In a recent article published in Science, Dart and colleagues reported that colonic T cells expressing the V γ 4 T-cell receptor (TCR) interact with epithelial butyrophilin-like (BTNL) proteins BTNL3 and BTNL8 in healthy individuals, and they observed a depletion of V γ 4 T cells in in ﬂ ammatory bowel diseases with renormalization associated with disease remission. This paper identiﬁes the BTNL/V γ 4 axis as a new disease modiﬁer in IBD

Kabelitz, Dietrich

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RESEARCH HIGHLIGHT OPENThe Vγ4/butyrophilin conspiracy: novel role of intraepithelialγδ T cells in chronic inflammatory bowel diseaseDieter Kabelitz 1✉Signal Transduction and Targeted Therapy           (2023) 8:433 ; https://doi.org/10.1038/s41392-023-01697-wIn a recent article published in Science, Dart and colleaguesreported that colonic T cells expressing the Vγ4 T-cell receptor(TCR) interact with epithelial butyrophilin-like (BTNL) proteinsBTNL3 and BTNL8 in healthy individuals, and they observed adepletion of Vγ4 T cells in inflammatory bowel diseases withrenormalization associated with disease remission.1 This paperidentifies the BTNL/Vγ4 axis as a new disease modifier in IBD.1Conventional T cells express the αβ TCR and recognize peptidespresented by MHC class I (CD8) or MHC class II (CD4) molecules. Incontrast, γδ T cells are MHC-independent and fulfill importantfunctions in immunosurveillance against stressed and infectedcells. Moreover, studies (so far mainly in the murine system) alsoindicate a crucial role of γδ T cells in monitoring tissue integrity.The best-studied subset of human γδ T cells expresses a Vγ9Vδ2-encoded TCR. These cells account for the majority of peripheralblood γδ T cells but are less frequent in tissues. Vγ9Vδ2 T cellsrecognize pyrophosphate molecules (“phosphoantigens”) in astrictly butyrophilin (BTN) 3A1 and 2A1-dependent manner.2 BTNand BTN-like molecules (BTNL) are Ig superfamily-member, B7-related transmembrane proteins with multiple roles in theimmune system and beyond. Phosphoantigens are overproducedin cancer cells due to a dysregulated mevalonate pathway(isopentenyl pyrophosphate, IPP), or accumulate in infected cellsupon infection with microbes producing (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP). Such phosphoantigens bindto intracellular regions of BTN3A1 and induce a conformationalrearrangement of BTN2A1/3A1 complex which is then recognizedby the Vγ9Vδ2 TCR (Fig. 1a). As a consequence, Vγ9Vδ2 T cells cankill a variety of cancer cells because they sense the increasedlevels of IPP, but they do not depend on recognition of tumor-specific or tumor-associated antigens. Together with the fact thatγδ T cells are not HLA-restricted, these features have raised arecent burst of interest to bring γδ T cells into clinical applicationin cancer immunotherapy.3Distinct functions have been identified for tissue-residentepithelial γδ T cells in the skin and intestine where they contributeto barrier surveillance but also to tissue integrity and repair.Elegant mouse models have discovered the essential role ofspecific BTNL proteins expressed in a tissue-selective manner forthe selection of specific γδ TCR repertoires. For instance, Skint1and Skint2 (BTNL-related molecules) are required for the selectionof dendritic epidermal Vγ5 γδ T cells, while Btnl6 and Btnl1 arerequired for the development of intestinal Vγ7 γδ T cells.4 Theintra-epithelial γδ T cells contribute by multiple pathways to localtissue repair and immune defense, for instance by the productionof soluble mediators like IGF1, KGF, IFNγ, GZMA/B, and others.While models relying on genetic deficiency of a given Btnl genehave clearly demonstrated the importance of tissue-specificselection of γδ T cells and their importance for tissue homeostasisand immune surveillance in mice, similar studies in humans arenaturally much more demanding. In a previous study from theHayday group, it was already shown that the human colon harborsintraepithelial Vγ4-expressing γδ T cells which can specificallyinteract with BTNL3/BTNL8 heterodimers expressed on the colonicepithelial cells (Fig. 1b).5 However, it remained unresolvedwhether the reciprocal liaison of intraepithelial Vγ4γδ T cells withepithelial BTNL3/BTNL8 ligands has any implications for chronicinflammatory bowel diseases (IBD) like Crohn’s disease (CD) andulcerative colitis (UC). Both diseases are growing in incidence andare essentially incurable, which raises the importance of newinsights into new pathways that might be druggable.In their recent paper, Dart and colleagues performed anunprecedented in-depth study to characterize the colonic γδ TCRrepertoire in healthy donors and IBD patients, and they investigatedthe impact of BTNL3 and BTNL8 on the selection of Vγ4 γδintraepithelial cells (IEL).1 These studies were performed on a total of173 colonic endoscopic biopsies including non-IBD controls, unin-flamed gut from patients with confirmed IBD, and inflamed lesionsfrom IBD patients. First, they identified among Vδ2-negative γδ IEL amajor subset (around 47%) that expressed Vγ4 and the integrinCD103 as a marker of IEL and tissue-resident memory (TRM) T cells.CD103 is a receptor for E-cadherin on epithelial cells. Thetranscriptomic analysis of CD103+Vγ4 T cells revealed an enrichmentof genes important for effector function, activation and migration(e.g., KIR2DLA, TIGIT, CCR9, GZMA, FGF9) when compared toCD103negVγ4 IEL. They used HEK392T cells transduced with BTNL3+BTNL8 to interrogate the activation of the CD103+Vγ4 IEL. Theyobserved a specific TCR down-regulation together with up-regulationof 41BB and GZMB but little if any cytokines like TNFα, IFNγ, and nomobilization of CD107 as a surrogate marker of cytotoxic activity.Importantly, these cells were also largely refractory to activation withphorbolester plus ionomycin, and thus displayed a selective pattern ofresponsiveness. Further characterization of the CD103+Vγ4 IELrevealed high expression of FcεRIγ (involved in signal transduction)and NK receptors NKp46 and NKG2C. These studies have thus farcharacterized in detail the phenotype and activation of Vγ4 IEL in thehealthy human colon.Next, Dart and colleagues expanded their study to IBD patients.Overall, they observed a significantly lower γδ T-cell frequency ininflamed as opposed to healthy gut. More detailed analysisrevealed a disproportionate loss of CD103+Vγ4 cells. To investi-gate if the decline of intestinal γδ T cells in IBD might be due toReceived: 22 September 2023 Revised: 10 October 2023 Accepted: 30 October 20231Institute of Immunology, Christian-Albrechts University Kiel and University Hospital Schleswig-Holstein Campus Kiel, Kiel, GermanyCorrespondence: Dieter Kabelitz (dietrich.kabelitz@uksh.de)www.nature.com/sigtransSignal Transduction and Targeted Therapy© The Author(s) 20231234567890();,:antigen-driven activation and subsequent deletion, they per-formed Vγ TCR repertoire analysis. Interestingly, this revealedincreasing diversity in IBD versus control, indicating that the loss ofVγ4 IEL was not driven by antigenic pressure.To obtain genetic evidence for the role of BTNL3+ BTNL8 forthe selection of Vγ4 cells in humans is difficult. The authors hadthe opportunity to analyze healthy donors homozygous for aBTNL8*3 copy number variation in which deletion causes an in-frame fusion of the BTNL8 N terminus to the BTNL3 C terminusresulting in inefficient cell surface expression. These individualshad strongly reduced numbers of colonic CD103+Vγ4 IEL, whichelegantly supports the notion that BTNL3 and BTNL8 are indeedrequired for the selection of human Vγ4 IEL. In line, they found in alarge CD cohort a significant association between BTNL3 loss offunction and penetrating (B3) disease.Finally, they addressed the question how the Vγ4 IEL compart-ment can be modulated in IBD. Vγ4 IEL cells express RNA encodingreceptors for inflammatory cytokines, and the authors found thatculturing primary gut T cells in IL12+ IL18 changed thephenotype of Vγ4 cells with significant reduction of CD103, TIGIT,NKp44, suggesting that the inflammatory cytokine milieu mighthave similar effects in vivo. Vice versa, they observed inmacroscopically healed mucosa from several UC patients anincreased proportion of CD103+Vγ4 IEL, indicating that these cellsmight recover locally upon effective treatment. Moreover, theirpreliminary investigation with 19 IBD patients suggests thatreconstitution of colonic CD103+Vγ4 IEL might be associated withsustained remission. As the authors discuss, monitoring of specificimmune subsets like CD103+Vγ4 cells (which can be performedupon routine endoscopic biopsy) might be a useful novelbiomarker to assess disease activity in IBD.Taken together, this study has uncovered novel insights into theintricate role of a specific human γδ T-cell subset in intestinalimmune surveillance. The selective depletion of CD103+Vγ4 cellsin IBD, linked with deficiencies in BTNL3/BTNL8 expression, andthe possible reconstitution of the γδ IEL compartment uponremission reported in this paper1 paves the way for similar studiesin other barrier diseases affecting, e.g., the lung or skin.ACKNOWLEDGEMENTSD.K. is supported by the Deutsche Forschungsgemeinschaft grant Ka 502/19-3. OpenAccess funding enabled and organized by DEAL project.AUTHOR CONTRIBUTIONSD.K. wrote the manuscript, drew the figure, and approved the final version of thearticle.FUNDINGOpen Access funding enabled and organized by Projekt DEAL.ADDITIONAL INFORMATIONCompeting interests: The author declares no competing interests.REFERENCES1. Dart, R. J. et al. Conserved γδ T cell selection by BTNL proteins limits progression ofhuman inflammatory bowel disease. Science 381, eadh0301 (2023).2. Rigau, M. et al. Butyrophilin 2A1 is essential for phosphoantigen reactivity by γδT cells. Science 367, eaay5516 (2020).3. Kabelitz, D., Serrano, R., Kouakanou, L., Peters, C. & Kalyan, S. Cancer immu-notherapy with γδ T cells: many paths ahead of us. Cell. Mol. Immunol. 17, 925–939(2020).4. Jandke, A. et al. Butyrophilin-like proteins display combinatorial diversity inselecting and maintaining signature intraepithelial γδ T cell compartments. Nat.Commun. 11, 3769 (2020).5. Melandri, D. et al. The γδTCR combines innate immunity with adaptive immunityby utilizing spatially distinct regions for agonist selection and antigen respon-siveness. Nat. Immunol. 19, 1352–1365 (2018).Fig. 1 Differential role of human γδ T-cell subsets in immune surveillance. a Vγ9Vδ2 T cells are the dominant subset of γδ T cells in peripheralblood. They recognize phosphoantigens (pAg) overproduced in transformed cells (isopentenyl pyrophosphate, IPP) or accumulating ininfected cells ((E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, HMBPP) in a strictly BTN3A1/2A1-dependent manner. Vγ9Vδ2 T cells playimportant roles in tumor defense and control of microbial infection. b Intestinal intraepithelial Vγ4Vδ1 T cells can directly interact with BTNL3/BTNL8 heterodimers expressed on epithelial cells. A subset of intestinal Vγ4 T cells expresses CD103 (αEβ7 integrin), a marker of IEL and TRMT cells. CD103 is a receptor for E-cadherin on endothelial cells. These cells are important for tissue surveillance and repair and arecompromised in IBD. Figure created with BioRender.comThe Vγ4/butyrophilin conspiracy: novel role of intraepithelial. . .Kabelitz2Signal Transduction and Targeted Therapy           (2023) 8:433 Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in anymedium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the CreativeCommons license, and indicate if changes were made. The images or other third partymaterial in this article are included in the article’s Creative Commons license, unlessindicated otherwise in a credit line to the material. If material is not included in thearticle’s Creative Commons license and your intended use is not permitted by statutoryregulation or exceeds the permitted use, you will need to obtain permission directlyfrom the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.© The Author(s) 2023The Vγ4/butyrophilin conspiracy: novel role of intraepithelial. . .Kabelitz3Signal Transduction and Targeted Therapy           (2023) 8:433 

The Vγ4/butyrophilin conspiracy: novel role of intraepithelial γδ T cells in chronic inflammatory bowel disease

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The Vγ4/butyrophilin conspiracy: novel role of intraepithelial γδ T cells in chronic inflammatory bowel disease

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