Generation of colitogenic Th17 CD4 T cells is enhanced by IL-17+γδ T cells

Mice

C57BL/6, B6 Ly5.1, B6 Thy1.1, B6 TCRβ-/-, B6 Rag1-/- mice were purchased from the Jackson Laboratory (Bar Harbor, ME). IL-17F-RFP Tg mice were previously described (12). IFNγ-YFP Yeti mice (13) were kindly provided from Dr. Markus Mohrs (Trudeau Institute). TCRβδ-/- mice were bred at the animal facility of the Lerner Research Institute. All animal procedures were conducted according to the guidelines of the Institutional Animal Care and Use Committee.

Cell sorting and adoptive transfer

Lymph node naive CD4 T cells were obtained as previously reported (14). CD25^neg CD44^low naive T cells were further sorted using a FACSAria cell sorter (BD Bioscience, San Jose, CA). 2.5 × 10⁵ naive T cells were transferred alone or in combination with sorted γδ T cells to TCRβ-/- or TCRβ/δ-/- mice. After T cell transfer, mice were weighed weekly and monitored for signs of disease.

FACS Analysis

Cells were stained with anti-CD4 (RM4-5), anti-γδ TCR (GL3), anti-IL-17A (17B7), anti-IFN-γ (XMG1.2), anti-Thy1.1 (HIS51), anti-CD45.1 (A20) (all Abs from eBioscience) and anti-CCR6 (140706, BD Pharmingen, San Diego, CA). Cells were acquired using a FACSCalibur or LSR II (BD Biosciences) and analyzed using FlowJo software (Treestar, Ashland, OR).

Ex vivo stimulation

Tissue cells were ex vivo stimulated with PMA (10ng/ml) and Ionomycin (1μM) for 4 hrs in the presence of 2 μM monensin (Calbiochem, San Diego, CA) during the last 2 hrs. Cells were immediately fixed with 4% paraformaldehyde, permeabilized, and stained with fluorescence conjugated antibodies. Isotype matched control Ab was used for a staining control.

Lamina Propria cell Isolation

Recipients were sacrificed at the indicated time points after T-cell transfer. Colons were isolated and cleaned in HBSS. Colons were cut into small pieces and resuspended in HBSS with 0.5μM EDTA and 15μg/mL Dithiothreitol (DTT) and shaken for 15 min twice at room temperature. Colons were then resuspended in cRPMI with 400μg/mL DNase I (Roche) and 1mg/mL collagenase D (Roche) and shaken at 37°C for 90 min. Supernatant and colon was passed through a 70μM strainer and washed. Cells were resuspended in a 33% Percoll gradient and spun at room temperature for 20 min. Pellet was collected, washed and used for further experiments.

Real time PCR

Colon tissue was disrupted using a TissueLyser II (Qiagen, Valencia, CA). Total RNA was extracted using an RNeasy column (Qiagen). cDNA was subsequently obtained using a SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA). Real time PCR was performed using gene specific primers and probe sets (Applied Biosystem, Foster City, CA) and ABI 7500 PCR machine (Applied Biosystem).

Histology

For H&E staining colon tissue was fixed in 10% Acetic Acid/60% Methanol. Slides were stained with hematoxylin and eosin. Colon tissues were scored in a blinded fashion as previously reported (15). For immunohistochemistry paraffin embedded colon sections were incubated overnight at 4°C in primary anti-CD3 (ab5690, Abcam, Cambridge, MA) followed by secondary biotinylated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA). Vectastain Elite ABC Kit and DAB (Vector Laboratories, Burlingame, CA) were used for color development, and processed for mounting using Clarifier I, Bluing Solution, Clear-Rite 3 and Mounting Media (all Richard-Allan, Kalamazoo, MI).

Statistical analysis

Results represent the mean ± SD. Statistical significance was determined by the Student's t-test (unpaired two-tailed) using the Prism 4 software (GraphPad, La Jolla, CA). A p value of <0.05 was considered statistically significant.

TCRβ-/- mice are highly susceptible but TCRβδ-/- mice are fully resistant to CD4-mediated colitis

To examine a role of γδ T cells in a CD4-mediated colitis model, naïve CD4 cells were transferred into TCRβ-/- and TCRβδ-/- mice. TCRβ-/- recipients developed severe weight loss with a massive cellular infiltration in the colon tissues, whereas TCRβδ-/- recipients displayed no sign of weight loss only with mild inflammation in the colon (Fig. 1A-1C). Consistent with the disease severity and histopathology, donor T cell accumulation in the lymphoid and gut tissues was markedly elevated in TCRβ-/- compared to TCRβδ-/- mice (data not shown). A dramatic increase in CD4 T cells that produce IL-17 was noticed in TCRβ-/- recipients (Fig. 1D and S1). Moreover, CD4 T cells producing IL-17F were also higher in TCRβ-/- than in TCRβδ-/- recipients (data not shown). Although the proportion of IFNγ+ CD4 T cells was found to be very high in resistant TCRβδ-/- mice (Fig. S1), the absolute numbers of IFNγ-producing CD4 T cells was much higher in colitic TCRβ-/- mice (data not shown). Therefore, severe colitis seen in TCRβ-/- mice is associated with enhanced Th17 differentiation as reported in IBD patients (16), strongly suggesting that the presence of γδ T cells in TCRβ-/- mice may promote Th17 differentiation and colitogenicity of CD4 T cells. Of note, T cell-deficient mice, particularly TCRβ-/-, are known to spontaneously develop colitis starting at ~4 months of age (17). However, both strains of mice used in these experiments were ~2 months of age and continuously gained weight without T cell transfer (Fig. 1A), demonstrating that the spontaneous colitis is not involved.

γδ T cell cotransfer renders TCRβδ-/- recipients susceptible to colitis

To directly test if γδ T cells are responsible for the enhanced pathology seen in TCRβ-/- mice, purified γδ T cells were cotransferred with CD4 T cells into TCRβδ-/- recipients. γδ T cell cotransfer rendered resistant TCRβδ-/- mice highly susceptible to colitis, thus the recipients developed a weight loss (Fig. 1A) and severe colitis (Fig. 1B and 1C). Donor CD4 T cell accumulation in the lymphoid and gut tissues was dramatically elevated to a level seen in TCRβ-/- recipients (data not shown). Furthermore, CD4 T cells producing IL-17 significantly increased in all tested tissues after γδ T cell cotransfer (Fig. 1D and S1), strongly supporting the hypothesis that Th17 differentiation and colitogenicity of CD4 T cells is greatly enhanced by γδ T cells in vivo. γδ T cell transfer alone without CD4 T cells did not result in any noticeable weight loss (data not shown), further supporting the notion that γδ T cells amplify Th17 differentiation, aggravating colitis. Notably, the onset of weight loss in TCRβ-/- recipients was substantially faster compared to that TCRβδ-/- mice receiving γδ T cells (Fig. 1A), which is probably attributed to high levels of γδ T cells generated in TCRβ-/- mice (17).

IL-17+ but not IL-17-γδ T cells promote T cell mediated colitis

To examine a mechanism underlying γδ T cell-mediated Th17 differentiation, we next measured γδ T cell cytokine production in TCRβ-/- mice before and after CD4 T cell transfer. Interestingly, γδ T cells that produce IL-17 significantly increased following T cell transfer (Fig. 2). The increase was specific for IL-17+ γδ T cell subsets since IFNγ+ γδ T cells remained unchanged. Moreover, a noticeable increase was observed even 2 weeks after transfer, suggesting that IL-17+ γδ T cells may play a role in altering the inflammatory responses elicited by T cells.

Phenotypic dichotomy associated with IFNγ- or IL-17-producing γδ subsets was recently reported: IFNγ-producing γδ T cells are CD27+ CCR6-, and IL-17-producing γδ T cells are CD27- CCR6+ (18, 19). When CCR6+ and CCR6- γδ T cells were sorted and examined for gene expression, the expression of il17a and il17f genes was primarily associated with CCR6+ γδ T cells, while ifng expression was primarily found in CCR6- γδ T cells (Fig. S2). To test if IL-17-producing and IFNγ-producing γδ T cells play different roles in T cell differentiation and colitis induction, FACS sorted CCR6+ or CCR6- γδ T cells were transferred into TCRβδ-/- recipients with CD4 T cells. Cotransfer of CCR6+ (i.e., IL-17+) γδ T cells resulted in a significant weight loss (Fig. 3A). By stark contrast, weight loss resulted from CCR6- γδ T cell cotransfer was relatively mild (Fig. 3A). Colitis and CD3+ cell infiltration was dramatically pronounced in mice that received CD4 plus CCR6+ γδ T cells compared to recipients of CD4 plus CCR6- γδ T cells or CD4 T cells alone (Fig. 3B and 3C). Notably, a difference in relative weight loss in CCR6+ γδ T cell recipients compared to the recipients of CD4 T cell alone was already noticeable 21 days post transfer (Fig. 3A). Moreover, the cellular infiltration in the colon of CCR6+ γδ T cell recipients was often extended into the outer muscular layer of the colon (Fig. 3B), suggesting more severe colitis after transfer of γδ T cells enriched for IL-17-producing cells. Importantly, both γδ T cell subsets were equally accumulated in the lamina propria of immunodeficient recipients when measured 5 weeks post transfer (data not shown), suggesting that enhanced susceptibility of CCR6+ γδ T cell recipients is not attributed to differential migration of γδ T cells in vivo. Consistent with colitis, IL-17 expression in CD4 T cells was significantly elevated in mice that received CCR6+ γδ compared to those received CCR6- γδ T cells (Fig. 3D and 3E), while IFNγ expression was comparable in all tested groups (data not shown). Moreover, the absolute numbers of IL-17+ CD4 T cells were significantly greater in CD4/CCR6+ γδ T cell recipients than those in CD4/CCR6- γδ T cell or CD4 T cell recipients (data not shown). Therefore, the presence of IL-17+ γδ T cells greatly enhance Th17 differentiation and subsequent development of T cell-mediated colitis.

Even though CCR6 expression marks IL-17+ γδ T cells, γδ T cells may acquire or lose IL-17-producing potentials after transfer into TCRβδ-/- recipients, where they undergo homeostatic proliferation (20). To verify this possibility purified CCR6+ and CCR6- γδ T cells were separately transferred into Rag-/- mice, allowed to proliferate, and examined for cytokine expression 2 weeks after transfer. Surprisingly, CCR6+ γδ T cells remained IL-17+ cells, while CCR6- γδ T cells failed to convert into IL-17+ cells and remain IFNγ-producing cells (Fig. 4). Furthermore, gene expression profiles measured by qPCR remained unchanged (data not shown). Therefore, $\mathrm{IL}\text{-}17\text{-}∕\mathrm{IFN}\tilde{\gamma }$ producing γδ T cell subsets are not reversible in vivo even during homeostatic proliferation; thus enhanced Th17 differentiation and colitis seen in CCR6+ γδ T cell recipients appears to be strongly associated with IL-17 produced by γδ T cells.

How IL-17-producing γδ T cells play such proinflammatory roles? Inflammatory IL-6/IL-23-producing DCs support Th17 immunity and T cell mediated colitis (21). Since IL-17 can induce those cytokines from DCs (22), it is possible that IL-17 produced by γδ T cells may induce factors that favor Th17 differentiation from DCs. Although IL-17-/- (or IL-17F-/-) CD4 T cells were shown to equally induce colitis (23, 24), the fact that RORγ-/- CD4 T cells fail to induce Th17 immunity and colitis strongly suggests both the importance of IL-17 and the redundancy between IL-17 and IL-17F in inducing colitis. Since IL-17-producing γδ T cells also express IL-17F (data not shown), it will be important to examine if IL-17 and IL-17F derived from γδ T cells play redundant (or synergistic) roles in supporting Th17 differentiation and colitis. We are currently investigating this issue.

γδ T cell functions are highly divergent in vivo. γδ T cells mediate a regulatory effect in preventing generation of autoantigen-specific Th17 CD4 T cells in an autoimmune uveitis model (25). In contrast, γδ T cells promote the generation of myelin-specific Th17 differentiation, exacerbating the development of EAE (26). In the context of intestinal inflammation, intestinal resident γδ T cells have been proposed to play exacerbating roles in colitis developed in TCRα-/- mice (27). It was recently reported that γδ T cells may augment autoimmune responses by suppressing Treg functions as well as preventing the generation of inducible Treg cells (26). Whether γδ T cell-mediated suppression of Treg functions plays a role in the current model system remains to be determined.

Collectively, our results suggest that IL-17+ γδ T cells promote in vivo Th17 differentiation and may contribute to subsequent development of intestinal inflammation. IL-17+ γδ T cell subsets may represent a potential target for treating chronic inflammatory disorders associated with Th17 effector cells such as IBD.