LA JOLLA INSTITUTE IDENTIFIES MOLECULAR SWITCH ENABLING IMMUNE CELLS TO BETTER FIGHT DISEASE
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mechanism that enables CD4 helper T cells to assume the more aggressive role of killer T cells in mounting an immune attack against AIDS, cancers.....could enable the development of more potent drugs for AIDS, cancer and many other diseases
PRESS RELEASE - full text article from Nature Immunology below following press release
LA JOLLA INSTITUTE IDENTIFIES MOLECULAR SWITCH ENABLING IMMUNE CELLS TO BETTER FIGHT DISEASE
Finding on Killer Cells Opens New Avenue for Combating AIDS, Cancer and other Diseases
SAN DIEGO - January 20, 2013. A research team led by the La Jolla Institute for Allergy & Immunology has discovered the mechanism that enables CD4 helper T cells to assume the more aggressive role of killer T cells in mounting an immune attack against viruses, cancerous tumors and other damaged or infected cells. The finding, made in collaboration with researchers from the RIKEN Institute in Japan, could enable the development of more potent drugs for AIDS, cancer and many other diseases based on using this mechanism to trigger larger armies of killer T cells against infected or damaged cells.
CD4 helper T cells, which normally assist other cells of the immune system during an infection, and CD8 killer T cells, which directly attack and eliminate infected cells, are two of the body's most important immune cells for defending against diseases. Earlier research studies have shown that helper T cells can become killer cells in some instances. However, the specific mechanism of action that allowed this to occur was not known until now.
We have identified the molecular switch that enables CD4 T cells to override their programming as helper cells and transform into cytolytic (killer) cells," said La Jolla Institute scientist and study co-leader Hilde Cheroutre, Ph.D. "Our team also showed that these transformed helper T cells represent a separate and distinct population of cells. They are not a subset of TH-1 helper cells as previously thought."
Jay A. Berzofsky, M.D., Ph.D., chief of the Vaccine Branch at the National Cancer Institute's Center for Cancer Research, called the finding "a major advance" that provides new understanding about the cell's lineage and basic mechanisms. Dr. Berzofsky was among the researchers whose work in the 1980s first demonstrated that helper cells could convert to killer cells.
"Understanding how these cells derive and what causes them to switch from helper T cells to cytolytic T cells is an important step to learning how to manipulate them in disease," he said, noting it could lead to novel approaches "either to turn these cells off in autoimmune disease or turn them on in infectious diseases."
He added that the finding could also have important implications in cancer. "We need all of the cytolytic machinery that we can get to try to destroy cancers," he said. "If we can learn to turn them on, I think it's reasonable to believe that these cytolytic T cells can play an important role in controlling cancer."
The findings were published today in Nature Immunology in a paper entitled "Transcriptional reprogramming of mature CD4 helper T cells generates distinct MHC class II-restricted cytotoxic T lymphocytes." Dr. Cheroutre is co-senior author on the study together with Dr. Ichiro Taniuchi of the RIKEN Research Center for Allergy and Immunology in Yokohama, Kanagawa, Japan. First authors on the paper are: Mohammad Mushtaq Husain, Ph.D., of the La Jolla Institute; Daniel Mucida, Ph.D., formerly of the La Jolla Institute, now at Rockefeller University; Femke van Wijk, Ph.D., formerly of the La Jolla Institute, now at the University Medical Center Utrecht, The Netherlands, and Sawako Muroi, of the RIKEN Institute.
Mitchell Kronenberg, Ph.D., La Jolla Institute president & chief scientific officer, said the study reflects the very successful collaboration between the La Jolla Institute and RIKEN in Japan, which have joined efforts on a number of projects over the years.
In the study, the researchers found that a certain transcription factor, which are molecules in the cell nucleus that control the activity of cells, continually suppresses the killer T cell lineage in helper T cells. Using mice, the team showed that turning off this transcription factor (ThPOK) enabled the helper cells in the body's peripheral areas, like the blood, spleen and the intestine, to override their original programming and to become killer T cells.
"While our work focused on the intestines, we found that helper T cells in all tissues of the body have the potential to become killer cells in response to recognition of viral, tumor or other antigens in the context of cytokines such as IL-15," said Dr. Cheroutre.
Jonathan Braun, M.D., chair of the Department of Pathology and Laboratory Medicine at UCLA's David Geffen School of Medicine, praised the study as laying the groundwork for using T helper cells in a much more aggressive manner. "Helper T cells are mainly understood for their role in regulating other immune cells," he said. "This work reveals how they themselves can be triggered to become the action cells in the immune response. This opens new possibilities for how to manipulate them therapeutically in disease."
Dr. Cheroutre said the transformation of CD 4 helper T cells into killer cells already occurs in the body naturally. "Our finding could help to explain a number of occurrences that we haven't really understood up to this point, such as why some people can be chronically infected with HIV without developing AIDS." In these instances, Dr. Cheroutre is convinced that CD4 helper T cells must be taking over the role of killer cells after the CD8 T cells become exhausted. "It's like the helper cells can come in as reinforcements to keep the virus under control. If we can develop ways to artificially trigger that process, we may be able to significantly help people with HIV and other chronic infections."
While scientists would want to trigger a larger army of virus-specific killer cells in the case of infections, the opposite would be true in inflammation-fueled autoimmune diseases, like rheumatoid arthritis or multiple sclerosis, said Dr. Cheroutre. "The CD4 T cells are the bad wolves in inflammatory diseases because they often trigger more pro-inflammatory cells which worsen these conditions," she said. "With this knowledge, we may be able to prevent that by coaxing the CD4 killer cells to become regulatory cells instead, which is another one of their potential functions. In regulatory mode, the CD4 T cells suppress the immune system. This suppression reduces inflammatory cells, which is what we want to do in autoimmune diseases."
However in cancer, the CD4 T cell's regulatory function becomes problematic because they inhibit the killer T cells from destroying cancerous cells.This is because of their built-in mechanism to keep T cells from attacking the body's own cells, said Dr. Cheroutre. "Cancer cells develop from our own cells and only look a little different from healthy cells," she explained. "The killer T cells can sense that they are different and decide to eliminate them. However, the CD4 regulatory T cells frequently suppress the killer T cells and prevent them from destroying the cancerous cells. This is often how cancer cells can escape the immune system's normal action of stamping out bad cells."
Dr. Cheroutre said she believes it may be possible, using the newly discovered mechanism, to turn the CD4 regulatory T cells into killer cells that would aid, rather than block, the immune system's attack on cancerous cells.
Transcriptional reprogramming of mature CD4+ helper T cells generates distinct MHC class II-restricted cytotoxic T lymphocytes
Published online 20 January 2013
Daniel Mucida1,8,9, Mohammad Mushtaq Husain1,9, Sawako Muroi2,9, Femke van Wijk1,8,9, Ryo Shinnakasu1,8, Yoshinori Naoe2,8, Bernardo Sgarbi Reis3, Yujun Huang1, Florence Lambolez1, Michael Docherty1,8, Antoine Attinger1,8, Jr-Wen Shui1, Gisen Kim1, Christopher J Lena1, Shinya Sakaguchi4, Chizuko Miyamoto2, Peng Wang5,8, Koji Atarashi6,8, Yunji Park1,8, Toshinori Nakayama7, Kenya Honda6,8, Wilfried Ellmeier4, Mitchell Kronenberg1, Ichiro Taniuchi2 & Hilde Cheroutre1
"Therefore, the new insights reported here defining CD4+ CTLs as a distinct subset of effector cells and identifying the mechanism that leads to the unique differentiation of these cells provide information to finally move forward the field of CD4+ CTLs and to specifically target these cells to enhance protective immunity or to prevent or treat inflammatory diseases and immunological pathology."
TCRαß thymocytes differentiate into either CD8αß+ cytotoxic T lymphocytes or CD4+ helper T cells. This functional dichotomy is controlled by key transcription factors, including the helper T cell master regulator ThPOK, which suppresses the cytolytic program in major histocompatibility complex (MHC) class II-restricted CD4+ thymocytes. ThPOK continues to repress genes of the CD8 lineage in mature CD4+ T cells, even as they differentiate into effector helper T cell subsets. Here we found that the helper T cell fate was not fixed and that mature, antigen-stimulated CD4+ T cells terminated expression of the gene encoding ThPOK and reactivated genes of the CD8 lineage. This unexpected plasticity resulted in the post-thymic termination of the helper T cell program and the functional differentiation of distinct MHC class II-restricted CD4+ cytotoxic T lymphocytes.
CD4+ T cells are commonly classified as 'helper' T cells on the basis of their roles in providing help to promote or dampen cellular and humoral immune responses. In contrast, CD8αß+ cytotoxic T lymphocytes (CTLs) provide direct protective immunity by killing infected or transformed cells. The helper T cell program is initially induced during thymic development, during which thymocytes expressing a major histocompatibility complex (MHC) class II-reactive T cell antigen receptor (TCR) develop into the CD4+ helper T cell lineage, whereas thymocytes with specificity for MHC class I differentiate into the CD8+ CTL lineage. The functional programming, which coincides with but does not depend on the MHC restriction or expression of the coreceptor CD4 or CD8αß, is controlled by the action and counteraction of key transcription factors. Together with Tox and GATA-3, the helper T cell transcription factor ThPOK (cKrox; encoded by Zbtb7b (called 'Thpok' here)) first induces the CD4+ helper T cell fate and prevents thymocytes from differentiating into CD8+ CTLs1, 2, 3, 4, 5, 6. Runx3, a member of the Runx family of transcription factors, has the opposite effect and terminates CD4 expression while promoting differentiation into the CTL lineage7, 8. The CD8-CD4 lineage dichotomy persists in the periphery for mature T cells, in which ThPOK continues to suppress the cytotoxic fate of MHC class II-restricted CD4+ T cells even as they differentiate into effector helper T cell subsets controlled by additional transcription factors6.
That lineage separation, however, is not all encompassing, and reports have repeatedly indicated the presence of CD4+ T cells with cytolytic functions in various species, including humans and rodents9, 10, 11, 12. At steady state, populations of the effector cells that reside in the intestine as intraepithelial lymphocytes (IELs) show enrichment for cytotoxic T cells, including CD4+ T cells13, 14, 15, whereas under inflammatory conditions, including viral infections, autoimmune disorders and in response to tumor antigens, many cytolytic CD4+ T cell populations expand in the blood and peripheral tissues9, 10, 11, 12, 16, 17. Their widespread abundance and participation in various beneficial as well as pathogenic adaptive immune responses9, 10, 11, 12, 16, 17 underscore the physiological relevance of cytolytic CD4+ effector cells. However, the generation of convincing evidence directly linking them to specific aspects of the adaptive immune response has been difficult, as they have been viewed merely as functional variants of the well-defined T helper type 1 (TH1) subtype12, 18. The lack of a defined gene signature has also greatly impaired progress in elucidating the biology of cytolytic CD4+ T cells and in designing clinical strategies that specifically target these cells in various diseases.
Here we provide proof indicating that cytotoxic CD4+ T cells represent a distinct subset of effector cells that can be defined by the absence of the master regulator ThPOK, which maintains the helper T cell fate in all subsets of classical CD4+ helper T cells by continuously suppressing the CD8+ CTL lineage program6. Nevertheless, we also found that the ThPOK- CD4+ CTLs originated from ThPOK-expressing progenitor cells that initially committed to the ThPOK-controlled helper T cell lineage during thymic selection. Our findings therefore challenge the view that the helper T cell program in CD4+ T cells is fixed and show that mature CD4+ T cells can lose ThPOK expression post-thymically and can be functionally reprogrammed to become MHC class II-restricted CTLs. We also identify the Thpok silencer as the transcriptional switch that terminated Thpok transcription and by default drove the derepression of the CTL program in mature CD4+ effector cells. At steady state, CD4+ CTLs remained immunologically quiescent even in the continuous presence of their cognate antigens. However, in response to restimulation in the context of interleukin 15 (IL-15), CD4+ CTLs greatly increased their inflammatory and cytolytic functions and differentiated into potent killer effector cells. Overall, our data demonstrate that CD4+ CTLs are not a simple variant of classical ThPOK-controlled TH1 cells but instead are distinct functional MHC class II-restricted effector cells that can be characterized by the loss of ThPOK expression and the derepression of aspects of the gene-expression program of the CD8+ CTL lineage.
Not all mature CD4+ T cells express ThPOK
The reported cytolytic activity of mature CD4+ T cells is inconsistent with the idea that ThPOK continuously suppresses the CTL program in all mature, MHC class II-restricted CD4+ T cells6 and suggests that these cells might not be under the negative control of ThPOK. To investigate this, we analyzed ThPOK expression in mature T cells isolated from reporter mice with sequence encoding green fluorescent protein (GFP) knocked in to the ThPOK locus (Thpok-GFP mice)19. As expected, CD4+ Thpok-GFP lymphocytes isolated from the spleen or mesenteric lymph nodes (mLNs), which are mostly naive T cells, were GFP+ (Fig. 1a), which indicated that they all expressed ThPOK, as is typical of mature cells of the CD4+ helper T cell lineage. Conversely, all cells in the CD8+ fraction were GFP- (Fig. 1a), consistent with the absence of ThPOK expression in cells of the CTL lineage. Unexpectedly, many of the Thpok-GFP CD4+ effector T cells that accumulated in the intestine at steady state were GFP- (Fig. 1b,c), which indicated that like their CD8+ counterparts, they did not express ThPOK. Notably, most of the GFP- CD4+ cells were in the subset of double-positive (DP) IELs that coexpressed CD4 and CD8α without CD8ß20 (Fig. 1b-d). Consistent with the lack of ThPOK-mediated suppression, these CD4+CD8α+CD8ß- cells also had functional features similar to those of mature CD8+ CTLs, including abundant expression of granzyme (Fig. 1e,f) and substantial expression of the activation-induced degranulation marker CD107a (LAMP-1), a glycoprotein present in the membrane of cytotoxic granules and exposed on the surface of activated cytolytic cells21 (Fig. 1g,h). The induction of CD107a by the DP subset was similar to that of typical TCRαß+ CD8+ CTLs, whereas activated CD4+CD8α- single-positive (SP) IELs or helper T cells from the spleen did not induce this cytolytic marker (Fig. 1g,h). Moreover, activated DP cells also effectively killed target cells in vitro, as measured by the release of lactate dehydrogenase after lysis of target cells (Fig. 1i and Supplementary Fig. 1a). In sum, these data demonstrated that in normal mice, not all CD4+ effector cells expressed ThPOK and, furthermore, that those ThPOK- CD4+ lymphocytes expressed CD8α (but not CD8ß) and had cytolytic activity that closely resembled that of mature CD8+ CTLs.
Mature ThPOK- CD4+ T cells derive from ThPOK+ thymocytes
ThPOK is the master regulator of the helper T cell lineage and is first expressed in the thymus, where it counteracts Runx3 and suppresses the CTL fate of MHC class II-restricted thymocytes4, 5, 6. The absence of ThPOK expression associated with cytotoxicity in mature CD4+ T cells could suggest that they might have originated from ThPOK- progenitors. To investigate this, we designed a fate-mapping mouse model in which we tracked previous ThPOK expression in mature T cell subsets (Fig. 2a). Inactivation of Thpok transcription in MHC class I-specific thymocytes of the CD8+ CTL lineage is mediated by repressive factors, such as Runx proteins, that bind to the Thpok silencer DNA element22, 23. In MHC class II-restricted cells, however, ThPOK itself binds to the Thpok silencer and prevents Runx-mediated silencing, which results in a positive feedback loop with continuous expression of Thpok (Fig. 2a). A fate marker that reports any previous activity of the Thpok silencer in thymocytes is therefore an accurate reporter for the lineage origin and initial commitment of mature CD4+ T cells. On the basis of that rationale, we designed a mouse strain with transgenic expression of Cre recombinase, 'ThPOK-Cre', in which a Thpok silencer inserted into a Cd4 enhancer-promoter locus controls expression of a transgene encoding Cre (Fig. 2a). In contrast to the widely used strain of mice that express Cre under the control of the Cd4 enhancer-promoter starting at the CD4+CD8αß+ thymocyte stage, in ThPOK-Cre mice, the Thpok silencer prevents Cd4 enhancer-promoter activity in immature thymocytes and cells of the CD8 lineage, which results in Cre expression exclusively in mature CD4+ SP thymocytes committed to the helper T cell lineage (Fig. 2a). Consistent with that, in progeny of a cross of ThPOK-Cre mice with reporter mice expressing yellow fluorescent protein (YFP) from the ubiquitous Rosa26 promoter (Rosa26-YFP), we found that only cells derived from thymocytes that were committed to the CD4+ helper T cell lineage were marked by the expression of the YFP reporter after Cre-mediated recombination (Fig. 2b,c). In those progeny of the Rosa26-YFP x ThPOK-Cre cross, CD8+ SP thymocytes and mature peripheral CD8+ T cells, including CD4-CD8+ mucosal T cells, did not express YFP, whereas, as expected, most CD4+ SP lymph node T cells and IELs were YFP+. Notably, CD8α-expressing CD4+ IELs were also YFP+, to an extent similar to that of conventional CD4+ SP cells (Fig. 2b,c), which indicated that they had induced ThPOK-dependent Cre expression and therefore that the mature ThPOK- CD4+ T cells must have been derived from progenitor cells that expressed ThPOK at an earlier stage.
ThPOK+CD4+ helper T cells lose ThPOK expression
The idea that the mature ThPOK- CD4+ cells previously expressed ThPOK, together with the observation that those cells mainly accumulated among effector cells, suggested that the loss of ThPOK expression might have been the result of a post-thymic activation or maturation process that mature T cells undergo in the periphery. To assess this, we adoptively transferred highly purified ThPOK-expressing GFP+ naive Thpok-GFP CD4+ T cells into recipient mice deficient in recombination-activating gene 1 (Rag1-/- mice). The lymphopenic conditions in this model induced strong proliferation and differentiation of the donor cells, which accumulated mainly as cells of the TH17 subset of helper T cells (characterized by the expression of the transcription factor RORγt and the cytokine IL-17) in the large intestine, and notably, also as DP effector cells, especially in the small intestine of the Rag1-/- hosts (Fig. 3a,b). Donor cells in the spleen and mLNs were GFP+ (Fig. 3c), which indicated that they continued to express ThPOK. In contrast, many of the Thpok-GFP CD4+ T cells that accumulated as effector cells in the intestine did not have detectable expression of GFP (Fig. 3c), which indicated that they had substantially diminished or complete loss of ThPOK expression. Consistent with the observations obtained with immunologically replete mice, the loss of GFP expression was again greatest in those CD4+ cells that also reinduced CD8α expression, although a small fraction of CD4+ SP IELs were also GFP- (Fig. 3c,d). Serial transfer of those SP donor cells into a second set of Rag1-/- recipients generated many more DP cells (Fig. 3e), which indicated that the reexpression of CD8α on mature CD4+ T cells followed the loss of ThPOK expression and further indicated that the Cd8a locus in conventional CD4+ helper T cells might be constitutively suppressed by ThPOK. To analyze this, we used chromatin immunoprecipitation (ChIP) combined with tiling arrays of cells from genetically engineered mice that express Flag-hemagglutinin epitope-tagged ThPOK (FH-ThPOK) from the Thpok locus19. FH-ThPOK associated with the E8I enhancer element24 in the Cd8a locus in SP CD4+ thymocytes (Fig. 3f). We also confirmed that result in mature CD4+ SP T cells by ChIP assay (Supplementary Fig. 2a), which suggested that in mature CD4+ thymocytes and lymphocytes, ThPOK prevented CD8α expression by direct suppression of the E8I enhancer element in the Cd8a locus, and also that the expression of CD8α without CD8ß in mature ThPOK- CD4+ T cells was driven by derepression of the E8I enhancer. In support of that finding, both the frequency of CD8α-expressing CD4+ IELs and CD8 expression itself were much lower in E8I-deficient mice than in wild-type mice (Fig. 3g), a finding also true for the progeny of E8I-deficient cells versus wild type donor cells in Rag1-/- recipients of an equal number of both genotypes of donor cells (Fig. 3h and Supplementary Fig. 2b). Overall the data indicated that naive ThPOK+ CD4+ helper T cell cells might have lost expression of ThPOK in the periphery and consequently regained expression of ThPOK-suppressed genes such as Cd8a.
ThPOK loss coincides with CTL differentiation
Although subsets of polarized CD4+ effector cells are controlled by unique transcription factors, such as T-bet for TH1, GATA-3 for TH2 and RORγt for TH17 cells, ThPOK continues to function as a master regulator of the helper T cell lineage and continues to suppress the CTL program. Consistent with that, naive Thpok-GFP donor spleen cells that differentiated into TH17 effector cells in the intestine of the Rag1-/- recipient mice remained GFP+ (Fig. 4a), which indicated that their gene-expression profile was still regulated by the ThPOK. To examine the effect of the loss of ThPOK expression on the gene-expression pattern of CD4+ effector cells, we analyzed gene microarrays generated from RNA isolated from sorted ThPOK+ or ThPOK- SP and DP effector cells from normal unmanipulated Thpok-GFP reporter mice19 (Fig. 4b) and from Rag1-/- recipients of naive Thpok-GFP CD4+ SP donor cells (Fig. 4c and Supplementary Fig. 3a). Notably, we found that DP and SP ThPOK- CD4+ T cells had a unique but similar gene-expression pattern that differed from that of SP ThPOK+ CD4+ cells, although in the transfer experiments, the donor cells originated from the same pool of naive lymphocytes and differentiated in the same host environment (Fig. 4b,c and Supplementary Fig. 3a). As expected, many of the ThPOK+ CD4+ SP cells isolated from the intestine had a distinct TH17 gene-expression pattern, whereas expression of those genes was barely detectable in the ThPOK- and DP subsets (Fig. 4b-d). Furthermore, RT-PCR analysis of genes characteristic of TH17 cells, including those encoding the cytokines IL-17A, IL-17F and IL-22, the cytokine receptor IL-23R and the TH17 hallmark nuclear transcription factor RORγt, confirmed that ThPOK- CD4+ effector cells were distinct from TH17 cells (Fig. 4e,f and Supplementary Fig. 3b). Moreover, analysis of genes characteristic of other CD4+ helper T cell types, such as TH1 and TH2 cells, demonstrated that the gene-expression pattern of CD4+ SP lymphocytes and DP lymphocytes that had lost ThPOK expression did not resemble the patterns of the known helper T cell effector subsets (Supplementary Fig. 3c).
In contrast, in addition to reexpressing CD8α, ThPOK- CD4+ effector cells also expressed many other genes typically associated with the CD8+ CTL program. This included the expression of various genes encoding cytolytic proteins, such as several granzymes and perforin, as well as interferon-γ (IFN-γ) and several receptors expressed by natural killer cells and mature CD8+ CTLs (Fig. 4b,c,g,h and Supplementary Fig. 3d,e). Of particular note was their expression of the cytotoxicity-related, MHC class I-restricted, T cell-associated molecule CRTAM and the CD2 family member CD244 (2B4), both known to promote the cytolytic function and IFN-γ production of CD8+ T cells25, 26, 27.
The reciprocal expression in mature CD4+ effector cells of either ThPOK or CTL signature genes (Fig. 4i) confirmed the hypothesis that ThPOK continuously suppressed the CTL program in conventional helper T cell cells but also demonstrated that the differentiation of CD4+ CTL effector cells coincided with the post-thymic loss of ThPOK. In agreement with that, after enforced expression of ThPOK via retroviral transduction, CD4+ donor cells no longer differentiated into CTLs in vivo (Fig. 4j), whereas transfection of a construct encoding Thpok with a spontaneous mutation found in the helper-deficient mouse strain4 did not prevent the differentiation of helper T cells into CD4+ CTLs (Supplementary Fig. 3f). These data indicated that the loss of ThPOK coincided with derepression of the CTL phenotype in mature CD4+ effector cells.
Thpok silencer derepression terminates Thpok expression
In thymocytes committed to the CD8+ CTL lineage, ThPOK expression is switched off at the transcriptional level by the Thpok silencer22. Consequently, in mice with germline deletion of this silencer (ThpokSΔ/SΔ mice), MHC class I-restricted thymocytes mature as CD4-expressing T cells in the periphery22. To determine if the Thpok silencer engages in a similar role to terminate Thpok expression in mature MHC class II-restricted precursors of CD4+ CTLs, we analyzed the peripheral maturation of CD4+ T cells in ThpokSΔ/SΔ mice. To eliminate the MHC class I-restricted CD4-expressing cells in these mice, we crossed the ThpokSΔ/SΔ mice with MHC class I-deficient (ß2-microglobulin-deficient (B2m-/-)) mice, which abrogated the development of MHC class I-restricted CD4+CD8+ T cells. Notably, we found considerable enhancement of all CD4+ populations, including DP IELs, in B2m-/- mice, in the absence of mature CD8+ T cells (Fig. 5a); however, ThpokSΔ/SΔB2m-/- mice had considerably fewer mature MHC class II-restricted CD8α-expressing CD4+ T cells (Fig. 5a). These observations indicated that the Thpok silencer was a critical genomic switch in the process of the differentiation of CD4+ CTLs that turned off Thpok transcription in mature CD4+ T cells. We further confirmed that idea with CD4+ T cells in which a loxP-flanked Thpok silencer (ThpokSfl/Sfl) was deleted at the mature stage by transfection of the cells with a retroviral construct containing sequence encoding Cre linked to GFP. The transfer of transfected ThpokSfl/Sfl CD4+ T cells (which expressed Cre (GFP+) and consequently had deletion of the Thpok silencer) into Rag1-/- recipient mice resulted in impaired accumulation of CD8α-expressing CD4+ CTLs in the intestine of the recipients (Fig. 5b), which further emphasized the critical role of the Thpok silencer in terminating Thpok expression as part of the CTL-differentiation process of CD4+ effector cells. In contrast to ThPOK, which suppresses the repressive activity of the Thpok silencer, the zinc-finger transcription factor MAZR is known to activate the Thpok silencer28, which results in MAZR-induced negative regulation of Thpok transcription. Consistent with the participation of MAZR in reactivation of the Thpok silencer in mature CD4 T cells, MAZR-deficient CD4+ donor cells transferred into Rag1-/- recipient mice formed fewer CD8α-expressing CD4+ CTLs than did their wild-type counterparts in the intestine of the recipients (Fig. 5c,d). These results indicated that transcriptional regulation of Thpok was key for control of the helper T cell phenotype of mature CD4+ T cells and that reactivation of its silencer led to termination of the helper T cell program and, conversely, to the functional differentiation of MHC class II-restricted CD4+ CTLs.
CD4+ CTL differentiation is driven in vivo by antigen
The loss of ThPOK observed in progeny of naive ThPOK+ donor cells suggested that induction of the CTL program in mature CD4+ T cells coincided with an activation or maturation process. The reexpression of CD8α as well as the cytolytic functional differentiation of mature CD4+ T cells was reminiscent of the cytotoxic-lineage differentiation of positively selected CD8+ SP thymocytes mediated by IL-7 (ref. 29). Despite that, however, we found significantly more CD8α-expressing CD4+ CTLs in mice deficient in the receptor for IL-7 than in wild-type mice (Fig. 6a), which indicated that the in vivo differentiation of mature CD4+ CTLs was not an IL-7-driven process. Similar to other effector cells in the intestine, DP cells were not present in germ-free mice (Supplementary Fig. 4a) and seemed to be present in normal numbers in germ-free mice reconstituted with specific pathogen-free microorganisms (Supplementary Fig. 4b), which indicated that some microbial factors directly or indirectly promoted the accumulation of CD4+ CTLs in the intestine. In contrast, unlike classic CD4+ TH17 effector cells, they did not increase in number in the intestine of germ-free mice monocolonized with segmented filamentous bacteria alone30 (Supplementary Fig. 4b) or in response to an infection with Citrobacter rodentium, a pathogen known to induce a strong TH17 response in the large intestine (Supplementary Fig. 4c), whereas they were present in normal numbers in the intestine of mice deficient in the adaptor MyD88. These observations indicated that the microbial conditions required for the steady-state accumulation of CD4+ CTLs in the intestine were probably not established by a single microorganism and did not depend on IL-1 signals or signals induced by Toll-like receptors (Supplementary Fig. 4d). Published reports have indicated that CD4+ CTLs are antigen-experienced cells that differentiate in response to repeated activation signals10, 12. In agreement with that, DP cells with a CTL phenotype isolated from Rag1-/- recipients of naive spleen CD4+ SP T cells also showed evidence of strong or repeated activation and considerable uptake of the thymidine analog BrdU but only weak staining for the active cell-cycle marker Ki67 (Fig. 6b), which indicated that they were resting cells that had previously been activated and had previously intensely proliferated. In addition, CD4+ CTLs also had higher expression of the activation marker CD69 than did CD4+ SP effector helper T cells (Fig. 6b), which suggested that the activation process that coincided with the loss of ThPOK expression and derepression of the CTL program also coincided with those CD4+ effector cells that received strong or repeated activation signals. To directly investigate that, we analyzed the effector differentiation of monoclonal OT-II CD4+ T cells (which have transgenic expression of a TCR specific for ovalbumin amino acids 323-339 (OVA(323-339)) in response to continuous activation in vivo with their cognate peptide OVA(323-339) presented by I-Ab. T cells migrate as effector cells only to peripheral tissues, and in agreement with that, in the absence of OVA antigen, very few OT-II CD4+ T cells accumulated in the intestine of OT-II mice. Similarly, after transfer of naive OT-II CD4+ T cells into Rag1-/- recipient mice, only a limited number of the donor cells migrated to the intestine of the recipient mice in the absence of OVA antigen (data not shown). In contrast, after mice were fed an OVA-containing diet for at least 4 weeks, a large number of activated OT-II CD4+ cells accumulated in the intestine of the OT-II mice and the Rag1-/- recipients, and, notably, many reexpressed CD8α (Fig. 6c), whereas those that remained SP had a tendency to express the transcription factor Foxp3 (Fig. 6c). To determine if the derepression of CD8α expression on the responder OT-II CD4+ T cells coincided with the loss of ThPOK and their differentiation into CTLs, we isolated cells from OT-II Thpok-GFP reporter mice, transferred those naive donor cells into OVA-fed Rag1-/- recipient mice and analyzed the phenotype and function of the OVA-responding cells that accumulated as effector cells in the intestine of the hosts. As expected, many OT-II Thpok-GFP DP cells accumulated in the small intestine but, notably, they were all GFP- (Fig. 6d), which indicated that they had lost ThPOK expression. Furthermore, in addition to the reexpression of CD8α, OT-II GFP- effector cells also newly induced the expression of typical cytolytic markers, such as 2B4 and granzyme B (Supplementary Fig. 5a). Notably, they had an antigen-specific cytolytic response when restimulated in vitro with the OT-II TCR-specific peptide OVA(323-339) (Fig. 6e) but not when restimulated with the MHC class I-restricted OVA peptide SIINFEKL (amino acids 257-264; Supplementary Fig. 5b).
Despite their functional potential, OT-II ThPOK- CD4+ CTLs remained immunologically quiescent in vivo even in the continuous presence of their cognate antigen in the diet. That finding was similar to results obtained with MHC class I-restricted CD8αß+ CTLs, which also seem to be inactive under steady-state conditions31. After challenge with excess amounts of IL-15, however, as in active celiac disease18, 32, CD8+ CTLs become pathogenic killer cells33, 34 that also considerably upregulate secretion of the inflammatory cytokines IFN-γ and tumor-necrosis factor (TNF)18,35,36,. To determine if CD4+ CTLs might be as responsive to IL-15 as CD8+ CTLs, we restimulated the diet-induced OT-II ThPOK- CD4+ CTLs in vitro in the context of IL-15. As expected, the addition of IL-15 resulted in higher CD8α expression37; however, IL-15 alone or together with the irrelevant MHC class I-restricted peptide SIINFEKL had no effect on the functional differentiation or maturation of the MHC class II-restricted OT-II CD4+ CTLs (Supplementary Fig. 5b). In contrast, the addition of IL-15 resulted in considerably enhanced immune responses of OVA(323-339)-stimulated OT-II ThPOK- CD4+ CTLs, as measured by the substantal upregulation of CD107a expression and the much greater production of IFN-γ and TNF (Fig. 6e). These data therefore demonstrated that antigen-induced CD4+ CTLs generated in the absence of inflammation were poised to exert potent effector functions when reactivated by their cognate antigen in the context of the inflammatory cytokine IL-15. We confirmed those data with polyclonal ThPOK- CD4+ CTLs isolated from the intestine of normal lymphocyte-sufficient mice and showed that unmanipulated CD4+ CTLs analyzed ex vivo also had the potential in response to polyclonal activation and to increase their cytolytic and inflammatory immune responses when exposed to IL-15. Although IL-15 alone supported short-term survival of wild-type CD4+ CTLs and CD8αß+ CTLs, this cytokine alone did not induce cytolytic effector functions in these polyclonal cells (Supplementary Fig. 5c). In contrast, similar to results obtained with the dietary antigen-induced OT-II ThPOK- CD4+ CTLs or normal CD8αß+ CTLs (Supplementary Fig. 5d), the addition of IL-15 also resulted in considerably enhanced cytolytic and inflammatory functions of wild-type polyclonal TCR-stimulated CD4+ CTLs, whereas it had only a negligible effect on ThPOK+ CD4+ helper T cells (Fig. 6f). These data emphasizes the likely pathogenic potential of antigen-induced ThPOK- CD4+ CTLs, which were poised to kill under conditions in which IL-15 was abundant.