A Novel T Cell Subset with a Potential Role in Inflammatory Bowel Disease

CD8 βα + δγ T Cells: A Novel T Cell Subset with a Potential Role in Inflammatory Bowel Disease

This information is current as Mohammad Kadivar, Julia Petersson, Lena Svensson and of September 24, 2021. Jan Marsal J Immunol published online 14 November 2016 http://www.jimmunol.org/content/early/2016/11/12/jimmun ol.1601146 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 14, 2016, doi:10.4049/jimmunol.1601146 The Journal of Immunology

CD8ab+ gd T Cells: A Novel T Cell Subset with a Potential Role in Inﬂammatory Bowel Disease

Mohammad Kadivar,* Julia Petersson,* Lena Svensson,*,1 and Jan Marsal*,†,1

gd T cells have been attributed a wide variety of functions, which in some cases may appear as contradictory. To better understand the enigmatic biology of gd T cells it is crucial to define the constituting subpopulations. gd T cells have previously been categorized into two subpopulations: CD8aa+ and CD82 cells. In this study we have defined and characterized a novel subset of human gd T-cells expressing CD8ab. These CD8ab+ gd T cells differed from the previously described gd T cell subsets in several aspects, including the degree of enrichment within the gut mucosa, the activation status in blood, the type of TCRd variant used in blood, and small but significant differences in their response to IL-2 stimulation. Furthermore, the novel subset expressed cytotoxic mediators and CD69, and produced IFN-g and TNF-a. In patients with active inflammatory bowel disease the mucosal +

frequencies of CD8ab gd T cells were signiﬁcantly lower as compared with healthy controls, correlated negatively with the Downloaded from degree of disease activity, and increased to normal levels as a result of anti–TNF-a therapy. In conclusion, our results demonstrate that CD8ab+ gd T cells constitute a novel lymphocyte subset, which is strongly enriched within the gut and may play an important role in gut homeostasis and mucosal healing in inﬂammatory bowel disease. The Journal of Immunology, 2016, 197: 000–000.

cells can be subdivided into conventional and uncon- gd T cells constitute a distinct subset of T cells involved in both

ventional T cells, based on their function and expression of innate and adaptive responses. In mouse thymus, conventional http://www.jimmunol.org/ TCRs and coreceptors (1). Conventional T cells express TCRab+ cells differentiate through sequential stages, including a T 2 2 the ab variant of the TCR together with either the CD4 or CD8ab CD4 CD8 (double negative [DN]), a double positive, and fi- coreceptor. The unconventional T cells on the other hand have nally, a CD4+ or CD8+ single positive stage. TCRgd+ cells deviate been postulated to express either the ab TCR or the gd TCR, early from this path and develop from DN thymocytes (5). As together with the coreceptor CD8aa homodimer or no coreceptor heterogeneity of gd T cells is becoming increasingly recognized, it at all (1, 2), although rare CD4+ gd T cells also have been de- has been suggested that they comprise more than a single lineage scribed (3, 4). and that different gd subsets descend from precursors at various branching points (6). Indeed, in humans TCRgd+ cells have been identified among both DN and double positive thymocytes (7). by guest on September 24, 2021 *Immunology Section, Lund University, S-22184 Lund, Sweden; and †Department of Human gd T cells can also be stratified according to which of the Gastroenterology, Skane˚ University Hospital, S-22185 Lund, Sweden different variable Vd segments they use in their rearranged TCR. 1L.S. and J.M. contributed equally to this work. In contrast to ab T cells the usage in the gd T cell population is ORCIDs: 0000-0003-1499-191X (M.K.); 0000-0001-8578-4876 (J.P.); 0000-0001- largely restricted to two TCR segments, namely the Vd1 and Vd2. 6927-979X (L.S.); 0000-0003-4808-0014 (J.M.). Vd1+ cells are found mainly at mucosal sites, can recognize stress- Received for publication July 1, 2016. Accepted for publication October 13, 2016. induced self-molecules (8) and lipid Ags presented by CD1c/d (9), This work was supported by the Swedish Research Council (K2010-80P-21592-01-4 and have been shown to have immune regulatory and tumor sur- and K2010-80X-215917-01-4), the Olle Engkvist Byggma¨stare Foundation, the + IngaBritt and Arne Lundberg Research Foundation, the Gyllenstiernska Krapperup veillance functions (9). Vd2 cells on the other hand are primarily Foundation, the Nanna Svartz Foundation, and the Crafoord Foundation (to L.S.), and found in the circulation, can recognize nonpeptidic phosphoanti- the O¨ sterlund Foundation (to L.S. and J.M.). This work was also supported by the gens, and are typically antimicrobial and anti-inflammatory in Hedlund Foundation, the Julin Foundation, the Bengt Ihre Stipend, the Swedish Society for Medical Research, the Royal Physiographic Society of Lund, Skane˚ character (9). University Hospital donations, the Swedish Society of Medicine, an investigator- In healthy adults, 1–10% of circulating T cells are gd T cells, initiated study grant from AbbVie, research support from the Healthcare Region of and they comprise up to 40% of the intraepithelial lymphocytes Southern Sweden, and grants to researchers in public health care from the Swedish government (ALFSKANE-539811) (to J.M.). (IEL) in the gut (10). gd T cells have been implicated in protective M.K. contributed to study concept and design, performed experiments, analyzed and immunity against microbes, pathogenesis of several immune- interpreted data, wrote and revised the manuscript, and performed statistical analyses; mediated diseases including inflammatory bowel disease (IBD) as J.P. performed experiments; L.S. contributed to concept and design, analyzed and well as immune regulation, tumor surveillance, and repair of ep- interpreted data, wrote and revised the manuscript, supervised the study, and obtained funding; J.M. obtained ethical approval, contributed to study concept and design, ithelial damage in the gut (2, 11, 12). Thus, results from studies on analyzed and interpreted data, wrote and revised the manuscript, provided human the role of gd T cells diverge considerably, which lends further experimental material and recruited subjects, supervised the study, and obtained funding. support to the idea of distinct gd T cell subsets with different functionalities (13). Address correspondence and reprint requests to Dr. Jan Marsal and Dr. Lena Svensson, Immunology Section, Lund University, BMC D14, S-22184 Lund, Sweden. E-mail One of the hallmarks of IBD is a profuse infiltration of T cells addresses: [email protected] (J.M.) and [email protected] (L.S.) into the gut mucosa (10). Crohn’s disease (CD) and ulcerative The online version of this article contains supplemental material. colitis (UC) are chronic relapsing and remitting inflammatory Abbreviations used in this article: CD, Crohn’s disease; DN, double negative; HBI, disorders of the gut, collectively called IBD. The pathogenesis of Harvey–Bradshaw Index; IBD, inflammatory bowel disease; IEL, intraepithelial IBD is still not fully understood, but is thought to involve a lymphocyte; MFI, mean fluorescence intensity; MHC-I, MHC class I. dysregulated immune response to a subset of commensal bacteria Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 in genetically susceptible individuals (10, 14). To what extent and

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601146 2 ROLE OF A NOVEL gd T CELL SUBSET IN IBD in which way different immune cell subsets contribute to the path- T cells were stained first. For cytokine staining, cells were stimulated with ogenic process and subsequent mucosal healing upon treatment re- phorbol 12-myristate 13-acetateand (PMA) (10 ng/ml; Sigma-Aldrich) and mains to be clarified. ionomycin (100 ng/ml; Sigma-Aldrich) for 4 h in the presence of brefeldin A (BioLegend) and monensin (Sigma-Aldrich) for the last 3 h. For intra- In the current study, we have identified a novel human T cell subset cellular staining, cells were fixed and permeabilized according to the which coexpresses TCRgd and CD8ab. We found this novel subset manufacturer’s instructions using Transcription Factor Buffer Set (BD to be present in both blood and intestine, with a marked enrichment Pharmingen) after staining for T cell surface markers, followed by staining in the latter. Furthermore, we found that the CD8ab+ gd T cells have for IFN-g, IL-17, TNF-a, Ki67, granzyme B, and perforin. In both pro- cedures cells were initially incubated with Fixable Viability Dye eFluor cytotoxic potential, respond to IL-2, produce IFN-g and TNF-a,and 450 (eBioscience) before surface staining. Multicolor flow cytometry and + that the large majority are TCRVd1 . Analyzing intestinal biopsies sorting were performed using LSR II, FACSAria II machines (BD Bio- we found a decrease in the proportion of CD8ab+ gd T cells in sciences) or MoFlo Astrios (Beckman Coulter). Flow cytometry results patients with IBD compared with healthy controls. Also, we found were analyzed using the FlowJo data analysis software (Tree Star). For a negative correlation between CD8ab+ gd T cells and disease normalized mean fluorescence intensity (MFI), the MFI of the negative control sample was subtracted from the MFI of the stained sample. activity in IBD. However, after anti–TNF-a treatment of the IBD patients the percentage of these cells increased to the levels of RT-PCR analysis and primers healthy controls. Expression levels of TCRa, TCRd, CD8a, CD8b and GAPDH genes were analyzed using real-time RT-PCR. Whole RNA was extracted from double- sorted (i.e., sorted and resorted) cells using RNeasy Plus Mini Kit (Qia- Materials and Methods gen). cDNA was synthesized using Maxima First Strand cDNA Synthesis Peripheral blood and intestinal samples Kit for quantitative RT-PCR with dsDNase (Thermo Scientific). The kit Downloaded from Blood samples and ileoclonic biopsies were collected from healthy controls contains double-strand–specific DNase to eliminate potential contaminat- (n = 5–20 depending on type of analysis) and patients with ulcerative ing genomic DNA. Real-time PCR was performed using SsoFast EvaGreen colitis (n =5)orCD(n = 10–11). Patients with moderate to severe CD Supermix (Bio-Rad). The primers were as follows: TCRd (Vd2-Cd) 59- (Harvey–Bradshaw Index [HBI] score of $7) were treated with adalimumab GCA GGA GTC ATG TCA GCC AT-39 and 59-GAC AAG CGA CAT TTG (monoclonal anti–TNF-a Ab) for 3 mo (160 mg s.c. at start and 80 mg TTC CA-39; TCRa (TCRa2) 59-TCC GCC AAC CTT GTC ATC TC-39 after 2 wk, followed by 40 mg every second week) and analyzed again and 59-ATC GGT GAA TAG GCA GAC AGA CTT-39; CD8a (CD8a2) 59- following treatment. The study was performed in accordance with the GGT TGT ACT ATG CTG TTA TGA G-39 and 59-TTT TTC GGA TGC declaration of Helsinki and was approved by the regional ethics com- TGT TTA CC-39; CD8b (CD8b2) 59-TCA TTC TCA ATC TCA CAA GC- http://www.jimmunol.org/ 9 9 9 9 mittees in Lund and Linko¨ping, Sweden (Dnr. 2011/60 and 2011-201-31, 3 and 5 -GAA GGA AAT CAA CCA CAC TC-3 ; GAPDH 5 -TGC ACC respectively). Written informed consent was obtained from all subjects ACC AAC TGC TTA GC-39 and 59-GGC ATG GAC TGT GGT CAT before they were included. GAG-39. The RT-PCR–amplified products were examined by 2% agarose gel electrophoresis, stained with ethidium bromide. Isolation of PBMCs PCR products sequencing Blood samples were collected in heparin-coated tubes (Becton Dickinson). PBMCs were isolated from whole blood by density gradient centrifugation RT-PCR products were extracted from 2% agarose gel after electrophoresis using Lymphoprep (Axis-Shield). using GeneJET Gel Extraction Kit (Thermo Scientific). The purified RT-PCR products were mixed with primer in Mix2Seq tubes (Eurofins

Isolation of mucosal leukocytes Genomics) according to the manufacturer’s instructions. Sequencing was by guest on September 24, 2021 done by Eurofins Genomics. Six to eight pinch biopsies were incubated and stirred at 37˚C for 45 min in HBSS-EDTA (without Ca2+ or Mg2+) buffer containing 5 mM EDTA, Fecal calprotectin 15 mM Hepes, 2% FBS and antibiotics. Tissues were then washed at 37˚C Fecal samples were analyzed by ELISA according to the manufacturer’s for 20 min with 15 mM Hepes-HBSS buffer. The cell suspensions present € after the EDTA treatment and washing steps were kept for analysis of IEL. instructions (Buhlmann). Concentration was determined relative to stan- The remaining tissues were enzymatically digested with 2 mg/ml collage- dard curves and expressed as microgram per gram of feces. nase Ia (Sigma-Aldrich), 20 U/ml DNase I (Sigma-Aldrich), and 100 mM Statistics CaCl2 at 37˚C for 1 h in 1.5 ml microtubes on a shaker. The resulting cell suspension was diluted in R10 medium containing RPMI 1640 (Invitrogen), Statistical analyses were performed using GraphPad Prism software 10% FCS, and antibiotics, passed through a 100 mm cell strainer, and kept (GraphPad). for analysis of lamina propria mononuclear cells. IL-2 stimulation Results A novel subset of human TCRgd+ T cells expresses both CD8a Isolated PBMCs or sorted gd T cells (106 cells/ml) were cultured at 37˚C, and CD8b 5% CO2, in six-well plates with RPMI 1640 (Invitrogen) supplemented with 10% FBS (Invitrogen) and antibiotics. To study the effect of IL-2 Previous studies have described gd T cells to be primarily either stimulation, PBMCs or sorted gd T cells were cultured for 48 h with or 2 + 2 2 without 50 ng/ml recombinant IL-2 (Novartis). Surface and intracellular CD4 CD8aa or CD4 CD8 (1, 2, 11). In this study, using flow markers were analyzed by flow cytometry. cytometry, we identified a subset of gd T cells coexpressing CD8a and CD8b in healthy human gut biopsies (Fig. 1A). The levels of Abs CD8a and CD8b expression were distinct but significantly lower Fluorochrome-conjugated Abs against CD3 (clone UCHT1), CD8a (RPA-T8), than for TCRab+ T cells (Fig. 1D, 1E). CD25 (BC96), CD122 (TU27), CD132 (TUGh4), CD56 (HCD56), TCRab To confirm this initial data we investigated the expression levels (IP26), TCRgd (B1), TCRVd2 (B6), CD69 (FN50), and IL-17 (BL168) were of CD8a and CD8b mRNA in sorted peripheral blood gd T cells. obtained from BioLegend. Abs to CD8b (SIDI8BEE) and IFN-g (4S.B3) were + purchased from eBioscience. Abs to CD8b (2ST8.5H7), Ki67 (B56), TNF-a In agreement with the flow cytometry data, we found that TCRgd (6401.1111), granzyme B (GB11), FasL (Nok-1), and perforin (gG9) were T cells indeed do express both CD8a and CD8b mRNA (Fig. 1B, obtained from BD Biosciences. TCRVd1 Ab (TS8.2) was purchased from 1C). As a positive and negative control for CD8 a/b expression, Thermo Fisher Scientific. we used sorted TCRab+ T cells and a human prostate cancer cell Flow cytometry line (PC3), respectively. PC3 cells did not show any CD8b ex- + + pression (Fig. 1C) whereas sorted TCRab T cells confirmed clear For flow cytometry analysis of CD8ab gd T cells, we designed four + + different Ab panels: two panels for identifying T cell subpopulations and expression (Fig. 1B). Sorted TCRab and TCRgd T cells did not characterizing their surface markers and two panels for phenotyping and show any TCRd and TCRa mRNA expression, respectively, evaluating the function of CD8ab+ gd T cells. The surface markers of confirming the complete purity of these sorted cell populations The Journal of Immunology 3

equal or even lower expression of the Vd1 compared with the Vd2 segment (Fig. 2A). These results show that the novel subpopulation of CD8ab+ gd T cells predominantly uses the Vd1 segment regardless of their in vivo localization, which distinguishes them from the CD8aa+ and CD82 gd T cell subsets that in blood are enriched for Vd2 expression. To conversely examine the subpopulation composition within the Vd1+ and Vd2+ populations, we first gated on Vd1+ and Vd2+ cells, respectively, and analyzed the proportions of cells being CD8ab+, CD8aa+, and CD82. This analysis showed that the Vd1+ population is made up of a mixture of CD8ab+, CD8aa+, and CD82 gd T cells, whereas the Vd2+ population contains very few CD8ab+ gd T cells (Supplemental Fig. 1B). To examine the activation state of the different T cell subpopulations we stained for CD69, which is upregulated early upon lymphocyte activation (15). Interestingly, a significantly larger proportion of blood CD8ab+ gd T cells was CD69+ compared with both the other gd T cell subpopulations and ab T cells. In the gut most T cells were CD69+ and there were no significant Downloaded from http://www.jimmunol.org/

FIGURE 1. A novel subset of human gd T cells that expresses both CD8a and CD8b.(A) Representative flow cytometry plots of gut IEL from healthy control showing gating strategies applied on live CD3+ cells for identification of CD8ab+ T cells. (B) Representative agarose gel electro- by guest on September 24, 2021 phoresis of real time RT-PCR amplification products showing that both TCRab+ and TCRgd+ cells expressed CD8a and CD8b (n = 5). Purity of sorted TCRab+ and TCRgd+ cells was checked by expression of TCRa or TCRd. GAPDH was used as housekeeping gene. (C) Relative expression level of CD8b mRNA in TCRgd+ cells. PC3 cells (human prostate cancer cell line) were used as biological negative control (n = 5–7). Graphs show the MFI of CD8a (D)andCD8b (E)inTCRab+CD8ab+ and CD8ab+ gd T cells (n =7). Values are means 6 SD. Mann–Whitney U test, *p , 0.05, **p , 0.01, ***p , 0.001.

(Fig. 1B). All the PCR product sizes were assessed by gel electrophoresis, and the specificity of the RT-PCR assay was confirmed by amplicon sequencing for the products of CD8a and CD8b primers (data not shown). Together, these findings demonstrate that a human CD8ab+ gd T cell subpopulation exists in addition to the previously described CD82 and CD8aa+ gd T cell subpopulations. CD8ab+ gd T cells are predominantly Vd1+ gd T cell expression of Vd1orVd2 correlates with tissue localization and Ag recognition, and characterizes the two most common and biologically important subtypes of human gd T cells (9). To characterize the novel subset in this context, gd T cells were isolated from blood, gut epithelium, and gut lamina propria and FIGURE 2. CD8ab+ gd T cells are predominantly TCRVd1+.(A)Per- stained for Vd1 and Vd2. There was a clear predominance of Vd1 + + 2 2 + + centage of TCRVd1 ,TCRVd2 ,andTCRVd1 Vd2 cells among CD8ab , usage among CD8ab gd T cells in all three compartments ex- + 2 + 2 CD8aa , and CD8 gd T cells in blood (closed circles) (n = 10), and LP amined (Fig. 2A, Supplemental Fig. 1A). CD8aa and CD8 gd and IEL compartments (open circles) (n = 5). (B)PercentageofCD69+ cells T cells from gut epithelium and lamina propria showed a similar in different subsets of T cells in blood (closed circles) (n = 10) and gut (open pattern, that is, substantially more frequent Vd1expressionas circles) (n = 5). Values are means 6 SD. Paired-ANOVA and Fisher’s post compared with Vd2. However, in blood these two subsets showed hoc test, *p , 0.05, **p , 0.01, ***p , 0.001. LP, lamina propria. 4 ROLE OF A NOVEL gd T CELL SUBSET IN IBD

denotes cells with cytotoxic activity (17–19). Thus, we investigated the expression of CD56 on CD8ab+ gd T cells and found that the proportion of CD56+ cells among peripheral blood and intestinal CD8ab+ gd T cells, similar to the other gd T cell subsets, is higher compared with CD4+ T helper cells (Fig. 4). Taken together, analyses of four different cytotoxicity-related markers suggest that CD8ab+ gd T cells possess cytotoxic potential. CD8ab+ gd T cells produce IFN-g and TNF-a but not IL-17 The cytokines produced by T cells are thought to characterize their function. It has previously been shown that subsets of murine gd T cells produce IFN-g, TNF-a, and IL-17 (20, 21). Thus, we investigated the production of these cytokines by different subsets of human gd T cells. The results showed that CD8ab+ gd T cells, similar to the other gd T cell subsets, produce IFN-g and TNF-a but not IL-17 (Fig. 5). In blood, a greater proportion of CD8ab+ gd T cells compared with CD4+ T helper cells showed IFN-g and TNF-a production (Fig. 5C, 5D). However, in both blood and gut + + the expression levels of IFN-g in IFN-g CD8ab gd T cells were Downloaded from lower than in Th1 cells (Supplemental Fig. 1D). CD8ab+ gd T cells represent a functionally distinct subset Human circulating gd T cells comprise a mixture of naive and effector/memory phenotype cells (22, 23). It has been suggested that IL-2 signaling can induce a TCR-independent differentiation http://www.jimmunol.org/ of some unactivated gd T cells in the periphery (24). We cultured PBMCs from healthy donors with or without IL-2 to examine the effects of IL-2 on different subsets of gd T cells in the absence of TCR stimulation. Our results showed that the percentages of CD8aa+ and CD8ab+ gd T cells were increased with IL-2 stimulation, whereas the percentage of CD82 gd T cells was decreased (Fig. 6A, Supplemental Fig. 2A). However, IL-2 stimulation was associated with increased expression of the

proliferation marker Ki67 in all three gd T cell subsets (Fig. 6B, by guest on September 24, 2021 Supplemental Fig. 2B). Interestingly, IL-2 signaling increased IFN-g and TNF-a production in the CD8ab+ gd T cell subset, decreased the same in CD82 gd T cells, and left the CD8aa+ gd T cell subset unchanged (Fig. 6C, 6D, Supplemental Fig. 2C, 2D). To examine whether these changes represented an indirect or a direct FIGURE 3. CD8ab+ gd T cells display cytotoxic properties. (A)Rep- resentative flow cytometry histograms show the expression of Granzyme B, Perforin, and FasL in peripheral blood and gut T cell subsets. Gray plots and black lines represent isotype controls and CD8ab+ gd T cells, respectively. (B) Expression of Granzyme B, Perforin, and FasL demonstrated for different subsets of T cells in blood (gray bars) (n =11),andgut(blackbars) (n = 10). Conventional T helper cells (TCRab+CD4+ cells) represent non- cytotoxic T cells. Values are means 6 SD. Paired-ANOVA and Fisher’s post hoc test, *p , 0.05, **p , 0.01, ***p , 0.001. FasL, Fas ligand. differences in CD69 expression among the subpopulations (Fig. 2B, Supplemental Fig. 1C). CD8ab+ gd T cells display cytotoxic properties The conventional CD8ab+ ab T cells constitute the main cytotoxic T cell population in the body. In addition, it has been shown that gd T cells can be cytotoxic (16). We therefore examined whether the novel CD8ab+ gd T cells have cytotoxic potential. Staining for cytotoxic mediators (granzyme B, perforin, and the apoptosis-inducing protein Fas ligand), we found that all examined gut lymphocyte subsets expressed considerably lower levels of these as compared FIGURE 4. A large proportion of CD8ab+ gd T cells expresses CD56. with the corresponding lymphocyte subset in blood (Fig. 3). However, A + ( ) Representative flow cytometry plots show the expression of CD56 on the levels of cytotoxic mediators expressed by the novel CD8ab and CD32 cells, CD82,CD8aa+,andCD8ab+ gd T cells in blood. (B) Percentage the other gd T cell subsets were significantly higher compared with of CD56+ cells in different subsets of T cells in peripheral blood (closed cir- + CD4 ab T cells, in both blood and gut (Fig. 3B). It has been reported cles) (n = 11) and gut (open circles) (n = 7). Values are means 6 SD. Paired- that expression of CD56 on both TCRab+ and TCRgd+ T cells ANOVA and Fisher’s post hoc test, *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 5

epithelium, and lamina propria of healthy subjects. In blood, CD8ab+ gd T cells comprised a very small percentage of CD3+ T cells and in the lamina propria it was somewhat higher (mean 0.3 6 0.5% and 0.7 6 0.5%, respectively) (Fig. 7A). In contrast, a sub- stantial proportion of the intestinal IEL was constituted by CD8ab+ gd T cells (6.4 6 4.3%) (Fig. 7A), albeit with inter- individual variation. Furthermore, colon IEL contained higher percentages of CD8ab+ gd T cells out of total CD3+ cells than ileum (Fig. 7B). In addition, the percentage of CD8ab+ cells among gd T cells was increased within the gut compared with blood, whereas the op- posite was true for CD82 gd T cells, and no difference was seen for the CD8aa+ gd T cell subset (Fig. 7C–E). When the frequencies of the different subsets within the gd T cell population were compared between intestinal segments, no signiﬁcant differences were seen (Supplemental Fig. 3). Finally, although gd Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 5. CD8ab+ gd T cells from healthy individuals produce IFN-g and TNF-a but not IL-17. Representative flow cytometry plots show the production of IFN-g and IL-17 (A) or IFN-g and TNF-a (B) in intestinal TCRab+CD4+, and CD82, CD8aa+, and CD8ab+ gd T cells. Percentage of IFN-g+ (C), TNF-a+ (D), and IL-17+ (E) cells in different subsets of T cells in peripheral blood (closed circles) and gut (open circles) are shown (n = 4–11). Values are means 6 SD. Paired-ANOVA and Fisher’s post hoc test, *p , 0.05, **p , 0.01, ***p , 0.001. effect, we performed the same experiments with sorted gd Tcells and found equivalent or even slightly increased IL-2 responsiveness, suggesting the effect to be direct (Fig. 6E). Furthermore, we investigated the expression of the IL-2 receptor complex subunits CD25, + CD122, and CD132. CD8ab gd T cells expressed CD122 and FIGURE 6. The CD8ab+ gd T cell subpopulation is a functionally CD132 (which together make up the intermediate affinity IL-2 distinct subset in terms of IL-2 response. Isolated PBMCs or sorted receptor), but not CD25 (Fig. 6F), showing that these cells have gd T cells were cultured for 48 h in the presence or absence of IL-2. Iso- the potential to respond directly to IL-2. These results suggest that lated PBMCs or sorted gd T cells were restimulated with PMA and ion- the three subpopulations of gd T cells described here differ not omycin, and IFN-g and TNF-a producing gd T cells identified by staining of intracellular cytokines. (A) Differences in absolute percentage points for only phenotypically but also functionally in regards to their re- 2 + + sponse to IL-2 stimulation. Finally, the expression levels of CD25, CD8 , CD8aa , and CD8ab gd T cells with versus without IL-2 stimulation (n = 14). Differences in absolute percentage points for Ki67+ (B), CD122, and CD132 were similar on the three gd T cell subsets IFN-g+ (C) and TNF-a+ (D) cells in different subsets of gd T cells in the (Fig. 6F, Supplemental Fig. 2E), which argues that as yet unrec- presence or absence of IL-2 stimulation (n = 5–14). (E) Sorted gd Tcells ognized intrinsic cellular differences, other than receptor protein were cultured for 48 h in the presence or absence of IL-2, and analyzed for levels, are likely responsible for the differential IL-2 responses. IFN-g and TNF-a expression. The same experiment was performed in CD8ab+ gd T cells are enriched within the gut parallel with PBMC cultures, and the graph shows the comparison of the two conditions. (F) Expression of CD25 (IL-2Ra), CD122 (IL-2Rb)and + To examine the distribution of CD8ab gd T cells, we analyzed CD132 (IL-2Rg) on CD8ab+ gd T cells in peripheral blood (n =6). the frequency of these cells in the peripheral blood, the intestinal ANOVA and Fisher’s post hoc test, *p , 0.05, **p , 0.01, ***p , 0.001. 6 ROLE OF A NOVEL gd T CELL SUBSET IN IBD

significant (Fig. 8B). Together these results suggest that the frequency of CD8ab+ gd T cells in the gut mucosa decreases in active IBD and the more severe the inflammation, the lower the frequency of CD8ab+ gd T cells. Anti–TNF-a treatment increases the percentage of intestinal CD8ab+ gd T cells in patients with Crohn’s disease We examined CD patients given anti–TNF-a therapy (adalimumab) to find out whether the decrease in percentage of CD8ab+ gd T cells in inflamed intestinal tissues changes during treatment. Blood and intestinal samples from CD patients were analyzed before and after 3 mo of adalimumab treatment (Fig. 9). There were no major effects on the percentages of CD8ab+ gd T cells in peripheral blood (Fig. 9). However, our results showed that the percentage of CD8ab+ gd T cells increased during anti–TNF-a treatment in both the epithelial and lamina propria compartments of inflamed ileum as well as inflamed colon (Fig. 9). The frequency of CD8ab+ gd T cells increased both when analyzed as the per- + centage out of total CD3 cells and out of total gd T cells (Fig. 9). Downloaded from In contrast, such changes were not observed for the CD82 and CD8aa+ gd T cell subsets (Supplemental Fig. 4C). FIGURE 7. The novel CD8ab+ gd T cell subset is enriched within the gut. Percentage of CD8ab+ gd T cells out of CD3+ cells (A) and out of Discussion TCRgd+ cells (C) in peripheral blood (n = 20), LP (n = 20), and intestinal gd T cells have previously been defined as being primarily either epithelium (n = 20) of healthy controls. (B) Percentage of CD8ab+ gd + 2 + CD8aa or CD8 (1, 2). In addition, uncommon CD4 gd T cells http://www.jimmunol.org/ + T cells out of CD3 cells in different segments of the gut (n = 6–11). have been reported (3, 4). Here, we describe a subset of human gd Percentage of CD82 (E) and CD8aa+ gd T cells (D) out of TCRgd+ cells T cells expressing the CD8ab heterodimer. This novel T cell in peripheral blood (n = 20), LP (n = 20), and intestinal epithelium (n = 20) d + of healthy controls. Values are means 6 SD. ANOVA and Fisher’s post subset was predominantly TCRV 1 , expressed cytotoxic media- hoc test, *p , 0.05, **p , 0.01, ***p , 0.001. LP, lamina propria. tors, CD69, IFN-g, and TNF-a, and responded to IL-2 stimulation. Importantly, the CD8ab+ gd T cells differed from previously known gd T cell subsets in several aspects: the degree of en- T cells in general are known to be prevalent within the gut, the richment within the gut mucosa, in their response to IL-2, and in results show that the degree of enrichment of CD8ab+ gd T cells their activation status and TCRVd variant usage in blood. In-

+ by guest on September 24, 2021 in the gut is higher than for the other gd T cell subpopulations. triguingly, the frequency of CD8ab gd T cells in the gut was + decreased in IBD patients compared with healthy controls, cor- The frequency of intestinal CD8ab gd T cells decreases related negatively with the degree of disease activity, and in- during inflammation creased upon anti-TNF therapy to the level of healthy controls. Given that gd T cells have been implicated in the pathogenesis of As gd T cells are not MHC-restricted and are not thought to a number of immune-related diseases but also immune regulation interact with MHC class I (MHC-I), an obvious question is what and epithelial repair in the gut we went on to address a potential function the CD8ab coreceptor, which normally binds to MHC-I, role of CD8ab+ gd T cells in IBD. We examined the frequency of has on these cells. Of note, CD8ab+ gd T cells have also been CD8ab+ gd T cells among CD3+ cells in the blood, the gut lamina identified in other species under certain circumstances, that is in propria and gut epithelium of IBD patients and healthy controls TCRb2/2 mice (26). Interestingly, both CD8aa and CD8ab have (Fig. 8A). There were no significant differences in the percentage been reported to bind to MHC-I and I-like molecules with similar of peripheral blood CD8ab+ gd T cells in IBD patients versus affinity, but CD8ab was more effective than CD8aa in promoting healthy controls (Fig. 8A). However, the percentage of CD8ab+ TCR activation (27, 28). In addition, CD8 has been suggested to gd T cells in the epithelial compartment of both the small and play a role in TCR-independent immune responses of gd T cells, large intestine in IBD patients was significantly lower compared myeloid cells, and NK cells (29). There is data suggesting that the with healthy controls (Fig. 8A). This difference was seen in the gd TCR recognizes Ags without the support of an Ag presenting lamina propria compartment too, but here the results were not molecule, functioning similarly as Abs, but there are also studies statistically significant in all gut segments. showing involvement of MHC-like molecules where CD8ab po- The reduction in percentage of CD8ab+ gd T cells in the gut tentially could play a role (21). Additionally, some of these MHC- during inflammation could potentially be explained by an infil- like molecules are stress-induced and do not appear to present tration of ab T cell subsets into the inflamed tissues. Therefore, variable ligands (9). The field is thus not sufficiently elucidated we analyzed the percentage of CD8ab+ gd T cells out of total gd and some of the divergent results can likely be explained by gd T cells and correlated this with the disease activity of the patients T cell heterogeneity. as measured by the HBI (25) and fecal calprotectin. Indeed, the Our results showing that the large majority of CD8ab+ gd percentage of CD8ab+ gd T cells showed significant negative T cells express TCRVd1 suggest that these cells may have tumor correlation with both the HBI and fecal calprotectin levels surveillance, antimicrobial, or immune regulatory functions sim- (Fig. 8C), whereas the other gd T cell subsets did not show any ilar to what has been shown for other Vd1+ gd T cells (9). significant changes (Supplemental Fig. 4A, 4B). When comparing However, Vd1+ gd T cells comprise several subsets as shown in the proportions of CD8ab+ gd T cells out of gd T cells in IBD this study, and properties demonstrated for Vd1+ cells as a whole patients and healthy controls there was a trend toward decreased may not be extrapolated to all subsets constituting this population. numbers in IBD, however the differences were not statistically Interestingly, Vd1+ gd T cells are currently being explored for The Journal of Immunology 7 adoptive cell therapy strategies against cancer and researchers are gd T cells are a distinct lymphocyte subset and that they differ in looking for ways to optimize the initial results (16, 30). A po- anatomical distribution compared with other gd Tcells.The tential way to improve cell therapies is to define and select the mechanisms behind the enrichment of CD8ab+ gd T cells within most efficient subset(s), in which results from this study may help. the gut epithelium could comprise an enhanced ability to extravasate Another interesting application for cell therapy is treatment of and home to or be retained in this compartment, an enhanced ca- immune-mediated diseases. Indeed, encouraging results have pacity for survival, an increased proliferative activity, or facilitated come from animal models (31, 32) and phase I clinical trials have differentiation from other gd T cells. Dissecting these alternatives shown regulatory T cell therapy to be safe in patients with graft and defining their relative contribution requires further study. versus host disease (33) and type 1 diabetes (34). In addition, cell The specific enrichment of the CD8ab+ gd T cell subset in the therapy using regulatory T cells is under promising development gut tissues raises questions regarding its functionality and role. for treatment of CD (35). Given that certain Vd1+ gd T cells have Our results demonstrate that CD8ab+ gd T cells have cytotoxic immune regulatory functions in tumor microenvironments (16) potential, which suggests a role in killing infected cells, tumor and that a regulatory phenotype could be induced in blood-derived cells, or cells stressed due to additional reasons such as inflam- Vd1+ T cells using an anti-Vd1 Ab (36), it is conceivable, together mation (2). Before epithelial cells are shed into the gut lumen they with our data suggesting a role for Vd1+CD8ab+ gd T cells in undergo apoptosis, which is initiated as they approach the area of IBD, that selected Vd1+ gd T cell subsets could be explored for shedding, and a decreased apoptotic rate has been suggested to be adoptive cell therapy against IBD. associated with increased susceptibility to adenocarcinoma (39). Reactivation of the human CMV represents a common problem The relatively high frequency of CD8ab+ gd T cells in the IEL in the clinical management of IBD; it may drive mucosal in- compartment and their cytotoxic ability together indicate a role in Downloaded from flammation and is associated with therapy resistance (37). Inter- the induction of epithelial cell apoptosis and preserving homeo- estingly, Vd1+ gd T cells have been suggested to have a protective stasis. The explanation for levels of cytolytic markers being lower role during CMV reactivation in immunocompromised patients in gut as compared with blood we can only speculate on. It is (38). Thus, the novel CD8ab+ gd T cell subset may have a central conceivable that as a result of the more tolerogenic milieu of the role in controlling latent CMV infection in IBD patients. gut, lymphocytes receive signals to be less cytotoxic in the gut as

It is well known that the intestinal epithelium embraces large compared with the blood compartment. In addition, it is possible http://www.jimmunol.org/ numbers of gd T cells as compared with peripheral blood (10). that in situations where stronger cytotoxicity is warranted in the Conspicuously, our results showed that the enrichment of CD8ab+ gut, that is infections or neoplasia, lymphocytes upregulate their gd T cells in the gut significantly exceeded that of the whole gd cytotoxic potential. T cell population. Indeed, when compared with blood the previ- The results demonstrating that CD8ab+ gd T cells produce IFN-g ously recognized CD8aa+ and CD82 gd IEL subsets were the and TNF-a, suggest a role in cell-mediated immune responses. It is same and lower in percentage, respectively, whereas the novel well known that mucosal IFN-g and TNF-a levels are increased in CD8ab+ gd T cell subset was significantly more frequent in both IBD and drive inflammation (40). These two cytokines have also the gut lamina propria and epithelium. This suggests that CD8ab+ been shown to play important roles in gut mucosa homeostasis by by guest on September 24, 2021

FIGURE 8. The percentage of CD8ab+ gd T cells is decreased in inﬂamed intestine of IBD patients. Percentage of CD8ab+ gd T cells in peripheral blood, LP and IEL compartments of healthy controls (white bars) and IBD patients (gray bars) out of CD3+ cells (A)andoutofTCRgd+ cells (B)(n = 6–20). Values are means 6 SD. Mann–Whit- ney U test, *p , 0.05, **p , 0.01, ***p , 0.001. (C) Correlation of HBI and fecal calprotectin with percentages of intestinal CD8ab+ out of TCRgd+ cells (mean of frequencies for IEL and LP lymphocytes) in patients with CD (n =10). The p values were calculated using the Spearman correlation method. LP, lamina propria. 8 ROLE OF A NOVEL gd T CELL SUBSET IN IBD

Hence, depletion of or deficiency in gd T cells in the rodent 2,4,6- trinitrobenzene sulfonic acid and dextran sulfate sodium models of IBD exacerbates and prolongs intestinal inflammation (48, 52). Murine studies have also shown that gd T cells can alleviate inflammation in the gut by regulating ab T cell responses (12). Of note, in our study CD8ab+ gd T cells showed negative correlation with disease activity, whereas the other gd T cell subsets did not show any significant changes. These data collectively suggest that CD8ab+ gd T cells play a homeostatic role in the human intestinal mucosa, and that a disturbance in their balance with other T cell subsets may either be caused by or lead to intestinal inflammation. As a result of anti–TNF-a treatment of CD patients we observed a specific increase in the percentage of their mucosal CD8ab+ gd T cells, whereas those of CD8aa+ and CD82 gd T cell subsets remained the same or decreased. As these changes occurred in parallel with the event of mucosal healing our results suggest that CD8ab+ gd T cells play a role in tissue healing in CD patients during anti–TNF-a treatment. This is in agreement with previous

studies proposing a role for gd T cells in mucosal wound healing Downloaded from (2, 50, 52, 53). This study deﬁnes CD8ab+ gd T cells as a novel, distinct, and functionally active subset of human T cells, which is strongly enriched within the gut mucosa and may have important functions in regulating inﬂammation and wound healing in human IBD. By

improved subset delineation these results could contribute to the http://www.jimmunol.org/ development of adoptive cell therapy strategies.

Acknowledgments FIGURE 9. Anti–TNF-a treatment increases the percentage of intestinal We thank Bengt Johansson-Lindbom and Vasileios Bekiaris for discussions CD8ab+ gd T cells in patients with CD. (A) Changes in absolute percentage and critical reading of the manuscript, Erik Hertervig for providing human points of CD8ab+ gd T cells out of CD3+ and TCRgd+ cells after 3 mo of experimental material and recruitment of subjects, Emma Nilsson for pro- anti–TNF-a therapy (n = 6–10). (B) Percentages of CD8ab+ gd T cells before viding human experimental material, Sven Almer for discussions and eth- and after anti–TNF-a therapy, in peripheral blood, and the lamina propria and ical approval, Ann Tornberg for help in coordinating the study, and finally, IEL compartments of inflamed ileum and inflamed colon (n = 6–10). Values the staff at the endoscopy unit and the outpatient clinic at the Section for by guest on September 24, 2021 are means 6 SD. Wilcoxon test, *p , 0.05, **p , 0.01, ***p , 0.001. LP, Gastroenterology at Skane˚ University Hospital in Lund for supporting lamina propria. endoscopies, handling of human experimental material, and patient com- munication. regulating proliferation and apoptosis of epithelial cells (41, 42). Intriguingly, several cytokines including TNF-a have in later years Disclosures been shown to have dichotomous roles and depending on the im- J.M. has served as consultant for AbbVie, Merck, Sharp & Dohme, UCB Pharma, Hospira, Takeda, Ferring, Tillotts, and Bristol-Myers Squibb. J.M. munological status of the patient or the phase of the inflammatory has received investigator-initiated study grants from AbbVie, Hospira, and process they may have both protective and proinflammatory func- Ferring. The other authors have no financial conflicts of interest. tions (43). Indeed, under certain circumstances TNF-a supports the repair process re-establishing the integrity of the epithelium (44). Finally, McMenamin et al. (45) have described a population of IFN- References g-producing gd T cells with Ag-specific immunosuppressive 1. Hayday, A., E. Theodoridis, E. Ramsburg, and J. Shires. 2001. Intraepithelial lymphocytes: exploring the third way in immunology. Nat. Immunol. 2: 997–1003. properties. Taken together, given the microanatomical localization 2. Vantourout, P., and A. Hayday. 2013. Six-of-the-best: unique contributions of gd + of intestinal CD8ab gd T cells, these cells could play multiple T cells to immunology. Nat. Rev. Immunol. 13: 88–100. important roles in IBD. 3. Prinz, I., B. Silva-Santos, and D. J. Pennington. 2013. Functional development of gd T cells. Eur. J. Immunol. 43: 1988–1994. Interestingly, IL-2 stimulation led to an increase in the per- 4. Ziegler, H., C. Welker, M. Sterk, J. Haarer, H. G. Rammensee, R. Handgretinger, centages of TNF-a+ and IFN-g+ cells within the CD8ab+ gd and K. Schilbach. 2014. Human peripheral CD4(+) Vd1(+) gdT cells can de- T cell subset, whereas the proportion of TNF-a and IFN-g pro- velop into abT cells. Front. Immunol. 5: 645. + 5. Eberl, G., and D. R. Littman. 2004. Thymic origin of intestinal alphabeta T cells ducing cells remained the same or decreased in the CD8aa and revealed by fate mapping of RORgammat+ cells. Science 305: 248–251. CD82 gd T cell subsets. In contrast, Ribot et al. (24) showed that 6. Carpenter, A. C., and R. Bosselut. 2010. Decision checkpoints in the thymus. + + Nat. Immunol. 11: 666–673. Vd1 and Vd2 gd T cells responded equally to IL-2 in terms of 7. Fahl, S. P., F. Coffey, and D. L. Wiest. 2014. Origins of gd T cell effector IFN-g and TNF-a production. The differential responses of the subsets: a riddle wrapped in an enigma. J. Immunol. 193: 4289–4294. different gd T cell subsets to IL-2 stimulation in our study further 8. Groh, V., A. Steinle, S. Bauer, and T. Spies. 1998. Recognition of stress- + induced MHC molecules by intestinal epithelial gammadelta T cells. Science support that CD8ab gd T cells are a functionally distinct subset. 279: 1737–1740. We observed decreased percentages of CD8ab+ gd T cells in 9. Adams, E. J., S. Gu, and A. M. Luoma. 2015. Human gamma delta T cells: inflamed mucosa of IBD patients, and that the percentage of evolution and ligand recognition. Cell. Immunol. 296: 31–40. + 10. Marsal, J., and W. W. Agace. 2012. Targeting T-cell migration in inflammatory intestinal CD8ab gd T cells correlated negatively with the se- bowel disease. J. Intern. Med. 272: 411–429. verity of CD activity. gd T cells have been assigned a protective 11. Cheroutre, H., F. Lambolez, and D. Mucida. 2011. The light and dark sides of intestinal intraepithelial lymphocytes. Nat. Rev. Immunol. 11: 445–456. role in IBD (46–48) and implicated in maintaining the integrity of 12. Hayday, A., and R. Tigelaar. 2003. Immunoregulation in the tissues by gam- the intestinal epithelium by producing growth factors (49–51). madelta T cells. Nat. Rev. Immunol. 3: 233–242. The Journal of Immunology 9

13. Huang, Y., N. Jin, C. L. Roark, M. K. Aydintug, J. M. Wands, H. Huang, T cells preserves b-cell function in type 1 diabetes in children. Diabetes Care 35: R. L. O’Brien, and W. K. Born. 2009. The influence of IgE-enhancing and IgE- 1817–1820. suppressive gammadelta T cells changes with exposure to inhaled ovalbumin. 35. Canavan, J. B., C. Scotta`, A. Vossenka¨mper, R. Goldberg, M. J. Elder, I. Shoval, J. Immunol. 183: 849–855. E. Marks, E. Stolarczyk, J. W. Lo, N. Powell, et al. 2016. Developing in vitro 14. Cohen, L. B., R. M. Nanau, F. Delzor, and M. G. Neuman. 2014. Biologic expanded CD45RA+ regulatory T cells as an adoptive cell therapy for Crohn’s therapies in inflammatory bowel disease. Transl. Res. 163: 533–556. disease. Gut 65: 584–594. 15. Radulovic, K., and J. H. Niess. 2015. CD69 is the crucial regulator of intestinal 36. Hua, F., N. Kang, Y. A. Gao, L. X. Cui, D. N. Ba, and W. He. 2013. Potential inflammation: a new target molecule for IBD treatment? J. Immunol. Res. 2015: regulatory role of in vitro-expanded Vd1 T cells from human peripheral blood. 497056. Immunol. Res. 56: 172–180. 16. Siegers, G. M., and L. S. Lamb, Jr. 2014. Cytotoxic and regulatory properties of 37. Pillet, S., B. Pozzetto, C. Jarlot, S. Paul, and X. Roblin. 2012. Management of circulating Vd1+ gd T cells: a new player on the cell therapy field? Mol. Ther. cytomegalovirus infection in inflammatory bowel diseases. Dig. Liver Dis. 44: 22: 1416–1422. 541–548. 17.Alexander,A.A.,A.Maniar,J.S.Cummings,A.M.Hebbeler,D.H.Schulze, 38. Knight, A., A. J. Madrigal, S. Grace, J. Sivakumaran, P. Kottaridis, B. R. Gastman, C. D. Pauza, S. E. Strome, and A. I. Chapoval. 2008. Isopentenyl S. Mackinnon, P. J. Travers, and M. W. Lowdell. 2010. The role of Vd2-negative pyrophosphate-activated CD56+ gammadelta T lymphocytes display potent antitumor gd T cells during cytomegalovirus reactivation in recipients of allogeneic stem activity toward human squamous cell carcinoma. Clin. Cancer Res. 14: 4232–4240. cell transplantation. Blood 116: 2164–2172. 18. Urban, E. M., H. Li, C. Armstrong, C. Focaccetti, C. Cairo, and C. D. Pauza. 39. Grossmann, J., K. Walther, M. Artinger, P. Rummele,€ M. Woenckhaus, and 2009. Control of CD56 expression and tumor cell cytotoxicity in human J. Scho¨lmerich. 2002. Induction of apoptosis before shedding of human intestinal Vgamma2Vdelta2 T cells. BMC Immunol. 10: 50. epithelial cells. Am. J. Gastroenterol. 97: 1421–1428. 19. Qin, G., Y. Liu, J. Zheng, Z. Xiang, I. H. Ng, J. S. Malik Peiris, Y. L. Lau, and 40. Neurath, M. F. 2014. Cytokines in inflammatory bowel disease. Nat. Rev. W. Tu. 2012. Phenotypic and functional characterization of human gd T-cell Immunol. 14: 329–342. subsets in response to influenza A viruses. J. Infect. Dis. 205: 1646–1653. 41. Nava, P., S. Koch, M. G. Laukoetter, W. Y. Lee, K. Kolegraff, C. T. Capaldo, 20. Sutton, C. E., S. J. Lalor, C. M. Sweeney, C. F. Brereton, E. C. Lavelle, and N. Beeman, C. Addis, K. Gerner-Smidt, I. Neumaier, et al. 2010. Interferon- K. H. Mills. 2009. Interleukin-1 and IL-23 induce innate IL-17 production from gamma regulates intestinal epithelial homeostasis through converging beta- gammadelta T cells, amplifying Th17 responses and autoimmunity. Immunity catenin signaling pathways. Immunity 32: 392–402. 31: 331–341. 42. Capaldo, C. T., N. Beeman, R. S. Hilgarth, P. Nava, N. A. Louis, E. Naschberger, Downloaded from 21. Wu, Y. L., Y. P. Ding, Y. Tanaka, L. W. Shen, C. H. Wei, N. Minato, and M. Sturzl,€ C. A. Parkos, and A. Nusrat. 2012. IFN-g and TNF-a-induced GBP-1 W. Zhang. 2014. gd T cells and their potential for immunotherapy. Int. J. Biol. inhibits epithelial cell proliferation through suppression of b-catenin/TCF sig- Sci. 10: 119–135. naling. Mucosal Immunol. 5: 681–690. 22. Dieli, F., F. Poccia, M. Lipp, G. Sireci, N. Caccamo, C. Di Sano, and A. Salerno. 43. Bamias, G., D. Corridoni, T. T. Pizarro, and F. Cominelli. 2012. New insights 2003. Differentiation of effector/memory Vdelta2 T cells and migratory routes in into the dichotomous role of innate cytokines in gut homeostasis and inflam- lymph nodes or inflammatory sites. J. Exp. Med. 198: 391–397. mation. Cytokine 59: 451–459. 23. Turchinovich, G., and D. J. Pennington. 2011. T cell receptor signalling in gd 44. Mizoguchi, E., Y. Hachiya, M. Kawada, K. Nagatani, A. Ogawa, K. Sugimoto,

cell development: strength isn’t everything. Trends Immunol. 32: 567–573. A. Mizoguchi, and D. K. Podolsky. 2008. TNF receptor type I-dependent acti- http://www.jimmunol.org/ 24. Ribot, J. C., S. T. Ribeiro, D. V. Correia, A. E. Sousa, and B. Silva-Santos. 2014. vation of innate responses to reduce intestinal damage-associated mortality. Human gd thymocytes are functionally immature and differentiate into cytotoxic Gastroenterology 134: 470–480. type 1 effector T cells upon IL-2/IL-15 signaling. J. Immunol. 192: 2237–2243. 45. McMenamin, C., C. Pimm, M. McKersey, and P. G. Holt. 1994. Regulation of 25. Harvey, R. F., and J. M. Bradshaw. 1980. A simple index of Crohn’s-disease IgE responses to inhaled antigen in mice by antigen-speciﬁc gamma delta activity. Lancet 1: 514. T cells. Science 265: 1869–1871. 26. French, J. D., C. L. Roark, W. K. Born, and R. L. O’Brien. 2009. Gammadelta 46. Hoffmann, J. C., K. Peters, S. Henschke, B. Herrmann, K. Pﬁster, J. Westermann, T lymphocyte homeostasis is negatively regulated by beta2-microglobulin. and M. Zeitz. 2001. Role of T lymphocytes in rat 2,4,6-trinitrobenzene sulphonic J. Immunol. 182: 1892–1900. acid (TNBS) induced colitis: increased mortality after gammadelta T cell de- 27. Chang, H. C., K. Tan, and Y. M. Hsu. 2006. CD8alphabeta has two distinct pletion and no effect of alphabeta T cell depletion. Gut 48: 489–495. binding modes of interaction with peptide-major histocompatibility complex 47. Inagaki-Ohara, K., T. Chinen, G. Matsuzaki, A. Sasaki, Y. Sakamoto, class I. J. Biol. Chem. 281: 28090–28096. K. Hiromatsu, F. Nakamura-Uchiyama, Y. Nawa, and A. Yoshimura. 2004.

28. Cheroutre, H., and F. Lambolez. 2008. Doubting the TCR coreceptor function of Mucosal T cells bearing TCRgammadelta play a protective role in intestinal by guest on September 24, 2021 CD8alphaalpha. Immunity 28: 149–159. inflammation. J. Immunol. 173: 1390–1398. 29. Gibbings, D., and A. D. Befus. 2009. CD4 and CD8: an inside-out coreceptor 48. Kuhl,€ A. A., N. N. Pawlowski, K. Grollich, C. Loddenkemper, M. Zeitz, and model for innate immune cells. J. Leukoc. Biol. 86: 251–259. J. C. Hoffmann. 2007. Aggravation of intestinal inflammation by depletion/ 30. Poggi, A., and M. R. Zocchi. 2014. gd T lymphocytes as a first line of immune deficiency of gammadelta T cells in different types of IBD animal models. defense: old and new ways of antigen recognition and implications for cancer J. Leukoc. Biol. 81: 168–175. immunotherapy. Front. Immunol. 5: 575. 49. Boismenu, R., and W. L. Havran. 1994. Modulation of epithelial cell growth by 31. Ermann, J., P. Hoffmann, M. Edinger, S. Dutt, F. G. Blankenberg, J. P. Higgins, intraepithelial gamma delta T cells. Science 266: 1253–1255. R. S. Negrin, C. G. Fathman, and S. Strober. 2005. Only the CD62L+ subpop- 50. Chen, Y., K. Chou, E. Fuchs, W. L. Havran, and R. Boismenu. 2002. Protection ulation of CD4+CD25+ regulatory T cells protects from lethal acute GVHD. of the intestinal mucosa by intraepithelial gamma delta T cells. Proc. Natl. Acad. Blood 105: 2220–2226. Sci. USA 99: 14338–14343. 32. Issa, F., J. Hester, R. Goto, S. N. Nadig, T. E. Goodacre, and K. Wood. 2010. Ex 51. Komano, H., Y. Fujiura, M. Kawaguchi, S. Matsumoto, Y. Hashimoto, S. Obana, vivo-expanded human regulatory T cells prevent the rejection of skin allografts P. Mombaerts, S. Tonegawa, H. Yamamoto, S. Itohara, et al. 1995. Homeostatic in a humanized mouse model. Transplantation 90: 1321–1327. regulation of intestinal epithelia by intraepithelial gamma delta T cells. Proc. 33. Trzonkowski, P., M. Bieniaszewska, J. Juscinska, A. Dobyszuk, A. Krzystyniak, Natl. Acad. Sci. USA 92: 6147–6151. N. Marek, J. Mysliwska, and A. Hellmann. 2009. First-in-man clinical results of 52. Tsuchiya, T., S. Fukuda, H. Hamada, A. Nakamura, Y. Kohama, H. Ishikawa, the treatment of patients with graft versus host disease with human ex vivo K. Tsujikawa, and H. Yamamoto. 2003. Role of gamma delta T cells in the expanded CD4+CD25+CD127- T regulatory cells. Clin. Immunol. 133: 22–26. inflammatory response of experimental colitis mice. J. Immunol. 171: 5507– 34. Marek-Trzonkowska, N., M. Mysliwiec, A. Dobyszuk, M. Grabowska, 5513. I. Techmanska, J. Juscinska, M. A. Wujtewicz, P. Witkowski, W. Mlynarski, 53. Komori, H. K., T. F. Meehan, and W. L. Havran. 2006. Epithelial and mucosal A. Balcerska, et al. 2012. Administration of CD4+CD25highCD127- regulatory gamma delta T cells. Curr. Opin. Immunol. 18: 534–538.