y ago and again during the past 1,000 y.

Animals
COVID-19
Chromosomes, Human, Pair 12
Evolution, Molecular
Genetic Predisposition to Disease
Haplotypes
Humans
Neanderthals
Quantitative Trait Loci


A direct RT-qPCR approach to test large numbers of individuals for SARS-CoV-2

Abstract: SARS-CoV-2 causes substantial morbidity and mortality in elderly and immunocompromised individuals, particularly in retirement homes, where transmission from asymptomatic staff and visitors may introduce the infection. Here we present a cheap and fast screening method based on direct RT-qPCR to detect SARS-CoV-2 in single or pooled gargle lavages ("mouthwashes"). This method detects individuals with large viral loads (Ct≤29) and we use it to test all staff at a nursing home daily over a period of three weeks in order to reduce the risk that the infection penetrates the facility. This or similar approaches can be implemented to protect hospitals, nursing homes and other institutions in this and future viral epidemics.

Maricic T
Nickel O
Aximu-Petri A
Essel E
Gansauge M
Kanis P
Macak D
Richter J
Riesenberg S
Bokelmann L
Zeberg H
Meyer M
Borte S
Pääbo S

COVID-19
COVID-19 Nucleic Acid Testing
Humans
Mass Screening
Real-Time Polymerase Chain Reaction
SARS-CoV-2

Clinical Trial
Journal Article
Research Support, Non-U.S. Gov't


Denisovan DNA in Late Pleistocene sediments from Baishiya Karst Cave on the Tibetan Plateau

Abstract: A late Middle Pleistocene mandible from Baishiya Karst Cave (BKC) on the Tibetan Plateau has been inferred to be from a Denisovan, an Asian hominin related to Neanderthals, on the basis of an amino acid substitution in its collagen. Here we describe the stratigraphy, chronology, and mitochondrial DNA extracted from the sediments in BKC. We recover Denisovan mitochondrial DNA from sediments deposited ~100 thousand and ~60 thousand years ago (ka) and possibly as recently as ~45 ka. The long-term occupation of BKC by Denisovans suggests that they may have adapted to life at high altitudes and may have contributed such adaptations to modern humans on the Tibetan Plateau.

Zhang D
Xia H
Chen F
Li B
Slon V
Cheng T
Yang R
Jacobs Z
Dai Q
Massilani D
Shen X
Wang J
Feng X
Cao P
Yang MA
Yao J
Yang J
Madsen DB
Han Y
Ping W
Liu F
Perreault C
Chen X
Meyer M
Kelso J
Pääbo S
Fu Q

Animals
Caves
DNA, Ancient
DNA, Mitochondrial
Geologic Sediments
Hominidae
Humans
Phylogeny
Tibet


Denisovan ancestry and population history of early East Asians

Abstract: We present analyses of the genome of a ~34,000-year-old hominin skull cap discovered in the Salkhit Valley in northeastern Mongolia. We show that this individual was a female member of a modern human population that, following the split between East and West Eurasians, experienced substantial gene flow from West Eurasians. Both she and a 40,000-year-old individual from Tianyuan outside Beijing carried genomic segments of Denisovan ancestry. These segments derive from the same Denisovan admixture event(s) that contributed to present-day mainland Asians but are distinct from the Denisovan DNA segments in present-day Papuans and Aboriginal Australians.

Massilani D
Skov L
Hajdinjak M
Gunchinsuren B
Tseveendorj D
Yi S
Lee J
Nagel S
Nickel B
Devièse T
Higham T
Meyer M
Kelso J
Peter BM
Pääbo S

Animals
Asian People
DNA, Ancient
Evolution, Molecular
Female
Hominidae
Humans
Mongolia
Population
Skull


The major genetic risk factor for severe COVID-19 is inherited from Neanderthals

Abstract: A recent genetic association study1 identified a gene cluster on chromosome 3 as a risk locus for respiratory failure after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A separate study (COVID-19 Host Genetics Initiative)2 comprising 3,199 hospitalized patients with coronavirus disease 2019 (COVID-19) and control individuals showed that this cluster is the major genetic risk factor for severe symptoms after SARS-CoV-2 infection and hospitalization. Here we show that the risk is conferred by a genomic segment of around 50 kilobases in size that is inherited from Neanderthals and is carried by around 50% of people in south Asia and around 16% of people in Europe.

Animals
Asia
COVID-19
Case-Control Studies
Chromosomes, Human, Pair 3
Europe
Genetic Predisposition to Disease
Genetic Variation
Genome-Wide Association Study
Haplotypes
Hospitalization
Humans
Linkage Disequilibrium
Multigene Family
Neanderthals
Phylogeny
Severe Acute Respiratory Syndrome


A Neanderthal Sodium Channel Increases Pain Sensitivity in Present-Day Humans

Abstract: The sodium channel Nav1.7 is crucial for impulse generation and conduction in peripheral pain pathways [1]. In Neanderthals, the Nav1.7 protein carried three amino acid substitutions (M932L, V991L, and D1908G) relative to modern humans. We expressed Nav1.7 proteins carrying all combinations of these substitutions and studied their electrophysiological effects. Whereas the single amino acid substitutions do not affect the function of the ion channel, the full Neanderthal variant carrying all three substitutions, as well as the combination of V991L with D1908G, shows reduced inactivation, suggesting that peripheral nerves were more sensitive to painful stimuli in Neanderthals than in modern humans. We show that, due to gene flow from Neanderthals, the three Neanderthal substitutions are found in ∼0.4% of present-day Britons, where they are associated with heightened pain sensitivity.

Zeberg H
Dannemann M
Sahlholm K
Tsuo K
Maricic T
Wiebe V
Hevers W
Robinson HPC
Kelso J
Pääbo S

Adult
Aged
Amino Acid Substitution
Animals
Female
Humans
Male
Middle Aged
Mutation
NAV1.7 Voltage-Gated Sodium Channel
Neanderthals
Pain
Xenopus laevis


Human Stem Cell Resources Are an Inroad to Neandertal DNA Functions

Abstract: Induced pluripotent stem cells (iPSCs) from diverse humans offer the potential to study human functional variation in controlled culture environments. A portion of this variation originates from an ancient admixture between modern humans and Neandertals, which introduced alleles that left a phenotypic legacy on individual humans today. Here, we show that a large iPSC repository harbors extensive Neandertal DNA, including alleles that contribute to human phenotypes and diseases, encode hundreds of amino acid changes, and alter gene expression in specific tissues. We provide a database of the inferred introgressed Neandertal alleles for each individual iPSC line, together with the annotation of the predicted functional variants. We also show that transcriptomic data from organoids generated from iPSCs can be used to track Neandertal-derived RNA over developmental processes. Human iPSC resources provide an opportunity to experimentally explore Neandertal DNA function and its contribution to present-day phenotypes, and potentially study Neandertal traits.

Dannemann M
He Z
Heide C
Vernot B
Sidow L
Kanton S
Weigert A
Treutlein B
Pääbo S
Kelso J
Camp JG

Alleles
Animals
Brain
Cell Line
DNA
Haplotypes
Humans
Neanderthals
Phenotype
Pluripotent Stem Cells
RNA
Stem Cells


A high-coverage Neandertal genome from Chagyrskaya Cave

Abstract: We sequenced the genome of a Neandertal from Chagyrskaya Cave in the Altai Mountains, Russia, to 27-fold genomic coverage. We show that this Neandertal was a female and that she was more related to Neandertals in western Eurasia [Prüfer et al., Science 358, 655-658 (2017); Hajdinjak et al., Nature 555, 652-656 (2018)] than to Neandertals who lived earlier in Denisova Cave [Prüfer et al., Nature 505, 43-49 (2014)], which is located about 100 km away. About 12.9% of the Chagyrskaya genome is spanned by homozygous regions that are between 2.5 and 10 centiMorgans (cM) long. This is consistent with the fact that Siberian Neandertals lived in relatively isolated populations of less than 60 individuals. In contrast, a Neandertal from Europe, a Denisovan from the Altai Mountains, and ancient modern humans seem to have lived in populations of larger sizes. The availability of three Neandertal genomes of high quality allows a view of genetic features that were unique to Neandertals and that are likely to have been at high frequency among them. We find that genes highly expressed in the striatum in the basal ganglia of the brain carry more amino-acid-changing substitutions than genes expressed elsewhere in the brain, suggesting that the striatum may have evolved unique functions in Neandertals.

Mafessoni F
Grote S
de Filippo C
Slon V
Kolobova KA
Viola B
Markin SV
Chintalapati M
Peyrégne S
Skov L
Skoglund P
Krivoshapkin AI
Derevianko AP
Meyer M
Kelso J
Peter B
Prüfer K
Pääbo S

Animals
Biological Evolution
Female
Fossils
Gene Expression Regulation
Genetic Variation
Genome
Humans
Inbreeding
Neanderthals
Population Density
Russia


The Neandertal Progesterone Receptor

Abstract: The hormone progesterone is important for preparing the uterine lining for egg implantation and for maintaining the early stages of pregnancy. The gene encoding the progesterone receptor (PGR) carries introgressed Neandertal haplotypes with two missense substitutions and a mobile Alu element. These Neandertal gene variants have reached nearly 20% frequency in non-Africans and have been associated with preterm birth. Here, we show that one of the missense substitutions appears fixed in Neandertals, while the other substitution as well as the Alu insertion were polymorphic among Neandertals. We show that two Neandertal haplotypes carrying the PGR gene entered the modern human population and that present-day carriers of the Neandertal haplotypes express higher levels of the receptor. In a cohort of present-day Britons, these carriers have more siblings, fewer miscarriages, and less bleeding during early pregnancy suggesting that the Neandertal progesterone receptor alleles promote fertility. This may explain their high frequency in modern human populations.

Alleles
Alu Elements
Animals
Female
Fertility
Genetic Introgression
Haplotypes
Humans
Neanderthals
Pregnancy
Premature Birth
Receptors, Progesterone


Initial Upper Palaeolithic Homo sapiens from Bacho Kiro Cave, Bulgaria

Abstract: The Middle to Upper Palaeolithic transition in Europe witnessed the replacement and partial absorption of local Neanderthal populations by Homo sapiens populations of African origin1. However, this process probably varied across regions and its details remain largely unknown. In particular, the duration of chronological overlap between the two groups is much debated, as are the implications of this overlap for the nature of the biological and cultural interactions between Neanderthals and H. sapiens. Here we report the discovery and direct dating of human remains found in association with Initial Upper Palaeolithic artefacts2, from excavations at Bacho Kiro Cave (Bulgaria). Morphological analysis of a tooth and mitochondrial DNA from several hominin bone fragments, identified through proteomic screening, assign these finds to H. sapiens and link the expansion of Initial Upper Palaeolithic technologies with the spread of H. sapiens into the mid-latitudes of Eurasia before 45 thousand years ago3. The excavations yielded a wealth of bone artefacts, including pendants manufactured from cave bear teeth that are reminiscent of those later produced by the last Neanderthals of western Europe4-6. These finds are consistent with models based on the arrival of multiple waves of H. sapiens into Europe coming into contact with declining Neanderthal populations7,8.

Hublin JJ
Sirakov N
Aldeias V
Bailey S
Bard E
Delvigne V
Endarova E
Fagault Y
Fewlass H
Hajdinjak M
Kromer B
Krumov I
Marreiros J
Martisius NL
Paskulin L
Sinet-Mathiot V
Meyer M
Pääbo S
Popov V
Rezek Z
Sirakova S
Skinner MM
Smith GM
Spasov R
Talamo S
Tuna T
Wacker L
Welker F
Wilcke A
Zahariev N
McPherron SP
Tsanova T

Animals
Asia
Bone and Bones
Bulgaria
Caves
DNA, Ancient
DNA, Mitochondrial
Europe
Fossils
History, Ancient
Human Migration
Humans
Neanderthals
Phylogeny
Tool Use Behavior
Tooth


Single-cell-resolution transcriptome map of human, chimpanzee, bonobo, and macaque brains

Abstract: Identification of gene expression traits unique to the human brain sheds light on the molecular mechanisms underlying human evolution. Here, we searched for uniquely human gene expression traits by analyzing 422 brain samples from humans, chimpanzees, bonobos, and macaques representing 33 anatomical regions, as well as 88,047 cell nuclei composing three of these regions. Among 33 regions, cerebral cortex areas, hypothalamus, and cerebellar gray and white matter evolved rapidly in humans. At the cellular level, astrocytes and oligodendrocyte progenitors displayed more differences in the human evolutionary lineage than the neurons. Comparison of the bulk tissue and single-nuclei sequencing revealed that conventional RNA sequencing did not detect up to two-thirds of cell-type-specific evolutionary differences.

Khrameeva E
Kurochkin I
Han D
Guijarro P
Kanton S
Santel M
Qian Z
Rong S
Mazin P
Sabirov M
Bulat M
Efimova O
Tkachev A
Guo S
Sherwood CC
Camp JG
Pääbo S
Treutlein B
Khaitovich P

Animals
Brain
Evolution, Molecular
Humans
Immunohistochemistry
Macaca
Neurons
Pan paniscus
Pan troglodytes
RNA-Seq
Single-Cell Analysis
Transcriptome


Organoid single-cell genomic atlas uncovers human-specific features of brain development

Abstract: The human brain has undergone substantial change since humans diverged from chimpanzees and the other great apes1,2. However, the genetic and developmental programs that underlie this divergence are not fully understood. Here we have analysed stem cell-derived cerebral organoids using single-cell transcriptomics and accessible chromatin profiling to investigate gene-regulatory changes that are specific to humans. We first analysed cell composition and reconstructed differentiation trajectories over the entire course of human cerebral organoid development from pluripotency, through neuroectoderm and neuroepithelial stages, followed by divergence into neuronal fates within the dorsal and ventral forebrain, midbrain and hindbrain regions. Brain-region composition varied in organoids from different iPSC lines, but regional gene-expression patterns remained largely reproducible across individuals. We analysed chimpanzee and macaque cerebral organoids and found that human neuronal development occurs at a slower pace relative to the other two primates. Using pseudotemporal alignment of differentiation paths, we found that human-specific gene expression resolved to distinct cell states along progenitor-to-neuron lineages in the cortex. Chromatin accessibility was dynamic during cortex development, and we identified divergence in accessibility between human and chimpanzee that correlated with human-specific gene expression and genetic change. Finally, we mapped human-specific expression in adult prefrontal cortex using single-nucleus RNA sequencing analysis and identified developmental differences that persist into adulthood, as well as cell-state-specific changes that occur exclusively in the adult brain. Our data provide a temporal cell atlas of great ape forebrain development, and illuminate dynamic gene-regulatory features that are unique to humans.

Kanton S
Boyle MJ
He Z
Santel M
Weigert A
Sanchís-Calleja F
Guijarro P
Sidow L
Fleck JS
Han D
Qian Z
Heide M
Huttner WB
Khaitovich P
Pääbo S
Treutlein B
Camp JG

Animals
Biological Evolution
Brain
Genomics
Humans
Macaca
Organoids
Pan troglodytes
Pluripotent Stem Cells
Single-Cell Analysis
Species Specificity


Simultaneous precise editing of multiple genes in human cells

Abstract: When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.

Riesenberg S
Chintalapati M
Macak D
Kanis P
Maricic T
Pääbo S

CRISPR-Cas Systems
Chromosomes
DNA Breaks, Double-Stranded
DNA End-Joining Repair
DNA-Activated Protein Kinase
Gene Editing
HEK293 Cells
Humans
INDEL Mutation
Oligonucleotides
Recombinational DNA Repair
Sequence Deletion


A genetic analysis of the Gibraltar Neanderthals

Abstract: The Forbes' Quarry and Devil's Tower partial crania from Gibraltar are among the first Neanderthal remains ever found. Here, we show that small amounts of ancient DNA are preserved in the petrous bones of the 2 individuals despite unfavorable climatic conditions. However, the endogenous Neanderthal DNA is present among an overwhelming excess of recent human DNA. Using improved DNA library construction methods that enrich for DNA fragments carrying deaminated cytosine residues, we were able to sequence 70 and 0.4 megabase pairs (Mbp) nuclear DNA of the Forbes' Quarry and Devil's Tower specimens, respectively, as well as large parts of the mitochondrial genome of the Forbes' Quarry individual. We confirm that the Forbes' Quarry individual was a female and the Devil's Tower individual a male. We also show that the Forbes' Quarry individual is genetically more similar to the ∼120,000-y-old Neanderthals from Scladina Cave in Belgium (Scladina I-4A) and Hohlenstein-Stadel Cave in Germany, as well as to a ∼60,000- to 70,000-y-old Neanderthal from Russia (Mezmaiskaya 1), than to a ∼49,000-y-old Neanderthal from El Sidrón (El Sidrón 1253) in northern Spain and other younger Neanderthals from Europe and western Asia. This suggests that the Forbes' Quarry fossil predates the latter Neanderthals. The preservation of archaic human DNA in the warm coastal climate of Gibraltar, close to the shores of Africa, raises hopes for the future recovery of archaic human DNA from regions in which climatic conditions are less than optimal for DNA preservation.

Bokelmann L
Hajdinjak M
Peyrégne S
Brace S
Essel E
de Filippo C
Glocke I
Grote S
Mafessoni F
Nagel S
Kelso J
Prüfer K
Vernot B
Barnes I
Pääbo S
Meyer M
Stringer C

Animals
DNA, Ancient
Gibraltar
History, Ancient
Humans
Neanderthals
Oligonucleotide Array Sequence Analysis


Nuclear DNA from two early Neandertals reveals 80,000 years of genetic continuity in Europe

Abstract: Little is known about the population history of Neandertals over the hundreds of thousands of years of their existence. We retrieved nuclear genomic sequences from two Neandertals, one from Hohlenstein-Stadel Cave in Germany and the other from Scladina Cave in Belgium, who lived around 120,000 years ago. Despite the deeply divergent mitochondrial lineage present in the former individual, both Neandertals are genetically closer to later Neandertals from Europe than to a roughly contemporaneous individual from Siberia. That the Hohlenstein-Stadel and Scladina individuals lived around the time of their most recent common ancestor with later Neandertals suggests that all later Neandertals trace at least part of their ancestry back to these early European Neandertals.

Peyrégne S
Slon V
Mafessoni F
de Filippo C
Hajdinjak M
Nagel S
Nickel B
Essel E
Le Cabec A
Wehrberger K
Conard NJ
Kind CJ
Posth C
Krause J
Abrams G
Bonjean D
Di Modica K
Toussaint M
Kelso J
Meyer M
Pääbo S
Prüfer K

Animals
Cell Lineage
Cell Nucleus
DNA
Europe
Evolution, Molecular
Fossils
Genome
Germany
Mitochondria
Neanderthals


Publisher Correction: Lipidome determinants of maximal lifespan in mammals

Abstract: A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Bozek K
Khrameeva EE
Reznick J
Omerbašić D
Bennett NC
Lewin GR
Azpurua J
Gorbunova V
Seluanov A
Regnard P
Wanert F
Marchal J
Pifferi F
Aujard F
Liu Z
Shi P
Pääbo S
Schroeder F
Willmitzer L
Giavalisco P
Khaitovich P


Macrophage-Dendritic Cell-T-Cell Tetrads Orchestrate Antitumor Immunity and Response to Checkpoint Blockade

Abstract: Immune checkpoint inhibitors (ICIs) elicit durable responses in only a subset of patients with solid tumors, underscoring the need to define the cellular architectures that govern effective antitumor immunity. Here we identify a spatially organized multicellular immune unit comprising macrophages, cDC1s, CD4+ T-cells, and CD8+ T-cells that emerges in response to anti-CTLA-4 or dual checkpoint blockade. We term these structures tetrads. Using multiplexed imaging and spatial transcriptomics in mouse and human tumors, we show that tetrads assemble early during immune priming, depend on the ICOS-ICOSL pathway, and are enriched for ICOS+ Th1-like CD4+ T cells and ICOSLhigh cDC1s. CD8+ T-cells within tetrads exhibit an activated, non-terminally differentiated state, while tetrad-associated macrophages display an interferon-γ-responsive program that sustains CD8+ T-cell function and prevents dysfunction. Functionally, ICOSL+ cDC1s are required for tumor eradication in vivo. In patients with bladder cancer treated with neoadjuvant dual checkpoint blockade, tetrad, but not triad or dyad formation correlates with clinical response. These findings establish tetrads as a fundamental cellular unit coordinating antitumor immunity and responsiveness to ICIs.

bioRxiv : the preprint server for biology

Chaib M
Aminu M
Herbrich S
Arabi M
Xuan Y
Basi A
Casasent A
Macaluso MD
Hu KH
Gubin M
Chen X
Mancuso JJ
Basu S
Jindal S
Burks J
Watowich S
Wu J
Allison JP
Sharma P


Editor's Note: Analysis of Immune Signatures in Longitudinal Tumor Samples Yields Insight into Biomarkers of Response and Mechanisms of Resistance to Immune Checkpoint Blockade

Chen PL
Roh W
Reuben A
Cooper ZA
Spencer CN
Prieto PA
Miller JP
Bassett RL
Gopalakrishnan V
Wani K
De Macedo MP
Austin-Breneman JL
Jiang H
Chang Q
Reddy SM
Chen WS
Tetzlaff MT
Broaddus RJ
Davies MA
Gershenwald JE
Haydu L
Lazar AJ
Patel SP
Hwu P
Hwu WJ
Diab A
Glitza IC
Woodman SE
Vence LM
Wistuba II
Amaria RN
Kwong LN
Prieto V
Davis RE
Ma W
Overwijk WW
Sharpe AH
Hu J
Futreal PA
Blando J
Sharma P
Allison JP
Chin L
Wargo JA


Local T(reg) cell loss reprogrammes systemic antitumour immunity without breaking tolerance


Cancer-induced nerve injury promotes resistance to anti-PD-1 therapy

Abstract: Perineural invasion (PNI) is a well-established factor of poor prognosis in multiple cancer types1, yet its mechanism remains unclear. Here we provide clinical and mechanistic insights into the role of PNI and cancer-induced nerve injury (CINI) in resistance to anti-PD-1 therapy. Our study demonstrates that PNI and CINI of tumour-associated nerves are associated with poor response to anti-PD-1 therapy among patients with cutaneous squamous cell carcinoma, melanoma and gastric cancer. Electron microscopy and electrical conduction analyses reveal that cancer cells degrade the nerve fibre myelin sheets. The injured neurons respond by autonomously initiating IL-6- and type I interferon-mediated inflammation to promote nerve healing and regeneration. As the tumour grows, the CINI burden increases, and its associated inflammation becomes chronic and skews the general immune tone within the tumour microenvironment into a suppressive and exhaustive state. The CINI-driven anti-PD-1 resistance can be reversed by targeting multiple steps in the CINI signalling process: denervating the tumour, conditional knockout of the transcription factor mediating the injury signal within neurons (Atf3), knockout of interferon-α receptor signalling (Ifnar1-/-) or by combining anti-PD-1 and anti-IL-6-receptor blockade. Our findings demonstrate the direct immunoregulatory roles of CINI and its therapeutic potential.

Baruch EN
Gleber-Netto FO
Nagarajan P
Rao X
Akhter S
Eichwald T
Xie T
Balood M
Adewale A
Naara S
Sathishkumar HN
Islam S
McCarthy W
Mattson BJ
Ferrarotto R
Wong MK
Davies MA
Jindal S
Basu S
Roversi K
Nikpoor AR
Ahmadi M
Ahmadi A
Harwood C
Leigh I
Gong D
Tallón de Lara P
Tao DL
Davidson TM
Ajami NJ
Futreal A
Rai K
Kochat V
Castillo M
Gunaratne P
Goepfert RP
Hernandez SD
Khushalani NI
Wang J
Watowich SS
Calin GA
Migden MR
Yuan M
Liu N
Ye Y
Hwang WL
Vermeer PD
D'Silva NJ
Bunimovich YL
Yaniv D
Burks JK
Gomez J
Dougherty PM
Tsai KY
Allison JP
Sharma P
Wargo JA
Myers JN
Talbot S
Gross ND
Amit M

Animals
Programmed Cell Death 1 Receptor
Mice
Humans
Drug Resistance, Neoplasm
Female
Male
Interleukin-6
Melanoma
Interferon Type I
Tumor Microenvironment
Receptor, Interferon alpha-beta
Stomach Neoplasms
Skin Neoplasms
Peripheral Nerve Injuries
Carcinoma, Squamous Cell
Inflammation
Signal Transduction
Immune Checkpoint Inhibitors
Neurons
Neoplasms
Neoplasm Invasiveness

Journal Article
Research Support, N.I.H., Extramural


Tumour cells mimic erythroblasts to hijack iron from bone marrow macrophages


Neutrophils are dispensable for Shigella control: macrophages take centre stage


Anti-CTLA-4 generates greater memory response than anti-PD-1 via TCF-1

Abstract: The effects of T cell differentiation arising from immune checkpoint inhibition targeting cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) on the immunological memory response remain unclear. Our investigation into the effects of anti-CTLA-4 and anti-PD-1 on memory T cell formation in mice reveals that memory T cells generated by anti-CTLA-4 exhibit greater expansion, cytokine production, and antitumor activity than those from anti-PD-1. Notably, anti-CTLA-4 preserves more T cell factor-1 (TCF-1)+ T cells during priming, while anti-PD-1 leads to more thymocyte selection-associated high mobility group box (TOX)+ T cells. Experiments using conditional Tcf7- or Tox-knockout mice highlight that TCF-1 is essential for the memory response generated by anti-CTLA-4, whereas TOX deletion alone in T cells has no effect on the response to anti-PD-1. Deepening our understanding of how checkpoint inhibition affects memory response is crucial for advancing our understanding of the enduring impacts of these immunotherapies on the immune system.

Mok S
Liu H
Ağaç Çobanoğlu D
Anang NAS
Mancuso JJ
Wherry EJ
Allison JP

Animals
Mice
Immunologic Memory
CTLA-4 Antigen
Programmed Cell Death 1 Receptor
Mice, Knockout
Hepatocyte Nuclear Factor 1-alpha
Immune Checkpoint Inhibitors
Memory T Cells
Mice, Inbred C57BL


Neoantigen-specific cytotoxic Tr1 CD4 T cells suppress cancer immunotherapy

Abstract: CD4+ T cells can either enhance or inhibit tumour immunity. Although regulatory T cells have long been known to impede antitumour responses1-5, other CD4+ T cells have recently been implicated in inhibiting this response6,7. Yet, the nature and function of the latter remain unclear. Here, using vaccines containing MHC class I (MHC-I) neoantigens (neoAgs) and different doses of tumour-derived MHC-II neoAgs, we discovered that whereas the inclusion of vaccines with low doses of MHC-II-restricted peptides (LDVax) promoted tumour rejection, vaccines containing high doses of the same MHC-II neoAgs (HDVax) inhibited rejection. Characterization of the inhibitory cells induced by HDVax identified them as type 1 regulatory T (Tr1) cells expressing IL-10, granzyme B, perforin, CCL5 and LILRB4. Tumour-specific Tr1 cells suppressed tumour rejection induced by anti-PD1, LDVax or adoptively transferred tumour-specific effector T cells. Mechanistically, HDVax-induced Tr1 cells selectively killed MHC-II tumour antigen-presenting type 1 conventional dendritic cells (cDC1s), leading to low numbers of cDC1s in tumours. We then documented modalities to overcome this inhibition, specifically via anti-LILRB4 blockade, using a CD8-directed IL-2 mutein, or targeted loss of cDC2/monocytes. Collectively, these data show that cytotoxic Tr1 cells, which maintain peripheral tolerance, also inhibit antitumour responses and thereby function to impede immune control of cancer.

Sultan H
Takeuchi Y
Ward JP
Sharma N
Liu TT
Sukhov V
Firulyova M
Song Y
Ameh S
Brioschi S
Khantakova D
Arthur CD
White JM
Kohlmiller H
Salazar AM
Burns R
Costa HA
Moynihan KD
Yeung YA
Djuretic I
Schumacher TN
Sheehan KCF
Colonna M
Allison JP
Murphy KM
Artyomov MN
Schreiber RD

Animals
Female
Humans
Male
Mice
Antigens, Neoplasm
Cancer Vaccines
CD4-Positive T-Lymphocytes
Cell Line, Tumor
Chemokine CCL5
Dendritic Cells
Granzymes
Histocompatibility Antigens Class I
Histocompatibility Antigens Class II
Immunotherapy
Interleukin-10
Mice, Inbred C57BL
Neoplasms
T-Lymphocytes, Regulatory
Receptors, Immunologic
Membrane Glycoproteins
Cytotoxicity, Immunologic
Immune Tolerance
CD8-Positive T-Lymphocytes

Journal Article
Research Support, N.I.H., Intramural


Post-immunotherapy CTLA-4 Ig treatment improves antitumor efficacy

Abstract: Immune checkpoint therapies (ICT) improve overall survival of patients with cancer but may cause immune-related adverse events (irAEs) such as myocarditis. Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin fusion protein (CTLA-4 Ig), an inhibitor of T cell costimulation through CD28, reverses irAEs in animal models. However, concerns exist about potentially compromising antitumor response of ICT. In mouse tumor models, we administered CTLA-4 Ig 1) concomitantly with ICT or 2) after ICT completion. Concomitant treatment reduced antitumor efficacy, while post-ICT administration improved efficacy without affecting frequency and function of CD8 T cells. The improved response was independent of the ICT used, whether CTLA-4 or PD-1 blockade. The frequency of Tregs was significantly decreased with CTLA-4 Ig. The resulting increased CD8/Treg ratio potentially underlies the enhanced efficacy of ICT followed by CTLA-4 Ig. This paradoxical mechanism shows that a CTLA-4 Ig regimen shown to reduce irAE severity does not compromise antitumor efficacy.

Mok S
Ağaç Çobanoğlu D
Liu H
Mancuso JJ
Allison JP

Animals
Mice
Immunotherapy
CTLA-4 Antigen
Immune Checkpoint Inhibitors
CD8-Positive T-Lymphocytes
T-Lymphocytes, Regulatory
Cell Line, Tumor
Abatacept
Female
Humans
Mice, Inbred C57BL
Neoplasms
Programmed Cell Death 1 Receptor


Pancreatic Epithelial IL17/IL17RA Signaling Drives B7-H4 Expression to Promote Tumorigenesis

Abstract: IL17 is required for the initiation and progression of pancreatic cancer, particularly in the context of inflammation, as previously shown by genetic and pharmacological approaches. However, the cellular compartment and downstream molecular mediators of IL17-mediated pancreatic tumorigenesis have not been fully identified. This study examined the cellular compartment required by generating transgenic animals with IL17 receptor A (IL17RA), which was genetically deleted from either the pancreatic epithelial compartment or the hematopoietic compartment via generation of IL17RA-deficient (IL17-RA-/-) bone marrow chimeras, in the context of embryonically activated or inducible Kras. Deletion of IL17RA from the pancreatic epithelial compartment, but not from hematopoietic compartment, resulted in delayed initiation and progression of premalignant lesions and increased infiltration of CD8+ cytotoxic T cells to the tumor microenvironment. Absence of IL17RA in the pancreatic compartment affected transcriptional profiles of epithelial cells, modulating stemness, and immunological pathways. B7-H4, a known inhibitor of T-cell activation encoded by the gene Vtcn1, was the checkpoint molecule most upregulated via IL17 early during pancreatic tumorigenesis, and its genetic deletion delayed the development of pancreatic premalignant lesions and reduced immunosuppression. Thus, our data reveal that pancreatic epithelial IL17RA promotes pancreatic tumorigenesis by reprogramming the immune pancreatic landscape, which is partially orchestrated by regulation of B7-H4. Our findings provide the foundation of the mechanisms triggered by IL17 to mediate pancreatic tumorigenesis and reveal the avenues for early pancreatic cancer immune interception. See related Spotlight by Lee and Pasca di Magliano, p. 1130.

Castro-Pando S
Howell RM
Li L
Mascaro M
Faraoni EY
Le Roux O
Romanin D
Tahan V
Riquelme E
Zhang Y
Kolls JK
Allison JP
Lozano G
Moghaddam SJ
McAllister F

V-Set Domain-Containing T-Cell Activation Inhibitor 1


Molecular Pathways and Cellular Subsets Associated with Adverse Clinical Outcomes in Overlapping Immune-Related Myocarditis and Myositis

Abstract: Immune checkpoint therapies (ICT) can induce life-threatening immune-related adverse events, including myocarditis and myositis, which are rare but often concurrent. The molecular pathways and immune subsets underlying these toxicities remain poorly understood. To address this need, we performed single-cell RNA sequencing of heart and skeletal muscle biopsies obtained from living patients with cancers treated with ICTs and admitted to the hospital with myocarditis and/or myositis (overlapping myocarditis plus myositis, n = 10; myocarditis-only, n = 1) or ICT-exposed patients ruled out for toxicity utilized as controls (n = 9). All biopsies were obtained within 96 hours of clinical presentation. Analyses of 58,523 cells revealed CD8+ T cells with a cytotoxic phenotype expressing activation/exhaustion markers in both myocarditis and myositis. Furthermore, the analyses identified a population of myeloid cells expressing tissue-resident signatures and FcγRIIIa (CD16a), which is known to bind IgG and regulate complement activation. Immunohistochemistry of affected cardiac and skeletal muscle tissues revealed protein expression of pan-IgG and complement product C4d, which were associated with the presence of high-titer serum autoantibodies against muscle antigens in a subset of patients. We further identified a population of inflammatory IL1B+TNF+ myeloid cells specifically enriched in myocarditis and associated with greater toxicity severity and poorer clinical outcomes. These results provide insight into the myeloid subsets present in human immune-related myocarditis and myositis tissues and nominate new targets for investigation into rational treatments to overcome these high-mortality toxicities. See related Spotlight by Fankhauser et al., p. 954.

Siddiqui BA
Palaskas NL
Basu S
Dai Y
He Z
Yadav SS
Allison JP
Sheth RA
Tummala S
Buja M
Bhattacharjee MB
Iliescu C
Rawther-Karedath A
Deswal A
Wang L
Sharma P
Subudhi SK

Humans
Myocarditis
Myositis
Male
Female
Middle Aged
Immune Checkpoint Inhibitors
Aged
Neoplasms
CD8-Positive T-Lymphocytes
Adult
Receptors, IgG
Muscle, Skeletal
Single-Cell Analysis


A Modular Trial of Androgen Signaling Inhibitor Combinations Testing a Risk-Adapted Strategy in Patients with Metastatic Castration-Resistant Prostate Cancer

Abstract: To determine the efficacy and safety of risk-adapted combinations of androgen signaling inhibitors and inform disease classifiers for metastatic castration-resistant prostate cancers. In a modular, randomized phase II trial, 192 men were treated with 8 weeks of abiraterone acetate, prednisone, and apalutamide (AAPA; module 1) and then allocated to modules 2 or 3 based on satisfactory (≥50% PSA decline from baseline and <5 circulating tumor cell/7.5 mL) versus unsatisfactory status. Men in the former were randomly assigned to continue AAPA alone (module 2A) or with ipilimumab (module 2B). Men in the latter group had carboplatin + cabazitaxel added to AAPA (module 3). Optional baseline biopsies were subjected to correlative studies. Median overall survival (from allocation) was 46.4 [95% confidence interval (CI), 39.2-68.2], 41.4 (95% CI, 33.3-49.9), and 18.7 (95% CI, 14.3-26.3) months in modules 2A (n = 64), 2B (n = 64), and 3 (n = 59), respectively. Toxicities were within expectations. Of 192 eligible patients, 154 (80.2%) underwent pretreatment metastatic biopsies. The aggressive-variant prostate cancer molecular profile (defects in ≥2 of p53, RB1, and PTEN) was associated with unsatisfactory status. Exploratory analyses suggested that secreted phosphoprotein 1-positive and insulin-like growth factor-binding protein 2-positive macrophages, druggable myeloid cell markers, and germline pathogenic mutations were enriched in the unsatisfactory group. Adding ipilimumab to AAPA did not improve outcomes in men with androgen-responsive metastatic castration-resistant prostate cancer. Despite the addition of carboplatin + cabazitaxel, men in the unsatisfactory group had shortened survivals. Adaptive designs can enrich for biologically and clinically relevant disease subgroups to contribute to the development of marker-informed, risk-adapted therapy strategies in men with prostate cancer.

Clinical cancer research : an official journal of the American Association for Cancer Research

Aparicio AM
Tidwell RSS
Yadav SS
Chen JS
Zhang M
Liu J
Guo S
Pilié PG
Yu Y
Song X
Vundavilli H
Jindal S
Zhu K
Viscuse PV
Lebenthal JM
Hahn AW
Soundararajan R
Corn PG
Zurita-Saavedra A
Subudhi SK
Zhang J
Wang W
Huff C
Troncoso P
Allison JP
Sharma P
Logothetis CJ

Antineoplastic Combined Chemotherapy Protocols

Journal Article
Randomized Controlled Trial
Clinical Trial, Phase II


ICOS costimulation in combination with CTLA-4 blockade remodels tumor-associated macrophages toward an antitumor phenotype

Abstract: We have previously demonstrated synergy between ICOS costimulation (IVAX; ICOSL-transduced B16-F10 cellular vaccine) and CTLA-4 blockade in antitumor therapy. In this study, we employed CyTOF and single-cell RNA sequencing and observed significant remodeling of the lymphoid and myeloid compartments in combination therapy. Compared with anti-CTLA-4 monotherapy, the combination therapy enriched Th1 CD4 T cells, effector CD8 T cells, and M1-like antitumor proinflammatory macrophages. These macrophages were critical to the therapeutic efficacy of anti-CTLA-4 combined with IVAX or anti-PD-1. Macrophage depletion with clodronate reduced the tumor-infiltrating effector CD4 and CD8 T cells, impairing their antitumor functions. Furthermore, the recruitment and polarization of M1-like macrophages required IFN-γ. Therefore, in this study, we show that there is a positive feedback loop between intratumoral effector T cells and tumor-associated macrophages (TAMs), in which the IFN-γ produced by the T cells polarizes the TAMs into M1-like phenotype, and the TAMs, in turn, reshape the tumor microenvironment to facilitate T cell infiltration, immune function, and tumor rejection.

Sharma N
Fan X
Atolagbe OT
Ge Z
Dao KN
Sharma P
Allison JP

Humans
CTLA-4 Antigen
Tumor-Associated Macrophages
Neoplasms
CD8-Positive T-Lymphocytes
Phenotype
Tumor Microenvironment
Inducible T-Cell Co-Stimulator Protein


The future of targeting cytotoxic T-lymphocyte-associated protein-4: Is there a role?

Abstract: The 2022 yearly Think Tank Meeting in Siena, Tuscany (Italy), organized by the Italian Network for Tumor Biotherapy (NIBIT) Foundation, the Parker Institute for Cancer Immunotherapy and the World Immunotherapy Council, included a focus on the future of integrating and expanding the use of targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). The conference members exchanged their views on the lessons from targeting CTLA-4 and compared the effect to the impact of blocking Programmed cell death protein 1 (PD1) or its ligand (PDL1). The increasing experience with both therapeutic approaches and their combination suggests that targeting CTLA-4 may lead to more durable responses for a sizeable proportion of patients, though the specific mechanism is not entirely understood. Overcoming toxicity of blocking CTLA-4 is currently being addressed with different doses and dose regimens, especially when combined with PD1/PDL1 blocking antibodies. Novel therapeutics targeting CTLA-4 hold the promise to reduce toxicities and thus allow different combination strategies in the future. On the whole, the consent was that targeting CTLA-4 remains an important strategy to improve the efficacy of cancer immunotherapies.

European journal of cancer (Oxford, England : 1990)

Di Giacomo AM
Lahn M
Eggermont AM
Fox B
Ibrahim R
Sharma P
Allison JP
Maio M

Humans
CTLA-4 Antigen
T-Lymphocytes, Cytotoxic
Neoplasms
Italy
Immunotherapy


Exercise Training Reduces the Inflammatory Response and Promotes Intestinal Mucosa-Associated Immunity in Lynch Syndrome

Abstract: Lynch syndrome (LS) is a hereditary condition with a high lifetime risk of colorectal and endometrial cancers. Exercise is a non-pharmacologic intervention to reduce cancer risk, though its impact on patients with LS has not been prospectively studied. Here, we evaluated the impact of a 12-month aerobic exercise cycling intervention in the biology of the immune system in LS carriers. To address this, we enrolled 21 patients with LS onto a non-randomized, sequential intervention assignation, clinical trial to assess the effect of a 12-month exercise program that included cycling classes 3 times weekly for 45 minutes versus usual care with a one-time exercise counseling session as control. We analyzed the effects of exercise on cardiorespiratory fitness, circulating, and colorectal-tissue biomarkers using metabolomics, gene expression by bulk mRNA sequencing, and spatial transcriptomics by NanoString GeoMx. We observed a significant increase in oxygen consumption (VO2peak) as a primary outcome of the exercise and a decrease in inflammatory markers (prostaglandin E) in colon and blood as the secondary outcomes in the exercise versus usual care group. Gene expression profiling and spatial transcriptomics on available colon biopsies revealed an increase in the colonic mucosa levels of natural killer and CD8+ T cells in the exercise group that were further confirmed by IHC studies. Together these data have important implications for cancer interception in LS, and document for the first-time biological effects of exercise in the immune system of a target organ in patients at-risk for cancer.

Deng N
Reyes-Uribe L
Fahrmann JF
Thoman WS
Munsell MF
Dennison JB
Murage E
Wu R
Hawk ET
Thirumurthi S
Lynch PM
Dieli-Conwright CM
Lazar AJ
Jindal S
Chu K
Chelvanambi M
Basen-Engquist K
Li Y
Wargo JA
McAllister F
Allison JP
Sharma P
Sinha KM
Hanash S
Gilchrist SC
Vilar E

Colorectal Neoplasms, Hereditary Nonpolyposis

Clinical Trial
Journal Article
Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural


Cancer Immunology and Immunotherapy Showcased in the AACR Cancer Progress Report 2023


Inflammation induced by tumor-associated nerves promotes resistance to anti-PD-1 therapy in cancer patients and is targetable by interleukin-6 blockade

Abstract: While the nervous system has reciprocal interactions with both cancer and the immune system, little is known about the potential role of tumor associated nerves (TANs) in modulating anti-tumoral immunity. Moreover, while peri-neural invasion is a well establish poor prognostic factor across cancer types, the mechanisms driving this clinical effect remain unknown. Here, we provide clinical and mechniastic association between TANs damage and resistance to anti-PD-1 therapy. Using electron microscopy, electrical conduction studies, and tumor samples of cutaneous squamous cell carcinoma (cSCC) patients, we showed that cancer cells can destroy myelin sheath and induce TANs degeneration. Multi-omics and spatial analyses of tumor samples from cSCC patients who underwent neoadjuvant anti-PD-1 therapy demonstrated that anti-PD-1 non-responders had higher rates of peri-neural invasion, TANs damage and degeneration compared to responders, both at baseline and following neoadjuvant treatment. Tumors from non-responders were also characterized by a sustained signaling of interferon type I (IFN-I) - known to both propagate nerve degeneration and to dampen anti-tumoral immunity. Peri-neural niches of non-responders were characterized by higher immune activity compared to responders, including immune-suppressive activity of M2 macrophages, and T regulatory cells. This tumor promoting inflammation expanded to the rest of the tumor microenvironment in non-responders. Anti-PD-1 efficacy was dampened by inducing nerve damage prior to treatment administration in a murine model. In contrast, anti-PD-1 efficacy was enhanced by denervation and by interleukin-6 blockade. These findings suggested a potential novel anti-PD-1 resistance drived by TANs damage and inflammation. This resistance mechanism is targetable and may have therapeutic implications in other neurotropic cancers with poor response to anti-PD-1 therapy such as pancreatic, prostate, and breast cancers.

Baruch EN
Nagarajan P
Gleber-Netto FO
Rao X
Xie T
Akhter S
Adewale A
Shajedul I
Mattson BJ
Ferrarotto R
Wong MK
Davies MA
Jindal S
Basu S
Harwood C
Leigh I
Ajami N
Futreal A
Castillo M
Gunaratne P
Goepfert RP
Khushalani N
Wang J
Watowich S
Calin GA
Migden MR
Vermeer P
D'Silva N
Yaniv D
Burks JK
Gomez J
Dougherty PM
Tsai KY
Allison JP
Sharma P
Wargo J
Myers JN
Gross ND
Amit M


Immune checkpoint therapy-current perspectives and future directions

Abstract: Immune checkpoint therapy (ICT) has dramatically altered clinical outcomes for cancer patients and conferred durable clinical benefits, including cure in a subset of patients. Varying response rates across tumor types and the need for predictive biomarkers to optimize patient selection to maximize efficacy and minimize toxicities prompted efforts to unravel immune and non-immune factors regulating the responses to ICT. This review highlights the biology of anti-tumor immunity underlying response and resistance to ICT, discusses efforts to address the current challenges with ICT, and outlines strategies to guide the development of subsequent clinical trials and combinatorial efforts with ICT.

Sharma P
Goswami S
Raychaudhuri D
Siddiqui BA
Singh P
Nagarajan A
Liu J
Subudhi SK
Poon C
Gant KL
Herbrich SM
Anandhan S
Islam S
Amit M
Anandappa G
Allison JP

Humans
B7-H1 Antigen
Immunotherapy
Neoplasms
Clinical Trials as Topic
Immune Checkpoint Inhibitors

Journal Article
Review
Research Support, Non-U.S. Gov't


Neoadjuvant chemotherapy plus nivolumab with or without ipilimumab in operable non-small cell lung cancer: the phase 2 platform NEOSTAR trial

Abstract: Neoadjuvant ipilimumab + nivolumab (Ipi+Nivo) and nivolumab + chemotherapy (Nivo+CT) induce greater pathologic response rates than CT alone in patients with operable non-small cell lung cancer (NSCLC). The impact of adding ipilimumab to neoadjuvant Nivo+CT is unknown. Here we report the results and correlates of two arms of the phase 2 platform NEOSTAR trial testing neoadjuvant Nivo+CT and Ipi+Nivo+CT with major pathologic response (MPR) as the primary endpoint. MPR rates were 32.1% (7/22, 80% confidence interval (CI) 18.7-43.1%) in the Nivo+CT arm and 50% (11/22, 80% CI 34.6-61.1%) in the Ipi+Nivo+CT arm; the primary endpoint was met in both arms. In patients without known tumor EGFR/ALK alterations, MPR rates were 41.2% (7/17) and 62.5% (10/16) in the Nivo+CT and Ipi+Nivo+CT groups, respectively. No new safety signals were observed in either arm. Single-cell sequencing and multi-platform immune profiling (exploratory endpoints) underscored immune cell populations and phenotypes, including effector memory CD8+ T, B and myeloid cells and markers of tertiary lymphoid structures, that were preferentially increased in the Ipi+Nivo+CT cohort. Baseline fecal microbiota in patients with MPR were enriched with beneficial taxa, such as Akkermansia, and displayed reduced abundance of pro-inflammatory and pathogenic microbes. Neoadjuvant Ipi+Nivo+CT enhances pathologic responses and warrants further study in operable NSCLC. (ClinicalTrials.gov registration: NCT03158129 .).

Cascone T
Leung CH
Weissferdt A
Pataer A
Carter BW
Godoy MCB
Feldman H
William WN Jr
Xi Y
Basu S
Sun JJ
Yadav SS
Rojas Alvarez FR
Lee Y
Mishra AK
Chen L
Pradhan M
Guo H
Sinjab A
Zhou N
Negrao MV
Le X
Gay CM
Tsao AS
Byers LA
Altan M
Glisson BS
Fossella FV
Elamin YY
Blumenschein G Jr
Zhang J
Skoulidis F
Wu J
Mehran RJ
Rice DC
Walsh GL
Hofstetter WL
Rajaram R
Antonoff MB
Fujimoto J
Solis LM
Parra ER
Haymaker C
Wistuba II
Swisher SG
Vaporciyan AA
Lin HY
Wang J
Gibbons DL
Jack Lee J
Ajami NJ
Wargo JA
Allison JP
Sharma P
Kadara H
Heymach JV
Sepesi B

Clinical Trial, Phase II
Journal Article
Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural
Adaptive Clinical Trial


Author Correction: Neoadjuvant relatlimab and nivolumab in resectable melanoma

Amaria RN
Postow M
Burton EM
Tetzlaff MT
Ross MI
Torres-Cabala C
Glitza IC
Duan F
Milton DR
Busam K
Simpson L
McQuade JL
Wong MK
Gershenwald JE
Lee JE
Goepfert RP
Keung EZ
Fisher SB
Betof-Warner A
Shoushtari AN
Callahan M
Coit D
Bartlett EK
Bello D
Momtaz P
Nicholas C
Gu A
Zhang X
Korivi BR
Patnana M
Patel SP
Diab A
Lucci A
Prieto VG
Davies MA
Allison JP
Sharma P
Wargo JA
Ariyan C
Tawbi HA