Input:
LMP1 Upregulates IL10 and Constitutively Activates Stat3
To identify cytokines that may contribute to the increased survival and growth of lymphomas, the expression levels of a panel of cytokines were screened on CD19+ MACS-purified B cells, using an RPA probe set for IL4, IL5, IL10, IL13, IL15, IL9, IL2, IL6, and IFNgamma. Expression levels were quantified with a phosphorimager and normalized to the ribosomal housekeeping gene L32. None of the tested cytokines were detected in wild-type lymphocytes, therefore cytokine:L32 ratios were set to 1 in the mouse B cell lymphoma line 967. Transcription of IL10, IL15, and IFNgamma were reproducibly detected in LMP1 transgenic lymphocytes and lymphoma cells and was higher than in the B cell lymphoma cell lines 967 and K46mu (Figure 5A). There was no significant difference in the expression of IL15 and IFNgamma between LMP1 transgenic lymphocytes and lymphoma cells, suggesting that upregulation of IL15 and IFNgamma is induced by LMP1 expression in healthy lymphocytes but is not a unique property of malignant lymphocytes. Strikingly, IL10, a B lymphocyte stimulatory cytokine, was increased 1.5- to 5-fold in the wild-type and LMP1 transgenic lymphoma cells compared to LMP1 transgenic lymphocytes (Figure 5A). Production of IL15 and IFNgamma has been associated with induction of cytotoxic effector responses in cells latently infected with EBV [33,34]. However, transformation and growth properties induced by EBV are associated with the upregulation of IL10 [35-38]; hence, the effects of IL10 upregulation on the growth properties of the lymphoma cells were further examined. Immunoblot analysis indicated that LMP1 transgenic lymphocytes and wild-type and LMP1 transgenic lymphoma cells had corresponding increased levels of phosphorylated alpha and beta isoforms of activated Stat3, a target of the IL10 receptor (Figure 5B). However, when comparing the same lymphomas, there was no correlation between the levels of IL10 induction and the levels of Stat3 activation. This suggests that the activation of Stat3 is not solely induced by IL10 or that Stat3 activation may be constitutive. Additionally, there was no correlation between the levels of LMP1 expression and the levels of IL10 induction (Figures 1A and 5A). This indicates that the induction of IL10 is a general property associated with enhanced survival and may only be indirectly affected by LMP1. Neutralizing antibodies to IL10 did not affect the survival of lymphoma cells as determined by the MTS assay (unpublished data), suggesting constitutive activation of Stat3. This was confirmed by immunoblot analysis such that in the presence of anti-IL10 neutralizing antibodies, pStat3 levels remained activated in lymphoma cells isolated from wild-type and LMP1 transgenic lymphomas (Figure 5C). Exogenous addition of IL10 enhanced pStat3 activation above constitutive levels, indicating that lymphoma cells are responsive to IL10 treatment (Figure 5C). This means that although the lymphoma cells have constitutive Stat3 activation, it may be further enhanced by IL10 induction. The neutralizing effect of the anti-IL10 antibody was confirmed by pre-incubation of IL10 with anti-IL10 antibody compared to a rat IgG1 isotype control (Figure 5C).
Nuclear translocation of pStat3 is a consequence of activation, and nuclear pStat3 was not detected by immunohistochemistry staining of spleen sections from control mice. However, nuclear pStat3 was detectable in LMP1 transgenic mice and wild-type lymphomas and was detected more homogeneously in LMP1 transgenic lymphomas (Figure 5D). The constitutive activation of pStat3 and abundant nuclear Stat3 suggests that Stat3 signaling contributes to LMP1-mediated lymphoma development.

detected

detected

detected

detected

detected

detected

detected

detected

detected

IFNgamma

expression

expression

IFNgamma

expression

Production

Production

IFNgamma

expression

translocation

Nuclear

detected

nuclear

detectable

nuclear

detected

nuclear

Neutralizing

neutralizing effect

phosphorylated

Upregulates

Activates

higher

Transcription

upregulation

upregulation

IFNgamma

induced

upregulation

expression

increased

upregulation

upregulation

increased

phosphorylated

activated

induction

activation

activation

induced

activation

induction

induction

activation

activated

enhanced

activation

activation

activation

enhanced

activation

consequence

translocation

activation

activation

activation

activation

target

correlation

induction

activation

correlation

expression

induction

affected

induction

Transcription

Transcription

Transcription

IFNgamma


Input:
LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway
LMP1 transformation of rodent fibroblasts requires activation of PI3K and Akt [5]. Additionally, activated pAkt is frequently detected in NPC and the neoplastic Reed-Sternberg cells of classical HD [39,40]. To determine if Akt signaling is activated in LMP1 transgenic mice, pAkt and several of its targets were assessed by immunoblotting of splenic CD19+ MACS-purified B cells. LMP1 transgenic B cells had increased levels of pAkt compared to wild-type lymphocytes; however, progression to lymphoma in both LMP1-positive and -negative lymphoma cells did not further increase pAkt levels. The Akt target glycogen synthase kinase 3 (GSK3) is inactivated by phosphorylation; however, increased phosphorylated GSK3 was not detected in the transgenic lymphocytes and was almost absent in the lymphoma samples (Figure 6A). This finding indicates that GSK3 is not a target of activated Akt in the LMP1 transgenic lymphocytes and lymphoma cells. Similarly, activation of Akt without phosphorylation of GSK3 has been previously shown in EBV-positive HD [40]. In contrast, the wild-type lymphocytes lacked activated Akt but did have detectable phosphorylated GSK3. This further suggests that additional pathways are involved in the regulation of GSK3.
To identify other potential Akt targets, immunoblot analysis for p-mTOR was performed. Activated p-mTOR was not increased in LMP1 transgenic lymphocytes or lymphoma cells, indicating that this pathway is not affected by LMP1-induced Akt activation and does not contribute to lymphoma development (Figure 6B). Akt is also known to phosphorylate and induce the degradation of the pro-apoptotic Forkhead family of transcription factors, leading to cell cycle progression and survival in some human tumors [41,42]. Immunoblot analysis of splenic B cells did not consistently detect p-FoxO1 levels, a signal that targets FoxO1 for degradation. Hence, degradation of FoxO1 was assessed by detection of total FoxO1 levels. Immunoblot analysis indicated that total FoxO1 levels were greatly decreased in wild-type and LMP1 transgenic lymphomas (Figure 6B), suggesting that inhibition of the Forkhead signaling pathway is an important target of Akt in lymphoma development. However, considering that Akt activation did not induce FoxO1 degradation in LMP1 transgenic B cells, Akt may not be the sole regulator of FoxO1, and it may be that progression to lymphoma requires modulation of multiple pathways.
The Forkhead family of transcription factors is known to induce the expression of the Cdk inhibitor p27 [43,44]. LMP1-transformed rodent fibroblasts have decreased expression of p27, upregulation of Cdk2, and subsequent phosphorylation and inactivation of the tumor suppressor gene Rb [45]. To investigate whether LMP1 affected cell cycle regulation through the Rb pathway in B cells, immunoblot analyses for pRb, Cdk2, and p27 were performed on splenic CD19+ MACS-purified B cells. LMP1 transgenic B cells had enhanced levels of pRb with concomitant stabilization of total Rb levels and Cdk2 compared to wild-type B lymphocytes (Figure 6C). Progression to lymphoma in both wild-type and LMP1 transgenic lymphoma cells led to increased levels of Rb, correspondingly high levels of Cdk2, and decreased levels of p27 (Figure 6C). These data indicate that the Rb pathway is deregulated in LMP1 transgenic lymphocytes and that lymphoma cells are distinguished by loss of FoxO1 and decreased p27.

target

Akt

glycogen synthase kinase 3

target

Akt

targets

Akt

expression

inactivated

target

decreased

degradation

decreased

loss

decreased

phosphorylated

phosphorylation

phosphorylated

requires

activation

activation

Akt

activated

pAkt

increased

pAkt

increase

pAkt

increased

phosphorylated

activated

Akt

activation

Akt

activated

Akt

Activated

p-mTOR

increased

Activated

induced

activation

activation

Akt

activation

Akt

induce

activation

degradation

enhanced

pRb

enhanced

led

increased

increased

Rb

high levels

regulation

affected

Activated

induced

targets

regulator

Akt


Input:
LMP1 Promotes Tumor Growth and Survival through Activation of Akt, NFkappaB, and Stat3 Pathways
To explore which pathways were required for the enhanced growth and survival of LMP1-induced lymphomas, splenocytes from wild-type and LMP1 transgenic mice were cultured in the presence of inhibitors for Akt, NFkappaB, Stat3, mTOR, or MAPK and assayed for growth and survival by the MTS assay. As previously shown, wild-type lymphocytes were not viable in culture and could not be tested with the inhibitors. However, the enhanced viability of LMP1 transgenic lymphocytes was effectively blocked by treatment with triciribine, BAY11-7085, cucurbitacin I, and slightly with SB203580, but not by treatment with rapamycin, U0126, or AG490 (Figure 7). Triciribine inhibits the activation of Akt and at 20 muM has been shown to induce growth arrest in cancer cells with aberrant Akt activity [46]. The effects of triciribine on cell growth of the transgenic lymphocytes and lymphomas were apparent as low as 1 muM, suggesting that activation of Akt is required for the survival and growth of LMP1 transgenic lymphocytes and lymphoma cells (Figure 7). The effects of the inhibitors were assessed by identifying phosphorylated Akt, Stat3, and total levels of IkappaBalpha (Figure 8). Treatment with triciribine effectively blocked phosphorylation of Akt, and phosphorylated Akt was no longer detected past 5 muM. Phosphorylated Stat3 and IkappaBalpha were still present at 25 muM. These findings suggest that triciribine specifically targets Akt and that Akt activation is required for the enhanced viability of the transgenic lymphocytes and lymphoma cells.
Inhibition of NFkappaB signaling rapidly induces cell death of EBV-transformed lymphocytes [17,18]. BAY11-7085, an inhibitor of NFkappaB signaling, also greatly decreased the viability of the LMP1 transgenic lymphocytes and lymphoma cells at doses as low as 1 muM, and at 5 muM the cells were completely nonviable (Figure 7). This is well within the reported IC50 of 10 muM. Phosphorylated Akt, Stat3, and total IkappaBalpha were still present up to treatment with 15 muM and then were no longer detected (Figure 8). This finding suggests that inhibition of NFkappaB can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells without significant effects on activation of Akt or Stat3.
Cucurbitacin I inhibits activation of Stat3 by suppressing the activation of its kinase JAK2. It has been shown to selectively inhibit the growth of tumors with constitutively activated Stat3 [47]. Similarly, LMP1 transgenic lymphocytes and lymphoma cells were susceptible to cucurbitacin I treatment starting at 0.1 muM, a dose that corresponds closely to the reported IC50 of 500 nM (Figure 7) [47]. Phosphorylated Akt, Stat3, and total IkappaBalpha were not detectable past 1 muM and at higher doses all protein levels were greatly decreased, indicative of the total loss of viability (Figure 8). A second reported inhibitor of Stat3, AG490, had no effect on growth (Figure 7), but activation of Akt, Stat3, or levels of IkappaBalpha were also not affected (Figure 8). These findings suggest that inhibition of Stat3 can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells, but Stat3 inhibition also has considerable crossover effects on Akt and NFkappaB signaling.
LMP1 has also been shown to activate JNK and p38 MAPK pathways [13,48], and LMP1 transgenic lymphocytes were mildly susceptible to growth inhibition by SB202190, an inhibitor of p38 MAPK, but not U0126, an inhibitor of MEK1/2 activity. However, effects of SB202190 were only apparent at high doses (>10 muM), much higher than the reported IC50 of 0.35 muM, suggesting that p38 MAPK does not significantly contribute to the enhanced viability in LMP1 transgenic lymphocytes or lymphoma cells (Figure 7). Interestingly, both wild-type and LMP1 transgenic lymphomas were similarly susceptible to triciribine, BAY11-7085, and cucurbitacin I treatments, but not SB202190, AG490, or U0126 treatment, suggesting that activation of Akt, NFkappaB, and Stat3 but not MAPK pathways are characteristics associated with malignant transformation (Figure 7). rapamycin, an inhibitor of mTOR, did not affect the viability of the transgenic lymphocytes or lymphoma cells, confirming that mTOR is not targeted by Akt activation in LMP1 transgenic lymphocytes or malignant lymphoma cells (Figure 7).

inhibitors

Akt

inhibitors

inhibitors

mTOR

inhibits

activation

blocked

phosphorylation

inhibits

activation

decreased

Akt

decreased

decreased

IkappaBalpha

inhibitor

inhibition

inhibition

inhibitor

inhibitor

inhibitor

mTOR

phosphorylated

Akt

phosphorylated

phosphorylation

Akt

phosphorylated

Akt

Phosphorylated

Phosphorylated

IkappaBalpha

Phosphorylated

Akt

Phosphorylated

Phosphorylated

Akt

Phosphorylated

activation

Akt

activation

Akt

detected

phosphorylated

activation

Akt

present

Phosphorylated

present

IkappaBalpha

detected

Phosphorylated

detected

IkappaBalpha

activation

Akt

activation

activation

activated

detectable

Akt

detectable

detectable

IkappaBalpha

targets

Akt

effects

activation

affected

IkappaBalpha

targeted

mTOR


Input:
This study defines the oncogenic properties of LMP1 in promoting B cell lymphomagenesis. LMP1 transgenic mice have a higher incidence of lymphoma [26] and the progression to lymphoma correlates with higher expression levels of LMP1 (Figure 1A and 1B), suggesting that LMP1 is directly involved in tumor development. Table 1 summarizes the biological and molecular properties that were identified in wild-type and LMP1 transgenic lymphomas. Although many of the molecular properties studied were similar between wild-type and LMP1 transgenic lymphomas, there were distinguishing biological properties, namely the ability of LMP1 transgenic lymphomas to induce higher levels of survival and proliferation. Interestingly, although LMP1 transgenic mice develop lymphomas in the same B-1a cell type as spontaneous wild-type lymphomas (Figure 2), some signaling effects induced by LMP1 may explain the enhanced promotion to lymphomagenesis. Since CD40-deficient mice have decreased numbers of IgMhighIgDlow cells, a phenotype associated with B-1, marginal zone, and memory B cells, the mimicry of CD40 signaling by LMP1 could possibly contribute to the expansion of B-1 cells [20]. It is noteworthy that expression of LMP1 in transgenic mice has been shown to inhibit the formation of GCs [23,49], preventing typical B-2 cells from antigen-driven selection and expansion. The lack of GC reactions may contribute to the bias of LMP1 transgenic mice towards B-1 cell lymphomas. Interestingly, LMP2 signaling also favors development of B-1 cells, but this occurs in the absence of transformation. These results suggest that the mimicry of B cell receptor signaling by LMP2 promotes B-1 cell differentiation but not transformation [24,50,51]. This promotion of B-1 differentiation may account for the ability of LMP2 to exacerbate autoimmunity and bypass anergy induction [52,53]. In contrast, the preponderance of tumors of B-1a origin does not reflect effects of LMP1 signaling on B cell differentiation, as splenic B cells from healthy LMP1 transgenic mice contain similar numbers of B-1 and B-2 cells as wild-type mice. In support of this lack of effect, the differentiation of B-1 versus B-2 cells is thought to be independent of CD40 signaling [54].
B-1 cells constitute the predominant lymphocyte population in the peritoneal and pleuropericardial cavities, while B-2 cells are mainly found in the spleen, lymph node, and peripheral blood. B-1 cells produce the main source of IgM and IgA antibodies in serum, which are involved in T cell-independent responses to common microbial antigens. Importantly, B-1 cells have the unique capacity to self replenish and are also predisposed to transformation [28,29]. Clonal expansion of B-1 cells can be detected in aging mice above 18 mo of age, and B-1 cells are thought to be the murine progenitor of B cell chronic lymphocytic leukemia [30]. The data presented in this study indicate that although LMP1 is expressed in all B lymphocytes in the transgenic mice, malignancy develops in this specific subset of B cells. The elevated expression of LMP1 in B-1a cells and the activation of specific pathways apparently induce malignant growth. These same pathways can also become sporadically activated in aged mice and also result in lymphoma development. This is similar to EBV-associated cancers in vivo, where pathways that are activated by LMP1 are also activated in the less prevalent EBV-negative forms of the cancers [40,55-58]. Thus, the contribution of EBV and LMP1 to tumor development is apparently the continuous activation of pathways that can also be sporadically activated and contribute to tumor development.
The lymphomas were marked by the upregulation of IL10, constitutive activation of Stat3 signaling, and a requirement for activation of Akt, NFkappaB, and Stat3 pathways (Figures 5 and 7). Induction of IL10 is associated with the transformation of B-1 lymphomas in mice [59,60] and is frequently associated with EBV-positive B cell malignancies acting as a B cell growth factor [35-38]. In addition, LMP1 has been shown to stimulate IL10 expression in Burkitt lymphoma cell lines [61,62]. This suggests that although Stat3 is constitutively activated in the lymphoma cells, the induction of IL10 may further enhance Stat3 activation or may contribute to other IL10-responsive signaling pathways.
LMP1 activates both the canonical and non-canonical pathways of NFkappaB signaling [14,63-65], and inhibition of NFkappaB blocked the survival of LMP1 transgenic lymphocytes and LMP1-positive and -negative lymphoma cells. NFkappaB and PI3K signaling are crucial for CD40-induced proliferation, and mice deficient for cRel or the p85 regulatory subunit of PI3K are unresponsive to mitogenic stimuli, including CD40 ligation [66-68]. We have previously shown that cRel is specifically activated in both wild-type and LMP1 transgenic lymphomas, suggesting that activation of cRel is associated with B cell transformation [27]. Our observations suggest that similar to CD40-induced proliferation, LMP1 induces proliferation through PI3K-mediated activation of Akt and activation of NFkappaB components such as cRel. CD40 also induces downregulation of the cell cycle inhibitor p27 through a PI3K-dependent manner, and the LMP1 lymphoma cells also had decreased levels of p27 with phosphorylation of Rb and increased Cdk2 (Figure 6C) [66]. Although LMP1 has been shown to deregulate the Rb pathway in epithelial cells [69], to our knowledge this is the first demonstration of this property in B lymphocytes.
The requirement for Akt activation was confirmed by the striking inhibition of lymphoma viability by triciribine, an Akt inhibitor. However, the activated pAkt did not lead to phosphorylation and inactivation of the downstream target GSK3 (Figure 6A). This effect has also been described in EBV-positive HD biopsies [40]. In contrast, rapamycin, U0126, and SB202190 did not affect the survival of LMP1 transgenic lymphocytes or the wild-type and LMP1 transgenic lymphoma cells (Figure 7A, 7C, and 7E). This lack of effect by rapamycin confirmed the absence of activated p-mTOR levels (Figure 6B). These findings suggest that other Akt targets contribute to malignant progression. One key target is likely the inhibition of the Fox01 transcription factors. Repression of the pro-apoptotic transcription factor FoxO1 in a PI3K-dependent manner can inhibit expression of bcl6, a transcription factor necessary for GC formation [49,70]. It has been shown that overstimulation of CD40 signaling with agonistic antibodies inhibits GC formation [71]. Similarly, due to mimicry of CD40 signaling, transgenic LMP1 mice are also defective in GC formation [23,49]. The constitutive signaling by LMP1 likely blocks GC formation through downregulation of bcl6. Interestingly, clinical studies indicate that expression of LMP1 and bcl6 are mutually exclusive in non-HD and classical HD [72,73]. Thus, the LMP1 transgenic lymphomas mirror aspects of EBV-induced HD. Although the activation of Akt and the lack of Fox01 in the lymphoma cells suggest that LMP1 affects bcl6 and GC formation through this pathway, regulation of other Forkhead targets involved in cell cycle progression, such as p27 and CyclinD2, likely contribute to malignant transformation. Indeed, loss of FoxO1 expression in lymphoma cells correlated with a loss of p27 (Figure 6B and 6C). CyclinD2 has also been shown to be upregulated by LMP1 through release of FoxO1-mediated repression [70].
In summary, in this transgenic model of lymphomagenesis, LMP1 promotes malignancy in B-1a cells, a population that is predisposed to clonal expansion with age. The malignant lymphocytes were distinguished by constitutively active Stat3 signaling, decreased p27, and activated Akt and NFkappaB pathways, properties that are associated with promoting the growth and survival of B lymphocytes. Importantly, Akt, NFkappaB, and Stat3 pathways were critically required for the growth and survival of malignant lymphocytes as well as healthy LMP1 transgenic lymphocytes. The growth of EBV-transformed lymphocytes also requires activation of NFkappaB, and these studies provide insight into how LMP1 contributes to EBV-associated transformation. The transgenic lymphomas mirror multiple aspects of EBV-induced tumors and suggest that in vivo these properties of LMP1 are major factors in the development of cancer.

ligation

expression

expression

expressed

expression

expression

expression

expression

expression

expression

deficient

deficient

cRel

deficient

downregulation

decreased

inhibitor

Akt

inactivation

absence

activated

inhibition

Repression

inhibit

Repression

expression

downregulation

exclusive

expression

expression

lack

loss

expression

loss

decreased

phosphorylation

Rb

phosphorylation

higher

expression

elevated

expression

upregulation

Induction

stimulate

expression

activated

induction

enhance

induction

activation

activation

activated

cRel

activation

cRel

induces

activation

induces

activation

activation

Akt

activation

cRel

induces

downregulation

increased

requirement

activation

activation

Akt

activated

pAkt

lead

activated

phosphorylation

lead

activated

inactivation

activated

p-mTOR

activation

Akt

correlated

loss

loss

upregulated

target

Akt

inhibition

affects

pathway

pathway

regulation

regulation


Input:
Transgenic mice.
Generation of LMP1 mice under the Ig heavy chain promoter and enhancer have been described previously and were maintained as heterozygotes on a Balbc background [26]. LMP1 mice were genotyped by Southern blot and PCR analysis of tail DNA as described previously [26]. Spleen and liver sections were fixed in 4% paraformaldehyde and embedded in paraffin, and 5-mum sections were stained with hematoxylin and eosin for histopathological analysis. Lymphomas were passaged by intraperitoneal injection of 1 x 108 splenocytes into SCID mice and sacrificed upon development of an extended abdomen. Animals were housed in the Association for Assessment and Accreditation for Animal Care-approved animal facility at the University of North Carolina at Chapel Hill. All protocols were approved by the Institutional Animal Care and Use Committee.


Input:
Isolation and growth of B cells.
Splenocytes were prepared by homogenizing spleen tissue with two frosted slides and debris was filtered through a 100-mum cell strainer. Erythrocytes were lysed using 0.8% ammonium chloride solution (StemCell Technologies) for 10 min on ice and washed twice with PBS. B cells were isolated using CD19-MACS beads according to the manufacturer's instructions (Miltenyi Biotec) and grown in Iscove's medium supplemented with heat-inactivated 10% fetal bovine serum and antibiotic/antimycotic (GIBCO). Splenocytes isolated from SCID-passaged lymphomas consisted of 80%-90% B cells as determined by flow cytometry, and were hence not further purified with CD19-MACS beads. Splenocytes were seeded at 1.25 x 106 cells/ml and where applicable, recombinant mouse IL4 was added at 100 ng/ml, recombinant mouse IL10 at 10 ng/ml, and rat IgG1 anti-mouse IL10 and rat IgG1 isotype control at 10 mug/ml (R&D Systems). For BrdU incorporation assays, splenocytes were pulsed for 24 h with 10 muM BrdU 1 d post-harvest.


Input:
MTS assay.
The 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) cell cytotoxicity/proliferation assays were performed using the CellTiter 96 aqueous one-solution cell proliferation assay (Promega), according to manufacturer's instructions. For IL4 studies, splenocytes were cultured for 3 d in the presence or absence of 100 ng/ml IL4. Cells were seeded on day 3 in triplicate in a 96-well plate at 2.5 x 106 cells/ml at 100 mul per well. MTS reagent was added for 4 h and absorbance was read at 540 nm; values plotted were subtracted from blanks. For neutralization assays, splenocytes were seeded at 5 x 106 cells/ml at 100 mul per well on day of harvest and IL4, rat IgG1 anti-mouse IL4, or rat IgG1 isotype control (R&D Systems) were added at the concentrations indicated in the figures. Cultures were pulsed for 4 h with MTS reagent 1 d post-seeding. For inhibitor studies, splenocytes were seeded at 1 x 107 cells/ml at 100 mul per well on day of harvest, and inhibitors were added at the concentrations indicated in the figures. The inhibitors BAY11-7085, rapamycin, triciribine, U0126, SB202190, and cucurbitacin I were purchased from EMD Biosciences. For non-malignant splenocyte cultures, B cell activation was induced with 10 mug/ml of goat F(ab') anti-mouse IgM (Jackson ImmunoResearch). Cultures were pulsed for 4 h with MTS reagent 1 d post-seeding.


Input:
Immunohistochemistry.
Paraffin-embedded spleen sections were deparaffinized in Histoclear (National Diagnostics) and rehydrated in graded ethanol. Sections were antigen retrieved by microwaving in citrate buffer (pH 6.0) for 15 min (LMP1 staining) and 10 min (pStat3 staining). For LMP1 staining, sections were blocked with 1% BSA and 0.1% cold fish skin gelatin followed with streptavidin/biotin block (Vector Labs). Rat IgG anti-LMP1 (clones 8G3 and 1G6, Ascenion) were used at 1:10 dilution from tissue culture supernatants, followed with 8 mug/ml biotinylated mouse F(ab') anti-rat IgG (H+L) pre-adsorbed to mouse serum (Jackson ImmunoResearch) and 2 mug/ml streptavidin-alkaline phosphatase conjugate (Jackson ImmunoResearch). Stains were developed with BCIP/NBT and counterstained in Nuclear Fast Red (Dako). Phospho-Stat3 was detected with 4 mug/ml of pStat3 antibody (Tyr705, Cell Signaling) and detected with anti-rabbit Poly-HRP IHC detection kit (Chemicon).


Input:
Flow cytometry.
One million splenocytes were stained with the appropriate primary antibody unconjugated or conjugated to FITC, PE, or APC diluted in stain buffer (PBS with 3% FBS). For BrdU detection, the FITC-BrdU flow kit was used as instructed by the manufacturer (BD Bioscience). Briefly, cells were exposed to EMA (Molecular Probes) for exclusion of dead cells, stained for surface antigens, fixed in paraformaldehyde, and permeabilized with saponin. To expose BrdU epitopes, cells were treated with Dnase and stained with FITC-conjugated anti-BrdU antibody. Flow cytometry was performed on FACScalibur using the CellQuest program (Becton Dickinson). Further analysis was conducted on the Summit v4.2 program (Dako).


Input:
Rnase protection assay.
Total RNA was isolated from CD19+ MACS-purified B cells using the Rneasy midi purification kit with Dnase treatment, according to the manufacturer's instructions (Qiagen). The mCK-1 probe template set (BD Biosciences) was labeled using the In Vitro Transcription Kit according to the manufacturer's instructions (BD Biosciences). Briefly, 50 ng of mCK-1 probe set was labeled with [alpha-32P]UTP using T7 RNA polymerase and purified using Sephadex G-50 columns (NucAway Spin column, Ambion). The labeled probe was quantitated using a scintillation counter, and 6 x 105 cherenkov cpm was used to hybridize to 4 mug of total RNA using the RPA kit (BD Biosciences). Samples were denatured at 90 degreesC and hybridized at 56 degreesC overnight. Single-stranded RNA was digested with a mixture of Rnase A and T1, and precipitated using isopropanol. Protected probes were resolved on a denaturing 4.75% acrylamide gel, dried, and imaged using a phosphorImager (Molecular Dynamics). Densitometry was performed using the ImageQuant TL v2005 software (GE Healthcare).


Input:
PCR analysis of kappa chain rearrangement.
DNA was isolated from splenocytes using the Dneasy Tissue Kit (Qiagen), with Rnase treatment. PCR reactions contained 100 ng of genomic DNA, 0.2 muM each primer, 0.2 mM dNTPs, and 2.5 U Taq DNA polymerase (NEB) performed in 1X ThermoPol buffer (NEB). Primers used were Vkappacon and Jkappa5-1degrees and have been described previously [74]. PCR conditions were 94 degreesC for 2 min, 40 cycles of 94 degreesC for 30 s, 63 degreesC for 90 s, and 72 degreesC for 1 min, followed by 1 cycle of 72 degreesC for 5 min.


Input:
Immunoblot analysis.
Whole cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate) supplemented with 2 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1:100 protease/phosphatase inhibitor cocktails (Sigma). Crude lysates were centrifuged at 13,000 rpm for 10 min at 4 degreesC and the supernatants were collected for further analysis. Protein concentrations were determined with the Bio-Rad DC protein assay system. Lysates were boiled in the presence of 2.5% beta-mecaptoethanol, separated by denaturing SDS-PAGE, and transferred to 0.45-mum Optitran membranes (Schleicher & Schuell) in a Bio-Rad transfer unit. Membranes were immunoblotted with the appropriate primary antibody followed by horseradish peroxidase-tagged secondary antibodies (Amersham Biosciences and Dako) and detected with the SuperSignal West Pico System (Pierce).


Input:
Antibodies.
FITC-conjugated goat anti-mouse IgM, rat IgG2akappa anti-mouse IgD (clone 11-26), rat IgG1kappa anti-mouse kappa (clone 187.1), rat IgG2bkappa anti-mouse lambda (clone JC5-1); PE-conjugated goat anti-mouse IgG; un-conjugated goat anti-mouse kappa and anti-mouse lambda were purchased from Southern Biotech. APC-conjugated rat IgG2akappa anti-mouse CD19 (clone 6D5), PE-conjugated mouse IgG2akappa anti-LMP1 (clone S12), un-conjugated mouse IgG2a anti-Rb (clone 2), and anti-Cdk2 (clone 55) were purchased from BD Bioscience. PE-conjugated rat IgG2akappa anti-mouse CD5 (clone 53-7.3) was purchased from eBioscience. Rat anti-LMP1 (clones 8G3, 1G6, 7E10, and 7G8) was purchased from Ascenion. Rabbit anti-pAkt (Ser473), anti-pGSK3alpha/beta (Ser21/9), anti-pStat3 (Tyr705), anti-pStat6 (Tyr641), anti-pmTOR (Ser2448), anti-Stat6, anti-Akt, anti-FoxO1, and anti-p27 were purchased from Cell Signaling. Rabbit anti-Stat3 (H-190), anti-IkappaBalpha (C-21), and goat anti-beta actin (I-19) were purchased from Santa Cruz Biotechnology. Mouse IgG1kappa anti-GSK3 was purchased from Upstate Biotechnology. Rabbit anti-pRb (Thr373) was purchased from EMD Biosciences.


Input:
High LMP1 Expression Correlates with the Development of Lymphoma
LMP1 expression is shown by (A) immunoblotting of purified B cells (CD19+) and (B) immunohistochemistry staining of spleen tissue from wild-type (WT) and LMP1 transgenic mice.
(A) Lymphomas are identified with a number (1-7). Arrows indicate the LMP1-specific band and its degradation products as well as a non-specific band. Actin was used as a loading control.
(B) White and red pulps are shown, but this architecture is lost upon development of lymphoma. Scale bar, 20 mum.

Expression

expression

High

Expression


Input:
LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion
(A) Flow cytometry analysis of splenocytes from a WT or LMP1 transgenic lymphoma for the pan-B cell (CD19), B-1a cell (CD5), and Ig heavy chain (IgM and IgD) and light chain (kappa and lambda) markers. Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 4. This analysis was repeated on four other LMP1 transgenic lymphomas (1, 2, 3, and 6) showing a similar B-1a phenotype, of which lymphomas 2 and 4 were also doubly positive for kappa and lambda light chains.
(B) Flow cytometry analysis of WT or LMP1 transgenic splenocytes for B-1a (CD19+CD5+) and B-1b or B2 subsets (CD19+CD5-). Percentages of B-1a and B-1b or B2 subsets are shown in each quadrant. This analysis was repeated on three other WT and two other LMP1 transgenic mice with similar results.
(C) Immunoblot analysis for kappa and lambda light chains of B cells (CD19+) purified from WT and LMP1 transgenic mice. Actin was used as a loading control.


Input:
LMP1 Promotes B Cell Survival and Proliferation In Vitro
(A) MTS assay of splenocytes from WT and LMP1 transgenic mice. Splenocytes were cultured in the presence (grey bars) or absence (black bars) of IL4 for 3 d. The results are the mean +/- SEM of triplicate samples averaged from multiple mice where "n" the number of mice analyzed is as follows: n = 2 for WT lymphocytes and WT lymphomas, n = 11 for LMP1 transgenic lymphocytes, and n = 13 for LMP1 transgenic lymphomas.
(B) EMA exclusion of CD19+ gated splenocytes from WT and LMP1 transgenic mice showing percentage of viable B cells cultured with (white bars) or without (black bars) IL4 for 2 d.
(C) Flow cytometry analysis for incorporated BrdU in WT and LMP1 transgenic lymphoma cells cultured with or without IL4 for 2 d. Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 2. Percentages of cells in each quadrant are shown.


Input:
Wild-Type and LMP1 Transgenic Lymphoma Cells Survive Independently of IL4/Stat6 Signaling in Culture
(A) Rnase protection assay for IL4 mRNA from purified B cells (CD19+) from WT and LMP1 transgenic splenocytes. The L32 and GAPDH housekeeping genes were used as a loading control. Arrow indicates the position of the protected probe.
(B and C) Immunoblot analysis of WT and LMP1 transgenic mice for activated pStat6 in (B) purified B cells (CD19+) at the time of harvest or in (C) whole splenocytes cultured with or without IL4. (B) Actin was used as a loading control, and the white line indicates that intervening lanes have been spliced out.
(D and E) MTS assay of (D) WT lymphocytes and (E) LMP1 transgenic lymphoma cells cultured with IL4, a neutralizing antibody to IL4, or a rat IgG isotype control at the indicated concentrations. Shown are the results from LMP1 transgenic lymphoma 3. The results are the mean +/- SEM of triplicate samples from a single representative experiment that was repeated twice with similar results.

activated


Input:
LMP1 Upregulates IL10 Expression and Constitutively Activates Stat3
(A) Relative expression of IL10, IL15, and IFNgamma mRNA in WT and LMP1 transgenic B cells (CD19+), as detected with an Rnase protection assay. Mouse lymphoma cell lines 967 and K46mu were used as controls. Expression levels were quantified with a phosphorimager and values were normalized to the ribosomal housekeeping gene L32. The cytokine:L32 ratio was set to 1 in the mouse B cell lymphoma line 967.
(B and C) Immunoblot analysis of activated pStat3 in purified B cells (CD19+) from WT and LMP1 transgenic mice (B) at the time of harvest, and (C) 4 h after culture with or without IL10, a neutralizing antibody to IL10, or a rat IgG1 isotype control. (C) Shown are the results for WT lymphoma 1 and LMP1 transgenic lymphoma 1. Arrows indicate the positions of the alpha and beta isoforms of Stat3. Actin was used as a loading control.
(D) Immunohistochemistry detection of activated nuclear pStat3 in the spleens of WT and LMP1 transgenic mice. Scale bar, 20 mum.

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Input:
LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway
(A and B) Immunoblot analysis of purified B cells (CD19+) from the spleens of WT and LMP1 transgenic mice for Akt signaling, probing for (A) activated pAkt and downstream targets, including inactivated pGSK3alpha/beta, and (B) activated p-mTOR, and total levels of FoxO1. Arrows indicate the positions of alpha and beta isoforms of GSK3. The white line indicates that intervening lanes have been spliced out.
(C) Immunoblot analysis for cell cycle proteins regulating the Rb pathway, probing for activated pRb, and total levels of Cdk2 and the Cdk inhibitor p27. Actin was used as a loading control.

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Input:
Akt, NFkappaB, and Stat3 Signaling Are Required for the Growth and Survival of Lymphoma Cells
MTS assay of splenocytes from (A and B) WT or (C and D) LMP1 transgenic lymphomas and (E and F) LMP1 transgenic lymphoctyes. Splenocytes were cultured with or without inhibitors of NFkappaB (BAY11), mTOR (rapamycin), Akt (triciribine), MEK1/2 (U0126), p38 (SB202190), or Stat3 (cucurbitacin I and AG490) at the indicated concentrations. The results are the mean +/- SEM of triplicate samples. Shown are the results for (A and B) WT lymphoma 1, (C and D) LMP1 transgenic lymphoma 2, and (E and F) one out of two LMP1 transgenic mice analyzed. This analysis was repeated with LMP1 transgenic lymphoma 4 yielding similar results.

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Input:
Analysis of Akt, NFkappaB, and Stat3 Pathways in Contribution to the Growth and Survival of Lymphoma Cells
(A-D) Immunoblot analysis of wild-type and LMP1 transgenic lymphomas for Akt, NFkappaB, and Stat3 signaling after treatment with (A) an Akt inhibitor, triciribine, (B) an NFkappaB inhibitor, BAY11-7085, and the Stat3 inhibitors (C) cucurbitacin I and (D) AG490, at the indicated concentrations. Arrows indicate the positions of alpha and beta isoforms of Stat3. Actin was used as a loading control.

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Input: Summary of Analysis Performed on Wild-Type and LMP1 Transgenic Lymphomas

Transgenic


Input:
Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs
Activation of naive CD8+ T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs). CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes. We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) gamma expression, respectively. In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation. Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-gamma, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.

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Input: Naive CD8+T cells differentiate into effector CTLs with the ability to lyse antigen-bearing target cells by exocytosis of lytic granules containing perforin and granzymes, and to produce inflammatory cytokines such as IFN-gamma and TNF upon restimulation through the TCR (1,2). In vivo experiments have elucidated many critical parameters governing the development and evolution of primary CTL responses (3,4). In this study, we have used in vitro systems such as those developed to study CD4+T cell differentiation to define the molecular basis of effector CTL differentiation (5,6).The T-box transcription factors Eomesodermin (Eomes) and T-bet are needed for important aspects of effector and memory CTL differentiation (7). In uninfected mice, compound deletion of theTbx21(encoding T-bet) andeomesodermingenes is associated with a selective loss of CD8+T cells with an IL-2Rbeta-high, memory phenotype (8). Mice deficient for both T-bet and Eomes in T cells have impaired expression of cytolytic mediators, manifest poor cytolytic activity, and fail to control acute lymphocytic choriomeningitis virus infection (9). Nevertheless, the specific roles of T-bet and Eomes in clonal expansion and CTL differentiation have not yet been resolved: in particular, it is not known whether these transcription factors function redundantly to control effector CD8+T cell differentiation, and whether they do so directly by targeting specific effector cytokine and cytolytic genes.Runx proteins, a family of three DNA-binding transcription factors, control thymocyte differentiation and the CD4/CD8 lineage decision (10-13). Runx3 and perforin mRNA are expressed by double-positive (DP) thymocytes and CD8+single-positive (SP) thymocytes but not in CD4+SP cells (14). Although Runx3 is not expressed in naive CD4+T cells, its expression is up-regulated during Th1 cell differentiation, and Runx3 influences Th1 cell differentiation and function through direct regulation of theIl4andIfngcytokine genes (15,16). In contrast, all three Runx proteins are expressed in mature CD8+T cells (10,12), and Runx3-deficient CD8+T cells show reduced cytolytic activity (12,13). We therefore tested whether Runx3 influenced cytolytic T cell differentiation.In this report, we show that Runx3 and T-box factors synergistically regulate CTL differentiation and function. T-bet is induced quickly upon TCR stimulation and is required for early programming of cytokine production (17), whereas Eomes is induced later during differentiation and sustains IFN-gamma expression. Runx3 is required for Eomes and perforin expression, and both Eomes and Runx3 bind at thePrf1locus; in contrast, perforin expression is unaffected in T-bet-deficient cells. T cells lacking Runx3 show decreased expression of IFN-gamma and granzyme B, and Runx3 also binds the promoter regions of theIfngandGzmbgenes. Collectively, these results provide evidence for a complex transcriptional network in which Runx3 is a primary regulator ofGzmbexpression but synergizes with T-bet and Eomes, respectively, to promote transcription of theIfngandPrf1genes.

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Input:
A cell culture system to monitor effector CTL differentiation
We used a simple cell culture system to examine the kinetics of effector gene expression during CD8+ T cell differentiation. Naive CD8+ T cells from P14 TCR transgenic mice were activated for 2 d with anti-CD3 and anti-CD28 or with splenic APCs in the presence of Gp33 peptide, and were cultured in media containing 100 U/ml of recombinant human IL-2 (rhIL-2). We used TCR transgenic mice for these experiments because they provide a reliable source of CD8+ T cells that are truly naive; however, we chose not to stimulate cells with antigen in most experiments so as to avoid contamination with proteins and nucleic acids derived from APCs. There were only minor differences in gene expression during differentiation induced by antigen/APC versus anti-CD3/anti-CD28, and the major conclusions presented in this report are the same for both activating conditions.
Under our culture conditions, activated CD8+ T cells expanded exponentially and accumulated for >8 d. We limited our analysis to the first 6-8 d after activation, a period that coincides with clonal expansion of CD8+ T cells after activation in vivo.


Input:
Distinct expression kinetics of perforin and granzyme B during CTL development in culture
Our experiments revealed clear differences in the kinetics of perforin, granzyme B, and cytokine expression during CD8+ T cell activation (Fig. 1). Naive T cells showed detectable expression of perforin mRNA as well as perforin protein (Fig. 1, A-D). Relative to its expression in naive T cells, perforin (Prf1) mRNA expression did not increase appreciably at day 2 but showed a reproducible decrease at day 4, followed by robust reexpression between days 4 and 8 (Fig. 1, A-D). In contrast, granzyme B (Gzmb) mRNA was low or undetectable in naive T cells but was strongly up-regulated by day 2 after stimulation and increased progressively until day 6 (Fig. 1, A and B); similarly, granzyme B protein was expressed by day 4 and remained high until day 6 (Fig. 1 E). As expected, a small fraction of naive T cells expressed the cytokines IFN-gamma and TNF in response to stimulation, and this capacity increased significantly in differentiated cells (Fig. 1 E; see also Fig. 2 A).
We evaluated antigen-dependent cytolytic function in a short-term assay in which target cell death was measured within 2 h (Fig. 1 F). By limiting the duration of TCR stimulation, this strategy minimizes cytolysis secondary to new gene expression during the period of the assay. Naive T cells did not display significant cytolytic function in this short-term assay (unpublished data), most likely because they express immature (unprocessed) forms of perforin and lack the capacity to degranulate (18, 19). Even after activation for 2 or 4 d, the cells showed poor cytolytic activity (Fig. 1 F), in striking contrast to their capacity for efficient cytokine production (Fig. 1 E). Only cells cultured until day 6 displayed robust cytotoxicity, as judged by their ability to induce apoptosis in a large number of target cells (Fig. 1 F).
These results show that after a strong priming stimulus through TCRs and co-stimulatory receptors in vitro, granzyme B expression and the ability to produce effector cytokines are programmed early, whereas perforin expression and cytolytic function are induced later, during the phase of clonal expansion in IL-2. Therefore, the two major effector functions of CTL, cytokine production and cytolytic activity, are not intrinsically coregulated.

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Input:
Distinct kinetics of T-bet and Eomes expression during CTL differentiation
The T-box transcription factors T-bet and Eomes have been linked to the regulation of genes encoding effector cytokines (e.g., Ifng) and genes important for cytolytic function (e.g., Prf1 and GzmB) (20). We investigated the kinetics of expression of these transcription factors in our in vitro cultures (Fig. 1, A-D). T-bet mRNA and protein were not detectable in naive CD8+ T cells, but were strongly induced upon TCR priming (day 2) and remained expressed through day 6 of differentiation (Fig. 1, A and C; quantified in Fig. 1, B and D). In contrast, Eomes expression was low or undetectable at both the mRNA and protein levels in naive CD8+ T cells, and TCR priming in culture had only a modest effect on its expression at day 2 (Fig. 1, A and C). Strong induction of Eomes mRNA and protein was only observed at day 4 and later (Fig. 1, A and C). T-bet mRNA expression slightly preceded the expression of GzmB mRNA; similarly, Eomes mRNA and protein were expressed ~1 d ahead of the reexpression of perforin mRNA and protein, respectively (Fig. 1, B and D).
This detailed kinetic analysis suggested that, under our culture conditions, T-bet and Eomes contribute to distinct aspects of gene transcription during CTL differentiation. T-bet is required early for IFN-gamma production, and our data suggested that Eomes might not function during this early period but rather might contribute later to the control of perforin expression. Our data seemed most consistent with a model in which TCR signals induce T-bet, which in turn induces IFN-gamma (17) and possibly granzyme B; subsequently, Eomes is induced during the period of clonal expansion in IL-2 and activates perforin expression.

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Input:
Perforin and granzyme B expression are not appreciably regulated by T-bet
To test the model outlined in the previous paragraph directly, we compared the expression of IFN-gamma, perforin, and granzyme B in CD8+ T cells from WT and Tbx21 (T-bet)-deficient mice. As expected (17, 21), naive Tbx21-/- CD8+ T cells produced IFN-gamma poorly upon activation (Fig. 2 A). Notably, this deleterious effect of T-bet deficiency was only observed in differentiating CD8+ T cells until day 4 of culture but was almost completely mitigated by day 6 (Fig. 2 A). This most likely reflected compensation by Eomes, which was strongly induced between days 4 and 6 (Fig. 1). In contrast, T-bet-deficient T cells cultured for 6 d showed no defect in perforin mRNA expression (Fig. 2 B, compare lanes 1 and 4). We consistently observed a modest reduction in GzmB mRNA in T-bet-deficient T cells (Fig. 2 B, compare lanes 1 and 4), which did not translate into a decrease in expression of granzyme B protein (Fig. 2 C).
To examine the role of Eomes, we transduced naive CD8+ T cells from WT and Tbx21-/- mice with retroviruses containing internal ribosome entry site (IRES)-GFP that were either empty or encoded a strongly transactivating version of Eomes (Eo-VP16) (8), and expanded them for 6 d under our culture conditions. Eo-VP16, but not the empty GFP retrovirus, increased perforin expression in both WT and T-bet-deficient CD8+ T cells (Fig. 2 B, lanes 2, 3, 5, and 6). As expected, Eo-VP16 also rescued the early defect in IFN-gamma production observed in T-bet-deficient CD8+ T cells (Fig. 2 D). However, Eo-VP16 did not induce GzmB mRNA expression in either WT or T-bet-deficient cells; thus, the partial T-bet dependence of GzmB mRNA expression cannot be compensated for by Eo-VP16.

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Input:
Runx3 controls multiple aspects of the CTL differentiation program, in part through induction of Eomes
Because Runx3 is highly expressed in peripheral CD8+ T cells, and because of the T-bet-Runx3 cooperation we observed earlier in CD4+ T cells (15), we examined the role of Runx3 in effector CTL differentiation. We isolated CD8+ T cells from Runx3-/- (KO) mice of the outbred ICR background and their WT Runx3+/+ littermates by positive selection with anti-CD8 magnetic beads (Figs. S1 and S2, available at http://www.jem.org/cgi/content/full/jem.20081242/DC1). Strikingly, Runx3-/- CD8+ T cells were strongly impaired in their ability to differentiate into effector CTLs, as judged by expression of perforin, granzyme B, and IFN-gamma (Fig. 3). Compared with WT T cells, perforin mRNA and protein expression were essentially undetectable in Runx3-/- T cells at day 6 of culture (Fig. 3, A and B). Runx3-/- T cells also had no detectable Eomes expression; in contrast, T-bet expression was unimpaired (Fig. 3 A). Furthermore, Runx3 was required for maximal production of IFN-gamma, but not TNF or IL-2, by CD8+ T cells restimulated at day 6 (Fig. 3 C).
We previously reported that Th1 cell differentiation was regulated through a feed-forward loop in which T-bet is up-regulated early and induces Runx3, after which T-bet and Runx3 cooperate to induce IFN-gamma and silence IL-4, thus promoting stable differentiation toward the Th1 lineage (15, 22). Because (a) Runx3 appeared necessary for Eomes induction (Fig. 3 A), (b) the kinetics of Eomes expression paralleled those of perforin expression (Fig. 2), and (c) overexpression of Eo-VP16 in either WT or T-bet-deficient T cells led to an increase in both perforin and IFN-gamma expression (Fig. 2, B and D), we asked whether CTL differentiation was also potentially regulated by a feed-forward loop involving these same two classes of Runx and T-box transcription factors. Specifically, we asked whether Runx3, which was necessary for Eomes induction, then cooperated with Eomes to regulate transcription of the effector CTL markers perforin, IFN-gamma, and granzyme B.
To test this hypothesis, we used chromatin immunoprecipitation (ChIP) assays to ask whether Eomes and Runx3 bound regulatory regions of the Prf1, Ifng, and Gzmb genes (Fig. 3 D). Both proteins associated with gene regulatory regions in differentiated CTLs. Runx3 bound to the Prf1 and Gzmb transcription start sites (TSS); to a known IL-2 responsive enhancer located near -1 kb of the Prf1 gene (23); to the distal CTL-specific DNase I hypersensitive site 9 in the Prf1 locus (24); to the Ifng promoter near the TSS, as previously reported for Th1 cells (10); and to several DNase I hypersensitive sites in the Ifng locus (Fig. 3 D and not depicted) (25). Eomes bound primarily to the Prf1 TSS and the -1 kb enhancer; this binding was substantially greater than that observed at the promoter of the Il2rb gene, a known direct target of Eomes (8), and comparable to that observed at the Ifng TSS, a known target of T-box proteins in both Th1 and CD8+ T cells (Fig. 3 D) (17).
To determine whether Runx3 controlled the expression of CTL effector genes through its induction of Eomes, we retrovirally expressed Runx3 and Eo-VP16 in CD8+ T cells from Runx3-/- mice. Because of the limited number of CD8+ T cells in these mice, and because we saw no difference between Runx3-/- CD8+CD4- SP and CD8+CD4+ DP cells in our previous experiments, we used total Runx3-/- CD8+ T cells without further fractionation as recipients for retroviral transduction. Reconstitution of Runx3-/- CD8+ T cells with Runx3 restored expression of Eomes as well as perforin, granzyme B, and IFN-gamma (Fig. 4, A and B). In addition, Runx3-/- T cells showed a compensatory up-regulation of Runx1, which was suppressed upon reconstitution with Runx3, indicating that Runx1 is a target of repression by Runx3. Notably, Eo-VP16 did not up-regulate perforin expression when expressed in Runx3-/- cells, even though it restored the capacity to induce IFN-gamma expression upon TCR restimulation (Fig. 4, A and B). This result suggests strongly that perforin expression requires Runx3 and Eomes.
As expected from their defect in perforin and granzyme B expression, Runx3-/- CD8+ T cells showed defective cytolytic activity in a mixed lymphocyte reaction (12). However, TCR-stimulated Runx3-/- CD8+ cells were as effective as WT cells in killing tumor cells in a redirected CTL assay (12). Furthermore, CD8+ cells from the peritoneal cavity of Runx3-/- mice immunized with certain tumor cells effectively killed these targets (13). Therefore, although activation of the perforin/granzyme B machinery is defective in Runx3-/- CD8+ cells, these cells are not entirely devoid of cytolytic activity and could still effectively kill targets, possibly by alternative mechanisms such as the Fas-Fas ligand pathway.

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Input:
Runx3 and T-box factors control a complex program of transcriptional regulation during CTL differentiation
Collectively, these data provide evidence that Runx3, together with T-box factors, orchestrates a complex program of transcriptional regulation in differentiating CTL (Fig. 4 C). Runx3 is present in naive CD8+ T cells before activation (12). It represses Runx1 and has a positive role in the induction of Eomes, granzyme B, perforin, and IFN-gamma. Runx3 binds to promoters and putative regulatory regions of the latter three genes, suggesting a direct effect on gene expression. Additional experiments are needed to determine whether Eomes and Runx1 are also direct target genes of Runx3.
Surprisingly, Runx3 contributed to the optimal expression of TNF, IL-2, and IFN-gamma at day 4 (Fig. S2). For TNF and IL-2, the requirement for Runx3 subsides by day 6 (Fig. 3 C), possibly because of compensation by Runx1, which is derepressed in Runx3-/- cells (Fig. 4 A). Runx3 continues to be required for IFN-gamma expression even at day 6, perhaps because of its role in the induction of Eomes expression (Fig. 4 A).
An unexpected finding was that the two T-box transcription factors, T-bet and Eomes, are up-regulated with very different kinetics in CD8+ T cells under our culture conditions and have nonredundant roles in the subsequent expression of key effector proteins (Fig. 4 C). T-bet is needed early to confer on activated CD8+ T cells the competence to produce IFN-gamma upon restimulation, but its function is less important at later times. Eomes, which is induced late and functions downstream of Runx3, may substitute for T-bet in promoting the acute expression of IFN-gamma in restimulated CTLs (8). Indeed, T-bet and Eomes both contribute to perforin expression in NK cells (8, 26), and Eomes induces granzyme B as effectively as T-bet in developing Th2 cells (7); thus, the relative roles of these T-box transcription factors vary depending on cell type.
Surprisingly, however, Eomes and T-bet appeared nonredundant in their ability to induce two other markers of CTL function, Prf1 and Gzmb (Fig. 4 C). Rather, T-bet and Eomes were involved in regulating granzyme B and perforin expression, respectively: up-regulation of T-bet and Eomes mRNA and protein closely preceded up-regulation of Gzmb and Prf1 mRNA and protein, respectively. T-bet had no role in perforin expression under our culture conditions, and Eo-VP16 did not affect granzyme B expression when expressed in T-bet-/- or Runx3-/- cells. Because conventional Eomes-deficient mice die before precursor cells can be isolated for bone marrow transfers (27) and because T cells conditionally deficient in Eomes have only recently been described (9), we were unable to introduce Runx3 into Eomes-deficient CD8+ T cells to test formally whether Eomes cooperated with Runx3 to induce perforin expression.
Collectively, our data are consistent with a transcriptional network in which preexisting Runx3 cooperates with the induced T-box factors T-bet and Eomes and IL-2Rbeta signals (unpublished data) to orchestrate CTL differentiation (Fig. 4 C). Our data recall the "feed-forward" interaction between T-bet and Runx3 that we previously described in CD4+ (Th1) T cells (15) but are distinct in two respects: in differentiating Th1 cells, T-bet is induced by TCR signals and IFN-gamma, and in turn induces Runx3 (15), whereas in differentiating CD8+ T cells, preexisting Runx3 is required to induce the T-box transcription factor Eomes. Whole-genome experiments in these and other systems will be required to establish whether cooperation between T-box and Runx family transcription factors is a general feature of cellular differentiation programs.

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binds

perforin

regulatory regions

binds

IFN-gamma

regulatory regions

target

Eomes

target

present

expression

granzyme B

expression

perforin

expression

IFN-gamma

expression

TNF

expression

expression

IFN-gamma

expression

IFN-gamma

expression

Eomes

produce

IFN-gamma

expression

IFN-gamma

expression

perforin

expression

granzyme B

expression

perforin

expression

perforin

expression

granzyme B

expressed

expression

perforin

represses

subsides

requirement

derepressed

deficient

Eomes

deficient

Eomes

deficient

Eomes

has a positive role

induction

induction

Eomes

induction

granzyme B

induction

perforin

induction

IFN-gamma

contributed

expression

requirement

TNF

requirement

required

expression

induction

expression

up-regulated

T-bet

up-regulated

Eomes

needed

T-bet

produce

induced

Eomes

promoting

Eomes

expression

promoting

T-bet

expression

contribute

T-bet

expression

contribute

Eomes

expression

induces

Eomes

granzyme B

induces

granzyme B

T-bet

induce

Eomes

induce

Eomes

Gzmb

induce

T-bet

induce

T-bet

Gzmb

up-regulation

T-bet

up-regulation

Eomes

preceded

up-regulation

up-regulation

up-regulation

Gzmb

up-regulation

induce

Eomes

expression

induce

expression

induced

T-bet

induced

Eomes

induced

T-bet

IFN-gamma

induces

T-bet

required

induce

induce

Eomes

effect

expression

role

induction

functions downstream

Eomes

regulating

T-bet

expression

regulating

Eomes

expression

had no role

T-bet

expression

affect

expression

interaction

T-bet

T-bet

interaction

T-bet

interaction

T-bet

interaction


Input:
Antibodies and reagents.
The following antibodies used for intracellular or surface stains were obtained from eBioscience: anti-IL-2, anti-IFN-gamma, anti-TNF, anti-granzyme B, anti-CD8, anti-CD25, and anti-CD44. Anti-CD69 was purchased from BD. For ChIP experiments, the anti-Eomes antibody was obtained from Abcam and the anti-Runx3 antibody was produced by the Groner laboratory. The following antibodies were used for immunoblotting: antiperforin (Abcam), anti-Eomes (Abcam), and anti-Pol-II (Santa Cruz Biotechnology, Inc.). The T-bet antibody was provided by L. Glimcher (Harvard School of Public Health, Boston, MA).
The following reagents were used for the experiments presented in this report: Annexin V-FITC Apoptosis Detection Kit (BD), CD8 Negative Isolation Kit (Invitrogen), CD8 MicroBeads (Miltenyi Biotec), and SYBR Green PCR Core Reagents (Applied Biosystems). The Gp33 peptide (KAVYNFATC) was synthesized by the Tufts University Core Facility, and 10 mM of stock solutions was prepared in DMSO.


Input:
Isolation and culture of primary CD8+ T cells.
CD8+ T cells from 4-8-wk-old Tcra-/- x P14 TCR transgenic (Taconic), C57BL/6J WT, or Tbx21-/- (The Jackson Laboratory) mice were purified (>95% purity) by negative selection (Invitrogen) from pooled spleen and lymph node cells. CD8+ T cells from Runx3-/- mice on the ICR background were purified by positive selection (Miltenyi Biotec). All mice were maintained in specific pathogen-free barrier facilities and used according to protocols approved by the Immune Disease Institute and the Harvard Medical School Animal Care and Use Committees. For stimulation, purified CD8+ T cells were cultured at 106 cells/ml (10 ml) in T25 flasks coated with 1 mug/ml each of anti-CD3 (clone 2C11) and anti-CD28 (clone 37.51) by pretreatment with 300 mug/ml goat anti-hamster IgG. After 48 h, cells were removed from the TCR stimulation and recultured at a concentration of 5 x 105 cells/ml in media supplemented with 100 U/ml rhIL-2. Every 24 h, viable cells were counted and readjusted to 5 x 105 cells/ml with fresh media containing the corresponding amount of rhIL-2.


Input:
Isolation of CD8+ T cells from Runx3-/- mice.
Runx3-deficient T cells fail to silence CD4 expression normally (Fig. S1) (12, 13). We therefore further fractionated the positively selected CD8+ T cells from Runx3 KO mice into CD8+CD4- SP or CD8+CD4+ DP cells by separation using anti-CD4 magnetic beads. This yielded a Runx3 KO SP "enriched" population that contained 75% CD8+CD4- cells and a KO DP enriched population that contained 85% CD8+CD4+ cells (Fig. S1). The cells were stimulated with anti-CD3+ anti-CD28 for 2 d before removing them from the TCR stimulus and culturing them in media containing 100 U/ml IL-2. As previously reported, TCR-induced proliferation of Runx3-/- CD8+ T cells was severely impaired, irrespective of CD4 expression (Fig. S1) (12, 13). However, the Runx3-/- cells showed cell-surface expression patterns indicative of activated cells, including up-regulation of CD25 and CD69 (Fig. S1). As expected from their ability to up-regulate CD25, Runx3-/- CD8+ T cells responded to IL-2 supplementation after day 2 and efficiently expanded until day 6 of the culture period, albeit at slower rates compared with WT cells (Fig. S1). Although a fraction of the KO DP cells silenced CD4 expression after activation, the ratio of SP/DP cells in each enriched population remained constant thereafter, and we did not observe any major differences between these two populations throughout the culture period, indicating that in terms of effector CTL differentiation and under our culture conditions, Runx3-/- CD8+ T cells that also coexpress CD4 are indistinguishable from those that do not. The data presented in Fig. S2 are from Runx3 KO SP cells, whereas those shown in Figs. 3 and 4 are from total Runx3 KO CD8 cells.

expression

expression

expression

coexpress

deficient

silence

deficient

expression

silenced

expression

up-regulation

up-regulation

up-regulate


Input:
FACS-based cytotoxicity assay.
To measure cytotoxicity, EL4 thymoma target cells were loaded with 0 or 1 muM Gp33 peptide for 2 h before a 2-h coincubation with P14 CD8+ T cells at the effector-to-target ratios indicated in the figures in 96-well round-bottom plates. After the coincubation period, cells were stained with Annexin V-FITC and anti-CD8-allophycocyanin. Data analysis was performed with FlowJo software (Tree Star, Inc.); EL4 target cells (CD8-negative events) were gated, and the percentage of Annexin V+ target cells was determined.


Input:
Cytokine and surface marker staining.
To assess cytokine production, cells were restimulated with 10 nM PMA + 1 muM ionomycin for 6 h (unless indicated otherwise in the figures), and intracellular cytokine stains were performed as previously described (28). To detect expression of surface molecules, cells were washed in PBS, resuspended in FACS wash buffer (3% FBS, 0.1% sodium azide, 30 mM Hepes, 1x PBS) containing the antibodies indicated in the figures at previously optimized concentrations, incubated for 15 min at room temperature (RT), washed, and resuspended in 2% formaldehyde fixative solution before acquisition on a FACSCalibur (BD).


Input:
Retroviral transduction of primary CD8+ T cells.
For transduction experiments, viral supernatants were generated by calcium phosphate transfection of Phoenix cells and concentration by overnight centrifugation at 6,000 g. At ~42 h after the initial TCR activation of 106 CD8+ T cells per well in 12-well plates, the culture media was removed and replaced with complete media supplemented with 8 mug/ml polybrene containing fresh plus concentrated virus. The plates were centrifuged at 700 g for 1 h at RT before returning to 37degreesC for an additional 5 h. Retroviral constructs for Eomes-VP16 and the MIG control empty vector were a gift from S.L. Reiner (University of Pennsylvania, Philadelphia, PA) (8).


Input:
ChIP and real-time PCR analysis.
20 x 106 CD8+ T cells per immunoprecipitation were fixed by adding a 1/10th volume of fixation solution (11.1% formaldehyde, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Hepes) to 1 volume of culture media and were incubated for 10 or 30 min at RT. Fixation was stopped with 120 mM glycine on ice for 5 min. Fixed cells were washed 2x with cold PBS, 1x with cold solution I (10 mM Tris [pH 7.5], 10 mM EDTA, 0.5 mM EGTA, 1% Triton X-100), and 1x with cold solution II (10 mM Tris [pH 7.5], 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). After washes, cell pellets were resuspended at 40 x 106 cells/ml in ChIP lysis buffer (150 mM NaCl, 25 mM Tris [pH 7.5], 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate plus protease and phosphatase inhibitors), and chromatin was sheared with a sonicator to yield 0.5-1-kb DNA fragments. After preclearing the sheared chromatin with protein A-sepharose beads and removing 5% as input chromatin, immunoprecipitation was performed by adding optimized antibody amounts (per 20 x 106 cell equivalents: 2.5 mug anti-Eomes, 1:100 dilution anti-Runx3), followed by overnight incubation at 4degreesC; protein A-sepharose beads were added for the last 3 h of the incubation period. Beads were washed 2x with RIPA buffer (50 mM Tris [pH 8], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% deoxycholate), 1x with high salt buffer (50 mM Tris [pH 8], 500 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS), and 1x with TE buffer. After the last wash, DNA was eluted by resuspending the beads in elution buffer (1% SDS, 100 mM NaHCO3). Both input and ChIP chromatin were then treated with RNase A (5 mug total) for 1 h at 37degreesC, followed by the addition of proteinase K (100 mug total) and overnight incubation at 65degreesC to reverse cross-linking. DNA was then purified with QIAquick columns (Gel Extraction Kit; QIAGEN) according to the manufacturer's instructions and resuspended in a 50-mul volume. For real-time PCR detection of immunoprecipitated targets using the SYBR Green PCR Kit, a standard curve was obtained with serial dilutions of input DNA for each sample, and 1 mul ChIP DNA was used per PCR reaction (performed in duplicates). Melt curves and agarose gels were analyzed to ensure amplification of specific target sequences. Refer to Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081242/DC1) for a list of primer sets. The data are presented as the number of immunoprecipitated target sequences relative to input chromatin, assuming two copies of target sequence per cell equivalent used for the ChIP.


Input:
Northern and Western blot analyses.
RNA isolation and Northern blot analysis was performed as previously described (29). In brief, 10 mug of total RNA was loaded per lane and transferred to positively charged nylon membranes (Hybond-N+; GE Healthcare), which was confirmed by ethidium bromide staining of ribosomal RNA species on the membrane. Membranes were hybridized with 1 ng/ml alpha-[32P]dCTP-labeled trichloroacetic acid precipitable probe in ExpressHyb hybridization buffer (Clontech Laboratories, Inc.). All cDNA probes were confirmed to have the appropriate single-copy specificity under these conditions using genomic Southern blot analysis. Band intensities were acquired by phosphorimaging analysis.
For Western analysis, whole-cell protein lysates were obtained from CD8+ T cells at the time points indicated in the figures during clonal expansion in 100 U/ml IL-2 with lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1% NP-40) by resuspending samples in 10 mul per 106 cells and incubating on ice for 30 min in the presence of protease inhibitors. Immunoblot analysis was performed with the antibodies indicated in the figures after SDS-PAGE (10-30 mug of total protein was loaded per well). Quantification of detected protein was performed with an Intelligent Dark Box unit (LAS-3000; Fujifilm) and normalized for loading with the amount of RNA Pol-II detected in each lane.


Input:
Online supplemental material.
Fig. S1 shows the characterization of peripheral CD8+ T cells from Runx3-/- mice. Fig. S2 shows effector protein expression by Runx3 WT and KO cells at day 4 of in vitro culture. Primer sequences used for ChIP experiments are shown in Table S1. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20081242/DC1.


Input: Kinetics of gene expression during CD8+ T cell differentiation. (A) Kinetics of Prf1, Gzmb, Tbx21 (T-bet), and Eomes mRNA expression in differentiating P14 CD8+ T cells analyzed by Northern blotting. RNA from day 7 Th1 cells was used as a control. Sizes of mRNA transcripts are indicated. (B) Quantification of relative mRNA amounts by phosphorimager analysis. (C) Kinetics of protein expression in differentiating P14 CD8+ T cells analyzed by immunoblotting. Sizes of protein bands are indicated. (D) Relative protein amounts quantified from the Western blots. (E) Intracellular staining for granzyme B, IFN-gamma, and TNF. Granzyme B staining was specific relative to an isotype control (not depicted). Cells were restimulated with PMA and ionomycin for 4 h. (F) FACS-based assay to measure cytolytic activity of P14 CD8+ T cells against EL4 targets loaded with 0 (-) or 1 (+) muM Gp33 peptide (effector-to-target ratio = 5:1). Percentage of Annexin V+ (apoptotic) target cells in the CD8-negative EL4 target population (dot plots) was determined (histograms). Cytolytic activity was blocked by incubation with 2 mM EGTA (not depicted), confirming involvement of the granule exocytosis (perforin-granzyme B) pathway. Data are representative of at least five (A-E) or three (F) independent experiments.

staining

granzyme B

staining

IFN-gamma

staining

TNF

staining

Granzyme B

mRNA expression

Eomes

mRNA expression

mRNA expression

Gzmb

mRNA expression


Input: Regulation of perforin, granzyme B, and IFN-gamma expression by T-bet and Eomes in differentiating CTLs. (A) IFN-gamma expression by WT (Tbx21+/+) and T-bet-deficient (Tbx21-/-) T cells. Naive CD8+ T cells, or cells activated and cultured for 4 or 6 d, were restimulated with PMA and ionomycin for 6 h, and IFN-gamma expression was assessed by intracellular staining. Numbers show the percentage of IFN-gamma+ cells. (B) Northern blot analysis of Prf1 and GzmB mRNA expression in WT or T-bet-deficient CD8+ T cells activated and either left uninfected (uninf) or transduced with retroviruses expressing Eomes-VP16 (Eo-VP16) or an empty IRES-GFP cassette (GFP). Total cellular RNA was analyzed on day 6 of culture. The frequency of transduced cells in the cultures was equivalent for both constructs (~65-70% GFP+ cells; not depicted). (C) Granzyme B and IFN-gamma expression by Tbx21+/+ and Tbx21-/- T cells analyzed in restimulated cells that had been cultured for 5 d. (D) IFN-gamma production by cells transduced with Eo-VP16 or control (GFP) retroviruses (RV) measured on day 4 after 6 h of restimulation with PMA and ionomycin. Numbers show the percentage of GFP+ IFN-gamma+ cells. Results are representative of three (A and C) or two (B and D) independent experiments.

expression

perforin

expression

granzyme B

expression

IFN-gamma

expression

IFN-gamma

expression

IFN-gamma

expressing

expression

Granzyme B

expression

IFN-gamma

production

IFN-gamma

deficient

T-bet

deficient

T-bet

Regulation

expression

T-bet

Regulation

expression

Eomes

mRNA expression

mRNA expression

GzmB


Input: Key role for Runx3 in effector CTL differentiation. (A) Western analysis of Runx3, Eomes, T-bet, and perforin expression in Runx3+/+ versus Runx3-/- CD8+ SP T cells differentiated for 6 d. beta-Actin was used as a loading control. (B) Northern blot analysis of Prf1 mRNA expression in Runx3+/+ versus Runx3-/- CD8+ T cells differentiated for 6 d. beta-Actin was used as a loading control. (C) Expression of granzyme B, IFN-gamma, TNF, and IL-2 by resting or restimulated (6 h) Runx3+/+ versus Runx3-/- CD8+ SP T cells differentiated for 6 d. The vertical gray line indicates the granzyme B MFI for WT GFP+ cells. Results in A-C are representative of two independent experiments. (D) ChIP analysis of binding of endogenous Runx3 and Eomes to the Prf1 locus. Enrichment of the indicated genomic regions was evaluated by real-time PCR of DNA from immunoprecipitated and input chromatin. The data are the means of duplicate measurements from two chromatin preparations from two independent CD8+ T cell differentiations. The efficiency of recovery of input for the -1-kb region of Prf1 was 0.97% for the Runx3 ChIP and 0.5% for the Eomes ChIP.

binding

binding

Eomes

expression

perforin

expression

expression

Eomes

expression

T-bet

Expression

granzyme B

Expression

IFN-gamma

Expression

TNF

Expression

mRNA expression


Input: Runx3 controls Eomes, perforin, granzyme B, and IFN-gamma expression in effector CTLs. Runx3+/+ or Runx3-/- CD8+ T cells were activated and transduced with retroviruses bearing an empty IRES-GFP cassette (GFP) or also encoding Eomes-VP16 (Eo-VP16) or Myc-Runx3 (Runx3). The frequency of transduced cells in the cultures was equivalent for all constructs (~75-90% GFP+ cells; not depicted). (A) Protein expression in whole-cell extracts (day 6) was analyzed by immunoblotting. Overexpression of Eomes-VP16 cannot be detected with the Eomes antibody, as the C-terminal epitope is within the region that has been replaced with the VP16 transactivation domain. (B) Expression of granzyme B and IFN-gamma after culture for 6 d and restimulation for 4 h with PMA and ionomycin was determined by intracellular staining. The percentage of positively stained cells is shown above the gate; the mean fluorescence intensity (MFI) of granzyme B staining for the total population is shown below the gate. The vertical gray lines indicate the MFI for WT GFP+ cells. Results are representative of at least two independent experiments. (C) Schematic diagram of the transcriptional network involving Runx3 and T-box factors. T-bet is induced by TCR signals and is essential for early IFN-gamma expression. Runx3 is present in naive CD8+ T cells and represses Runx1 and induces Eomes, perforin, granzyme B, and IFN-gamma expression. Eomes may participate in sustaining late IFN-gamma expression, whereas Runx3 and Eomes (but not T-bet) may cooperate to activate perforin expression. The dotted line indicates the partial effect of T-bet deficiency on Gzmb mRNA but not granzyme B protein expression.

expression

Eomes

expression

perforin

expression

granzyme B

expression

IFN-gamma

Expression

granzyme B

Expression

IFN-gamma

staining

granzyme B

expression

IFN-gamma

present

expression

Eomes

expression

perforin

expression

granzyme B

expression

IFN-gamma

expression

IFN-gamma

expression

perforin

expression

Gzmb

expression

granzyme B

represses

deficiency

T-bet

Overexpression

induced

T-bet

essential

T-bet

expression

induces

expression

activate

expression

activate

Eomes

expression

activate

T-bet

expression

controls

expression

participate

Eomes

expression

effect

deficiency

expression


Input:
Insights into the Regulation of TNF-alpha Production in Human Mononuclear Cells: The Effects of Non-Specific Phosphodiesterase Inhibition
OBJECTIVE
The objective of this study was to determine the effect of nonspecific phosphodiesterase inhibition on transcription factor activation and tumor necrosis factor-alpha (TNF-alpha) production in lipopolysaccharide (LPS)-stimulated human mononuclear cells.
INTRODUCTION
The production of TNF-alpha following LPS stimulation is one of the key steps in bacterial sepsis and inflammation. The mechanism by which phosphodiesterase inhibition alters TNF-alpha production in the presence of LPS remains unclear.
METHODS
Human mononuclear cells were stimulated with LPS (1 mug/mL), in the presence and absence of Pentoxifylline (PTX; 20 mM), a nonspecific phosphodiesterase inhibitor. Western blotting of phosphorylated cytoplasmic I-kappaBalpha, nuclear factor-kappaB p65 (NF-kappaB), and nuclear cAMP-response element binding protein (CREB) was performed. DNA binding of NF-kappaB and CREB was verified by electrophoretic mobility shift assay. TNF-alpha levels were determined in the supernatant of stimulated cells in the presence and absence Protein kinase A inhibition by an enzyme-linked immunosorbent assay (ELISA).
RESULTS
PTX was demonstrated to significantly reduce cytoplasmic I-kappaBalpha phosphorylation, nuclear p65 phosphorylation, and the DNA binding activity of NF-kappaB. In contrast, PTX markedly enhanced the phosphorylation and DNA binding activity of CREB. Cells concomitantly treated with PTX and LPS secreted similar levels of TNF-alpha in the presence and absence Protein kinase A inhibition.
DISCUSSION
The increased level of cAMP that results from phosphodiesterase inhibition affects cytoplasmic and nuclear events, resulting in the attenuation of NF-kappaB and the activation of CREB transcriptional DNA binding through pathways that are partially Protein kinase A-independent.
CONCLUSION
PTX-mediated phosphodiesterase inhibition occurs partially through a Protein kinase A-independent pathway and may serve as a useful tool in the attenuation of LPS-induced inflammation.

Production

TNF-alpha

production

tumor necrosis factor-alpha

production

TNF-alpha

production

TNF-alpha

secreted

TNF-alpha

reduce

phosphorylation

reduce

phosphorylation

phosphorylated

I-kappaBalpha

phosphorylation

I-kappaBalpha

phosphorylation

following

production

in the presence of

alters

Regulation

Production

effect

production

alters

production

presence

secreted

absence

secreted


Input:
The bacterial membrane component lipopolysaccharide (LPS) is capable of initiating phosphorylation and activation of multiple host intracellular protein kinases and transcription factors. Transcription factor activation, which results in the modulation of gene transcription and protein synthesis, is a critical element in the defense mechanism of the host immune system.1,2
LPS-induced transcription factor activation has been shown to be a key regulator of tumor necrosis factor-alpha (TNF-alpha) production.3 TNF-alpha is a potent pro-inflammatory cytokine involved in a wide spectrum of cellular responses. Furthermore, TNF-alpha synthesis can be attenuated in immune cells exposed to phosphodiesterase (PDE) inhibition after challenge by a variety of pro-inflammatory stimulants.4 The signaling mechanisms affected by PDE inhibition, which ultimately lead to the downregulation of TNF-alpha production, have not been well characterized in inflammatory cells.
Classically, it has been demonstrated that PDE inhibition results in the intracellular accumulation of the second messenger cyclic adenosine-3,5-monophosphate (cAMP) and subsequent activation of Protein kinase A (PKA).5 PKA activation then leads to the phosphorylation of the transcription factor cAMP-response element binding protein (CREB), transmission of signals into the nucleus, and the subsequent modulation of gene transcription.6 This apparently simple linear cascade does not fully explain the mechanism by which an elevation in the intracellular cAMP level exerts wide-ranging effects on multiple cellular functions. There is a growing body of evidence suggesting that cAMP may function through both PKA-dependent and -independent mechanisms.6-8
LPS-induced activation of the transcription factor nuclear factor-kappaB (NF-kappaB) has also been the focus of a great deal of research. It has been clearly demonstrated that agents that increase intracellular cAMP also inhibit NF-kappaB-dependent pro-inflammatory gene transcription, particularly of the TNF-alpha gene.9
Controversy exists regarding the exact mechanism(s) by which PDE inhibition down-regulates TNF-alpha production. Possibilities include, but are not limited to, inhibition of NF-kappaB DNA binding activity, downregulation of NF-kappaB transcriptional activity, increased CREB activation, and competition between NF-kappaB and CREB for common co-activators such as CREB binding protein (CBP). It is also not known whether these processes rely solely upon the activation of PKA.
Therefore, the objective of the present study is to determine the effects of nonspecific PDE inhibition with 1-[5-oxohexyl]-3,7-dimethylxanthine (Pentoxifylline; PTX) on NF-kappaB and CREB activation in vitro in human mononuclear cells. With the use of specific inhibitors, we also investigated the role of PKA in LPS-induced TNF-alpha production.

production

tumor necrosis factor-alpha

synthesis

TNF-alpha

production

TNF-alpha

production

TNF-alpha

production

TNF-alpha

attenuated

synthesis

downregulation

production

inhibit

dependent

down-regulates

production

lead

downregulation

induced

production

regulator

production

dependent

transcription

role

induced

transcription

TNF-alpha


Input: This study was approved by the Human Research Protections Program and the Institutional Review Board. Written consent to participate in the study was obtained from all volunteers prior to blood donation.Heparinized Vacutainers were purchased from Becton Dickinson (San Jose, CA). 1.5-mL Eppendorf centrifuge tubes were purchased from Fisher Scientific (Pittsburg, PA). Hank's balanced salt solution (HBSS) and RPMI 1640 were obtained from Irvine Scientific (Santa Ana, CA). LPS fromEscherichia coliserotype 0111:B4 and PTX were purchased from Sigma (St. Louis, MO). The PKA inhibitor, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoguinolinesulfonamide-2HCl] (H89), was purchased from Calbiochem (La Jolla, CA). Antibodies for phosphorylated CREB (serine 133), phosphorylated I-kappaBalpha (serines 32 and 36), phosphorylated NF-kappaB p65 subunit (serine 276), and secondary antibodies were purchased from Cell Signaling (Beverly, MA).Dextran T500 and Percoll were received from GE Healthcare/Amersham Biosciences (Piscataway, NJ). The enzyme-linked immunosorbent assay (ELISA) for TNF-alpha was obtained from Quantikine R&D Systems (Minneapolis, MN).Tris-glycine and DNA retardation gels, nitrocellulose membranes, and western blot running buffers were obtained from Invitrogen (Carlsbad, CA). NE-PER(R)nuclear and cytoplasmic extraction reagents, the BCA protein reagent kit, the Supersignal West Pico Chemiluminescent Kit, and the LightShift Chemiluminescent EMSA Kit were purchased from Pierce (Rockland, IL). CREB and NF-kappaB DNA oligonucleotide probes were synthesized by IDT (Coralville, IA). Nylon membranes were purchased from Roche Applied Sciences (Indianapolis, IN). Western blot band quantification was performed with the UN-SCAN-IT Gel Digitizing software (Silk Scientific, Orem, UT).

phosphorylated

CREB

serine 133

phosphorylated

I-kappaBalpha

serines 32

phosphorylated

I-kappaBalpha

phosphorylated

serine 276


Input:
Human Peripheral Blood Mononuclear Cell Isolation and Stimulation
Human mononuclear cells were isolated from the peripheral blood of four healthy human volunteers. The sample size was chosen from our previously published work, which demonstrated reliable results.10 Cell isolation and all subsequent experiments were conducted under sterile and pyrogen-free conditions. Blood collected in heparin tubes was incubated with Dextran T500 and the red cells were sedimented for 40 minutes at room temperature. The resultant serum was then washed with HBSS and separated by Percoll gradient centrifugation according to the manufacturer's instructions. Cell viability was assessed by trypan blue dye exclusion, with purity of greater than 95%. Isolated cells were resuspended in RPMI 1640 supplemented with 10% FBS and 5 mM HEPES at a concentration of 1 x 107cells/mL. Each subsequent experiment listed below was conducted on samples of 5 x 106 cells per treatment group. Isolated cells were stimulated with either HBSS as a negative control, LPS (1 mug/mL), PTX (20 mM), or concomitant LPS / PTX, at the above-mentioned concentrations, for 30 minutes at 37degreesC. Cells were placed on ice for 10 minutes to stop the reaction and the samples were then stored at -70degreesC for further analysis. The stimulation times and concentrations of LPS and PTX utilized in this study were determined in previous pilot studies done in our laboratory, which examined the effects of increasing concentrations of PTX on LPS-induced TNF-alpha production. LPS at a concentration of 1 mug/mL and PTX at a concentration of 20 mM were the minimum concentrations necessary to produce a reproducible and reliable up-regulation and down-regulation in TNF-alpha production, respectively, in quantitative assays (data not shown).

production

TNF-alpha

production

TNF-alpha

down-regulation

production

induced

production

up-regulation

production


Input:
Effect of PTX on LPS-induced TNF-alpha production by mononuclear cells
The concentration of TNF-alpha was quantitatively determined by ELISA in the media of cells exposed to the treatments described above after stimulation for up to a maximum of 18 hours, at 3-hour intervals. The results are expressed in pg/mL.

production

TNF-alpha

induced

production


Input:
SDS-PAGE and Immunoblotting (I-kappaBalpha, NF-kappaB p65, and CREB)
Following stimulation, PBMCs were washed with ice-cold PBS and centrifuged to collect a cell pellet. The pellet was resuspended in ice-cold SDS sample buffer supplemented with 100 mM dithiothreitol. Cytoplasmic and nuclear extracts were isolated with NE-PER nuclear and cytoplasmic extraction reagents with 1x Halt Protease Inhibitor Cocktail according to the manufacturer's instructions. Protein concentrations were determined with the BCA protein reagent kit for each sample according to a standardized curve for albumin. Cell lysates were collected by boiling the samples in a 100degreesC water bath for 5 minutes. Ten mug of protein per sample was separated by SDS-polyacrylamide gel electrophoresis through 8-16% tris-glycine polyacrylamide gradient gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% milk in Tris-buffered saline/Tween 20 (Fischer Scientific, Pittsburgh, PA) for 1 hour. Cytoplasmic extracts were incubated with phosphorylated I-kappaBalpha antibody (1:200), while nuclear extracts were incubated with either phosphorylated NF-kappaB p65 antibody (1:500) or phosphorylated CREB antibody (1:500) overnight at 4degreesC in separate experiments. The membranes were washed with Tris-buffered saline/Tween 20 and incubated for 1 hour at room temperature with the secondary antibody, horseradish peroxidase-linked anti-rabbit IgG diluted 1:2000 in blocking solution. After repeated washing of the membrane, the Supersignal West Pico Chemiluminescent Kit was applied for antibody detection per the manufacturer's instructions.
Western blot data is presented as a percentage of LPS stimulation. The percentage of LPS stimulation was calculated by dividing the mean band pixel total for each treatment arm divided by the mean pixel total of samples stimulated with LPS and multiplying by 100. Thus, LPS stimulation is reported as 100% and each of the treatment arms is reported as a percent of LPS stimulation.

phosphorylated

I-kappaBalpha

phosphorylated


Input:
NF-kappaB and CREB Electrophoretic Mobility Shift Assay (EMSA)
The non-radioactive LightShift Chemiluminescent EMSA Kit was used to detect DNA-protein interactions. The NF-kappaB 3' biotin end-labeled DNA oligonucleotide used as a probe for the EMSA was a 42-bp double stranded construct (5'-TTGTTACAA-GGGGACTTTCCGCTGGGGACTTTCCAGGGAGGC - 3') containing two tandemly repeated NF-kappaB binding sites (underlined). Specificity was determined by a competition assay with the addition of 200 molar excess of unlabeled double stranded NF-kappaB oligonucleotide. The CREB 3' biotin end-labeled DNA oligonucleotide used as a probe for the EMSA was a 23-bp double stranded construct (5'-TTT TCG AGC TCTGACGTCAGA-GC - 3') containing the CRE consensus sequence (underlined). Specificity was determined by a competition assay with the addition of 200 molar excess of unlabeled double stranded CREB oligonucleotide.
Nuclear extracts (10 mug) were incubated with 5 nM NF-kappaB or CREB probe (NF-kB: 1x binding buffer, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 0.1% NP40, 10% glycerol, and 50 ng/mul poly dI-dC, CREB: 1x binding buffer, 20 mM Tris, pH 7.5, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 0.10% NP40, 6% glycerol, 0.1 mg/mL BSA and 50 ng/mul poly dI-dC) and were then electrophoresed through a 6% DNA retardation gel at 100V for 90 minutes. The gels were electrophoretically transferred at 380mA for 1 hour on ice to a positively charged nylon membrane and immediately cross-linked for 15 minutes with a UV transilluminator equipped with a 312 nm bulb. Streptavidin-horseradish peroxidase conjugate and the LightShift Chemiluminescent Substrate were used to detect the biotin end-labeled DNA. The nylon membranes were exposed to x-ray film for 1-3 minutes for detection.


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Role of PKA on LPS-induced mononuclear cell TNF-alpha production
To evaluate the role of PKA on TNF-alpha production in mononuclear cells and its involvement in the attenuation of LPS-induced TNF-alpha production observed following PTX treatment, H89, a specific PKA inhibitor, was utilized. Isolated mononuclear cells were incubated according to the treatment groups described above in the presence and absence of pretreatment with H89 (10 muM) for 1 hour at 37degreesC. The dose of H89 was chosen based on previous work which demonstrated specific and complete inhibition of PKA at this concentration.10 Since the activity of this inhibitor is both specific and consistent at this specified dose, we did not include experiments with additional PKA inhibitors with this set of experiments. The TNF-alpha concentration in the supernatant was measured quantitatively by ELISA. The results are expressed in pg/mL.

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Statistical Analysis
The experimental results obtained in this study were derived from four separate experiments with healthy volunteer donors. Each assay was performed in duplicate or triplicate where appropriate. Data is presented as the mean +/- SEM. Statistical differences between groups were determined by one-way analysis of variance (ANOVA) with a Bonferroni correction. Statistical significance was defined as P < 0.05.


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The effects of PTX on LPS-induced TNF-alpha production
To determine the duration of PDE inhibition on TNF-alpha production after LPS stimulation, time course studies were conducted. In accordance with prior work, PTX had a rapid and sustained effect on TNF-alpha production in vitro (Figure 1).

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