Circulating and Tissue-Resident CD4+ T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function Is Altered During Inflammation

1See Covering the Cover synopsis on page 1175.

2See Covering the Cover synopsis on page 1175.

3CD4+ T cell responses to intestinal bacteria are known to occur, however these responses remain poorly characterized in humans.Microbiota-reactive CD4+ T cells are prevalent and normal constituents of the human immune system that are functionally altered during IBD pathogenesis.The functional relevance of the detected T-cell responses in humans remains to be elucidated.T-cell responses to commensals might support intestinal homeostasis by producing barrier-protective cytokines and providing a large pool of T cells with potential cross-reactivity to pathogens.

4CD4+ T cell responses to intestinal bacteria are known to occur, however these responses remain poorly characterized in humans.

5Microbiota-reactive CD4+ T cells are prevalent and normal constituents of the human immune system that are functionally altered during IBD pathogenesis.

6The functional relevance of the detected T-cell responses in humans remains to be elucidated.

7T-cell responses to commensals might support intestinal homeostasis by producing barrier-protective cytokines and providing a large pool of T cells with potential cross-reactivity to pathogens.

8Vast numbers of microbes populate the gastrointestinal tract and contribute to digestion, epithelial barrier integrity, and development of appropriately educated mucosal immunity. Intestinal immune responses are tightly regulated to allow protective immunity against pathogens, while limiting responses to dietary antigens and innocuous microbes. The “mucosal firewall” prevents systemic dissemination of microbes by confining microbial antigens to the gut-associated lymphoid tissue. In the gut-associated lymphoid tissue, dendritic cells drive regulatory T-cell differentiation in response to dietary antigens and commensal bacteria. Nevertheless, vast numbers of potentially commensal-reactive effector and memory T cells populate intestinal mucosae. Recent evidence suggests that in mice, tolerance to commensal-derived antigens may be lost during pathogen-induced epithelial damage and subsequent transient exposure to commensals., In humans, circulating memory T cells recognize peptides derived from gut bacteria and can cross-react to pathogens, which can confer immunologic advantage during subsequent new infections., Although this process can be beneficial during homeostasis, deranged responses to commensals may promote inflammatory conditions, such as inflammatory bowel diseases (IBDs).

9IBDs (including Crohn’s disease and ulcerative colitis) result from a prolonged disturbance of gut homeostasis, the precise etiology of which is uncertain. One hypothesis is that, in genetically susceptible individuals, IBD may be triggered by intestinal dysbiosis that promotes aberrant immune stimulation. Indeed, in mouse models of colitis, intestinal microbiota promote inflammation in part by stimulating microbiota-reactive CD4+ T cells., Whether this drives IBD in humans, however, remains unknown.

10Although CD4+ T-cell responses to intestinal bacteria are known to occur in humans,,, several aspects of this topic are largely uncharacterized, including the frequency of human T cells in the gut and periphery that are reactive to phylogenetically distinct intestinal microbes; the T-cell receptor (TCR) diversity and clonotype sharing of these T cells; the functional phenotype of gut microbe-reactive T cells and their impact on tissue-resident cell populations; and how microbe-reactive T cells change during chronic intestinal inflammation. To address this knowledge gap, we extensively characterized CD4+ T-cell responses to intestinal microbiota in healthy individuals and IBD patients.

11Using 2 independent assays, we observed that for almost all enteric bacteria examined, bacteria-reactive CD4+ T cells were present at a frequency of 40−500 per million CD4+ T cells in adult peripheral blood. Bacteria-reactive T cells were also prevalent in the gut mucosa, with prominent enrichment for proteobacteria reactivity. Microbiota-responsive T cells showed a diverse TCR Vβ repertoire and potently stimulated inflammatory responses by intestinal epithelial and stromal cells. Intriguingly, T cells from IBD patients displayed a normal spectrum of microbial responses, but expressed high amounts of interleukin (IL) 17A, consistent with increased amounts of T-helper (Th) 17-polarizing cytokines in inflamed intestinal tissue. Collectively, these data demonstrate that microbiota-reactive CD4+ T cells are prevalent and normal constituents of the human immune system that are functionally altered during IBD pathogenesis.

12Leukoreduction chambers from healthy individuals were obtained from the National Blood Service (Bristol, UK). Peripheral EDTA blood samples were obtained from IBD patients attending the John Radcliffe Hospital Gastroenterology unit or from healthy in-house volunteers. IBD patients (n = 119; ulcerative colitis, n = 59; Crohn’s disease, n = 60) diagnosed by endoscopic, histologic, and radiologic criteria were recruited for the study. Healthy volunteers (n = 30) without any known underlying acute or chronic pathologic condition served as control donors. All donors provided informed written consent. The National Health Service Research Ethics System provided ethical approval (reference numbers 09/H0606/5 for IBD patients and 11/YH/0020 for controls; OCHRe ref 15/A237 for cord blood samples). For details regarding cell isolation, see Supplementary Experimental Procedures.

13CD154 detection was done as described previously., Briefly, cells were plated at 5 × 106/cm2 for 7−12 hours with heat-inactivated bacteria. Brefeldin A (5 μg/mL; Sigma Aldrich, St Louis, MO) was added at 2 hours. After 8−12 hours, cells were harvested and treated as described in the section on intracellular cytokine, CD154, and transcription factor staining. For magnetic cell separation enrichment of CD4+CD154+ T cells, see Supplementary Experimental Procedures.

14Memory CD4+ CD45RO+ CD45RA− T cells were enriched from peripheral blood mononuclear cells (PBMCs) with untouched memory CD4+ T cell enrichment kit (Miltenyi Biotec, Bergisch Gladbach, Germany), sorted to >97% purity on a FACS ARIA III (BD, San Jose, CA) using CD45RA and CD45RO expression, and were labeled with carboxy-fluorescein succinimidyl ester (CFSE) or violet proliferation dye (Invitrogen, Carlsbad, CA). CD14+ monocytes were isolated from PBMC using anti-CD14 microbeads (Miltenyi Biotec), irradiated (45 Gy) and then pre-incubated for 3 hours with bacterial lysates before T-cell co-culture. T cells were co-cultured with the irradiated autologous monocytes at a ratio of 2:1 for 5−7 days. Cells were cultured in RPMI-1640 supplemented with 2 mM glutamine, 1% (v/v) non-essential amino acids, 1% (v/v) sodium pyruvate, penicillin (50 U/mL), streptomycin (50 mg/mL; all from Invitrogen), and 5% (v/v) human serum (National Blood Service, Bristol, UK). CD14+ monocytes were irradiated (45 Gy) and then pre-incubated for 3 hours with bacterial lysates before T-cell co-culture. For major histocompatibility complex II blockade, 10 μg/mL of a pan-HLA class-II blocking antibody (HLA-DR, DP, DQ [Tü39]) was added 30 minutes before T-cell co-culture. T-cell lines were generated by sorting CFSElow inducible T-cell costimulator (ICOS)high CD4+ T cells after 7 days of stimulation and expanding them with IL2 (300 U/mL) and anti-CD3/CD28 beads (beads/T cell ratio, 1:4, Dynals) for 10−14 days. Supernatants were collected from 1 × 106 CD4+ T cells stimulated for 24 hours with phorbol myristate acetate (5 ng/mL) and ionomycin (500 ng/mL; Sigma).

15PBMCs and lamina propria mononuclear cells were stained according to standard protocols. For details, see Supplementary Experimental Procedures.

16For intracellular cytokine staining, cells were stained with fixable viability dye eFluor 780 (eBioscience, San Diego, CA) and surface markers, fixed with 2% formaldehyde (Merck, Kenilworth, NJ), and stained for cytokines in magnetic cell separation buffer containing 0.05% saponin (Sigma-Aldrich). Transcription factor expression was analyzed using the FoxP3 staining buffer set (eBioscience) according to manufacturer’s instructions.

17CCD18Co (non-transformed human colon fibroblasts; ATCC, Manassas, VA), and LIM1863 (human colon epithelial cells; a kind gift of Dr Robert Whitehead, Ludwig Institute for Cancer Research) cells were cultured in humidified incubators with 5% CO2 at 37°C in Dulbecco’s modified Eagle medium or RPMI media (Sigma) with 10% fetal calf serum (Sigma) and 100 U penicillin/0.1 mg/mL streptomycin. Cells were stimulated with 5% T-cell supernatants for 24 hours. Cytokine neutralization was achieved by supernatant pre-incubation for 1−2 hours with 10 μg/mL anti−IL17A (eBio64CAP17), anti−interferon (IFN) gamma (B27), anti−tumor necrosis factor (TNF)−α (Remicade; Janssen Biotech, Horsham, PA), and anti−IL22 (IL22JOP).

18Statistical analyses were performed with GraphPad Prism, version 6.0 for Macintosh (GraphPad Software, La Jolla, CA). Statistically significant P values were indicated as follows: NS, not significant; ∗P ≤.05; ∗∗P ≤.01; ∗∗∗P ≤.001; ∗∗∗∗P ≤.0001. Tests are specified in figure legends.

19CD154 (also known as CD40 ligand) is rapidly up-regulated by CD4+ T cells after antigen stimulation, irrespective of their differentiation phenotype, major histocompatibility complex alleles, or the precise nature of the antigenic epitope., We therefore used CD154 to detect naïve and memory CD4+ T-cell responses among PBMCs after stimulation with heat-inactivated bacteria (Figure 1A). Seven aerobic and anaerobic intestinal bacterial species representing the 4 dominant gut phyla were chosen: Escherichia coli, Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, and Ruminococcus obeum (Supplementary Figure 1A and Supplementary Table 1). These bacteria are common in the healthy intestine, but are altered in relative abundance during inflammation., Furthermore, we analyzed responses to Salmonella typhimurium and Clostridium difficile due to their association with IBD., T-cell responses to these bacteria were compared with those against well-characterized barrier surface-related microbes that drive robust Th17 responses (Staphylococcus aureus and Candida albicans) or strong Th1 responses (Mycobacterium tuberculosis)., The presence of a large pool of M tuberculosis-reactive memory Th1 cells in non-exposed individuals has been documented previously. The responses in healthy controls are directed toward non-tuberculous mycobacteria rather than toward M tuberculosis.,, The superantigen Staphylococcus enterotoxin B (SEB) was used as a positive stimulation control.

20Stimulation with enteric bacteria reproducibly induced detectable numbers of CD4+CD154+ T cells in the peripheral blood. CD4+ T cells reactive to S aureus, C albicans, and M tuberculosis were generally more abundant (Figure 1B and Supplementary Figure 1B and C).

21Activation markers CD69 and ICOS were up-regulated on activated antigen-reactive CD4+CD154+ T cells (Supplementary Figure 1D). Responses were major histocompatibility complex II−dependent (Supplementary Figure 1E), and lipopolysaccharide failed to induce CD154 expression, confirming that CD154 up-regulation was not a consequence of non-specific microbial responses (Figure 1B and Supplementary Figure 1C). Based on CD154+ cell frequencies, we calculated that enteric bacteria-reactive CD4+ T cells were present at precursor frequencies of 40−500 cells per 106 circulating CD4+ T cells for almost all enteric bacteria surveyed (Figure 1C and Supplementary Figure 1F).

22The newborn gut is primarily colonized with maternal vaginal and fecal bacteria after birth. To understand whether T-cell reactivity to microbes develops after birth, we compared CD154 expression in umbilical cord blood with adult blood after enteric bacteria stimulation. As expected, appreciable responses to microbiota were observed only in adult blood. However, CD4+CD154+ T-cell frequencies after SEB stimulation were similar between adult and cord blood (Figure 1D and Supplementary Figure 1G).

23Human memory T cells down-regulate the naïve marker CD45RA and produce cytokines more efficiently than naïve T cells., In healthy individuals, the majority of bacteria-reactive CD4+ T cells had a memory phenotype (>80%, on average), indicating that they had been primed in vivo (Figure 1E and F and Supplementary Figure 2A and B). Microbiota-reactive CD4+CD154+CD45RA− T cells expressed high amounts of TNF-α and IL2 when compared to CD4+CD154+CD45RA+ T cells (Figure 1G and Supplementary Figure 2C). Therefore, the circulating pool of memory CD4+ T cells contains numerous microbiota-reactive cells that arise after birth and produce cytokines, including TNF-α and IL2.

24Memory T cells express numerous adhesion molecules and chemokine receptors to access different tissues under steady-state and inflammatory conditions.,, For example, α4β7 integrin and chemokine receptor (CCR) 9 regulate T-cell migration to distinct parts of the gut. Blockade of α4β7 integrin has shown clinical efficacy for treating IBD, whereas CCR9 blockade yielded mixed results., To identify homing receptors expressed by bacteria-reactive CD4+ T cells, we enriched CD4+CD154+ T cells using magnetic beads to visualize rare enteric-bacteria-reactive T cells, and analyzed them by flow cytometry (Figure 2A). Microbiota-reactive T cells had a central memory phenotype, with >60% expressing high levels of CCR7 (Figure 2B). Furthermore, 5%−10% of CD4+CD154+ T cells expressed the gut-homing surface markers integrin β7 and CCR9 (Figure 2C and D). Relative to total memory CD4+ T cells and CD4+CD154− T cells, enteric bacteria-reactive T cells had high expression of the mucosa-homing receptors CCR4 and CCR6 (>60%), low expression of CCR10, and comparable expression of CXCR3 and CCR2 (Figure 2D and Supplementary Figure 2D). Microbiota-reactive CD4+ T cells also expressed high amounts of CD161, a marker enriched on Th17 cells (Figure 2D and E). The majority of memory CD4+CD154+ T cells co-expressed CCR7, CCR4, CD161, and CCR6 in various combinations, some of which also expressed integrin β7 (Figure 2E, pie chart). Therefore, circulating microbiota-reactive CD4+ T cells are equipped with several homing receptors that promote mucosal access.

25When comparing gut microbiota-reactive CD4+ T cells with those reactive to non-enteric organisms (including S aureus, M tuberculosis, and C albicans), enteric bacteria-reactive T cells were partially enriched only in CCR4 expression (Figure 2F). The homing receptor phenotype of enteric bacteria-reactive T cells is consistent with that of T cells reactive to a broad diversity of microbes located at barrier surfaces.

26The gut harbors >3 × 1010 CD4+ T cells, but their specificity is unknown., We therefore estimated the abundance of human microbiota-reactive CD4+ T cells in the gut by examining non-inflamed colon specimens using the CD154 assay (Figure 3A). Lamina propria CD4+ T cells showed a dominant effector memory and central memory phenotype and expressed both tissue-resident and gut-related markers, with 80% of cells being CD69+ (Supplementary Figure 3A).

27We next stimulated lamina propria mononuclear cells with microbial lysates or SEB. We combined intracellular CD154 detection with TNF-α staining to increase assay sensitivity, as lamina propria CD4+ T cells expressed low amounts of CD154 without stimulation (Supplementary Figure 3B and C). Compared with peripheral blood frequencies of unrelated donors, there were similar frequencies of S aureus and SEB reactivity, and reduced M tuberculosis-reactivity in the gut. However, gut CD4+ T cells were enriched in reactivity toward intestinal bacteria and C albicans (Figure 3B and Supplementary Figure 3D). Bacteria-reactive cells comprised 150−4000 cells per 106 gut-resident memory CD4+ T cells for all enteric bacteria tested. Given that peripheral blood contained 40−500 bacteria-reactive cells per 106 memory CD4+ T cells (for each bacteria tested), this suggests that bacteria-reactive T cells are 3- to 8-fold more frequent in gut tissue as compared with those in circulation. The strong enrichment of S typhimurium and E coli reactivity in the gut was confirmed by assessing CD154 and TNF-α expression in CD4+ T cells from donor-matched blood and intestinal tissue (Figure 3C). Because the gut harbors up to 3 × 1010 memory T cells (vs 5−10 × 109 in blood), many of which are bacteria-reactive, the absolute number of gut-resident microbiota-reactive CD4+ T cells is likely to be at least 10 times greater than that in peripheral blood.

28Gut-resident bacteria-reactive (CD154+TNF-α+) T cells produced high amounts of IFN-gamma, IL17A, and IL2, while production of IL22, granulocyte macrophage colony-stimulating factor, and IL4 was generally low (Figure 3D and Supplementary Figure 3E and F). Interestingly, lamina propria T cells showed increased IL17A expression and reduced IFN-gamma production relative to cells with similar reactivity in peripheral blood (Figure 3D and Supplementary Figure 3E).

29To assess the clonal diversity of circulating bacteria-reactive memory CD4+ T cells, we expanded CFSE-labeled CD4+ T cells using whole bacteria and autologous irradiated monocytes as antigen presenting cells (Supplementary Figure 4A). S aureus-, M tuberculosis-, and SEB-reactive T cells served as controls. Antigen-reactive T cells proliferated in a major histocompatibility complex II−dependent manner and were readily detectible after 3−6 days (Figure 4A and Supplementary Figure 4B−D). Proliferating cells expressed several activation markers including ICOS, CD25, and OX40 (Figure 4A and Supplementary Figure 4B, E, and F). Consistent with the CD154 assay, S aureus, M tuberculosis, and SEB strongly induced T-cell proliferation (Figure 4A and B and Supplementary Figure 3B and C).

30Flow cytometry analysis revealed a diverse TCR Vβ repertoire in bacteria-reactive T cells, similar to polyclonal stimulation with phytohemagglutinin but different to stimulation with SEB, which is known to activate a restricted Vβ repertoire (Figure 4C). To directly assess the clonal diversity of bacteria-reactive CD4+ T cells, we isolated CFSElow bacteria-reactive memory T cells and assessed TCR Vβ clonotypes by multiplex polymerase chain reaction and deep sequencing. One hundred and fifty to eight hundred clonotypes were detected for each reactivity (Supplementary Figure 4G). The largest clonal diversity was detected among E coli- and S typhimurium−reactive cells, consistent with frequencies observed in the CD154 assay (see Figure 1B and C and Supplementary Figure 1B and F). While closely related species (eg, E coli vs S typhimurium) had 3%−8% overlap in T-cell clonotypes, little clonotype sharing was observed between T cells reactive to more distantly related bacteria (Supplementary Figure 3H). Indeed, E coli- and B animalis−reactive CD4+ T-cell lines were strongly restimulated when cultured with autologous monocytes loaded with E coli or B animalis lysates, respectively. In contrast, E coli−reactive T cells responded weakly to the closely related S typhimurium, while B animalis−reactive T cells responded weakly to L acidophilus, F prausnitzii, and C difficile (Supplementary Figure 5A). These data confirm the low degree of cross-reactivity predicted from TCR Vβ sequencing.

31To functionally characterize circulating microbiota-reactive memory cells, we analyzed CFSElow cells using flow cytometry after stimulation with enteric bacteria for 6 days. Enteric microbiota-reactive cells produced Th1- and Th17-related cytokines, including IFN-gamma, IL17A, and IL22, but only low amounts of the Th2 cytokine IL4, comparable to cells reactive toward S aureus or C albicans (Figure 4D and Supplementary Figure 5B and C). In contrast, memory T cells reactive toward SEB, M tuberculosis, influenza vaccine components, or tetanus toxoid showed a polarized Th1 profile with low expression of IL17A (Figure 4D and Supplementary Figure 5B and C). Boolean gating revealed a high degree of functional heterogeneity in expanded microbiota-reactive memory T cells, with frequent co-expression of IL17A, IL22, and IFN-gamma (Figure 4E). Bacteria-reactive cells co-expressed the transcription factors RAR-related orphan receptor γt and T-box expressed in T cells, which are characteristic of Th17 and Th1 cells, respectively (Figure 4F and G and Supplementary Figure 5D and E). Intriguingly, a subset of CD4+ T cells reactive to F prausnitzii, L acidophilus, or B animalis produced the immunoregulatory cytokine IL10 in addition to IFN-gamma and IL17A (Supplementary Figure 5F). Compared to T cells that are reactive toward M tuberculosis or vaccine antigens, enteric microbiota-reactive T cells are functionally distinct and produce barrier-promoting and immunoregulatory cytokines.

32During periods of epithelial damage and exposure to commensals, activation of microbiota-reactive memory T cells could promote protective immune responses. To assess their tissue-modulating capabilities, cell-free supernatants of microbiota-reactive memory T cells were used to stimulate CCD18Co intestinal myofibroblasts and LIM1863 colonic epithelial cells. CCD18Co and LIM1863 cells were then assessed for expression of various immune response genes that were selected a priori to represent responses to major T-cell−derived cytokines. Both cell types responded by expressing several cytokine and chemokine genes known to be induced by IL17A (including IL1B, CSF2, IL6, CXCL1, and CXCL8), as well as IFN-gamma−inducible genes, including CXCL9, CXCL10, and CXCL11 (Figure 5A and B). Conversely, supernatants from SEB-stimulated memory T cells (which produce little IL17A) mainly induced IFN-gamma−dependent genes. Thus, stimulation of non-hematopoietic intestinal cells by microbiota-reactive T cells may promote recruitment and activation of myeloid cell populations to facilitate pathogen control and tissue repair.

33We next assessed the effects of individual cytokines in E coli−reactive T-cell supernatants using combinations of neutralizing antibodies. This experiment revealed distinct IFN-gamma− and IL17A-/TNF-α−dependent groups of response genes in both intestinal epithelial cells and fibroblasts. IFN-gamma blockade strongly reduced expression of several chemokine genes, including CXCL9, CXCL10, CXCL11, CCL2, and CCL7 (IFN-gamma−dependent module; Figure 5C and D). Intriguingly, single blockade of IL17A, IL22, or TNF-α did not affect stromal or epithelial cell activation (Figure 5C and D). However, combined blockade of IL17A and TNF-α influenced a large number of genes, including CSF2, IL1B, TNF, CXCL1, CXCL8, CXCL5, CXCL6, and CCL20 (IL17A/TNF-α−dependent module). Triple blockade of IFN-gamma, IL17A, and TNF-α completely inhibited stromal and epithelial cell activation. IL22 blockade did not affect cytokine or chemokine production, but attenuated induction of the antimicrobial peptide REG3G in LIM1863 cells. Given that the products of T-cell−stimulated stromal and epithelial cells are highly expressed in the inflamed mucosa of IBD patients (Figures 5E and 7D), this signature might reflect the activation of microbiota-reactive T cells after epithelial disruption, a key feature of IBD.

34IBD is thought to arise in part from aberrant adaptive immune responses to microbiota. Human CD4+ T cells in IBD have been functionally characterized mainly by polyclonal stimulation.,, Therefore, we evaluated microbiota-reactive CD4+ T-cell responses in IBD patients using the CD154 detection approach. Circulating microbiota-reactive CD4+ T-cell frequencies were decreased in IBD patients compared with healthy donors, which might reflect their selective recruitment to the inflamed gut (Figure 6A and Supplementary Figure 6A). However, intestinal memory CD4+ T cells from IBD patients did not display reciprocally higher frequencies of microbial specificity (Figure 6B and Supplementary Figure 6B). We next calculated the frequency of memory CD4+ T cells in inflamed mucosae using flow cytometry. Memory CD4+ T cells were present at higher frequencies in inflamed tissue from IBD patients compared with tissue from matched non-lesional sites of IBD patients and healthy controls (Figure 6C). These findings were confirmed using a previously published bioinformatics approach known as CIBERSORT in an independent cohort (Supplementary Figure 6C). Based on both approaches, memory CD4+ T cells are typically 2- to 4-fold more frequent in inflamed tissue from IBD patients compared with tissue from healthy controls. Because inflamed tissue contains a higher abundance of memory CD4+ T cells than healthy mucosa, it can be inferred that gut-resident microbiota-reactive CD4+ T cells are similarly enriched in patients with active IBD (Figure 6C, Supplementary Figure 6C).

35To evaluate functional alterations in microbiota-reactive CD4+ T cells in IBD, intracellular CD154 detection was combined with cytokine analysis. Compared with healthy controls, circulating microbiota-reactive CD4+ T cells from IBD patients displayed increased IL17A and IL2 production, but decreased expression of IFN-gamma (Figure 7A and Supplementary Figure 6D and E). Interestingly, increased IL17A production was observed in all enteric bacteria-reactive responses, but not in S aureus, M tuberculosis, or SEB responses (Figure 7A and Supplementary Figure 6E and F). These changes were observed in both Crohn’s disease and ulcerative colitis and were independent of disease activity or therapy (Supplementary Figure 6G). However, no difference in IL10 production was observed between healthy donors and IBD patients (Supplementary Figure 6H). IFN-gamma and IL17A co-expression is thought to identify pathogenic CD4+ T cells in mouse colitis models, so we assessed their co-expression in E coli−reactive memory CD4+ T cells. Compared with controls, IBD patients displayed significantly increased frequencies of IL-17A+IFN-gamma− cells and a marginal increase in IL17A+IFN-gamma+ cells, while the IL17A−IFN-gamma+ fraction was reduced significantly (Figure 7B). E coli−reactive CD4+ T cells from inflamed intestinal tissue showed an increase in IL17A single producers similar to that seen in peripheral blood (Figure 7C).

36Because the Th17-inducing cytokines IL1B, IL6, and IL23A were highly enriched in the inflamed intestinal tissue of IBD patients (Figure 7D), we reasoned that they might promote Th17 polarization of bacteria-reactive T cells. Indeed, treatment of microbiota-reactive CD4+ T cells from healthy donors and IBD patients with IL1β, IL6, or IL23 for 1 week during stimulation with E coli, S typhimurium, L acidophilus, or B animalis (CFSE dilution assay) resulted in a 1.5- to 2-fold increase in IL17A production (Figure 7E and Supplementary Figure 6I).

37Taken together, these experiments demonstrate that circulating and gut-resident microbiota-reactive CD4+ T cells express increased frequencies of IL17A in IBD. Intestinal tissues from patients with active IBD express gene modules driven by Th1/Th17-derived cytokines, suggesting that bacteria-reactive memory cells could contribute to the tissue response.

38The gastrointestinal tract harbors a large and diverse population of commensal bacteria, and how the immune system interacts with them is subject to intense investigation. Here we used 2 different methodologies to characterize microbiota-reactive CD4+ T-cell frequencies and phenotypes in the blood and intestinal tissue of healthy individuals and those with IBD. For each bacterial strain tested, the healthy CD4+ T-cell repertoire contains reactive cells at a frequency of 40−4000 per million, consistent with other antigen-reactive memory T cells. Microbiota-reactive CD4+ T cells were mainly of a memory phenotype, present in both blood and gut tissue, had a diverse TCR Vβ repertoire, and showed little clonotype sharing. Notably, microbiota-reactive CD4+ T cells were functionally heterogeneous in terms of homing receptor expression and effector functions and could stimulate intestinal cells via production of IL17A, IFN-gamma, IL22, and TNF-α. In addition, microbiota-reactive CD4+ T cells were recruited to sites of inflammation and showed increased IL17A production in patients with IBD.

39Characterizing T-cell responses to bacteria is technically challenging due to their complex antigenic makeup. We therefore used the CD154 and CFSE dilution assays, both of which exploit microbial complexity, to provide large numbers of antigens. The combination of CFSE dilution and TCR Vβ sequencing allowed us to quantify clonotype heterogeneity and sharing between different bacteria-reactive T cells. Given the phylogenetic similarity of several bacteria used in this study, the paucity of clonotype sharing was surprising. Nevertheless, enteric bacteria-reactive T cells could be cross-reactive to other antigens not assessed in this study, and may have been primed during immune responses to other targets. High interclonal and intraclonal functional heterogeneity in human CD4+ T-cell responses to microbes and vaccines was observed recently. However, clonotype sharing between different microbial stimuli has not been studied previously and requires further investigation.

40Microbiota-reactive CD4+ T cells showed substantial phenotypic and functional heterogeneity. The majority of circulating enteric bacteria-reactive CD4+ T cells co-expressed chemokine receptors, including CCR4, CCR6, and CCR7, while a smaller fraction expressed the gut-related homing receptors α4β7 and CCR9. These receptors promote access to secondary lymphoid organs and various mucosal tissues, including the intestine., In addition, circulating and gut-resident microbiota-reactive T cells displayed both Th17 and Th1 characteristics and, in some cases, produced IL10. Gut-resident cells showed a clear Th17 bias when compared to circulating populations, which was more pronounced in IBD.

41Based on our observations, we can speculate that continuous sampling of luminal antigens by intestinal dendritic cells causes low-level stimulation of gut-resident CD4+ T cells to produce cytokines that support epithelial integrity, barrier function, and intestinal homeostasis., Indeed, cytokine production by commensal-reactive CD4+ T cells might play a more significant role in supporting gut homeostasis than previously thought (Supplementary Figure 7). However, this homeostatic circuit might be disrupted in IBD due to dysbiotic changes and/or perturbed myeloid cell activity, causing inappropriate T-cell activation and a pathogenic imbalance of cytokine production.

42While IL17A is frequently cited as a pathogenic cytokine, it is also critical for promoting mucosal barrier function and protection from pathogens. Absence of IL17A was recently shown to increase epithelial injury and compromise barrier function in mouse models of colitis., Indeed, IL17A is a critical driver of neutrophil recruitment, and its absence could therefore exacerbate mucosal inflammation by facilitating bacterial invasion and dispersal. Notably, blockade of IL17A in Crohn’s disease caused disease exacerbation, despite being well tolerated and therapeutically effective in psoriasis. Thus, IL17A likely plays a key tissue-protective role in humans, suggesting that the increased Th17 polarization of microbiota-reactive T cells in IBD patients could reflect an effort to bolster tissue integrity.

43Host-microbial homeostasis depends on minimizing contact between micro-organisms and mucosal surfaces via the combined action of epithelial cells, mucus, IgA, antimicrobial peptides, and immune cells., Active immune responses to gut flora have been linked to disease. However, this concept should be revisited in light of our current findings and the observation that healthy individuals generate antibody responses to commensals., At least 2 plausible mechanisms could explain the genesis of these microbiota-reactive responses. First, mucosal dendritic cells constantly survey the luminal microenvironment and thereafter migrate to secondary lymphoid tissues to initiate B- and T-cell responses., Second, during gastrointestinal infections in mice, immune responses against commensals and pathogens are induced in parallel. Continuous luminal sampling of intestinal microbiota and periodic epithelial breaches during gastrointestinal infections might provide a plethora of memory T cells with potential reactivity toward newly encountered pathogens., Therefore, contrary to the notion that they promote inflammatory pathology, acquired commensal-reactive T-cell responses may be essential to promote barrier function and IL10-mediated immune regulation—2 cornerstones of intestinal homeostasis.