γδ T lymphocytes, dominant T cell subsets in the intestine, mediate both regulatory and pathogenic roles, yet the mechanisms underlying such opposing effects remain unclear. In this study, we identified a unique γδ T cell subset that coexpresses high levels of gut-homing integrins, CD103 and α4β7. They were exclusively found in the mesenteric lymph node after T cell–mediated colitis induction, and their appearance preceded the inflammation. Adoptive transfer of the CD103+α4β7high subsets enhanced Th1/Th17 T cell generation and accumulation in the intestine, and the disease severity. The level of generation correlated with the disease severity. Moreover, these cells were also found to be elevated in a spontaneous mouse model of ileitis. Based on the procolitogenic function, we referred to this subset as “inflammatory” γδ T cells. Targeting inflammatory γδ T cells may open a novel strategy to treat inflammatory diseases where γδ T cells play a pathogenic role including inflammatory bowel disease.

The γδ T cells occupy distinct immunologic niches in different tissues; they constitute only 1–2% of T cells in lymphoid tissues, whereas ∼50% of T cells in the intestine express the γδ TCR (1). Lymphoid γδ T cells are motile and circulate throughout the periphery, whereas intestinal γδ T cells are rather stationary and display limited mobility within the tissues (2, 3). Unlike αβ T cells, γδ T cells express “activated” phenotypes and exhibit effector functions, surveying malignant or virus-infected cells for ultimate elimination (1, 4). γδ T cells are also heterogeneous depending on the surface phenotypes and cytokine production. For example, subsets of lymphoid γδ T cells produce IL-17 or IFN-γ, and those cells express nonoverlapping surface markers such as CD27, NK1.1, and CCR6 (5, 6). IL-17+ γδ T cells are found in lymphoid, dermal, and nongut mucosal tissues such as the lung and reproductive organs, although they are not usually enriched in gut mucosal tissues (710). γδ T cells play highly diverse roles in immunity. γδ T cells support inflammation in many autoimmune inflammation models (11, 12). However, they also play protective roles in certain inflammatory conditions by regulating epithelial cell survival and regeneration (13). The cellular mechanisms underlying the opposing roles of γδ T cells remain largely unknown.

We previously reported that γδ T cells promote T cell–mediated colitis (14, 15). However, whether there is a particular γδ T cell subset mediating the pathogenic roles and, if so, what is the precise mechanism underlying their inflammatory functions remains obscure. In this article, we report that a subset of lymphoid γδ T cells in the gut draining mesenteric lymph node (mLN) and intestinal tissues express two key gut-homing integrin molecules, CD103 and α4β7, and that their appearance precedes the development of colitis. Adoptive transfer of CD103+α4β7high γδ T cell subsets isolated from the mLN dramatically enhances the accumulation of effector T cells producing IFN-γ or IL-17 in the intestine and exacerbates colonic inflammation. Importantly, the level of circulating CD103+α4β7high γδ T cells directly correlates with the level of Th1/Th17 CD4 T cell accumulation in the target colon tissues. Gene expression profiles using the Nanostring assay demonstrate that CD103+α4β7high γδ T cell subsets have distinct transcriptional profiles. Lastly, elevated accumulation of the subset is also found in a spontaneous model of chronic intestinal inflammation. Taken together, we propose that CD103+α4β7high γδ T cells represent a novel subset of “inflammatory” γδ (iγδ) T cells that may promote the development of chronic inflammation in the intestine.

C57BL/6-Rag1−/−, CD45.1 C57BL/6, and C57BL/6 TCRβ−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 Tcrd-eGFP mice were previously reported (16). Different ages of SAMP1/YitFc and age-matched AKR mice were also used. All mice were maintained under a specific pathogen-free facility located in the Lerner Research Institute and Case Western Reserve University. All animal experiments were performed in accordance with approved protocols for the Institutional Animal Care and Usage Committee.

Whole lymph node (LN) naive CD4 T cells were obtained as previously reported (17). CD25negCD44low naive T cells were further sorted using a FACSAria cell sorter (BD Biosciences, San Jose, CA). A total of 2.5 × 105 naive CD4 T cells were transferred to TCRβ−/− mice. After T cell transfer, mice were bled and analyzed for blood γδ T cells. In some experiments, various γδ T cell subsets were sorted from TCRβ−/− recipients 21 d after transfer and transferred to naive Rag1−/− recipients together with naive CD4 T cells. Weight loss was determined weekly. Colon tissues were fixed in 10% acetic acid/60% methanol and stained with H&E. Colon tissues were scored in a blinded fashion as previously reported (18).

Lamina propria (LP) or intraepithelial lymphocytes (IELs) were isolated as previously reported (19). Cells were stained with anti-CD4 (RM4-5), anti–IL-17A (eBio17B7), anti–IFN-γ (XMG1.2), anti-CD45.1 (A20), anti-CD69 (N418), anti-Vγ4 (UC3-10A6), anti-γδ TCR (GL3), anti-Ki67 (SolA15), anti-CD103 (M1/70), or anti-α4β7 (DATK32) (all Abs from eBioscience or Pharmingen). Anti-Vγ1 (2.11) (20) and anti-Vγ7 (UC1) (21) Abs were obtained from Dr. Rebecca O’Brien (National Jewish Health). Cells were acquired using an LSR II (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR). For intracellular staining, cells were separately harvested and ex vivo stimulated with PMA (10 ng/ml) and Ionomycin (1 μM) for 4 h in the presence of 2 μM of monensin (Calbiochem, San Diego, CA) during the last 2 h of stimulation. Cells were immediately fixed with 4% paraformaldehyde, permeabilized, and stained with fluorescence-conjugated Abs.

For measurements of gene expression by Nanostring, 100 ng of RNA was hybridized to the Mouse Immunology Panel at 65°C overnight, then processed on a GEN2 analysis system using the high-sensitivity protocol and high-resolution data capture. Raw counts were normalized using nSolver version 2.5 software and controls incorporated into the code set. Background counts were removed by subtracting the mean plus 2 SDs of the negative controls, and lane-specific differences in hybridization and binding intensity were corrected using the geometric mean of the positive controls. Gene expression was normalized to the geometric mean of four reference genes (Rpl19, Eef1g, Tbp, Polrb), which were expressed at stable levels in all samples.

Hierarchical clustering and heat map generation were performed in nSolver software. Statistical significance was determined by the Mann–Whitney U test (two-tailed) using the Prism 5 software (GraphPad, La Jolla, CA). A p value <0.05 was considered statistically significant.

In patients with inflammatory bowel disease (IBD), γδ T cell levels are elevated in both circulation and the gut tissues (22, 23). In mice, γδ T cells support T cell–induced colonic inflammation (14, 15). To investigate the mechanisms underlying proinflammatory functions of γδ T cells, we first tested whether γδ T cells express altered proliferative and migratory behaviors, contributing to the inflammatory processes. We particularly focused on two gut-homing integrins, α4β7 and CD103 (αE), that also pair with the β7 integrin. γδ T cells in the lymphoid tissues (mLN and peripheral LN [pLN]) contain subsets expressing CD103 and/or low levels of α4β7: CD103+α4β7low, CD103α4β7low, and CD103α4β7 subsets (Fig. 1A). In contrast, γδ T cells in the intestinal LP or IEL compartments were mainly CD103+α4β7 cells (Fig. 1A). The integrin expression of intestinal γδ T cells was not overlapping with that of mLN γδ T cells, whereas its expression of mLN and pLN γδ T cells overlapped (Fig. 1B). LP and IEL γδ T cells uniformly expressed CD69, whereas mLN γδ T cells were mainly CD69 (Fig. 1C) (24). The integrin expression pattern was substantially different from that of conventional CD4 T cells (Fig. 1A). The integrin expression of γδ T cells remained unaffected by the presence of αβ T cells (Fig. 1B). Because lymphoid and intestinal γδ T cells are thought to be separated in steady-state conditions (2, 3), we next examined γδ T cell phenotypes during inflammation.

FIGURE 1.

Gut-homing integrin expression of γδ T cells in steady-state conditions. (A) α4β7 and CD103 expression on γδ T cells or CD4 T cells is shown. (B) mLN, LP, IEL, and pLN cells from WT or TCRβ−/− mice were stained for γδ TCR, α4β7, and CD103. Integrin expression of mLN γδ T cells is shown in gray zebra plots. Overlaid dot plots represent integrin expression of LP, IEL, and pLN γδ T cells. (C) CD69 expression of mLN, SI-LP, and SI-IEL γδ T cells. Each experiment was carried out at least three times, with results similar to the representative examples shown.

FIGURE 1.

Gut-homing integrin expression of γδ T cells in steady-state conditions. (A) α4β7 and CD103 expression on γδ T cells or CD4 T cells is shown. (B) mLN, LP, IEL, and pLN cells from WT or TCRβ−/− mice were stained for γδ TCR, α4β7, and CD103. Integrin expression of mLN γδ T cells is shown in gray zebra plots. Overlaid dot plots represent integrin expression of LP, IEL, and pLN γδ T cells. (C) CD69 expression of mLN, SI-LP, and SI-IEL γδ T cells. Each experiment was carried out at least three times, with results similar to the representative examples shown.

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We previously reported that γδ T cells support the development of colitogenic effector cells (14, 15). To gain insight into the underlying mechanisms by which γδ T cells exert procolitogenic functions, we first monitored γδ T cell levels during the course of colitis developed in TCRβ−/− recipients after naive CD4 T cell transfer. The proportions of circulating γδ T cells dramatically increased starting at ∼2 wk and peaked around 4 wk after induction (Fig. 2A). Because colitis typically develops after ∼3 wk postinduction, these results raise the possibility that the increased presence of γδ T cells may be associated with the disease development.

FIGURE 2.

CD103+α4β7high γδ T cell subsets during colitic inflammation. (A) Groups of TCRβ−/− mice were induced for colitis by transferring naive CD4 T cells. The mice were weekly bled and stained for γδ T cells. Data shown are the mean ± SD of five mice. (B) TCRβ−/− mice were transferred with naive CD4 T cells. α4β7 and CD103 expression of circulating γδ T cells was determined by flow analysis. (C) γδ T cell integrin expression in the indicated tissues was determined after CD4 T cell transfer. (D) Integrin expression of γδ T cells in spleen and pLN was measured 3 wk after CD4 T cell transfer. Each experiment was carried out at least three times, with results similar to the representative examples shown.

FIGURE 2.

CD103+α4β7high γδ T cell subsets during colitic inflammation. (A) Groups of TCRβ−/− mice were induced for colitis by transferring naive CD4 T cells. The mice were weekly bled and stained for γδ T cells. Data shown are the mean ± SD of five mice. (B) TCRβ−/− mice were transferred with naive CD4 T cells. α4β7 and CD103 expression of circulating γδ T cells was determined by flow analysis. (C) γδ T cell integrin expression in the indicated tissues was determined after CD4 T cell transfer. (D) Integrin expression of γδ T cells in spleen and pLN was measured 3 wk after CD4 T cell transfer. Each experiment was carried out at least three times, with results similar to the representative examples shown.

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We next examined whether there is a particular γδ T cell subset or subsets that preferentially expand. We found that blood CD103+ γδ T cells highly expressing α4β7 significantly increased ∼2 wk after transfer (Fig. 2B, Supplemental Fig. 1). γδ T cells that express α4β7 only also increased, although the magnitude of increase was greater in CD103+α4β7high γδ T cell subsets. We also examined the integrin expression of γδ T cells in other lymphoid and intestinal tissues. α4β7 expression of γδ T cells was slightly reduced after T cell transfer (day 7). However, CD103+α4β7high γδ T cells began to appear in the mLN between days 7 and 12, continuously increased around day 21, and gradually disappeared from the mLN around day 35 after disease induction (Fig. 2C). The increase in CD103+α4β7high subsets was pronounced in the gut draining mLN (Fig. 2C), whereas they were hardly found in other lymphoid tissues such as pLN and spleen even at the peak of the inflammation (Fig. 2D). CD103+α4β7high LP γδ T cells (in both the small and large intestine) similarly increased starting around 12 d postinduction and remained high for the duration of the experiments (Fig. 2C). Similar levels of CD103+α4β7high IEL γδ T lymphocytes were also found in the small and large intestines (data not shown). Of note, their appearance never occurred without T cell transfer, that is, colitis induction. Thus, the generation (or differentiation) of CD103+α4β7high γδ T cell subsets appears to be associated with T cell activation and the development of colonic inflammation. It is also important to point out that integrin expression pattern of γδ T cells is substantially different from that of conventional CD4 T cells found in the mLN and inflamed gut tissues. Unlike γδ T cells, the majority of CD4 T cells were CD103α4β7high cells (Supplemental Fig. 2), suggesting that the mechanisms involved in the generation may be unique in γδ T cells.

γδ T cells cotransferred into Rag1−/− recipients together with CD4 T cells exacerbate colitis development (14). These γδ T cells similarly developed into CD103+α4β7high subsets in the mLN, but not pLN, of Rag1−/− recipients (Fig. 3). γδ T cells in the LP also displayed similar phenotypes as seen in TCRβ−/− recipients (Fig. 3). γδ T cells undergo homeostatic proliferation in lymphopenic hosts (25). Because γδ T cells transferred into Rag1−/− recipients did not develop into CD103+α4β7high γδ T cell subsets without CD4 T cells (Fig. 3), these results strongly suggest that integrin upregulation is not the result of homeostatic proliferation. Furthermore, the CD103+α4β7high subset developed in TCRβ−/− models is not an artifact resulting from the intestinal inflammation in TCRβ−/− mice in which γδ T cell level is higher than in wild type animals.

FIGURE 3.

Gut-homing expression on γδ T cells was dependent on CD4 T cell–mediated inflammation. Naive CD4 T cells and lymphoid γδ T cells were cotransferred into Rag1−/− mice. The mice were sacrificed 3 wk posttransfer, and γδ T cell expression of α4β7 and CD103 was determined by flow analysis. Data shown are representative of three independent experiments.

FIGURE 3.

Gut-homing expression on γδ T cells was dependent on CD4 T cell–mediated inflammation. Naive CD4 T cells and lymphoid γδ T cells were cotransferred into Rag1−/− mice. The mice were sacrificed 3 wk posttransfer, and γδ T cell expression of α4β7 and CD103 was determined by flow analysis. Data shown are representative of three independent experiments.

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We next tested proliferation behavior of various γδ T cell subsets and found that each subset displayed markedly different proliferation activity. As shown in Fig. 4A, α4β7high (CD103+ and CD103) γδ T cells in the mLN and intestine expressed high levels of Ki67, whereas Ki67 expression of α4β7low subsets was dramatically low. Ki67 expression of γδ T cell subsets in naive TCRβ−/− animals without T cell transfer remained low (data not shown). Thus, observed changes in γδ T cell subset frequencies are likely due to expansion of certain γδ T cell subsets rather than to contraction of other populations. These results suggest that both CD103+α4β7high cells and CD103α4β7+ subsets may be responsive to stimulation, although the nature of stimulating signals remains unclear. If this is an Ag-induced response, it is conceivable that proliferating T cells may express oligoclonal TCRs. The proportions of Vγ4+ and Vγ5+ cells among the γδ subsets in the mLN and LP were not drastically different (Fig. 4B and data not shown). The proportions of Vγ1- and Vγ7-expressing CD103+α4β7high cells were slightly higher than those of other subsets, although the difference was not statistically significant (Fig. 4C). Overall, the proliferation does not appear to be Ag driven.

FIGURE 4.

Characterization of γδ T cell subsets. (A) TCRβ−/− recipients of naive CD4 T cells were sacrificed 3 wk posttransfer, and γδ T cells in the mLN, small intestine (SI), and large intestine (LI) were examined for Ki67 expression. Based on the CD103 and a4b7 expression, each subset was numbered. (B) Vγ4 expression on each γδ T cell subset was measured. (C) Vγ1 and Vγ7 expression of the indicated mLN γδ T cell subsets were also measured. Data shown are the mean ± SD of five to seven individually tested mice from two independent experiments. Vγ expression is a representative of two or three independent experiments. ***p < 0.001, ****p < 0.0001.

FIGURE 4.

Characterization of γδ T cell subsets. (A) TCRβ−/− recipients of naive CD4 T cells were sacrificed 3 wk posttransfer, and γδ T cells in the mLN, small intestine (SI), and large intestine (LI) were examined for Ki67 expression. Based on the CD103 and a4b7 expression, each subset was numbered. (B) Vγ4 expression on each γδ T cell subset was measured. (C) Vγ1 and Vγ7 expression of the indicated mLN γδ T cell subsets were also measured. Data shown are the mean ± SD of five to seven individually tested mice from two independent experiments. Vγ expression is a representative of two or three independent experiments. ***p < 0.001, ****p < 0.0001.

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To compare gene expression profiles between different γδ subsets, we next performed a Nanostring analysis. Three γδ T cell subsets isolated from the mLN were used: CD103+α4β7high, CD103+α4β7low, and CD103α4β7low subsets. γδ T cells isolated from naive animals were included as a control. Gene expression pattern of CD103+α4β7high subsets was dramatically different from that of CD103+α4β7low subsets or naive γδ T cells (Fig. 5). In contrast, gene expression patterns of CD103+α4β7low cells were relatively similar to naive γδ T cells, although there appear to be genes specifically upregulated in these subsets (Fig. 5). Of note, genes highly expressed in CD103+α4β7high, but not in naive γδ T cells, are not overlapping with the genes highly expressed in CD103+α4β7low, but not on naive γδ T cells (Fig. 5). In particular, we identified 59 genes that are expressed >3-fold in CD103+α4β7high than in CD103+α4β7low subsets (Supplemental Table I). They include genes involved in cell death (Gzmb, Gzma, Fas, and Fasl), NK cell receptors (Cd244, Klra5, Klra7, Klrb1, Klrc1, Klrd1, and Klrk1), cytokines/receptors (Ifng, Il2, Il18, and Il12rb2), and chemokines/receptors (Ccl3, Ccl4, Ccl5, Ccr9, and Cx3cr1) (Supplemental Table I). Despite the enrichment in NK cell receptors, CD103+α4β7high cells did not express TCRVδ6.3 chain, a TCR repertoire heavily biased for NKTγδ T cells (26) (Supplemental Fig. 3). Seventeen of these genes were expressed >3-fold compared with any other γδ T cell subsets tested. These genes include Ccr9, Cd244, Cd8a, Gzma, Ifitm1, Klra5, and Tnfrsf8. Indeed, 60–70% of CD103+α4β7high mLN γδ T cells expressed CD8α (but not CD8β) subunit, an indication of CD8αα homodimer expression (Fig. 6A). We also found that 41 genes were found highly expressed in CD103+α4β7low subsets than in CD103+α4β7high subsets. These include cytokines/receptors (Csf1, Ebi3, Il1r, Il23r, and Il6ra), transcription factors (Rorc, Socs3, and Ikzf4), chemokine receptors (Ccr4, Ccr6, and Ccr8), and surface receptors (Cd274, Cd5, Tnfrsf4, Tnfsf8, and Tnfsf11) (Supplemental Table I). We measured cytokine production by different subsets of γδ T cells. Both CD103+α4β7high and α4β7+ subsets from mLN were highly enriched in IFN-γ+ cells, whereas the proportion of IFN-γ+ cells in CD103+ subsets or CD103α4β7 subsets was significantly lower (Fig. 6B and data not shown). Interestingly, little IL-17 expression was found in all subsets tested (Fig. 6B and data not shown). Likewise, CD103+α4β7high subsets in the IELs or LP expressed higher levels of IFN-γ compared with CD103+ subsets (Fig. 6C and data not shown).

FIGURE 5.

Gene expression profiles in different γδ T cell subsets. Different γδ T cell subsets (n = 3, CD103+α4β7high, CD103+α4β7low, and CD103α4β7low) were FACS sorted from the mLN of TCRβ−/− recipients after 3 wk post naive CD4 T cell transfer. Total γδ T cells from naive TCRβ−/− mice were also isolated and used as a control. RNA expression was compared by a Nanostring analysis.

FIGURE 5.

Gene expression profiles in different γδ T cell subsets. Different γδ T cell subsets (n = 3, CD103+α4β7high, CD103+α4β7low, and CD103α4β7low) were FACS sorted from the mLN of TCRβ−/− recipients after 3 wk post naive CD4 T cell transfer. Total γδ T cells from naive TCRβ−/− mice were also isolated and used as a control. RNA expression was compared by a Nanostring analysis.

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FIGURE 6.

CD103+α4β7high cells are phenotypically distinct. (A) TCRβ−/− mice were transferred with naive CD4 T cells and sacrificed 3 wk after the transfer. γδ T cells in the mLN and IEL were examined for CD8α and CD8β expression by flow analysis. Naive TCRβ−/− mice were included as a noninflammatory control. (B and C) mLN (B) and IEL (C) cells were harvested 3 wk after colitis induction and stimulated with PMA/Ionomycin for intracellular cytokine production. The data shown are representative of two independent experiments.

FIGURE 6.

CD103+α4β7high cells are phenotypically distinct. (A) TCRβ−/− mice were transferred with naive CD4 T cells and sacrificed 3 wk after the transfer. γδ T cells in the mLN and IEL were examined for CD8α and CD8β expression by flow analysis. Naive TCRβ−/− mice were included as a noninflammatory control. (B and C) mLN (B) and IEL (C) cells were harvested 3 wk after colitis induction and stimulated with PMA/Ionomycin for intracellular cytokine production. The data shown are representative of two independent experiments.

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To directly test whether γδ T cell subsets play different roles in modulating colitis development, we used a Rag1−/− mouse model. Rag1−/− mice that receive CD4 T cells plus γδ T cells develop exacerbated colitis compared with the disease induced by CD4 T cells alone (14, 15). Therefore, this is an advantageous model to directly compare pathogenic potentials of different γδ T cell subsets by separately transferring them into those mice. Naive CD4 T cells were first transferred into TCRβ−/− mice to generate different γδ T cell subsets (Fig. 2). Three weeks later, CD103+α4β7high or CD103+α4β7low subsets were isolated from the mLN or LP and separately transferred into naive Rag1−/− mice together with naive CD4 T cells. Strikingly, CD103+α4β7high mLN γδ T cell recipients had exacerbated colitis based on weight loss and colonic inflammation (Fig. 7A–C). CD103+α4β7high LP γδ T cell recipients also exhibited weight loss and inflammation; however, the disease was not as severe as for those recipients of mLN CD103+α4β7high cells (Fig. 7A–C). Recipients of CD103+α4β7low subsets experienced mild disease and inflammation comparable with the recipients of CD4 T cells alone (Fig. 7A–C). CD4 T cell expansion and accumulation in the inflamed tissues further supported the findings. CD4 T cell expansion was greater when mLN CD103+α4β7high subsets were used (Fig. 7D, 7E). T cell expression of IFN-γ and IL-17 was also enhanced when mLN CD103+α4β7high subsets were transferred (Fig. 7D, 7E). Inflammatory T cell accumulation in the colon tissues was strikingly increased in this condition (Fig. 7E). Migration of mLN CD103+α4β7high subset into the colon tissue was especially pronounced, further supporting their procolitogenic functions (Fig. 7F). Therefore, CD103+α4β7high γδ T cells generated in the gut draining mLN may directly enhance the generation of colitogenic Th1/Th17 type effector T cells and their subsequent infiltration into the colon.

FIGURE 7.

CD103+α4β7high mLN γδ T cells induce severe colitis after transfer. (A) CD103+α4β7high and CD103+α4β7low γδ T cells were FACS sorted from mLN and LP of TCRβ−/− mice that received naive CD4 T cells 3 wk earlier, and transferred into Rag1−/− mice together with naive CD4 T cells. Weight loss was monitored weekly. (B) H&E staining of the colon tissues. (C) Histology score. (D and E) The recipients were sacrificed 5 wk posttransfer, and donor CD4 T cell accumulation in the mLN (D) and colon (E) was determined. Cells were also ex vivo stimulated for intracellular IFN-γ and IL-17 expression. (F) CD103+α4β7high and CD103+α4β7low subsets derived from TcrdGFP TCRβ−/− mice were transferred into Rag1−/− mice together with naive CD4 T cells. GFP+ γδ T cell accumulation in the large intestine (LI)-LP and mLN was determined 5 wk after transfer. Data shown are the mean ± SD of six to eight individually tested mice from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7.

CD103+α4β7high mLN γδ T cells induce severe colitis after transfer. (A) CD103+α4β7high and CD103+α4β7low γδ T cells were FACS sorted from mLN and LP of TCRβ−/− mice that received naive CD4 T cells 3 wk earlier, and transferred into Rag1−/− mice together with naive CD4 T cells. Weight loss was monitored weekly. (B) H&E staining of the colon tissues. (C) Histology score. (D and E) The recipients were sacrificed 5 wk posttransfer, and donor CD4 T cell accumulation in the mLN (D) and colon (E) was determined. Cells were also ex vivo stimulated for intracellular IFN-γ and IL-17 expression. (F) CD103+α4β7high and CD103+α4β7low subsets derived from TcrdGFP TCRβ−/− mice were transferred into Rag1−/− mice together with naive CD4 T cells. GFP+ γδ T cell accumulation in the large intestine (LI)-LP and mLN was determined 5 wk after transfer. Data shown are the mean ± SD of six to eight individually tested mice from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

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We noticed that the proportion of CD103+α4β7high γδ T cells generated after T cell transfer varies, ranging from 5 to 15%. Whether the magnitude of CD103+α4β7high subset generation is linked to the severity of the disease was thus examined. TCRβ−/− recipients of CD4 T cells were divided based on the proportion of circulating CD103+α4β7high γδ T cells. If the level was >10% of the total γδ T cells anytime between days 14 and 24, they were considered the CD103+α4β7high γδ-high group (Fig. 8A). If the level remained <10%, then they were considered the CD103+α4β7high γδ-low group (Fig. 8A). Interestingly, the CD103+α4β7high γδ-high group displayed faster weight loss compared with the CD103+α4β7high γδ-low group (Fig. 8B), suggesting that CD103+α4β7high γδ T cell levels may be associated with the disease severity.

FIGURE 8.

The level of circulating CD103+α4β7high γδ T cell subsets correlates with disease severity. (A) TCRβ−/− mice (n = 18) received naive CD4 T cells, and blood CD103+α4β7high γδ T cells were monitored weekly. CD103+α4β7high γδ-high and CD103+α4β7high γδ-low groups were divided based on the level of circulating CD103+α4β7high γδ T cell levels. The mean SD of CD103+α4β7high γδ levels is shown. (B) Body weight loss from each group was compared. (C) The mice were sacrificed 25 d posttransfer. Circulating CD103+α4β7high γδ T cells were measured. mLN and LP cells were ex vivo stimulated to measure IFN-γ– and IL-17–producing CD4 T cells. Blood CD103+α4β7high γδ T cell levels were compared with cytokine-producing CD4 T cells in the tissues. *p < 0.05.

FIGURE 8.

The level of circulating CD103+α4β7high γδ T cell subsets correlates with disease severity. (A) TCRβ−/− mice (n = 18) received naive CD4 T cells, and blood CD103+α4β7high γδ T cells were monitored weekly. CD103+α4β7high γδ-high and CD103+α4β7high γδ-low groups were divided based on the level of circulating CD103+α4β7high γδ T cell levels. The mean SD of CD103+α4β7high γδ levels is shown. (B) Body weight loss from each group was compared. (C) The mice were sacrificed 25 d posttransfer. Circulating CD103+α4β7high γδ T cells were measured. mLN and LP cells were ex vivo stimulated to measure IFN-γ– and IL-17–producing CD4 T cells. Blood CD103+α4β7high γδ T cell levels were compared with cytokine-producing CD4 T cells in the tissues. *p < 0.05.

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TCRβ−/− recipients were next sacrificed 25 d postinduction. Blood CD103+α4β7high γδ T cell levels (day 25) were compared with the levels of IFN-γ+ or IL-17+ CD4 T cells accumulated in the mLN and colon. CD103+α4β7high blood γδ T cell levels directly correlated with IFN-γ+ and IL-17+ CD4 T cell accumulation in the colon (Fig. 8C). In contrast, the correlation was not observed in the mLN (Fig. 8C). These results suggest that the generation of CD103+α4β7high γδ T cells may be associated with the extent of colitic inflammation and inflammatory T cell accumulation in the target tissues, suggesting that this subset may serve as a disease marker.

We finally tested whether an inflammatory CD103+α4β7high γδ T cell subset is generated in the course of intestinal inflammation under nonlymphopenic settings. We used the SAMP/YitFc (SAMP) mouse model that develops spontaneous chronic ileitis. The SAMP mouse strain represents a well-established chronic model of IBD and provides an excellent system to study the initiation and progression of chronic intestinal inflammation (27). Because SAMP mice were derived from brother-sister mating of wild-type AKR (parental strain) mice, the phenotype occurs spontaneously, as in the human condition, without chemical, genetic, or immunologic manipulation. SAMP and age-matched (parental) control AKR mice were examined for γδ T cell expression of β7 integrins. CD103+α4β7high γδ T cells were increasingly found in both mLN and intestinal tissues of SAMP mice, especially after ∼12 wk of age, whereas the increase was absent in age-matched AKR control mice (Fig. 9). These results strongly suggest that CD103+α4β7high γδ T cells develop during spontaneous intestinal inflammation in nonlymphopenic hosts, and that their accumulation may be closely related to the disease development and inflammatory T cell accumulation in the target tissues.

FIGURE 9.

CD103+α4β7high γδ T cell levels in mice with spontaneous intestinal inflammation. SAMP1/YitFc mice and age-matched AKR control mice were examined for CD103+α4β7high γδ T cells in the mLN and large intestine (LI)-LP tissues. Data shown are the mean ± SD of four to five individually tested animals. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 9.

CD103+α4β7high γδ T cell levels in mice with spontaneous intestinal inflammation. SAMP1/YitFc mice and age-matched AKR control mice were examined for CD103+α4β7high γδ T cells in the mLN and large intestine (LI)-LP tissues. Data shown are the mean ± SD of four to five individually tested animals. *p < 0.05, **p < 0.01, ***p < 0.001.

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In this study, we report a novel γδ T cell subset that may play proinflammatory roles in generating colitogenic Th1/Th17 effector cells and in exacerbating intestinal inflammation. The subset coexpresses two key gut-homing integrins, CD103 and α4β7, and their appearance precedes the intestinal inflammation. The level of generation of this subset directly correlates with the disease severity and inflammatory T cell accumulation in the target tissues. Because of the inflammation-promoting property, we propose that the CD103+α4β7high subset be referred to as iγδ T cells.

iγδ T cells are first observed in the gut draining mLN, circulation, and intestinal tissues (both LP and IEL compartment), whereas they are not found in any other lymphoid tissues. Interestingly, the kinetics of their appearance is similar between the tissues, making the precise site of generation difficult to locate. We speculate that they are generated in the mLN and subsequently mobilized into the target tissues, where they likely perform inflammatory functions. It was recently reported from a study using photoconversion reporter animals that intestinal T cells can traffic to the peripheral lymphoid tissues as well as the brain (28, 29). However, iγδ T cells express not only two gut-homing integrin molecules, but also CCR9, a chemokine receptor directed to CCL25 constitutively expressed in the gut tissues (30, 31) (Supplemental Table I). Therefore, the possibility of gut resident γδ T cells migrating to the mLN is unlikely.

At steady-state conditions, intestinal γδ T cells are relatively homogeneous with CD103+α4β7low subsets, and they are thought to be protective, supporting intestinal epithelial cell regeneration and eliminating malignant or virus infected cells (32). During inflammation, intestinal γδ T cells become heterogeneous such that iγδ subsets infiltrating the gut tissues may attract inflammatory cells, interfere with gut resident γδ T cell functions, and provoke inflammation. Then, what is the mechanism by which iγδ T cells promote intestinal inflammation? From the Nanostring analysis, we found that they broadly express cytotoxic mediators and NK cell receptors, suggesting that they may provoke inflammatory responses by mediating cytotoxicity, although they may differ from unconventional NKTγδ cells because they hardly express TCRVδ6.3 (26). One possibility is that iγδ T cells may induce apoptosis of intestinal epithelial cells, impair gut barrier function, and increase the gut permeability. IELs isolated from mice undergoing intestinal graft-versus-host disease induce significantly more IEC apoptosis via a Fas-dependent mechanism when transferred (33). Consistent with this, iγδ T cells express significantly high levels of FasL mRNA compared with other γδ T cell subsets (data not shown). iγδ T cells also highly express the klrk1 (NKG2D), which interacts with stress-induced MICA and MICB molecules expressed on intestinal epithelial cells (34). This is a subject of ongoing investigation. It is interesting to note from the Nanostring analysis that genes that are highly upregulated in iγδ T cells are unique and are not expressed on any other γδ subsets tested. We also noted another set of genes that are upregulated in CD103+α4β7low γδ T cell subsets alone (Fig. 5). Importantly, the latter genes (upregulated in CD103+α4β7low subsets) were not overlapping with the former genes (highly expressed in iγδ T cells). The expression of these genes in naive γδ T cells was markedly low (Fig. 5), suggesting that both populations might have separately developed from naive γδ T cells after activation. In addition, they express chemokines capable of recruiting a variety of inflammatory cells expressing CCR1, CCR3, and CCR5 (35). Data from adoptive transfer of different γδ T cell subsets further support the notion because iγδ T cells isolated from the mLN dramatically promote the disease development and accumulation of colitogenic effector T cells in the target tissues.

The vitamin A metabolite, retinoic acid (RA), induces the expression of α4β7 and CCR9 on T cells, imprinting them with the gut tropism (36). In support, we previously showed that CD11b+ mLN DCs induce α4β7 expression on T cells via an RA-dependent mechanism (15). Therefore, γδ T cells residing in the mLN may respond to the RA available from the DC subsets and further develop into iγδ T cells. Whether this process involves γδ TCR activation remains to be tested. Because RA-mediated gut tropism is typically found in activated T cells, it is conceivable that activation may be required for iγδ T cell development. It is possible that TCR-independent activation likely mediated by inflammatory cytokines may promote the generation of iγδ T cells. Highly diverse Vγ expression of iγδ T cells indistinguishable between the γδ T cell subsets further supports this possibility. iγδ T cells express il12rb gene, whereas genes associated with Th17 type immunity including il23r, il1r, il6ra, rorc, and ccr6 were selectively depleted in iγδ T cells. In support of this, iγδ T cells expressed high levels of IFN-γ, but not IL-17, upon stimulation. Alternatively, γδ T cells in the mLN may undergo an iγδ T cell differentiation process in response to gut bacterial Ag stimulation (37).

Although the primary model system used in this study is TCRβ−/− mouse, in which γδ T cell levels in the lymphoid tissues are higher than conventional settings, it is important to note that γδ T cells exhibiting iγδ T cell phenotypes are similarly elevated in SAMP mice, especially after 12 wk of age. Consistent with these trends, the data in this article (Fig. 9), demonstrating a significant increase of iγδ T cells in both the mLN and the LP of SAMP mice >12 wk of age, compared with age-matched control AKR, suggest a potential involvement of iγδ T cells in the development, and possibly the perpetuation, of chronic intestinal inflammation that was observed in patients with IBD. Unlike the TCRβ−/− mouse model, iγδ T cells appear to exist in SAMP and AKR mice before overt disease development (Fig. 9). Based on published studies from our group and others, including a detailed phenotypic time course performed on mice derived from our specific mouse colony housed at Case Western Reserve University, critical time points in the development of SAMP ileitis have been established as follows: 3–4 wk (before histologic evidence of ileitis, but presence of small-intestinal epithelial permeability defect) (38), 7–9 wk (early, active phase of inflammation and dominant Th1 cytokine profile) (39), >12 wk (established, chronic inflammation and mixed Th1/Th2 cytokine profile); after 20 wk, no significant differences are observed in the severity of ileitis among SAMP mice (4042). It is thus possible that the pre-existing iγδ-like T cells may be further activated in SAMP mice by an unknown mechanism, contributing to the disease development in SAMP mice. The mechanism through which iγδ T cells acquire pathogenic functions in intestinal inflammation of SAMP mice is a subject of future investigation. In support of this, γδ T cell levels in IBD patients are increased in the circulation, as well as in the inflamed mucosa (22, 43). Epithelial barrier function is dysregulated in SAMP mice (27, 44), and iγδ T cells may induce apoptotic cell death of the epithelial cells, increasing gut permeability in this setting.

In conclusion, this study identified that there is a subset of γδ T cells that preferentially infiltrates the gut tissues and enhances colonic inflammation. Defining mechanisms by which iγδ T cells develop and mediate intestinal inflammation may uncover a novel strategy to treat inflammatory responses by targeting iγδ T cells.

We thank Dr. Bernard Malissen for kindly providing Tcrd-eGFP mice and Jennifer Powers for cell sorting.

This work was supported by the Crohn’s Colitis Foundation of America (B.M.) and the National Institutes of Health (Grants DK091222 and DK097948 to T.T.P.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

iγδ

inflammatory γδ

IBD

inflammatory bowel disease

IEL

intraepithelial lymphocyte

LP

lamina propria

mLN

mesenteric lymph node

pLN

peripheral LN

RA

retinoic acid.

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The authors have no financial conflicts of interest.

Supplementary data