Mardiyyah/TAPT_data_V2_split · Datasets at Hugging Face

PMCID stringclasses 24 values	Title stringclasses 24 values	Sentences stringlengths 2 40.7k
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Chain pairing analysis showed that cells from the T cell clusters mostly contained a single pair of chains (50-60%), with orphan (5-20%) and extra (5-10%) chains present in small fractions of cells (fig. S23A).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Notably, the frequency of extra α chains (extra VJ) was higher than that of β chains (extra VDJ), potentially due to more stringent and multi-layered allelic exclusion mechanisms at the TCRβ locus compared to TCRα (57).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	As expected, the NK and ILC clusters held no productive TCR chains.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Within the γδ T cell clusters, only a small proportion had a productive TCR chain, which may result from cytotoxic T cells co-clustering with γδ T cells.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	We also carried out γδ TCR sequencing in selected spleen, bone marrow and liver samples.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	The γδ TCR sequencing data was subjected to a customized analysis pipeline that we developed and optimised based on cellranger followed by contig re-annotation with dandelion (see Materials and Methods), facilitating the full recovery of γδ chains in our data.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	This analysis confirmed that the majority of productive γδ TCR chains originated from the ITGAD-expressing γδ T cells (fig. S23B), supporting the robust identification of this population.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	The Trm_Tgd population could not be confirmed by γδ TCR sequencing due to the lack of sample availability.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	We next examined V(D)J gene usage in relation to T cell identity.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	In the MAIT population, we detected significant enrichment of TRAV1-2, as expected (fig. S23C).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Selecting only the TRAV1-2+ cells (MAIT cluster and other clusters) revealed a notable tissue-specific distribution of TRAJ segments with TRAJ33 in spleen and liver, TRAJ12 in liver and TRAJ29/TRAJ36 in jejunum (fig. S23D).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	This suggests that there may be different antigens for MAIT cells in the spleen, liver and gut corresponding to the different metabolomes in these tissues.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	In addition, full analysis of the TCR repertoire of the MAIT cells revealed previously unappreciated diversity of V segment usage in the beta chain (fig. S23D).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	We then defined clonally related cells on the basis of identical CDR3 nucleotide sequences to investigate their TCR repertoires.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Using this approach, we found that clonally expanded cells were primarily from the resident memory T cell compartment, including the Th1/Th17 populations mentioned above (Fig. 4G and fig. S23E).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	As expected, these clonotypes were restricted to single individuals and within an individual they were distributed across tissues and subsets (Fig. 4H and fig. S23, F to H).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	We found a small number of isolated CD4+ T cell clones that shared Tregs and effector T cell phenotypes, possibly due to low levels of plasticity or due to (trans)differentiation from the same naive precursor cell in the periphery (fig. S23H).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Focusing on the most expanded clonotypes (>20 cells), the majority were widespread across five or more tissues, supporting the systemic nature of tissue-resident immune memory (Fig. 4G).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Moreover, we found that several clonotypes present across tissues consisted of a mixture of cells from the Tem/emra_CD8 and Trm/em_CD8 populations (fig. S23H), suggesting that a single naive CD8+ T cell precursor can give rise to diverse cytotoxic T cell states, which harbour immune memory across multiple non-lymphoid tissues, emphasizing the plasticity of phenotype and location within a clone.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	In summary, we have described 18 T/innate cell states in our data by integrating CellTypist logistic regression models, manual curation and V(D)J sequencing.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	This has yielded insights into the MAIT cell compartment and its antigen receptor repertoire distribution that differed between spleen, liver and gut.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	For the cytotoxic T cell memory compartment there was broad sharing of clones across gut regions for Trm_gut_CD8, and mixed Tem/emra_CD8 and Trm/em_CD8 T cell clonotypes with broad tissue distributions.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	After focusing on individual immune compartments, we next took a combined approach in order to better understand the immune landscape of selected tissues.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	As shown in Fig. 5A, each tissue has its own immune neighborhood, for example, while spleen and lymph nodes are rich in B cells, composition of their myeloid compartment varies.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	In particular, a large population of erythrophagocytic macrophages, known as red pulp macrophages, are evident in the spleen (in keeping with their role in red blood cell turnover), whereas lymph nodes are rich in dendritic cells.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	As expected, bone marrow uniquely contains progenitor populations.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Furthermore lung and liver contain significant numbers of monocytes, including CX3CR1+ nonclassical monocytes whereas these cells are absent from the jejunum, perhaps reflecting different degrees of vascularization.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	In contrast, the jejunum has an abundance of resident memory T cells (CD8+ T cells and Th1/Th17) as well as plasma cells.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Our long-term vision for CellTypist is to provide a reference atlas with deeply curated cell types publicly available to the community.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Therefore, via a semi-automatic process, we fed the identities of the 41 immune cell types identified in our dataset (including both shared and novel cell type labels) back into CellTypist, demonstrating how CellTypist can be updated and improved over time.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Combined with the initial 91 cell types and states included in the reference datasets, CellTypist now comprises a total of 101 annotated cell types (Fig. 5B).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Here, we present a multi-tissue study of immune cells across the human body within diverse organ donors.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	By sampling multiple organs from the same individuals, which allows for robust control of age, sex, medical history, drug exposure and sampling backgrounds, we reveal tissue-specific expression patterns across the myeloid and lymphoid compartments.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	We also introduce CellTypist, a publicly available and updatable framework for automated immune cell type annotation that, in addition to identifying major cell types, is able to perform fine-grained cell subtype annotation - normally a time-consuming process that requires expert knowledge.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	We developed CellTypist by integrating and curating data obtained from 19 studies performed across a range of tissues, with in-depth immune cell analysis comprising 91 harmonized cell type labels.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	However, as demonstrated here, for example in the γδ T cell compartment, manual curation following automated annotation still has a role to play in revealing specific cell subtypes that may be absent from the database/training set.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	To reduce the need for this, in the longer term, the CellTypist models will be periodically updated and extended to include further immune and non-immune sub-populations as more data become available.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Within the myeloid compartment, macrophages showed the most prominent features of tissue specificity.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Erythrophagocytic macrophages in the liver and spleen shared features related to iron-recycling (58) with macrophages in other locations, such as the mesenteric lymph nodes, suggesting that macrophages participate in iron metabolism across a range of tissues.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	In addition, we characterized subsets of migratory dendritic cells (CCR7+) revealing specific expression of CRLF2, CSF2RA and GPR157 in the lung and lung-draining lymph nodes, and expression of AIRE in the mesenteric and lung-draining lymph nodes.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	These migratory dendritic cell states are interesting targets for future in depth functional characterisation in the context of allergy, asthma and other related pathologies (59, 60).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	In the lymphoid compartment, we combined single-cell transcriptome and VDJ analysis, which allowed the phenotype of adaptive immune cells to be dissected using complementary layers of single cell genomics data.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Of note, we detected a subset of memory B cells expressing ITGAX (CD11c) and TBX21 (T-bet) that resemble ABCs previously reported to be expanded in ageing (48), following malaria vaccination (61) and in systemic lupus erythematosus (SLE) patients (62).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	In our data, these B cells did not show clonal expansion and at least 50% showed IgM subclass, suggesting that they may be present at low levels in healthy tissues and expand upon challenge as well as ageing.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	BCR analysis revealed isotype usage biased towards IgA2 in gut plasma cells, which may be related to structural differences (63) or higher resistance to microbial degradation as compared to IgA1 (64).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	In the T cell compartment, our results provided insights into the heterogeneity of T cell subtypes and their tissue adaptations.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Notably, we identified subsets of CD4+ Trm based on functional capacity for IFN-γ or IL-17 production that were mostly localized to intestinal sites, analogous to mouse CD4+ Trm generated from IL-17-producing effector T cells in the gut (65).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	We also identified different subsets of CD8+ Trm including a gut-adapted subset expressing CCR9, which mediates homing to intestinal sites via binding to CCL25 (66) and another Trm-like subset more targeted to lymphoid sites.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	TCR clone sharing between memory subtypes of CD8+ T cells suggests their origin from a common precursor, or their differentiation or conversion during migration or maintenance, such as conversion of effector memory T cells (Tem) to resident memory T cells (Trm) (56).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	We also identified distinct subsets of γδ T cells based on tissue-specific gene expression patterns, showing distinct integrin gene expression and tissue distributions.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	In summary, using this dataset of nearly 360,000 single cell transcriptomes (of which ~330,000 were immune cells) from donor-matched tissues from 12 deceased individuals, we have shown how a combination of CellTypist-based automated annotation, expert-driven cluster analysis and antigen receptor sequencing can synergize to dissect specific and functionally relevant aspects of immune cells across the human body.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	We have revealed previously unrecognized features of tissue-specific immunity in the myeloid and lymphoid compartments, and have provided a comprehensive framework for future cross-tissue cell type analysis.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Further investigation of human tissue-resident immunity is needed to determine the effect of important covariates such as underlying critical illness, donor age and gender as well as considering the immune cell activation status, to gain a defining picture of how human biology influences immune functions.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Our deeply characterised cross-tissue immune cell dataset has implications for the engineering of cells for therapeutic purposes and addressing cells to intended tissue locations, and for understanding tissue-specific features of infection as well as distinct modes of vaccine delivery to tissues.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Tissue was obtained from deceased organ donors via the Cambridge Biorepository for Translational Medicine (CBTM, https://www.cbtm.group.cam.ac.uk/), REC 15/EE/0152.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Detailed sample locations taken can be found in Fig. 1 and protocols are described in detail in Supplementary Materials.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Additional tissue samples were from Columbia University and were obtained from deceased organ donors at the time of organ acquisition for clinical transplantation through an approved protocol and material transfer agreement with LiveOnNY.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Six donors were processed with a uniform protocol at Cambridge university where solid tissues were cut into small pieces, then homogenised with enzymatic digestion for 2x 15 minute heating/mixing steps at 37°C.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	The remaining six donors were subjected to a tissue adapted protocol with the aim of improving immune cell recovery, and this protocol was harmonised as closely as possible between the two collection sites.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	For scRNA-seq experiments, single cells were loaded onto the channels of a Chromium chip (10x Genomics).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	cDNA synthesis, amplification, and sequencing libraries were generated using either the Single Cell 5′ Reagent (v1 and v2) (Cambridge University) or 3′ Reagent (v3) (Columbia University) Kit.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	TCRαβ, BCR and TCRγδ paired VDJ libraries were prepared from samples made with the 5′ Reagent kit.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	All libraries were sequenced on either a HiSeq 4000 or NovaSeq 6000 instrument.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	scRNA-seq data was aligned and quantified using the cellranger software (version 6.1.1, 10x Genomics Inc.).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Cells from hashtagged samples were demultiplexed using Hashsolo (67).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Cells with fewer than 1,000 UMI counts and 600 detected genes were excluded.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Doublets were detected using Scrublet (68).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Downstream analysis from data normalization to graph-based clustering were performed using Scanpy (version 1.6.0) (69), with details described in Supplementary Materials.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Data integration was done using BBKNN (70) and scVI (71), and the results were compared using kBET (72).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	scTCR-seq and scBCR-seq data were aligned and quantified using the cellranger-vdj software (version 2.1.1 and 4.0, respectively).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	For TCRγδ we implemented a customized pipeline (https://sc-dandelion.readthedocs.io/en/latest/notebooks/gamma_delta.html) due to cellranger being tuned towards alpha/beta TCR chains.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	scTCR-seq analysis including productive TCR chain pairing and clonotype detection was performed using the scirpy package (73).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Details of CellTypist, including cross-data cell type label harmonization and automated cell annotation, can be found in Supplementary Text.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Briefly, immune cells from 20 tissues of 19 studies were collected and harmonised into consistent labels.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	These cells were split into equal-sized mini-batches, and these batches were sequentially trained by the l2-regularized logistic regression using stochastic gradient descent learning.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Feature selection was performed to choose the top 300 genes from each cell type, and the union of these genes were supplied as the input for a second round of training.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	For single molecule FISH, samples were run using the RNAscope 2.5 LS fluorescent multiplex assay (automated).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Slides were imaged on the Perkin Elmer Opera Phenix High-Content Screening System, in confocal mode with 1 μm z-step size, using 20X (NA 0.16, 0.299 μm/pixel) and 40X (NA 1.1, 0.149 μm/pixel) water-immersion objectives.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	For flow cytometry, mononuclear cells (MNCs) were either stained ex vivo or post activation with PMA+I for two hours.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Cells were stained with the live/dead marker Zombie Aqua for 10 minutes at room temperature, and then washed with PBS+0.5%FCS, with the CD8 and B cell panels of antibodies.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	qPCR was performed in three spleen samples.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Cells were stained with the live/dead marker Zombie Aqua for 10 minutes at room temperature, and then washed with PBS+0.5%FCS, followed by staining with the antibodies at 4°C for 45 minutes.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Cell sorting was performed on a BD Fusion 4 laser sorter and RNA was extracted using a Zymo Research RNA micro kit.
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	For immunofluorescence, samples were fixed in 1% paraformaldehyde for 24 hours followed by 8 hours in 30% sucrose in PBS, and were stained for 2h at RT with the appropriate antibodies, washed three times in PBS and mounted in Fluoromount-G® (Southern Biotech).
PMC7612735	Cross-tissue immune cell analysis reveals tissue-specific features in humans.	Images were acquired using a TCS SP8 (Leica, Milton Keynes, UK) confocal microscope.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	The implementation of antiretroviral therapy (ART) has effectively restricted the transmission of Human Immunodeficiency Virus (HIV) and improved overall clinical outcomes.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	However, a complete cure for HIV remains out of reach, as the virus persists in a stable pool of infected cell reservoir that is resistant to therapy and thus a main barrier towards complete elimination of viral infection.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	While the mechanisms by which host proteins govern viral gene expression and latency are well-studied, the emerging regulatory functions of non-coding RNAs (ncRNA) in the context of T cell activation, HIV gene expression and viral latency have not yet been thoroughly explored.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Here, we report the identification of the Cytoskeleton Regulator (CYTOR) long non-coding RNA (lncRNA) as an activator of HIV gene expression that is upregulated following T cell stimulation.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Functional studies show that CYTOR suppresses viral latency by directly binding to the HIV promoter and associating with the cellular positive transcription elongation factor (P-TEFb) to activate viral gene expression.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	CYTOR also plays a global role in regulating cellular gene expression, including those involved in controlling actin dynamics.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Depletion of CYTOR expression reduces cytoplasmic actin polymerization in response to T cell activation.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	In addition, treating HIV-infected cells with pharmacological inhibitors of actin polymerization reduces HIV gene expression.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	We conclude that both direct and indirect effects of CYTOR regulate HIV gene expression.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	The introduction of antiretroviral therapy (ART) has successfully limited the spread of Human Immunodeficiency Virus (HIV) and improved patient clinical outcomes.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	However, a complete cure for HIV infection remains out of reach, as the transcriptionally silent but replication-competent provirus that is integrated into the host genome persists in long-lived cellular reservoirs, which are comprised of memory-resting CD4 T cells, as well as cells of myeloid lineages .
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	These reservoirs are highly stable and are resistant to both ART and the effects of the host immune surveillance, thus posing a significant obstacle to eradicating the HIV reservoirs.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	Consequently, in most people living with HIV, interrupting ART leads to rapid viral load rebound, usually within weeks after treatment cessation [3–6].
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	As T cell stimulation triggers activation of proviral transcription, one strategy that has been proposed to eliminate the HIV reservoirs is a “Shock-and-Kill” approach, which utilizes latency-reversing agents (LRAs) to first activate dormant HIV-infected T cells and facilitate cell death by viral cytopathic effects or immune-mediated killing.
PMC11075828	Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.	This step is done in the presence of ART, so there are no further rounds of HIV replication. [

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Chain pairing analysis showed that cells from the T cell clusters mostly contained a single pair of chains (50-60%), with orphan (5-20%) and extra (5-10%) chains present in small fractions of cells (fig. S23A).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Notably, the frequency of extra α chains (extra VJ) was higher than that of β chains (extra VDJ), potentially due to more stringent and multi-layered allelic exclusion mechanisms at the TCRβ locus compared to TCRα (57).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

As expected, the NK and ILC clusters held no productive TCR chains.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Within the γδ T cell clusters, only a small proportion had a productive TCR chain, which may result from cytotoxic T cells co-clustering with γδ T cells.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

We also carried out γδ TCR sequencing in selected spleen, bone marrow and liver samples.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

The γδ TCR sequencing data was subjected to a customized analysis pipeline that we developed and optimised based on cellranger followed by contig re-annotation with dandelion (see Materials and Methods), facilitating the full recovery of γδ chains in our data.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

This analysis confirmed that the majority of productive γδ TCR chains originated from the ITGAD-expressing γδ T cells (fig. S23B), supporting the robust identification of this population.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

The Trm_Tgd population could not be confirmed by γδ TCR sequencing due to the lack of sample availability.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

We next examined V(D)J gene usage in relation to T cell identity.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

In the MAIT population, we detected significant enrichment of TRAV1-2, as expected (fig. S23C).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Selecting only the TRAV1-2+ cells (MAIT cluster and other clusters) revealed a notable tissue-specific distribution of TRAJ segments with TRAJ33 in spleen and liver, TRAJ12 in liver and TRAJ29/TRAJ36 in jejunum (fig. S23D).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

This suggests that there may be different antigens for MAIT cells in the spleen, liver and gut corresponding to the different metabolomes in these tissues.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

In addition, full analysis of the TCR repertoire of the MAIT cells revealed previously unappreciated diversity of V segment usage in the beta chain (fig. S23D).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

We then defined clonally related cells on the basis of identical CDR3 nucleotide sequences to investigate their TCR repertoires.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Using this approach, we found that clonally expanded cells were primarily from the resident memory T cell compartment, including the Th1/Th17 populations mentioned above (Fig. 4G and fig. S23E).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

As expected, these clonotypes were restricted to single individuals and within an individual they were distributed across tissues and subsets (Fig. 4H and fig. S23, F to H).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

We found a small number of isolated CD4+ T cell clones that shared Tregs and effector T cell phenotypes, possibly due to low levels of plasticity or due to (trans)differentiation from the same naive precursor cell in the periphery (fig. S23H).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Focusing on the most expanded clonotypes (>20 cells), the majority were widespread across five or more tissues, supporting the systemic nature of tissue-resident immune memory (Fig. 4G).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Moreover, we found that several clonotypes present across tissues consisted of a mixture of cells from the Tem/emra_CD8 and Trm/em_CD8 populations (fig. S23H), suggesting that a single naive CD8+ T cell precursor can give rise to diverse cytotoxic T cell states, which harbour immune memory across multiple non-lymphoid tissues, emphasizing the plasticity of phenotype and location within a clone.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

In summary, we have described 18 T/innate cell states in our data by integrating CellTypist logistic regression models, manual curation and V(D)J sequencing.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

This has yielded insights into the MAIT cell compartment and its antigen receptor repertoire distribution that differed between spleen, liver and gut.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

For the cytotoxic T cell memory compartment there was broad sharing of clones across gut regions for Trm_gut_CD8, and mixed Tem/emra_CD8 and Trm/em_CD8 T cell clonotypes with broad tissue distributions.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

After focusing on individual immune compartments, we next took a combined approach in order to better understand the immune landscape of selected tissues.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

As shown in Fig. 5A, each tissue has its own immune neighborhood, for example, while spleen and lymph nodes are rich in B cells, composition of their myeloid compartment varies.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

In particular, a large population of erythrophagocytic macrophages, known as red pulp macrophages, are evident in the spleen (in keeping with their role in red blood cell turnover), whereas lymph nodes are rich in dendritic cells.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

As expected, bone marrow uniquely contains progenitor populations.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Furthermore lung and liver contain significant numbers of monocytes, including CX3CR1+ nonclassical monocytes whereas these cells are absent from the jejunum, perhaps reflecting different degrees of vascularization.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

In contrast, the jejunum has an abundance of resident memory T cells (CD8+ T cells and Th1/Th17) as well as plasma cells.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Our long-term vision for CellTypist is to provide a reference atlas with deeply curated cell types publicly available to the community.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Therefore, via a semi-automatic process, we fed the identities of the 41 immune cell types identified in our dataset (including both shared and novel cell type labels) back into CellTypist, demonstrating how CellTypist can be updated and improved over time.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Combined with the initial 91 cell types and states included in the reference datasets, CellTypist now comprises a total of 101 annotated cell types (Fig. 5B).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Here, we present a multi-tissue study of immune cells across the human body within diverse organ donors.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

By sampling multiple organs from the same individuals, which allows for robust control of age, sex, medical history, drug exposure and sampling backgrounds, we reveal tissue-specific expression patterns across the myeloid and lymphoid compartments.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

We also introduce CellTypist, a publicly available and updatable framework for automated immune cell type annotation that, in addition to identifying major cell types, is able to perform fine-grained cell subtype annotation - normally a time-consuming process that requires expert knowledge.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

We developed CellTypist by integrating and curating data obtained from 19 studies performed across a range of tissues, with in-depth immune cell analysis comprising 91 harmonized cell type labels.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

However, as demonstrated here, for example in the γδ T cell compartment, manual curation following automated annotation still has a role to play in revealing specific cell subtypes that may be absent from the database/training set.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

To reduce the need for this, in the longer term, the CellTypist models will be periodically updated and extended to include further immune and non-immune sub-populations as more data become available.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Within the myeloid compartment, macrophages showed the most prominent features of tissue specificity.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Erythrophagocytic macrophages in the liver and spleen shared features related to iron-recycling (58) with macrophages in other locations, such as the mesenteric lymph nodes, suggesting that macrophages participate in iron metabolism across a range of tissues.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

In addition, we characterized subsets of migratory dendritic cells (CCR7+) revealing specific expression of CRLF2, CSF2RA and GPR157 in the lung and lung-draining lymph nodes, and expression of AIRE in the mesenteric and lung-draining lymph nodes.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

These migratory dendritic cell states are interesting targets for future in depth functional characterisation in the context of allergy, asthma and other related pathologies (59, 60).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

In the lymphoid compartment, we combined single-cell transcriptome and VDJ analysis, which allowed the phenotype of adaptive immune cells to be dissected using complementary layers of single cell genomics data.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Of note, we detected a subset of memory B cells expressing ITGAX (CD11c) and TBX21 (T-bet) that resemble ABCs previously reported to be expanded in ageing (48), following malaria vaccination (61) and in systemic lupus erythematosus (SLE) patients (62).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

In our data, these B cells did not show clonal expansion and at least 50% showed IgM subclass, suggesting that they may be present at low levels in healthy tissues and expand upon challenge as well as ageing.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

BCR analysis revealed isotype usage biased towards IgA2 in gut plasma cells, which may be related to structural differences (63) or higher resistance to microbial degradation as compared to IgA1 (64).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

In the T cell compartment, our results provided insights into the heterogeneity of T cell subtypes and their tissue adaptations.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Notably, we identified subsets of CD4+ Trm based on functional capacity for IFN-γ or IL-17 production that were mostly localized to intestinal sites, analogous to mouse CD4+ Trm generated from IL-17-producing effector T cells in the gut (65).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

We also identified different subsets of CD8+ Trm including a gut-adapted subset expressing CCR9, which mediates homing to intestinal sites via binding to CCL25 (66) and another Trm-like subset more targeted to lymphoid sites.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

TCR clone sharing between memory subtypes of CD8+ T cells suggests their origin from a common precursor, or their differentiation or conversion during migration or maintenance, such as conversion of effector memory T cells (Tem) to resident memory T cells (Trm) (56).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

We also identified distinct subsets of γδ T cells based on tissue-specific gene expression patterns, showing distinct integrin gene expression and tissue distributions.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

In summary, using this dataset of nearly 360,000 single cell transcriptomes (of which ~330,000 were immune cells) from donor-matched tissues from 12 deceased individuals, we have shown how a combination of CellTypist-based automated annotation, expert-driven cluster analysis and antigen receptor sequencing can synergize to dissect specific and functionally relevant aspects of immune cells across the human body.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

We have revealed previously unrecognized features of tissue-specific immunity in the myeloid and lymphoid compartments, and have provided a comprehensive framework for future cross-tissue cell type analysis.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Further investigation of human tissue-resident immunity is needed to determine the effect of important covariates such as underlying critical illness, donor age and gender as well as considering the immune cell activation status, to gain a defining picture of how human biology influences immune functions.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Our deeply characterised cross-tissue immune cell dataset has implications for the engineering of cells for therapeutic purposes and addressing cells to intended tissue locations, and for understanding tissue-specific features of infection as well as distinct modes of vaccine delivery to tissues.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Tissue was obtained from deceased organ donors via the Cambridge Biorepository for Translational Medicine (CBTM, https://www.cbtm.group.cam.ac.uk/), REC 15/EE/0152.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Detailed sample locations taken can be found in Fig. 1 and protocols are described in detail in Supplementary Materials.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Additional tissue samples were from Columbia University and were obtained from deceased organ donors at the time of organ acquisition for clinical transplantation through an approved protocol and material transfer agreement with LiveOnNY.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Six donors were processed with a uniform protocol at Cambridge university where solid tissues were cut into small pieces, then homogenised with enzymatic digestion for 2x 15 minute heating/mixing steps at 37°C.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

The remaining six donors were subjected to a tissue adapted protocol with the aim of improving immune cell recovery, and this protocol was harmonised as closely as possible between the two collection sites.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

For scRNA-seq experiments, single cells were loaded onto the channels of a Chromium chip (10x Genomics).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

cDNA synthesis, amplification, and sequencing libraries were generated using either the Single Cell 5′ Reagent (v1 and v2) (Cambridge University) or 3′ Reagent (v3) (Columbia University) Kit.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

TCRαβ, BCR and TCRγδ paired VDJ libraries were prepared from samples made with the 5′ Reagent kit.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

All libraries were sequenced on either a HiSeq 4000 or NovaSeq 6000 instrument.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

scRNA-seq data was aligned and quantified using the cellranger software (version 6.1.1, 10x Genomics Inc.).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Cells from hashtagged samples were demultiplexed using Hashsolo (67).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Cells with fewer than 1,000 UMI counts and 600 detected genes were excluded.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Doublets were detected using Scrublet (68).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Downstream analysis from data normalization to graph-based clustering were performed using Scanpy (version 1.6.0) (69), with details described in Supplementary Materials.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Data integration was done using BBKNN (70) and scVI (71), and the results were compared using kBET (72).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

scTCR-seq and scBCR-seq data were aligned and quantified using the cellranger-vdj software (version 2.1.1 and 4.0, respectively).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

For TCRγδ we implemented a customized pipeline (https://sc-dandelion.readthedocs.io/en/latest/notebooks/gamma_delta.html) due to cellranger being tuned towards alpha/beta TCR chains.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

scTCR-seq analysis including productive TCR chain pairing and clonotype detection was performed using the scirpy package (73).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Details of CellTypist, including cross-data cell type label harmonization and automated cell annotation, can be found in Supplementary Text.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Briefly, immune cells from 20 tissues of 19 studies were collected and harmonised into consistent labels.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

These cells were split into equal-sized mini-batches, and these batches were sequentially trained by the l2-regularized logistic regression using stochastic gradient descent learning.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Feature selection was performed to choose the top 300 genes from each cell type, and the union of these genes were supplied as the input for a second round of training.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

For single molecule FISH, samples were run using the RNAscope 2.5 LS fluorescent multiplex assay (automated).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Slides were imaged on the Perkin Elmer Opera Phenix High-Content Screening System, in confocal mode with 1 μm z-step size, using 20X (NA 0.16, 0.299 μm/pixel) and 40X (NA 1.1, 0.149 μm/pixel) water-immersion objectives.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

For flow cytometry, mononuclear cells (MNCs) were either stained ex vivo or post activation with PMA+I for two hours.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Cells were stained with the live/dead marker Zombie Aqua for 10 minutes at room temperature, and then washed with PBS+0.5%FCS, with the CD8 and B cell panels of antibodies.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

qPCR was performed in three spleen samples.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Cells were stained with the live/dead marker Zombie Aqua for 10 minutes at room temperature, and then washed with PBS+0.5%FCS, followed by staining with the antibodies at 4°C for 45 minutes.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Cell sorting was performed on a BD Fusion 4 laser sorter and RNA was extracted using a Zymo Research RNA micro kit.

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

For immunofluorescence, samples were fixed in 1% paraformaldehyde for 24 hours followed by 8 hours in 30% sucrose in PBS, and were stained for 2h at RT with the appropriate antibodies, washed three times in PBS and mounted in Fluoromount-G® (Southern Biotech).

PMC7612735

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Images were acquired using a TCS SP8 (Leica, Milton Keynes, UK) confocal microscope.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

The implementation of antiretroviral therapy (ART) has effectively restricted the transmission of Human Immunodeficiency Virus (HIV) and improved overall clinical outcomes.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

However, a complete cure for HIV remains out of reach, as the virus persists in a stable pool of infected cell reservoir that is resistant to therapy and thus a main barrier towards complete elimination of viral infection.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

While the mechanisms by which host proteins govern viral gene expression and latency are well-studied, the emerging regulatory functions of non-coding RNAs (ncRNA) in the context of T cell activation, HIV gene expression and viral latency have not yet been thoroughly explored.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Here, we report the identification of the Cytoskeleton Regulator (CYTOR) long non-coding RNA (lncRNA) as an activator of HIV gene expression that is upregulated following T cell stimulation.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Functional studies show that CYTOR suppresses viral latency by directly binding to the HIV promoter and associating with the cellular positive transcription elongation factor (P-TEFb) to activate viral gene expression.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

CYTOR also plays a global role in regulating cellular gene expression, including those involved in controlling actin dynamics.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Depletion of CYTOR expression reduces cytoplasmic actin polymerization in response to T cell activation.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

In addition, treating HIV-infected cells with pharmacological inhibitors of actin polymerization reduces HIV gene expression.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

We conclude that both direct and indirect effects of CYTOR regulate HIV gene expression.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

The introduction of antiretroviral therapy (ART) has successfully limited the spread of Human Immunodeficiency Virus (HIV) and improved patient clinical outcomes.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

However, a complete cure for HIV infection remains out of reach, as the transcriptionally silent but replication-competent provirus that is integrated into the host genome persists in long-lived cellular reservoirs, which are comprised of memory-resting CD4 T cells, as well as cells of myeloid lineages .

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

These reservoirs are highly stable and are resistant to both ART and the effects of the host immune surveillance, thus posing a significant obstacle to eradicating the HIV reservoirs.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

Consequently, in most people living with HIV, interrupting ART leads to rapid viral load rebound, usually within weeks after treatment cessation [3–6].

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

As T cell stimulation triggers activation of proviral transcription, one strategy that has been proposed to eliminate the HIV reservoirs is a “Shock-and-Kill” approach, which utilizes latency-reversing agents (LRAs) to first activate dormant HIV-infected T cells and facilitate cell death by viral cytopathic effects or immune-mediated killing.

PMC11075828

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

This step is done in the presence of ART, so there are no further rounds of HIV replication. [