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Intestinal Peyer’s patches are essential lymphoid organs for the generation of T cell-dependent immunoglobulin (Ig) A production for gut homeostasis. Using IL-17 fate reporter mice we show here that endogenous TH17 cells in lymphoid organs of naïve mice home preferentially to the intestine and are maintained independently of IL-23. In Peyer’s patches such TH17 cells acquire a T follicular helper (TFH) phenotype and induce the development of IgA-producing germinal center B cells. Mice deficient in TH17 cells fail to generate antigen specific IgA responses, providing evidence that TH17 cells are the crucial subset required for high affinity T cell-dependent IgA production.
Disruption of mucosal homeostasis can lead not only to infections, but also chronic inflammatory diseases and cancer. Intestinal homeostasis is maintained by the immune system and the barrier function of epithelial cells. A large number of innate and adaptive immune cells reside in mucosal tissues and establish an immunological network to maintain healthy conditions. Amongst the adaptive immune cells, B cells producing IgA are an important player in maintenance of homeostasis and mucosal host defense1 and the lamina propria (LP) of the small intestine (SI) is home to a substantial proportion of TH17 cells present in non-immune mice.
IgA in the dimeric form is the dominant immunoglobulin isotype secreted into the intestinal lumen. The differentiation of T cell-dependent IgA-secreting B cells occurs in the Peyer’s patches (PP) of the small intestine. Selective deficiency of IgA is the most common form of primary immunodeficiency, with an incidence of approximately 1 in 600 individuals in the western world. Although symptoms are rarely severe, individuals with symptomatic selective IgA deficiency can suffer from recurrent pulmonary and gastrointestinal infections2. TH17 cells play a crucial role in the mucosal host defence as well as in the development of autoimmune diseases3. Under steady-state conditions TH17 cells are preferentially found in the lamina propria of the small intestine where their development depends on the presence of commensal microbiota, in particular on segmented filamentous bacteria (SFB)4. Interestingly SFB also stimulate a large amount of total intestinal IgA5.
The primary function of immune cells in the PP is surveillance of the intestinal lumen, which involves the induction of IgA antibody responses. IgA is important for the neutralization of toxins and response to pathogens, but also critically involved in shaping the diversity of the commensal microbiota6-7. Upon activation of B cells in the context of cognate T cell help, germinal centres (GCs) are generated and the induction of the activation-induced cytidine deaminase (AID) in GC B cells promotes somatic hypermutation and class-switch recombination of immunoglobulin genes. The majority of B cells in the PP differentiate into IgA-producing cells in the presence of T cell help, whereas T-independent IgA-producing B plasma cells, which are B220− can differentiate in the gut lamina propria without the generation of the germinal centers8-10. IgA-producing B cells in germinal centers undergo extensive somatic hypermutation10, resulting in higher antibody affinity.
Here we show that the majority of TH17 cells found in lymphoid organs of non-immune mice were dependent on gut microbiota and had a natural preference for the small intestine as upon adoptive transfer they selectively homed to this site. Intestinal TH17 cells underwent deviation towards a follicular helper T cell phenotype (TFH) in Peyer’s patches where they induced germinal centers (GC) and the development of host protective IgA responses. In marked contrast to pathogenic TH17 cells developing in the course of EAE, which are highly dependent on IL-2311-12, intestinal TH17 cells did not require IL-23 for their maintenance or for plasticity towards a TFH profile. Mice deficient in TH17 cells had a pronounced deficiency of antigen-specific intestinal IgA following immunization with cholera toxin, emphasising that TH17 cells are the helper T cell subset responsible for inducing the germinal center B cell switch towards high affinity, T cell-dependent IgA.
TH17 cells constitute approximately 0.1 % of CD4+ T helper cells in the peripheral lymph nodes (LN) and spleen in non-immune IL-17 fate reporter mice (Il17aCreR26ReYFP mice) in which IL-17-producing cells are permanently marked as eYFP+ cells12. This system is a powerful tool to track TH17 cells and investigate potential plasticity towards alternative effector functions, as detection of these cells does not depend on staining for intracellular IL-17. Flow cytometric analysis of eYFP+ TH17 cells from LN of Il17aCreR26ReYFP mice showed almost uniform surface expression of CCR6, IL-7Rα, CD25, CD103 and ICOS as well as expression of the signature cytokine IL-17 and the transcription factor RORγt (Fig.1a). Expression of CCR6 and CD103 suggested gut homing capacity, because the CCR6 ligand CCL20 is known to be expressed in the small intestine13. As intestinal TH17 cells are dependent on the gut microbiota and absent in germfree mice4, we compared the proportions of eYFP+ TH17 cells in LN, PP and LP of SPF or germfree Il17aCreR26ReYFP mice. eYFP+ TH17 cells were undetectable in PP and LP and also almost completely absent from LN of germfree Il17aCreR26ReYFP mice (Fig.1b). To test the homing properties of TH17 cells compared with other memory type T cells from non-immune mice, we sorted eYFP+ TH17 cells and eYFP− CD4+ T cells with an activated phenotype (CD44high) from LN of Il17aCreR26ReYFP mice (distinguished by expression of the allotypic marker CD45.1) and adoptively co-transferred them in a 1:1 ratio into Tcra-deficient hosts (CD45.2), which lack conventional CD4+ and CD8+ T cells. eYFP+ TH17 cells preferentially reconstituted the gut-associated tissues, such as the LP and PP of the small intestine, but not the peripheral lymph nodes where the cells had originally resided (Fig.1c,d). In contrast, eYFP− CD44high CD45.1+ non-TH17 cells preferentially seeded peripheral LN (Fig.1c,d). Thus, the majority of TH17 cells found in lymphoid organs of non-immune mice have gut-homing properties.
The preferential accumulation of TH17 cells in PP prompted us to examine the possibility that they might play a role in helping germinal center B cell differentiation. T follicular helper (TFH) cells reside in germinal centers and play an essential role in germinal center B cells differentiation and their distinguishing features are the expression of CXCR5, PD-1, IL-21, ICOS and the transcription factor Bcl614-16. We determined that about ~13-20% of eYFP+ TH17 cells as well as a similar proportion of eYFP− cells present in the PP of non-immune Il17aCreR26ReYFP mice expressed CXCR5 and PD-1, whereas PP eYFP+ γδ T cells did not express these TFH markers (Fig. 2a).
In order to verify the developmental origin of these cells, we sorted CXCR5− eYFP+ TH17 cells from LN of non-immune Il17aCreR26ReYFP mice, adoptively transferred them into Tcra-deficient hosts and subsequently determined expression of CXCR5 and PD-1 in different tissues of the adoptive hosts. Although eYFP+ TH17 cells homed to both PP and lamina propria of the adoptive hosts, conversion of eYFP+ cells to a TFH phenotype occurred exclusively in the PP environment (Fig. 2b). It should be noted that the small proportion of CD4+ eYFP− cells detected in the hosts after adoptive transfer are not donor derived T cells that lost eYFP expression, but CD4+ non-T cells present in the host.
In order to test to what extent plasticity of eYFP+ TH17 cells towards a TFH profile could occur in other tissues as a consequence of immunization, we induced EAE in Il17aCreR26ReYFP mice by immunizing with MOG+CFA. This induces an antibody response to MOG in addition to EAE and TH17 cells are thought to be involved in development of ectopic lymphoid follicles in the CNS17. Analysis of LN and spinal cord from Il17aCreR26ReYFP mice with EAE showed that about 4-7% of CD4+ T cells in the LN and 60% of CD4+ T cells in the spinal cord were eYFP+. However, about 2-4% of eYFP+ cells in LN, and none in the spinal cord, had a TFH signature (CXCR5+ PD-1high). In contrast, a substantial proportion (10-15%) of eYFP− CD4+ T cells had a TFH profile (Fig.2c). These observations suggest that TH17 plasticity towards TFH preferentially takes place in the PP environment.
We next analysed TH17 gene signatures in sorted CXCR5+ eYFP+ T cells isolated from PP (PP eYFP+ TFH), LN eYFP+ TH17 cells (LN TH17), non-TH17 PP eYFP− CXCR5+ TFH cells (PP TFH), as well as naïve CD4+ T cells, all isolated from non-immune Il17aCreR26ReYFP mice. eYFP+ CD4+ T cells with a TFH surface phenotype had down-regulated Rorc and Il17a mRNA and up-regulated the TFH signature genes Bcl6 and Il21, similar to non-TH17 related PP TFH cells (Fig.2d). Taken together, these data demonstrate that plasticity of TH17 towards a TFH-like phenotype takes place continuously in the PP environment under steady state conditions.
TH17 cell plasticity in autoimmune settings is strongly dependent on IL-2312. In order to test whether IL-23 is similarly involved in plasticity of intestinal TH17 cells towards TFH, we analysed Il17aCreR26ReYFP mice crossed onto a p19 (Il23a)-deficient background. Firstly, and in contrast to the well-defined role of IL-23 in TH17 maintenance in autoimmune settings, IL-23 was dispensable for the survival of intestinal TH17 cells, as similar numbers of TH17 cells were present in LN and PP of Il23a-deficient and wild-type Il17aCreR26ReYFP mice (Fig.3a). Furthermore, phenotypic conversion to a TFH phenotype occurred to the same extent in IL-23-deficient and wild-type Il17aCreR26ReYFP mice (Fig.3b) and TH17 cells with the TFH phenotype in Il23a−/− reporter mice upregulated Bcl6 and Il21 expression similar to TH17 cells from wild-type reporter mice (Fig.3c). In contrast, in accordance with previous observations12, TH17 cells were unable to deviate towards IFN-γ expression and did not express IL-22 in Il23a-deficient mice (Fig.3d). These data indicate that the intestinal, steady-state population of TH17 cells has distinct features from the TH17 cells elicited by immunization in the periphery.
The dependency of intestinal TH17 cells on commensal bacteria raises the possibility that TFH cells developing from gut homing ex-TH17 cells may be specialized for helping B cell IgA responses in PP germinal centers. We therefore analysed B cell expression of Aicda, which is required for somatic hypermutation, gene conversion and class-switch recombination of immunoglobulin genes. There was little Aicda expression in LN B cells of B6 mice, in line with the absence of germinal centers in mice that are kept under specific pathogen free (SPF) conditions. B cells in PP on the other hand are continuously stimulated by the commensal flora and expressed high levels of Aicda (Fig.4a). In absence of T cells in Tcra-deficient hosts, Aicda expression was very low (Fig.4a), as no germinal center B cells develop in the absence of T cell help. Transfer of eYFP+ TH17 cells, however, reconstituted Aicda expression to the level seen in B6 mice (Fig.4a).
Furthermore, B cell expression of the germinal center markers GL-7 and CD95, as well as expression of IgA, which was detected on B cells from wild-type, but not Tcra-deficient mice, was induced in PP B cells of Tcra-deficient mice upon TH17 transfer (Fig.4b,c). As a result, the concentration of IgA, but not that of other Ig isotypes, was substantially increased in serum of Tcra-deficient mice that had received TH17 cells, whereas Tcra -deficient mice that had not received adoptively transferred TH17 cells, as well as those which had received non-TH17 effector cells (eYFP− CD44high) showed no change in their IgA expression (Fig. 4d). These data strongly suggest that intestinal TH17 cells deviating to a TFH profile in the PP may be responsible for the induction of T cell-dependent IgA responses.
Previous reports suggested that Foxp3+ regulatory T (Treg) cells might adopt a TFH phenotype in PP8, 18-20. Since these studies had focused on Treg isolated from lymphoid organs, we first compared the homing as well as the adoption of a TFH phenotype in Treg and TH17 cells isolated from LN and spleen. Treg from B6.CD45.1 mice were isolated on the basis of high CD25 expression, which correlated well with Foxp3 expression (Fig.5a), and co-transferred with equal numbers of eYFP+ TH17 cells into Tcra-deficient hosts. Three months later, transferred Treg were preferentially found in LN, whereas transferred eYFP+ cells homed to LP and PP of the small intestine (Fig.5b). CD45.1+ donor Treg retained their Foxp3 expression and there was no indication that donor eYFP+ TH17 cells acquired Foxp3 expression in any location within the adoptive host (Fig.5c). Furthermore, while 15-30% of TH17 cells deviated to a TFH profile in PP, Treg cells did not acquire a TFH profile in any of the tissues examined (Fig.5d). As the poor homing of LN derived Treg to intestinal tissues might have precluded acquisition of a TFH phenotype in PP, we isolated RFPhigh Treg from LP and PP of Foxp3RFP and transferred them into Tcra-deficient hosts. Although we observed efficient homing of donor RFPhigh Treg into PP, these cells did not acquire a TFH profile in the adoptive hosts (Fig.5e). Furthermore, adoptive Treg transfer did not induce germinal center B cells and IgA production (Fig.5f,g). Taken together these data strongly suggest that the promotion of IgA class switching in GC B cells in the PP is a function of ex-TH17 derived TFH cells, whereas Treg neither adopt a TFH profile nor support IgA production.
After transfer into Tcra-deficient host, transferred eYFP+ TH17 cells expanded substantially, resulting in IgA production that far exceeded that seen in wild-type mice in steady-state. In Tcra-deficient hosts, lymphopenia may have resulted in unimpeded expansion to the commensal flora by transferred TH17 cells. However, adoptive transfer into intact wild-type hosts, which contain full niches of intestinal TH17 and TFH cells, does not lead to efficient engraftment of the small number of donor cells that can be isolated for transfer from nonmanipulated mice. The minimal differences in serum IgA concentration between T cell-deficient Tcra−/− mice and non-immune wild-type mice (see Fig.4d) suggest that under steady-state conditions most IgA expression is T cell-independent. In order to investigate a T cell-dependent IgA immune response in intact mice we immunized Il17aCreR26ReYFP mice with cholera toxin and evaluated the proportion of total eYFP+ cells and eYFP+ TFH cells in the PP, as well as antigen-specific IgA responses in serum and feces. The proportion of eYPF+ cells (5-10%) among total CD4+ T cells in the PP of cholera toxin-immunized Il17aCreR26ReYFP mice was similar that observed in non-immunized Il17aCreR26ReYFP mice (Fig.6a). However, 40-60% of the eYPF+ cells in the PP had acquired a TFH phenotype, compared with 13% at steady-state (Fig.6a and Fig.2a). We also observed a strong cholera toxin specific IgA response in the serum of immunized Il17aCreR26ReYFP mice (Fig.6a).
We used qPCR to assess markers associated with IgA switching in eYFP− and eYFP+ PP T cells from cholera toxin-immunized mice, but could not detect statistically significant differences in the two populations analyzed (Fig.6b). However, expression of ICOS and CD40L was consistently higher on eYFP+ cells than on eYFP− T cells, even before eYFP+ cells had acquired the CXCR5+ PD-1high TFH profile (Fig.6c,d).
To address if T cell-dependent IgA induction requires TH17 cells in an otherwise intact mouse, we generated bone marrow chimeras in which Tcra−/− hosts were reconstituted with whole bone marrow from Rorc−/− mice21 (Rorc−/− Tcra−/− chimeras), which do not develop TH17 cells22. Although Rorc is required for the development of lymphoid architecture in the mucosal immune system21, 23-24, the mucosal environment of these chimeric mice is not disturbed, as Rorc-expressing innate lymphoid cell types are present in the Tcra−/− hosts. Control Tcra−/− chimeras were reconstituted with bone marrow from wild-type hosts. Flow cytometric analysis of PP showed similar proportions of TFH cells in Rorc−/− and control Tcra−/− chimeras (Fig.7a). Rorc−/− Tcra−/− chimeras had a reduction of IL-17-producing CD4+ T cells compared to control Tcra−/− chimeras, whereas the proportion of Treg was comparable and IFNγ-, IL-4- and IL-13-producing CD4+ T cells were similar or even higher in Rorc−/− Tcra−/− chimeras compared to control Tcra−/− chimeras (Fig.7b). Serum isotype profiles in the two sets of chimeras prior to immunization were similar (Suppl.Fig.2). In order to test for T cell-dependent IgA production, we immunized Rorc−/− and control Tcra−/− chimeras with cholera toxin and analysed serum and fecal IgA amounts 10 days later. Control Tcra−/− chimeras mounted a strong cholera toxin-specific IgA response detectable in serum (Fig.7c) and faeces (Fig.7d). In contrast, Rorc−/− Tcra−/− chimeras had very low levels of cholera toxin-specific IgA, which were comparable to those observed in Tcra−/− mice (Fig.7c,d). Thus, these results show that TH17 cells are required for the germinal center switch to IgA production in PP.
TH17 cells are known to diversify their effector profile in response to different environmental conditions25. Here we describe the consequences of TH17 cell plasticity towards a TFH program in the small intestine PP environment, a process that promoted T cell-dependent IgA responses. Although previous studies demonstrated that TH17 cells could be reprogrammed to obtain TFH characteristics in vitro26, an in vivo demonstration of this phenomenon would not have been possible without an IL-17 fate reporter mouse (Il17aCreR26ReYFP mouse), as this allows identification of a TH17 origin irrespective of the production of the signature cytokine IL-17. Here we used the fate reporter mouse to demonstrate the deviation of TH17 cells towards a TFH phenotype under the influence of the PP environment, which results in substantial phenotypic and functional changes.
Thus, IL-17 -as well as RORγt expression, was extinguished in ex-TH17 cell-derived TFH, whereas IL-21 and Bcl-6 were strongly upregulated. Although IL-21 has been associated with in vitro generated TH17 cells3, expression of this cytokine was not detectable in TH17 cells from the intestine or lymphoid organs of non-immune mice.
TH17 cells are naturally found in the small intestine of non-immune pathogen free mice. Under steady-state conditions they are thought to contribute to gut barrier function via the stimulation of tight junction formation and anti-microbial peptides27-28. Our analysis of the Il17aCreR26ReYFP mice indicated that the majority of the few TH17 cells found in peripheral lymphoid organs probably have their developmental origin in the gut, which is also supported by the fact that germfree Il17aCreR26ReYFP mice lack both intestinal and most of the TH17 cells from lymphoid organs. It was particularly interesting to observe that the cytokine IL-23, a key factor for the development of TH17 responses with pathogenic features11-12, 29, is dispensable for the maintenance of intestinal TH17 cells and their deviation towards a TFH program. This confirms earlier suggestions that TH17 cells might develop towards either protective or pathogenic functions30.
Our data expand the functional repertoire of intestinal TH17 cells to include the induction of the GC B cell IgA response. In Tcra-deficient mice transferred with eYFP+ TH17 cells serum IgA levels were highly increased irrespective of deliberate immunization. This was presumably due to the exaggerated expansion of the transferred cells in the lymphopenic hosts and recall responses to commensal microbiota that may be less well controlled in T cell-deficient hosts. In fact, immunization with cholera toxin did not result in an antigen-specific IgA response these mice (data not shown). In contrast, immunization of Il17aCreR26ReYFP mice with cholera toxin resulted in a pronounced cholera toxin specific IgA response and more pronounced switching of eYFP+ towards a TFH phenotype.
In Rorc−/− Tcra−/− chimeras, serum IgA levels prior to immunization with cholera toxin were comparable to those seen in Tcra−/− mice as well as wild-type B6 mice (data not shown), suggesting that basal IgA levels in SPF mice may be mostly T cell-independent. TH17 cells were shown to be involved in upregulating the expression of the polymeric Ig receptor that transports IgA across the intestinal epithelium31. However, it seems this role can be attributed to IL-17 rather than to TH17 cells. An interesting possibility that remains to be addressed is whether RORγt+ ILCs contribute to T cell-independent IgA induction and/or upregulation of the IgA transporter. Since ex-TH17 derived TFH switch off IL-17, it is unlikely that they participate in this process and we have not detected changes in expression of polymeric Ig receptor in our various models, none of which were devoid of IL-17. Nevertheless, IgA responses to challenge with cholera toxin strongly depended on the presence of TH17 cells.
Plasticity of TH17 cells towards a TFH fate was restricted to the PP environment and not evident in peripheral LN. As intestinal IgA fulfils important roles in maintaining equilibrium with the commensal flora and efficient mucosal host defence7, this novel TH17 function provides another example of their crucial role in mucosal immunity. It is interesting to note that segmented filamentous bacteria, which are important stimulators of TH17 cell development, also drive germinal center formation and IgA production in PP4-5. IgA deficiency causes aberrant expansion of SFB4, 32-33 and one might speculate that a deficiency in TH17 cells would result in the same features.
The developmental relationship between TFH cells and other CD4+ T cell subsets remains a matter of debate (reviewed in15). Our data are compatible with a non-exclusive CD4+ T cell program that obtains input from multiple T cell subsets. TH2 cells have been shown able to acquire CXCR5 expression, resulting in the induction or inhibition of B cell differentiation and class switching in GC18, 34-36. IL-12-mediated STAT4 activation transiently induces a TFH transcriptional profile, followed by repression of the TFH gene signature by the TH1-specific transcription factor T-bet37. Induction of Bcl-6 requires ICOS38, which is highly expressed on TH17 cells. Temporal and spatial regulation of CXCR5 and Bcl-6 expression in interaction with dendritic cells and B cells promote TFH development38-40, but it remains unclear whether the TFH state resembles a terminal effector status or whether such cells can be redirected towards other T cell programs. Thus, it remains to be determined whether the extinction of a previous effector profile is complete following the acquisition of a TFH phenotype or whether each effector T cell subset contributes a unique feature of its original signature to the functional helper response in the germinal center reaction. Elucidation of these possibilities would be facilitated by fate reporter mice for each T cells subset, which would allow analysis of functional profiles irrespective of the expression of signature cytokines that currently defines their subset allocation.
The role of Treg in PP germinal center reactions remains controversial. In some cases it was argued that Treg converted to TFH to promote intestinal IgA responses41,18, whereas other studies suggested that Treg, expressing markers of TFH cells are essential to control, rather than promote the germinal center reaction19-20. It should be noted that depletion of Treg via antibodies to CD25 as used in a previous study on the role of Treg in intestinal IgA induction41, would also have depleted TH17 cells, which are homogenously CD25+. Our data did not confirm plasticity of Treg towards a TFH profile, nor a role in promoting IgA responses either in the transfer model or in bone marrow chimeras. For transfers we isolated the Treg population based either on high CD25 expression, which was shown to mark stable Treg42 or based on high RFP-Foxp3 expression in the Foxp3RFP mouse43. The discrepancy regarding Treg plasticity might be explained by technical issues with the Foxp3 reporter model that was previously used18, where plasticity was particularly prominent in cells expressing lower levels of Foxp3 or in transfers of mixtures of Foxp3+ and Foxp3− cells. Given the reciprocal relationship between Foxp3 and RORγt in Treg and TH17 cell development44, it is conceivable that Foxp3low Treg cells may have deviated towards a TH17 fate, thus mimicking their unique function in the intestinal immune response.
Our data highlight another facet of the host protective function of TH17 cells in mucosal tissues. At present it remains unclear what particular features ex-TH17 cells contribute to their interaction with B cells to promote IgA responses. Currently known genes that affect IgA are widely expressed in the PP environment and we did not detect differential expression in eYFP+ and eYFP− PP T cells for these markers. Interestingly, eYFP+ T cells display substantially higher levels of ICOS and CD40L, which might facilitate preferential contact with B cells. B cells in the PP patch environment are characterized by expression of the transcription factor RORα45 and compete for T cell help before entry into germinal centres. eYFP+ T cells may have preferential access. This, however, does not explain why TFH in PP of the Rorc−/− Tcra−/− chimeras, which do not face competition by ex-TH17 cells are still not competent to induce an IgA switch.
Given the prominent role of TH17 cells in autoimmunity, these cells seem as obvious targets for therapeutic intervention. However, understanding their role in maintaining intestinal barrier integrity is required in order to avoid disturbance of these beneficial functions.
IL-17 fate reporter (Il17aCreR26ReYFP) mice12 and Foxp3RFP mice with a bicistronic red fluorescent protein (RFP) reporter knocked into the Foxp3 locus43, as well as TCRα-deficient on a B6 background46 and p19−/− (Il23a−/−) mice (obtained from Dan Cua,Merck Research laboratories USA) were bred in the NIMR animal facility under specified pathogen free conditions. All animal experiments were done according to the NIMR Ethical Review committee and Home Office regulations. Some IL-17 fate reporter mice were raised in germ free (GF) conditions using caesarean section rederivation, as described at The European Mouse Mutant Archive http://www.emmanet.org/protocols/GermFree_0902.pdf. Briefly, day 20 post coitum uteri from donor females were transferred through a reservoir containing 1% VirkonS to the isolator housing the germ-free surrogate mothers. The microbiological status of the isolator was monitored every 3 weeks. Bones from Rorc(γt)GFP/GFP mice which are knockout for Rorc (γt)21 were obtained from Oliver Steinmetz (Hamburg).
anti-CCR6 (140708), CXCR5 (2G8), CD95 (Jo2), and anti-IgA (C10-3) were purchased from BD Biosciences. Anti-GL-7 (GL7) and RORγt (AFKJS-9) were obtained from eBioscience. Anti-CD4 (GK1.5), CD25(PC61), CD27 (LG.3A10), CD44 (IM7), CD45.1 (A20), CD103 (2E7), ICOS (C398.4A), IL-7Ra (A7R34), Podoplanin (8.1.1), PD-1 (29F.1A12), B220 (RA3-6B2), IL-17 (TC11-18H10.1) , IL-22 (AM22.3), IFNγ (XMG1.2), and TCR-β (H57-597) were from Biolegend.
Briefly, lymphocytes in the lamina propria were prepared by cutting the small intestine into 1cm pieces after removing Peyer’s patches, shaking for 20min at 37°C in 10ml IEL buffer (PBS supplemented with 10% FCS, 1mM pyruvate, 20μM HEPES, 10mM EDTA and penicillin /streptomycin mix, 10ug/ml Polymyxin B) to remove epithelial and intraepithelial cells and then digesting the remaining tissue using 1mg/ml collagenase D (Roche) and 10U/ml DNase1 (Sigma) at 37°C for 1 hr followed by 36.5% Percoll separation.
RNA was extracted from FACS sorted CD4 T cells using Trizol and reverse transcribed with Omniscript (Qiagen) according to the manufacturer’s protocol. The cDNA served as template for the amplification of genes of interest and the housekeeping gene (Hprt) by real-time PCR, using TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA); Hprt1 (Mm00446968_m1), Aicda (Mm00507774_m1), Bcl6 (Mm00477633_m1), Il17a (Mm00439619_m1), Il21 (Mm00517640_m1), Il22 (Mm00444241_m1), Prdm1 (Mm00476128_m1), Rorc (Mm01261019_g1), universal PCR Master Mix (Applied Biosystems, Warrington, UK) and the ABI-PRISM 7900 Sequence detection system (Applied Biosystems, Foster City, CA). Target gene expression was calculated using the comparative method for relative quantification upon normalisation to Hprt gene expression.
TCRα−/− mice were sublethally irradiated (500 rad) and reconstituted with B6 or Rorc−/− bone morrow. Before immunization with cholera toxin mice were deprived of food for 2 h and then given 0.25 ml of a solution containing eight parts HBSS and two parts 7.5% sodium bicarbonate by oral gavage (o.g.) to neutralize stomach acidity. After 30 min, mice were o.g. immunized with 25μg cholera toxin (List Biological Laboratories, USA). Concentrations of antibodies specific for cholera toxin were determined by ELISA with cholera toxin (List Biological Laboratories) as the capture agent.
Statistics were performed using a two-tailed Student T test.
This work was supported by the Medical Research Council UK (Ref. U117512792). JET was supported by a Research Fellowship from the Deutsche Forschungsgemeinschaft (TU 316/1-1) and MV is a recipient of a Boehringer Ingelheim Fonds PhD fellowship. We would like to thank the flow facility for expert cell sorting and Biological Services for breeding and maintenance of our mouse strains. Axenization was supported by EMMA, EU FP7 Capacities Specific Program. We would also like to thank Drs A and T Zal (MD Anderson, Houston) for advice with PP microscopy, Drs D Cua (Merck Research Laboratories, USA) for the Il23a−/− strain and Adrian Hayday (King’s College London) for Tcra−/− mice.
Contributions K.H. and B.S. conceived of the project and designed the experiments. K.H. did most of the experiments. M.V., J-E.T., and J.H.D. did specific experiments. J.D. established the germfree colony of reporter mice and O.M.S. supplied bone marrow from Rorc−/− mice, respectively. K.H. and B.S. wrote the paper.