Abstract
Article Figures and data Abstract Editor’s evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Transforming growth factor β (TGFβ) is an important differentiation factor for cytotoxic T lymphocytes (CTLs) and alters the expression levels of several of homing receptors during infection. SMAD4 is part of the canonical signaling network used by members of the transforming growth factor family. For this study, genetically modified mice were used to determine how SMAD4 and TGFβ receptor II (TGFβRII) participate in transcriptional programming of pathogen-specific CTLs. We show that these molecules are essential components of opposing signaling mechanisms, and cooperatively regulate a collection of genes that determine whether specialized populations of pathogen-specific CTLs circulate around the body, or settle in peripheral tissues. TGFβ uses a canonical SMAD-dependent signaling pathway to downregulate Eomesodermin (EOMES), KLRG1, and CD62L, while CD103 is induced. Conversely, in vivo and in vitro data show that EOMES, KLRG1, CX3CR1, and CD62L are positively regulated via SMAD4, while CD103 and Hobit are downregulated. Intravascular staining also shows that signaling via SMAD4 promotes formation of long-lived terminally differentiated CTLs that localize in the vasculature. Our data show that inflammatory molecules play a key role in lineage determination of pathogen-specific CTLs, and use SMAD-dependent signaling to alter the expression levels of multiple homing receptors and transcription factors with known functions during memory formation. Editor’s evaluation This manuscript provides important information concerning the impact of TGFB and SMAD factors on antiviral CD8 T cell differentiation and memory formation. The new and interesting data from in vivo performed experiments provide solid evidence in support of their claims. Overall, this is an important study with an impressive amount of data. The only weak point is the clarity of the overall goal/message of the study as stated in the introduction. https://doi.org/10.7554/eLife.76457.sa0 Decision letter Reviews on Sciety eLife’s review process Introduction Cytotoxic T lymphocytes (CTLs) make important contributions to protective immunity by eliminating cells that contain intracellular pathogens, however rapid mobilization to infected tissues is a decisive factor in immune control. Defining how activated CTLs differentiate into functionally specialized subsets of memory CD8 T cells is an important priority for vaccine development. Our study sheds new light on this important process by revealing how two intersecting signaling pathways control a major bifurcation in diffrentiation pathway of newly activated CTLs. Multiple subsets of CTLs can be distinguished using surface markers that influence their effector functions, survival properties, and capacity to localize in specific tissues. The major subsets include central memory CD8 T cells (TCM) that use the bloodstream to circulate through secondary lymphoid organs (SLOs), as well as some effector memory CD8 T cells (TEM) and tissue resident memory CD8 T cells (TRM) that distribute to peripheral tissues. Efforts to understand how this heterogeneity develops have inspired alternative models of CD8 T cell differentiation (Montacchiesi and Pace, 2021). Some studies suggest that antigen-specific CTLs follow a linear differentiation pathway by transitioning between subsets, while others favor a branching model of differentiation whereby multiple subsets of CTLs arise from pathways that diverge soon after antigen stimulation (Bannard et al., 2009). Although many studies show that environmental cues influence the transcriptional programs of pathogen-specific CTLs, the pathways that promote commitment to specific lineages are poorly defined. Our study focuses on a network of signaling pathways that are used by members of the transforming growth family (TGF). We show that two distinct signaling pathways influence the transcriptional programs of newly activated CTLs to provide customized responses during infections with different types of pathogens. The target genes are transcription factors and adhesion molecules that determine how CTLs distribute in different tissues. Pathogen-specific CTLs express many homing receptors that are dynamically regulated during infection. Under resting conditions, SLOs are surveyed by naive CD8 T cells that search for antigen-presenting cells bearing pathogen-derived peptides. Circulating leukocytes enter resting lymph nodes from wide blood vessels known as high endothelial venules (HEVs) (Mionnet et al., 2011). CD62L (L-selectin) is an adhesion molecule that interacts with carbohydrates expressed on the surface of activated endothelial cells (Steeber et al., 1998). Naive CD8 T cells leave the bloodstream after CD62L initiates an adhesion cascade that results in transendothelial migration (Ding et al., 2000). Inside the lymph node, signals from antigen receptor and costimulatory molecules induce clonal expansion and CD62L is replaced with adhesion molecules that facilitate migration to the site of infection. Cytokines that are released in infected tissues influence the transcriptional programs of activated CTLs. During the acute phase of infection, interleukin (IL)-12 works with other inflammatory molecules to promote formation of short-lived effector T cells (TEFF) that are primed for rapid effector functions (Chowdhury et al., 2011; Cui et al., 2009; Keppler et al., 2012). Some pathogens elicit robust inflammatory responses and generate large populations of terminally differentiated TEFF cells that express killer cell lectin-like receptor G1 (KLRG1) (Obar et al., 2011; Plumlee et al., 2013), which is a membrane-bound adhesion molecule with an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic tail (Ito et al., 2006; Rosshart et al., 2008; Tessmer et al., 2007). Although CTLs transiently express KLRG1 during antigen stimulation (Herndler-Brandstetter et al., 2018; Joshi et al., 2007), stable expression identifies CTLs that do not convert to a conventional memory phenotype (Dominguez et al., 2015; Plumlee et al., 2013). Most TEFF cells disappear as the infection resolves, leaving residual populations of memory CD8 T cells in the circulation and peripheral tissues. Intravascular (IV) staining shows that some surviving CTLs maintain KLRG1 expression inside the vasculature during the memory phase of infection (Hu et al., 2015). Similar populations of KLRG1+ CTLs that were detected after infections with other pathogens are referred to as long-lived effector cells (LLECs), or terminal effector memory CD8 T cells (TTEM) (Milner et al., 2020; Renkema et al., 2020). As inflammation resolves CD62L is reexpressed on TCM cells, while CD69+ TRM cells remain in the infected tissues (Mueller et al., 2013). How these subsets contribute to protective immunity varies according to the type of pathogen. TGFβ is an immunosuppressive cytokine with multiple functions during CD8 T cell differentiation. Members of the TGF cytokine family elicit cellular responses via both canonical and noncanonical signaling pathways (Derynck and Budi, 2019). Canonical signals are mediated by a cascade of structurally related molecules known as small (Sma) and mothers against decapentaplegic (Mad)-related (SMAD) proteins (Massagué, 2012). SMAD4 is an adaptor molecule that chaperones phosphorylated receptor-activated SMAD proteins (R-SMADs) into the nucleus to modulate gene expression. We previously used genetically modified mice to explore how pathogen-specific CTLs distribute in the lungs during infection with influenza A virus (IAV) and discovered that several adhesion molecules were regulated via the canonical SMAD signaling cascade (Hu et al., 2015). Extensive phenotypic changes indicated that loss of signaling via SMAD4 caused defects during formation of TEFF and TCM cells (Cao et al., 2015; Hu et al., 2015). To further define that mechanism that was responsible for these changes, we used multiple strains of genetically modified mice to explore how the SMAD signaling network influences genetic programming of pathogen-specific CTLs during infection. Our data show that SMAD4 and TGFβ receptor II (TGFβRII) use alternative signaling mechanisms to alter the expression levels of the same genes in opposite directions. The target genes include the T-box transcription factor EOMES, which is normally expressed during formation of TCM cells and downregulated by TGFβ during TRM development (Intlekofer et al., 2005; Mackay et al., 2015). We show that during transcriptional programming of newly activated CTLs, SMAD4 positively regulates EOMES expression and induces phenotypic changes that indicate progression toward a central memory phenotype, while formation of TRM cells is inhibited. Results SMAD4 modifies homing-receptor expression in the absence of TGFβ Several groups have used Cre-lox recombination to prevent expression of either SMAD4 (S4KO) or TGFβ receptor II (TR2KO) in peripheral CD8 T cells and found altered homing-receptor expression on antigen-experienced CTLs (Cao et al., 2015; Hu et al., 2015; Igalouzene et al., 2022; Wu et al., 2021; Zhang and Bevan, 2013). For these studies, different promoters were used to induce gene ablation which may account for some variations between the results. CD4-Cre, CD8-Cre, and the proximal Lck promoter (dLck-Cre) are all expressed during early stages of thymic development. When these promoters were to prevent SMAD4 expression in peripheral CD8 T cells, investigators reported modified effector responses after T cell activation (Cao et al., 2015; Liu et al., 2022; Wu et al., 2002). Since our goal was to avoid altering naive CD8 T cells during thymic development, we used the distal Lck promoter (dLck) to prevent SMAD4 expression. When we analyzed pathogen-specific CTLs during IAV infection, very few SMAD4-deficient CTLs expressed KLRG1 or CD62L, while abnormal CD103 expression was detected on CTLs in the spleen (Hu et al., 2015). These changes were unexpected, as CTLs displayed a reciprocal phenotype after the TGFβ receptor was ablated (Hu et al., 2015). We found that the mutation primarily influenced homing-receptor expression, since SMAD4-deficient CTLs proliferated and expressed IFNγ and TNFα at similar levels as wildtype CTLs (Hu et al., 2015). SMAD4 and TGFβRII are components of an interconnected signaling network. To further understand how these molecules control homing-receptor expression, we intercrossed S4KO and TR2KO mice to produce CTLs with both mutations (S4TR2-DKO). The mice were further crossed with OTI mice that express a transgenic antigen receptor that is specific for a peptide (SIINFEKL) derived from chicken ovalbumin (OVA) presented on H-2Kb (Hogquist et al., 1994). To analyze homing-receptor expression during infection, the mice were infected with recombinant pathogens (X31-OVA and LM-OVA) that express the SIINFEKL peptide derived from chicken OVA (Jenkins et al., 2006; Pope et al., 2001). For this study, two pathogens were used to compare homing-receptor expression under different inflammatory conditions. To elicit a mild inflammatory response in the lungs, mice were infected with IAV (X31-OVA) as neuraminidase activates TGFβ (Schultz-Cherry and Hinshaw, 1996). Other mice were infected with Listeria monocytogenes (LM) to elicit an inflammatory response that supports formation of CTLs that express KLRG1 (Plumlee et al., 2015). To monitor changes in homing-receptor expression during infection, congenically marked OTI-Ctrl (lack Cre), OTI-TR2KO; OTI-S4KO and OTI-S4TR2DKO cells were transferred to B6 mice 48 hr before infection. On different days post infection (dpi), donor cells in the lungs and spleens were analyzed for CD103, KLRG1, and CD62L expression (Figure 1A, B). Statistical comparisons are shown in the supplementary data (Supplementary file 2). Prior studies show that KLRG1 is negatively regulated by TGFβ (Sanjabi et al., 2009; Schwartzkopff et al., 2015). Since latent-TGFβ can be activated by viral neuraminidase (Schultz-Cherry and Hinshaw, 1996), only small numbers of OTI-TR2Ctrl cells expressed KLRG1 after IAV infection, whereas most OTI-TR2KO cells expressed KLRG1 at high levels (Figure 1A). In contrast, there was little difference between percentages OTI-TR2Ctrl and OTI-TR2KO that expressed KLRG1 during the early stages of infection with LM-OVA (Figure 1B), indicating minimal influence of TGFβ during bacterial infection. As the infection progressed, some OTI-TR2KO cells reexpressed CD62L indicating conversion to TCM phenotype. The OTI-S4KO cells mostly lacked KLRG1 and CD62L expression after infection with both pathogens, indicating a defect during formation of TEFF and TCM cells (Cao et al., 2015; Hu et al., 2015). The OTI-S4KO and OTI-S4TR2-DKO cells expressed KLRG1 and CD62L at similar levels, whereas the increase in CD103 expression was slightly more pronounced on S4KO cells than OTI-S4TR2-DKO cells. p values are shown in Supplementary file 2. These data are consistent with other studies and show that SMAD4 regulates activated CTLs via a TGFβ-independent pathway (Igalouzene et al., 2022; Wu et al., 2021). We also compared cell proliferation during IAV infection using carboxyfluorescein succinimidyl ester (CFSE) dilution and 5-bromo-2-deoxyuridine (BrdU) incorporation (Figure 1—figure supplement 1). The data show that OTI-S4TR2DKO and OTI-S4TR2Ctrl cells proliferated at similar rates, indicating normal T cell priming. Figure 1 with 4 supplements see all Download asset Open asset SMAD4 ablation alters homing-receptor expression without transforming growth factor β (TGFβ) receptor II. (A, B) Naive donor cells from OTI-Ctrl, OTI-TR2KO, OTI-S4KO, and OTI-S4TR2-DKO were transferred to B6 mice before infection. Heatmaps show percentages of donor cells that expressed KLRG1, CD103, CD62L, and CD127 on different day post infection. Data are means ± standard deviation (SD) (n = 3/group). Two independent experiments gave similar results. p values were calculated using two-way ANOVA and statistical comparisons were made using Tukey’s multiple comparisons test. (A) Donor cells in the lungs and spleens after i.n. infection with X31-OVA. (B) Donor cells in the spleens after i.v. infection with LM-OVA. (C, D) IV staining shows OTI-S4KO and OTI-S4Ctrl cells in the lungs at 34 dpi with LM-OVA given by i.n. inoculation. Data are means ± standard deviation (SD) (n = 3-4/group). Two independent experiments gave similar results. p values were calculated using Student’s t tests (C) Histograms (percentages) and bar graphs (cell numbers) show all donor cells from OTI-S4KO (dashed) and OTI-S4Ctrl mice (continuous line). (D) Contour plots show donor cells in the bloodstream (right panel) and lung tissue (left panel). We previously used IV staining to analyze CTLs during IAV infection and found some KLRG1+ CTLs inside the blood vessels of the lungs >40 dpi (Hu et al., 2015). Since very few SMAD4-deficient CTLs expressed KLRG1 during infection with LM-OVA, we investigated whether these cells were located in the vasculature or peripheral tissues. Fractalkine (CX3CL1) is a chemoattractant with adhesive properties (Ostuni et al., 2020) that alters CD8 T cell migration. Prior studies indicate that CTLs in the vasculature express the fractalkine receptor (CX3CR1) at high levels (Nishimura et al., 2002). Several groups used reporter mice to monitor changes CX3CR1 expression during infection with different pathogens and found that terminally differentiated TEFF cells expressed KLRG1 and CX3CR1 at high levels (Gerlach et al., 2016; Jung et al., 2000). To explore whether CX3CR1 is regulated via SMAD4, we crossed the CX3CR1GFP+ reporter with OTI-S4KO mice for transfer experiments. Naive OTI-Ctrl and OTI-S4KO cells were mixed (1:1 ratio) and transferred to B6 mice before infection with LM-OVA. After 30 days, donor cells were analyzed for KLRG1 and GFP (surrogate for CX3CR1) expression (Figure 1C, D and Figure 1—figure supplement 2). Fluorescently conjugated antibodies (CD8β) were injected 3 min before sacrifice, to distinguish CTLs in the blood vessels and peripheral tissues. The majority of OTI-Ctrl cells in the lungs expressed KLRG1 and GFP at high levels (overlaid histogram and bar graph) (Figure 1C) and were primarily located inside the blood vessels (Figure 1D). Conversely, OTI-SKO cells expressed KLRG1 and GFP at intermediate/low levels (Figure 1C), and some cells were located in the tissue (Figure 1D). Similar distributions of donor cells were found in the red and white pulp of the spleen (Figure 1—figure supplement 2). Since small numbers of SMAD4-deficient CTLs expressed KLRG1 and CX3CR1 at high levels, the data indicate that SMAD4 acts as a catalyst for terminal differentiation. Rodents lose large percentages of their body weight during IAV infection and the rate of recovery to normal size can be used to assess the severity of disease. After IAV infection, S4KO mice experienced more severe disease than the controls (Hu et al., 2015). Since OTI-S4KO and OTI-S4TR2-DKO cells displayed similar phenotypes during infection, we used weight loss to measure of protective immunity. S4TR2-DKO and S4TR2-Ctrl mice were infected with X31-OVA and weight changes were recorded daily (Figure 1—figure supplement 3). As expected, the S4TR2-DKO mice lost more weight and recovered from infection more slowly than the controls. After 30 days, the immune mice (and uninfected controls) were challenged with different strain of IAV (WSN-OVAI) (Figure 1—figure supplement 4). None of the immune mice experienced substantial weight loss after infection with WSN-OVAI, indicating that protection against reinfection was not dependent on SMAD4 or TGFβ. SMAD4 modifies homing-receptor expression independently of R-SMAD2/3 The TGF family includes two groups of cytokines, that signal via alternative branches of the SMAD cascade. TGFβ and activins signal via R-SMAD2/3, while bone morphogenic proteins and related growth differentiation factors signal via R-SMAD1/5/8 (Fink et al., 2003; Takimoto et al., 2010). SMAD4 facilitates signaling by both groups of cytokines and chaperones complexes of phosphorylated R-SMADs into the nucleus for gene regulation (Massagué et al., 2005). Since we found that SMAD4 was required for formation of TCM cells but not TRM cells, we used quantitive polymerase chain reaction (qPCR) to compare SMAD4 expression in these subsets and found only a moderate difference in transcript levels (Figure 2—figure supplement 1). When CTLs are stimulated with TGFβ, the receptor phosphorylates R-SMAD3 and induces CD103 expression (Yang et al., 1999). Prior studies show some redundancy within the SMAD signaling network. After cancer cells were stimulated with TGFβ, R-SMAD2 and R-SMAD3 were phosphorylated and formed heterodimers that entered the nucleus in the absence of SMAD4 (Fink et al., 2003; Li et al., 2008). Since our data show that SMAD4-deficient CTLs maintained a consistent phenotype in the presence and absence of TGFβRII, we investigated whether R-SMAD2/3 were required to modify homing-receptor expression. Mice with flox sites in the genes for R-SMAD2 (Ju et al., 2006) and R-SMAD3 (Li et al., 2008) were used to generate CTLs that lack either R-SMAD2 (S2KO) or R-SMAD3 (S3KO), and both mutations (S23DKO). S3KO mice were further bred with S4KO mice to produce CTLs with both mutations (S34DKO). The respective Cre-deficient littermates were used as controls. To study the effects of cytokine stimulation, naive CD8 T cells were isolated from SLO by negative selection and activated with plate-bound anti-CD3/CD28 plus recombinant IL-2 (20 u/ml). After 48 hr, the remaining CTLs were washed and cultured in fresh medium without T cell receptor (TcR) stimulation (48 hr). Replicate wells were supplemented with either rIL-2 alone or rIL-2 plus exogenous TGFβ (10 ng/ml). Selected wells were supplemented with an ALK5 inhibitor (SB431542) to block responses to any TGFβ in the media. At 96 hr after TcR stimulation, the cultured CTLs were analyzed for CD103 (Figure 2A) and CD62L expression (Figure 2B). Figure 2 with 2 supplements see all Download asset Open asset SMAD4 influences the fate decisions of pathogen-specific cytotoxic T lymphocytes (CTLs) independently of R-SMAD2/3. (A, B) Naive CD8 T cells from genetically modified mice were activated in vitro and stimulated with cytokines. Selected wells were supplemented with SB431542 (10 μM). Bars show CTLs stimulated with rIL-2 alone (no fill), rIL-2 plus SB431542 (gray fill), and rIL-2 plus transforming growth factor β (TGFβ) (hatched). Data are means ± standard deviation (SD) (3 mice per group). p values were calculated using Student’s t tests. Two independent experiments produced similar results. (A) Percentages of CTLs that expressed CD103 at 96 hr after T cell receptor (TcR) stimulation. (B) Percentages of CTLs that expressed CD62L at 96 hr after TcR stimulation. (C, D) Different strains of genetically modified (empty squares) and control mice (filled circles) were infected (i.v.) with LM-OVA and at 8 dpi OVA-specific CTLs in the spleen were analyzed with MHCI Data are means ± per group). p values were calculated using two-way ANOVA and statistical comparisons were made using Tukey’s multiple comparisons test. Two independent experiments produced similar results. (C) shows CTLs analyzed for KLRG1 expression. (D) shows CTLs analyzed for CD103 expression. Naive CD8 T cells normally downregulate CD103 during antigen stimulation and expression TRM cells are to TGFβ et al., and 2002). the control CTLs not express CD103 during with rIL-2 and ALK5 while this was during stimulation with TGFβ (Figure The same of CD103 expression was CTLs lacked R-SMAD2 (S2KO) or R-SMAD3 In contrast, and TR2KO cells both lacked CD103 expression under all whereas and cells expressed CD103 at is a that TGFβ signaling through with SMAD proteins et al., 1999). A found that SMAD4 interacts with and heterodimers that expression in CTLs et al., 2021). S4KO cells expressed CD103 at levels than cells during stimulation with CD103 expression S4KO cells were stimulated with TGFβ. Conversely, CD103 expression not and S4TR2-DKO cells were stimulated with TGFβ. These data show that R-SMAD2/3 are required for TGFβ to CD103 in the absence of When R-SMAD2 is R-SMAD3 with SMAD4 to CD103 expression. that R-SMAD2/3 entered the nucleus without SMAD4 (Figure 2—figure supplement 2). Our in vivo data show that of OTI-S4KO cells CD62L during the memory phase of infection, indicating a defect during formation of TCM cells. Since TGFβ TCM formation et al., 2013), we analyzed the cultured CTLs for CD62L (Figure 2B). and of the control CTLs reexpressed CD62L cultured with rIL-2 and the percentages to the ALK5 inhibitor was Conversely, only of control CTLs expressed CD62L cultured with TGFβ. Although of and cells expressed CD62L during with the numbers not the ALK5 or exogenous TGFβ, was TR2KO and cells expressed CD62L at high levels under all These data indicate that SMAD4 functions during regulation of CD62L, since TGFβ signaling was not to CD62L expression in the absence of recombinant cytokines are not to induce KLRG1 expression on cultured CTLs et al., we infected the mice with LM-OVA. The were by i.v. and MHCI were used to analyze OVA-specific CTLs in the spleens at 8 dpi (Figure and of the control CTLs expressed KLRG1 and the percentages not in the absence of either R-SMAD2 (S2KO) or R-SMAD3 percentages of CTLs expressed KLRG1 in the absence of R-SMAD2/3 or a TGFβ receptor and that expression was by TGFβ (Sanjabi et al., 2009). in the absence of SMAD4 and only small percentages of CTLs expressed KLRG1, while the of CD103 cells (Figure The phenotypic changes in cells were slightly The for this is but may the influence of a Our data show that SMAD4 promotes formation of terminally differentiated TEFF cells via a mechanism that not TGFβ, or R-SMAD2/3. The transcriptional of SMAD4-deficient CTLs is similar to TRM cells TGFβ is an important factor during development, but little is known the of SMAD4 during transcriptional programming of activated CTLs. To this we used to compare the transcriptional of SMAD4-deficient and control CTLs during IAV infection. effector cells are differentiated CTLs that have not to a phenotype, and can be using high expression in the absence of KLRG1, and CD103 (Plumlee et al., 2015). To compare similar populations of antigen-specific CTLs, naive OTI-S4KO and OTI-S4Ctrl cells were transferred to B6 mice before infection with X31-OVA and cells were isolated from the spleens at were analyzed in and were with the expressed genes were according to in OTI-S4KO cells (Figure and Figure data 1). We found that OTI-S4KO cells expressed EOMES and CD62L at levels than the while was (Figure Since CD103 is expressed on large numbers of TRM cells in we used gene to compare the transcriptional of OTI-S4KO cells with previously The plots show comparisons with TRM cells in the small (Milner et al., Figure and other tissues (Figure genes that are with TRM cells in the small were in OTI-S4KO cells. We found similar with TRM cells isolated from the and as well as CTLs that were stimulated with TGFβ in vitro et al., 2016; et al., et al., et al., 2012). These indicate that early signaling via SMAD4 differentiation toward a resident memory phenotype. Figure 3 Download asset Open asset The transcriptional of SMAD4-deficient cytotoxic T lymphocytes (CTLs) a TRM phenotype. OTI-S4Ctrl and OTI-S4KO were transferred to B6 mice before infection with X31-OVA. effector cells were analyzed using isolated from of were analyzed in = (A) expressed genes are according to in OTI-S4KO cells. (B) OTI-S4KO cells expressed and at levels while was induced. (C, D) The transcriptional of OTI-S4KO cells was compared with (C) with analyzed dpi (D) with TRM cells in and cells in TCM cells in spleen and in vitro activated CTLs and indicate graph) and S4KO cells graph) were analyzed for at and 96 hr after T cell receptor (TcR) stimulation. Bars are cells stimulated with rIL-2 alone (filled and rIL-2 plus transforming growth factor β (TGFβ) were analyzed in Two independent experiments gave similar results. CTLs were analyzed for Hobit at 96 hr after TcR stimulation. Bars show cells stimulated with rIL-2 alone (filled and rIL-2 plus ALK5 inhibitor were analyzed in Two independent experiments gave similar results. was used to measure in OTI-S4KO and OTI-S4Ctrl cells at with LM-OVA. were analyzed in Two independent experiments gave similar results. CTLs were analyzed for at 96 hr after TcR stimulation. Bars show CTLs stimulated with rIL-2 alone (filled and rIL-2 plus TGFβ were analyzed in Two independent experiments gave similar results. p values were calculated using Student’s t tests. Figure data 1 data for from cells and analyzed dpi with X31-OVA. Download We used to further that SMAD4 altered expression in