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Gamma delta T cells (γδT cells) play crucial roles in the immune response against tumors, yet their functional dynamics under different cancer therapies remain poorly understood. Laser Ablative Immunotherapy (LAIT) is a novel cancer treatment modality combining local photothermal therapy (PTT) and intratumoral injection of an immunostimulant, N-dihydrogalactochitosan (glycated chitosan, GC). LAIT has been shown to induce systemic antitumor immune responses in pre-clinical studies and clinical trials, eradicating both treated local tumors and untreated distant metastases. In this study, we used LAIT to treat breast tumors in a mouse model and investigated the effects of LAIT on tumor-infiltrating γδT cells using single-cell RNA sequencing (scRNAseq). We characterized the γδT cells from tumors in control, PTT, GC, and LAIT (PTT + GC) groups, by identifying six distinct subtypes: activated, cytotoxic, cycling cytotoxic, IFN-enriched, antigen-presenting, and IL17-producing γδT cells. Differential gene expression analysis revealed that LAIT significantly upregulated genes associated with T cell activation, leukocyte adhesion, and interferon signaling in treated tumor tissues while downregulating genes involved in protein folding and stress responses. LAIT also uniquely increased the proportion of IL17-producing γδT cells, which correlated with prolonged survival in breast cancer patients, as analyzed using TCGA data. Furthermore, the transcriptomic profiles of γδT cells in LAIT-treated tumors closely resembled those in immune checkpoint inhibitor (ICI)-treated patients, suggesting potential synergistic effects. Our findings indicate that LAIT modulates the γδT cell transcriptome, enhancing their antitumor capabilities and providing a basis for combining LAIT with ICI therapy to improve cancer treatment outcomes., Competing Interests: Declaration of competing interest Wei R. Chen is co-founder and an unpaid member of the Board of Directors of Immunophotonics, Inc. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The intestinal epithelium harbours a unique lymphocyte population, the intraepithelial lymphocytes (IELs). A large fraction of IELs is represented by γδ T cells. Their role in epithelial homeostasis and immune response is well documented, but a conclusive view of their developmental pathway is still missing. In this review, we discuss the existing literature as well as recent advances regarding the tissue adaptation of γδ IELs, both for the characteristic cytotoxic subset and the newly described noncytotoxic subset. We particularly highlight the environmental cues and the transcriptional regulation that equip γδ T cells with their IEL phenotype., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

Gamma-delta T cells (γδ T cells) play a crucial role in both innate and adaptive immunity within tumors, yet their presence and prognostic value in cancer remain underexplored. This study presents a large-scale analysis of γδ T cell receptor (γδ TCR) reads from 11,000 tumor samples spanning 33 cancer types, utilizing the TRUST4 algorithm. Our findings reveal extensive diversity in γδ TCR clonality and gene expression, underscoring the potential of γδ T cells as prognostic biomarkers in various cancers. We further demonstrate the utility of TCR gamma (TRG) and delta (TRD) gene expression from standard RNA-sequencing (RNA-seq) data. This comprehensive dataset offers a valuable resource for advancing γδ T cell research, with implications for enhanced immunotherapy approaches or alternative therapeutic strategies. Additionally, our centralized database supports translational research into the therapeutic significance of γδ T cells., Competing Interests: Declaration of interests J.R.C.-G. is a consultant for Anixa Biosciences and Alloy Therapeutics; holds stock options in Compass Therapeutics, Anixa Biosciences, and Alloy Therapeutics; has a patent licensed by Anixa Biosciences; owns intellectual property filed with Compass Therapeutics; and is a co-founder of Cellepus Therapeutics, a CAR T cell company., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intraepithelial lymphocytes (IELs) reside in the epithelial layer and protect against foreign pathogens, maintaining the epithelial barrier function in the intestine. Interactions between IEL and epithelial cells are required for IELs to function effectively; however, the underlying molecular machinery remains to be elucidated. In this study, we found that intestinal epithelium-specific deficiency of the clathrin adaptor protein (AP)-1B, which regulates basolateral protein sorting, led to a massive reduction in IELs. Quantitative proteomics demonstrated that dozens of proteins, including known IEL-interacting proteins (E-cadherin, butyrophilin-like 2, and plexin B2), were decreased in the basolateral membrane of AP-1B-deficient epithelial cells. Among these proteins, CD166 interacted with CD6 on the surface of induced IEL. CD166 knockdown, using shRNA in intestinal organoid cultures, significantly inhibited IEL recruitment to the epithelial layer. These findings highlight the essential role of AP-1B-mediated basolateral sorting in IEL maintenance and survival within the epithelial layer. This study reveals a novel function of AP-1B in the intestinal immune system., (© 2024. The Author(s).)

In naive individuals, sensory neurons directly detect and respond to allergens, leading to both the sensation of itch and the activation of local innate immune cells, which initiate the allergic immune response 1,2 . In the setting of chronic allergic inflammation, immune factors prime sensory neurons, causing pathologic itch 3-7 . Although these bidirectional neuroimmune circuits drive responses to allergens, whether immune cells regulate the set-point for neuronal activation by allergens in the naive state is unknown. Here we describe a γδ T cell-IL-3 signalling axis that controls the allergen responsiveness of cutaneous sensory neurons. We define a poorly characterized epidermal γδ T cell subset 8 , termed GD3 cells, that produces its hallmark cytokine IL-3 to promote allergic itch and the initiation of the allergic immune response. Mechanistically, IL-3 acts on Il3ra-expressing sensory neurons in a JAK2-dependent manner to lower their threshold for allergen activation without independently eliciting itch. This γδ T cell-IL-3 signalling axis further acts by means of STAT5 to promote neuropeptide production and the initiation of allergic immunity. These results reveal an endogenous immune rheostat that sits upstream of and governs sensory neuronal responses to allergens on first exposure. This pathway may explain individual differences in allergic susceptibility and opens new therapeutic avenues for treating allergic diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The PD-1-PD-L1 immune checkpoint helps to maintain self-tolerance and prevent the development of autoimmune diseases. Immune checkpoint inhibitors are successful immunotherapeutics for several cancers, but responding patients can develop immune-mediated adverse events. It is well established that PD-1 regulates CD4 and CD8 T-cell responses, but its role in controlling the activation of pathogenic γδ T cells is less clear. Here we examined the role of PD-1 in regulating γδ T cells in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. We found that PD-1 was highly expressed on CD27 - Vγ4 γδ T cells in the lymph node (LN) and CNS of mice with EAE. Treatment of mice with anti-PD-1 significantly augmented IL-17A-producing CD27 - Vγ4 γδ T cells in the LN and CNS and enhanced the severity of EAE. The exacerbating effect of anti-PD-1 on EAE was lost in Tcrd -/- mice. Conversely, ligation of PD-1 suppressed Il17a and Rorc gene expression and IL-17A production by purified Vγ4 γδ T cells stimulated via the TCR, but not with IL-1β and IL-23. Our study demonstrates that PD-1 regulates TCR-activated CD27 - Vγ4 γδ T cells, but that cytokine-activated IL-17A producing γδ T cells escape the regulatory effects of the PD-1-PD-L1 pathway., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

The prevalence of obesity in the United States has continued to increase over the past several decades. Understanding how diet-induced obesity modulates mucosal immunity is of clinical relevance. We previously showed that consumption of a high fat, high sugar "Western" diet (WD) reduces the density and function of small intestinal Paneth cells, a small intestinal epithelial cell type with innate immune function. We hypothesized that obesity could also result in repressed gut adaptive immunity. Using small intestinal intraepithelial lymphocytes (IEL) as a readout, we found that in non-inflammatory bowel disease (IBD) subjects, high body mass index correlated with reduced IEL density. We recapitulated this in wild type (WT) mice fed with WD. A 4-week WD consumption was able to reduce IEL but not splenic, blood, or bone marrow lymphocytes, and the effect was reversible after another 2 weeks of standard diet (SD) washout. Importantly, WD-associated IEL reduction was not dependent on the presence of gut microbiota, as WD-fed germ-free mice also showed IEL reduction. We further found that WD-mediated Farnesoid X Receptor (FXR) activation in the gut triggered IEL reduction, and this was partially mediated by intestinal phagocytes. Activated FXR signaling stimulated phagocytes to secrete type I IFN, and inhibition of either FXR or type I IFN signaling within the phagocytes prevented WD-mediated IEL loss. Therefore, WD consumption represses both innate and adaptive immunity in the gut. These findings have significant clinical implications in the understanding of how diet modulates mucosal immunity., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: T. Stappenbeck advises Janssen, Boehringer Ingelheim, Kallyope, Takada, and Roche. T.C. Liu has research contracts with Denali Therapeutics and Interline Therapeutics. All other authors declare no relevant competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Injuries to the retinal pigment epithelium (RPE) trigger immune responses, orchestrating interactions within the innate and adaptive immune systems in the outer retina and choroid. We previously reported that interleukin 17 (IL-17) is a pivotal signaling molecule originating from choroidal γδ T cells, exerting protective effects by mediating functional connections between the RPE and subretinal microglia. In this current study, we generated mice with aryl hydrocarbon receptor (AhR) knockout specifically in IL-17-producing cells. These animals had deficiency in IL-17 production from γδ T cells, and exhibited increased sensitivity to both acute and chronic insults targeting the RPE. These findings imply that IL-17 plays a crucial role as a signaling cytokine in preserving the homeostasis of the outer retina and choroid., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jiyang Cai reports was provided by The University of Oklahoma Health Sciences Center. Jiyang Cai reports was provided by The University of Oklahoma Health Sciences Center. Jiyang Cai reports a relationship with The University of Oklahoma Health Sciences Center that includes: Jiyang Cai has patent pending to NA. NA If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

TCRαβ + CD8αα + intraepithelial lymphocytes (CD8αα + αβ IELs) are a specialized subset of T cells in the gut epithelium that develop from thymic agonist selected IEL precursors (IELps). The molecular mechanisms underlying the selection and differentiation of this T cell type in the thymus are largely unknown. Here, we found that Bcl6 deficiency in αβ T cells resulted in the near absence of CD8αα + αβ IELs. BCL6 was expressed by approximately 50% of CD8αα + αβ IELs and by the majority of thymic PD1 + IELps after agonist selection. Bcl6 deficiency blocked early IELp generation in the thymus, and its expression in IELps was induced by thymic TCR signaling in an ERK-dependent manner. As a result of Bcl6 deficiency, the precursors of IELps among CD4 + CD8 + double-positive thymocytes exhibited increased apoptosis during agonist selection and impaired IELp differentiation and maturation. Together, our results elucidate BCL6 as a crucial transcription factor during the thymic development of CD8αα + αβ IELs.

Gamma/delta T (γδ T)cells possess a unique mechanism for killing tumors, making them highly promising and distinguished among various cell therapies for tumor treatment. This review focuses on the major histocompatibility complex (MHC)-independent recognition of antigens and the interaction between γδ T cells and solid tumor cells. A comprehensive review is provided regarding the classification of human gamma-delta T cell subtypes, the characteristics and mechanisms underlying their functions, as well as their r545egulatory effects on tumor cells. The involvement of γδ T cells in tumorigenesis and migration was also investigated, encompassing potential therapeutic targets such as apoptosis-related molecules, the TNF receptor superfamily member 6(FAS)/FAS Ligand (FASL) pathways, butyrophilin 3A-butyrophilin 2A1 (BTN3A-BTN2A1) complexes, and interactions with CD4, CD8, and natural killer (NK) cells. Additionally, immune checkpoint inhibitors such as programmed cell death protein 1/Programmed cell death 1 ligand 1 (PD-1/PD-L1) have the potential to augment the cytotoxicity of γδ T cells. Moreover, a review on gamma-delta T cell therapy products and their corresponding clinical trials reveals that chimeric antigen receptor (CAR) gamma-delta T therapy holds promise as an approach with encouraging preclinical outcomes. However, practical issues pertaining to manufacturing and clinical aspects need resolution, and further research is required to investigate the long-term clinical side effects of CAR T cells. In conclusion, more comprehensive studies are necessary to establish standardized treatment protocols aimed at enhancing the quality of life and survival rates among tumor patients utilizing γδ T cell immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhu, Ren, Xie, Chen, Liang, Jiang, Wang and Zheng.)

The mechanisms underlying tissue repair in response to damage have been one of main subjects of investigation. Here we leverage the wound-induced hair neogenesis (WIHN) models in adult mice to explore the correlation between degree of damage and the healing process and outcome. The multimodal analysis, in combination with single-cell RNA sequencing help to explore the difference in wounds of gentle and heavy damage degrees, identifying the potential role of toll-like receptor 9 (TLR9) in sensing the injury and regulating the immune reaction by promoting the migration of γδT cells. The TLR9 deficient mice or wounds injected with TLR9 antagonist have greatly impaired healing and lower WIHN levels. Inhibiting the migration of γδT cells or knockout of γδT cells also suppress the wound healing and regeneration, which can't be rescued by TLR9agonist. Finally, the amphiregulin (AREG) is shown as one of most important effectors secreted by γδT cells and keratinocytes both in silicon or in the laboratory, whose expression influences WIHN levels and the expression of stem cell markers. In total, our findings reveal a previously unrecognized role for TLR9 in sensing skin injury and influencing the tissue repair and regeneration by modulation of the migration of γδT cells, and identify the TLR9-γδT cells-areg axis as new potential targets for enhancing tissue regeneration., (© 2024. The Author(s).)

Butyrophilin (BTN) molecules are emerging as key regulators of T cell immunity; however, how they trigger cell-mediated responses is poorly understood. Here, the crystal structure of a gamma-delta T cell antigen receptor (γδTCR) in complex with BTN2A1 revealed that BTN2A1 engages the side of the γδTCR, leaving the apical TCR surface bioavailable. We reveal that a second γδTCR ligand co-engages γδTCR via binding to this accessible apical surface in a BTN3A1-dependent manner. BTN2A1 and BTN3A1 also directly interact with each other in cis, and structural analysis revealed formation of W-shaped heteromeric multimers. This BTN2A1-BTN3A1 interaction involved the same epitopes that BTN2A1 and BTN3A1 each use to mediate the γδTCR interaction; indeed, locking BTN2A1 and BTN3A1 together abrogated their interaction with γδTCR, supporting a model wherein the two γδTCR ligand-binding sites depend on accessibility to cryptic BTN epitopes. Our findings reveal a new paradigm in immune activation, whereby γδTCRs sense dual epitopes on BTN complexes., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Despite the considerable progress in acute myeloid leukemia (AML) treatment, relapse after allogeneic hematopoietic stem cell transplantation (HSCT) is still frequent and associated with a poor prognosis. Relapse has been shown to be correlated with an incomplete eradication of CD34+ leukemic stem cells prior to HSCT. Previously, we have shown that a novel CD34-directed, bispecific T-cell engager (BTE) can efficiently redirect the T-cell effector function toward cancer cells, thus eliminating leukemic cells in vitro and in vivo . However, its impact on γδ T-cells is still unclear. In this study, we tested the efficacy of the CD34-specific BTE using in vitro expanded γδ T-cells as effectors. We showed that the BTEs bind to γδ T-cells and CD34+ leukemic cell lines and induce target cell killing in a dose-dependent manner. Additionally, γδ T-cell mediated killing was found to be superior to αβ T-cell mediated cytotoxicity. Furthermore, we observed that only in the presence of BTE the γδ T-cells induced primary AML blast killing in vitro . Importantly, our results show that γδ T-cells did not target the healthy CD34 intermediate endothelial blood-brain barrier cell line (hCMEC/D3) nor lysed CD34+ HSCs from healthy bone marrow samples., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Gamma delta (γδ) T cells are a heterogeneous population of cells that play roles in inflammation, host tissue repair, clearance of viral and bacterial pathogens, regulation of immune processes, and tumor surveillance. Recent research suggests that these are the main skin cells that produce interleukin-17 (I-17). Furthermore, γδ T cells exhibit memory-cell-like characteristics that mediate repeated episodes of psoriatic inflammation. γδ T cells are found in epithelial tissues, where many cancers develop. There, they participate in antitumor immunity as cytotoxic cells or as immune coordinators. γδ T cells also participate in host defense, immune surveillance, and immune homeostasis. The aim of this review is to present the importance of γδ T cells in physiological and pathological diseases, such as psoriasis, atopic dermatitis, autoimmune diseases, cancer, and lymphoma.

The Bacillus Calmette Guerin (BCG) vaccine has been shown to induce non-specific protection against diseases other than tuberculosis in vaccinated individuals, attributed to the induction of trained immunity. We have previously demonstrated that BCG administration induces innate immune training in mixed peripheral blood mononuclear cells and monocytes in calves. Gamma Delta (γδ) T cells are non-conventional T cells that exhibit innate and adaptive immune system features. They are in higher proportion in the peripheral blood of cattle than humans or rodents and play an essential role in bovine immune response to pathogens. In the current study, we determined if BCG administration induced innate immune training in bovine γδ T cells. A group of 16 pre-weaned Holstein calves (2-4 d age) were enrolled in the study and randomly assigned to vaccine and control groups (n=8/group). The vaccine group received two doses of 10 6 colony forming units (CFU) BCG Danish strain subcutaneously, separated by 2 weeks. The control group remained unvaccinated. Gamma delta T cells were purified from peripheral blood using magnetic cell sorting three weeks after receiving the 1 st BCG dose. We observed functional changes in the γδ T cells from BCG-treated calves shown by increased IL-6 and TNF-α cytokine production in response to in vitro stimulation with Escherichia coli LPS and PAM3CSK4. ATAC-Seq analysis of 78,278 regions of open chromatin (peaks) revealed that γδ T cells from BCG-treated calves had an altered epigenetic status compared to cells from the control calves. Differentially accessible peaks (DAP) found near the promoters of innate immunity-related genes like Siglec14 , Irf4 , Ifna2 , Lrrfip1 , and Tnfrsf10d were 1 to 4-fold more accessible in cells from BCG-treated calves. MOTIF enrichment analysis of the sequences within DAPs, which explores transcription factor binding motifs (TFBM) upstream of regulatory elements, revealed TFBM for Eomes and IRF-5 were among the most enriched transcription factors. GO enrichment analysis of genes proximal to the DAPs showed enrichment of pathways such as regulation of IL-2 production, T-cell receptor signaling pathway, and other immune regulatory pathways. In conclusion, our study shows that subcutaneous BCG administration in pre-weaned calves can induce innate immune memory in the form of trained immunity in γδ T cells. This memory is associated with increased chromatin accessibility of innate immune response-related genes, thereby inducing a functional trained immune response evidenced by increased IL-6 and TNF-α cytokine production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Samuel, Diaz, Maina, Corbett, Tuggle and McGill.)

Objectives: γδ T-lymphocytes play a role in angiotensin II (AngII)-induced hypertension, vascular injury and T-cell infiltration in perivascular adipose tissue (PVAT) in mice. Mesenteric arteries of hypertensive mice and subcutaneous arteries from obese humans present similar remodeling. We hypothesized that γδ T-cell subtypes in mesenteric vessels with PVAT (MV/PVAT) from hypertensive mice and subcutaneous adipose tissue (SAT) from obese humans, who are prone to develop hypertension, would be similar., Methods: Mice were infused with AngII for 14 days. MV/PVAT T-cells were used for single-cell RNA-sequencing (scRNA-seq). scRNA-seq data (GSE155960) of SAT CD45 + cells from three lean and three obese women were downloaded from the Gene Expression Omnibus database., Results: δ T-cell subclustering identified six δ T-cell subtypes. AngII increased T-cell receptor δ variable 4 ( Trdv4 ) + γδ T-effector memory cells and Cd28high δ T EM -cells, changes confirmed by flow cytometry. δ T-cell subclustering identified nine δ T-cell subtypes in human SAT. CD28 expressing δ T-cell subclustering demonstrated similar δ T-cell subpopulations in murine MV/PVAT and human SAT. Cd28+ γδ NKT EM and Cd28high δ T EM -cells increased in MV/PVAT from hypertensive mice and CD28high δ T EM -cells in SAT from obese women compared to the lean women., Conclusion: Similar CD28 + δ T-cells were identified in murine MV/PVAT and human SAT. CD28 high δ T EM -cells increased in MV/PVAT in hypertensive mice and in SAT from humans with obesity, a prehypertensive condition. CD28 + δ T-lymphocytes could have a pathogenic role in human hypertension associated with obesity, and could be a potential target for therapy., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The contribution of γδ T cells to immune responses is associated with rapid secretion of interferon-γ (IFN-γ). Here, we show a perinatal thymic wave of innate IFN-γ-producing γδ T cells that express CD8αβ heterodimers and expand in preclinical models of infection and cancer. Optimal CD8αβ + γδ T cell development is directed by low T cell receptor signaling and through provision of interleukin (IL)-4 and IL-7. This population is pathologically relevant as overactive, or constitutive, IL-7R-STAT5B signaling promotes a supraphysiological accumulation of CD8αβ + γδ T cells in the thymus and peripheral lymphoid organs in two mouse models of T cell neoplasia. Likewise, CD8αβ + γδ T cells define a distinct subset of human T cell acute lymphoblastic leukemia pediatric patients. This work characterizes the normal and malignant development of CD8αβ + γδ T cells that are enriched in early life and contribute to innate IFN-γ responses to infection and cancer., (© 2024. The Author(s).)

Tumor-infiltrating lymphocytes (TILs) are associated with improved survival in patients with epithelial ovarian cancer. However, TIL evaluation has not been used in routine clinical practice because of reproducibility issues. The current study developed two convolutional neural network models to detect TILs and to determine their spatial location in whole slide images, and established a spatial assessment pipeline to objectively quantify intraepithelial and stromal TILs in patients with high-grade serous ovarian carcinoma. The predictions of the established models showed a significant positive correlation with the number of CD8 + T cells and immune gene expressions. Patients with a higher density of intraepithelial TILs had a significantly prolonged overall survival and progression-free survival in multiple cohorts. On the basis of the density of intraepithelial and stromal TILs, patients were classified into three immunophenotypes: immune inflamed, excluded, and desert. The immune-desert subgroup showed the worst prognosis. Gene expression analysis showed that the immune-desert subgroup had lower immune cytolytic activity and T-cell-inflamed gene-expression profile scores, whereas the immune-excluded subgroup had higher expression of interferon-γ and programmed death 1 receptor signaling pathway. The established evaluation method provided detailed and comprehensive quantification of intraepithelial and stromal TILs throughout hematoxylin and eosin-stained slides. It has potential for clinical application for personalized treatment of patients with ovarian cancer., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that presents significant therapeutic challenges due to the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. As a result, conventional hormonal and targeted therapies are largely ineffective, underscoring the urgent need for novel treatment strategies. γδT cells, known for their robust anti-tumor properties, show considerable potential in TNBC treatment as they can identify and eliminate tumor cells without reliance on MHC restrictions. These cells demonstrate extensive proliferation both in vitro and in vivo , and can directly target tumors through cytotoxic effects or indirectly by promoting other immune responses. Studies suggest that expansion and adoptive transfer strategies targeting Vδ2 and Vδ1 γδT cell subtypes have shown promise in preclinical TNBC models. This review compiles and discusses the existing literature on the primary subgroups of γδT cells, their roles in cancer therapy, their contributions to tumor cell cytotoxicity and immune modulation, and proposes potential strategies for future γδT cell-based immunotherapies in TNBC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Zhao, Ren, Gao, Han, Lu and Li.)

The intestinal epithelium, which segregates the highly stimulatory lumen from the underlying tissue, harbors one of the largest lymphocyte populations in the body, intestinal intraepithelial lymphocytes (IELs). IELs must balance tolerance, resistance, and tissue protection to maintain epithelial homeostasis and barrier integrity. This review discusses the ontogeny, environmental imprinting, T cell receptor (TCR) repertoire, and function of intestinal IELs. Despite distinct developmental pathways, IEL subsets share core traits including an epithelium-adapted profile, innate-like properties, cytotoxic potential, and limited TCR diversity. IELs also receive important developmental and functional cues through interactions with epithelial cells, microbiota, and dietary components. The restricted TCR diversity of IELs suggests that a limited set of intestinal antigens drives IEL responses, with potential functional consequences. Finally, IELs play a key role in promoting homeostatic immunity and epithelial barrier integrity but can become pathogenic upon dysregulation. Therefore, IELs represent intriguing but underexamined therapeutic targets for inflammatory diseases and cancer.

Intraepithelial lymphocytes (IELs) are T cells important for the maintenance of barrier integrity in the intestine. Colon IELs are significantly reduced in both MyD88-deficient mice and those lacking an intact microbiota, suggesting that MyD88-mediated detection of bacterial products is important for the recruitment and/or retention of these cells. Here, using conditionally deficient MyD88 mice, we show that myeloid cells are the key mediators of TCRαβ+ IEL recruitment to the colon. Upon exposure to luminal bacteria, myeloid cells produce sphingosine-1-phosphate (S1P) in a MyD88-dependent fashion. TCRαβ+ IEL recruitment may be blocked using the S1P receptor antagonist FTY720, confirming the importance of S1P in the recruitment of TCRαβ+ IELs to the colon epithelium. Finally, using the TNFΔARE/+ model of Crohn's-like bowel inflammation, we show that disruption of colon IEL recruitment through myeloid-specific MyD88 deficiency results in reduced pathology. Our results illustrate one mechanism for recruitment of a subset of IELs to the colon., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Sepsis is a dysregulated systemic immune response to infection i.e. responsible for ∼35% of in-hospital deaths at a significant fiscal healthcare cost. Our laboratory, among others, has demonstrated the efficacy of targeting negative checkpoint regulators (NCRs) to improve survival in a murine model of sepsis, cecal ligation and puncture (CLP). B7-CD28 superfamily member, V-domain immunoglobulin suppressor of T cell activation (VISTA), is an ideal candidate for strategic targeting in sepsis. VISTA is a 35 to 45 kDa type 1 transmembrane protein with unique biology that sets it apart from all other NCRs. We recently reported that VISTA-/- mice had a significant survival deficit post-CLP, which was rescued upon adoptive transfer of a VISTA-expressing pMSCV-mouse Foxp3-EF1α-GFP-T2A-puro stable Jurkat cell line (Jurkatfoxp3 T cells). Based on our prior study, we investigated the effector cell target of Jurkatfoxp3 T cells in VISTA-/- mice. γδ T cells are a powerful lymphoid subpopulation that require regulatory fine-tuning by regulatory T cells to prevent overt inflammation/pathology. In this study, we hypothesized that Jurkatfoxp3 T cells nonredundantly modulate the γδ T cell population post-CLP. We found that VISTA-/- mice have an increased accumulation of intestinal CD69low γδ T cells, which are not protective in murine sepsis. Adoptive transfer of Jurkatfoxp3 T cells decreased the intestinal γδ T cell population, suppressed proliferation, skewed remaining γδ T cells toward a CD69high phenotype, and increased soluble CD40L in VISTA-/- mice post-CLP. These results support a potential regulatory mechanism by which VISTA skews intestinal γδ T cell lineage representation in murine sepsis., Competing Interests: Conflict of interest statement. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Atherosclerosis poses a significant threat to human health, impacting overall well-being and imposing substantial financial burdens. Current treatment strategies mainly focus on managing low-density lipids (LDL) and optimizing liver functions. However, it's crucial to recognize that Atherosclerosis involves more than just lipid accumulation; it entails a complex interplay of immune responses. Research highlights the pivotal role of lipid-laden macrophages in the formation of atherosclerotic plaques. These macrophages attract lymphocytes like CD4 and CD8 to the inflamed site, potentially intensifying the inflammatory response. γδ T lymphocytes, with their diverse functions in innate and adaptive immune responses, pathogen defense, antigen presentation, and inflammation regulation, have been implicated in the early stages of Atherosclerosis. However, our understanding of the roles of γδ T cells in Atherosclerosis remains limited. This mini-review aims to shed light on the characteristics and functions of γδ T cells in Atherosclerosis. By gaining insights into the roles of γδ T cells, we may uncover a promising strategy to mitigate plaque buildup and dampen the inflammatory response, thereby opening new avenues for effectively managing this condition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Xu, Chen, Fan, Saito and Cao.)

The accumulation of intraepithelial lymphocytes (IELs) is a histopathological feature of canine chronic enteropathy (CE), and IELs are considered the cells of origin of intestinal T-cell lymphoma (ITCL). However, the pathogenic mechanism of IEL activation in CE remains unclear. This study hypothesized that the expression of proinflammatory cytokines, associated with cytotoxic T/NK-cell activation, is upregulated in CE and ITCL, and examined the expression of IFN-γ , IL-2 , IL-12p35, IL-12p40 , IL-15 , and IL-21 and the downstream signal transducers and activators of transcription (STAT) pathway in the duodenal mucosa of dogs without lesions ( n = 11; NC), with IEL - CE ( n = 19; CE without intraepithelial lymphocytosis), IEL + CE ( n = 29; CE with intraepithelial lymphocytosis), and with ITCL ( n = 60). Quantitative polymerase chain reaction (PCR) revealed that IFN-γ and IL-21 were higher in IEL + CE than in IEL - CE or NC. Western blot revealed upregulation of STAT1 and STAT3 in IEL + CE. Double-labeling immunohistochemistry revealed a positive correlation between the Ki67 index of CD3 + T-cells and IFN-γ expression levels. Immunohistochemistry revealed a higher ratio of p-STAT1-positive villi in IEL + CE and ITCL than IEL - CE and NC, which positively correlated with IFN-γ expression levels. Among the 60 ITCL cases, neoplastic lymphocytes were immunopositive for p-STAT1 in 28 cases and p-STAT3 in 29 cases. These results suggest that IFN-γ and IL-21 contribute to the pathogenesis of IEL + CE, and IFN-γ may be involved in T-cell activation and mucosal injury in CE. STAT1 and STAT3 activation in ITCL cells suggests a role for the upregulation of the STAT pathway in the pathogenesis of ITCL., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Chemotherapy and radiotherapy frequently lead to intestinal damage. The mechanisms governing the repair or regeneration of intestinal damage are still not fully elucidated. Intraepithelial lymphocytes (IELs) are the primary immune cells residing in the intestinal epithelial layer. However, whether IELs are involved in intestinal epithelial injury repair remains unclear. Here, we found that IELs rapidly infiltrated the intestinal crypt region and are crucial for the recovery of the intestinal epithelium post-chemotherapy. Interestingly, IELs predominantly promoted intestinal regeneration by modulating the proliferation of transit-amplifying (TA) cells. Mechanistically, the expression of CD160 on IELs allows for interaction with herpes virus entry mediator (HVEM) on the intestinal epithelium, thereby activating downstream nuclear factor kappa (NF-κB) signaling and further promoting intestinal regeneration. Deficiency in either CD160 or HVEM resulted in reduced proliferation of intestinal progenitor cells, impaired intestinal damage repair, and increased mortality following chemotherapy. Remarkably, the adoptive transfer of CD160-sufficient IELs rescued the Rag1 deficient mice from chemotherapy-induced intestinal inflammation. Overall, our study underscores the critical role of IELs in intestinal regeneration and highlights the potential applications of targeting the CD160-HVEM axis for managing intestinal adverse events post-chemotherapy and radiotherapy., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: Mucosal T cells play a major role in inflammatory bowel disease (IBD). However, their immunometabolism during intestinal inflammation is poorly understood. Due to its impact on cellular metabolism and proinflammatory immune cell function, we here focus on the enzyme ATP citrate lyase (ACLY) in mucosal T cell immunometabolism and its relevance for IBD., Design: ACLY expression and its immunometabolic impact on colitogenic T cell function were analysed in mucosal T cells from patients with IBD and in two experimental colitis models., Results: ACLY was markedly expressed in colon tissue under steady-state conditions but was significantly downregulated in lamina propria mononuclear cells in experimental dextran sodium sulfate-induced colitis and in CD4 + and to a lesser extent in CD8 + T cells infiltrating the inflamed gut in patients with IBD. ACLY-deficient CD4 + T cells showed an impaired capacity to induce intestinal inflammation in a transfer colitis model as compared with wild-type T cells. Assessment of T cell immunometabolism revealed that ACLY deficiency dampened the production of IBD-relevant cytokines and impaired glycolytic ATP production but enriched metabolites involved in the biosynthesis of phospholipids and phosphatidylcholine. Interestingly, the short-chain fatty acid butyrate was identified as a potent suppressor of ACLY expression in T cells, while IL-36α and resolvin E1 induced ACLY levels. In a translational approach, in vivo administration of the butyrate prodrug tributyrin downregulated mucosal infiltration of ACLY high CD4 + T cells and ameliorated chronic colitis., Conclusion: ACLY controls mucosal T cell immunometabolism and experimental colitis. Therapeutic modulation of ACLY expression in T cells emerges as a novel strategy to promote the resolution of intestinal inflammation., Competing Interests: Competing interests: MFN has served as an advisor for Pentax, Giuliani, MSD, Abbvie, Janssen, Takeda and Boehringer. Moreover, MFN serves as an associated editor of the journal Gut. The remaining authors disclose no conflicts., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

γδ T cells play an important role in disease control in acute myeloid leukemia (AML) and have become an emerging area of therapeutic interest. These cells represent a minor population of T lymphocytes with intrinsic abilities to recognize antigens in a major histocompatibility complex-independent manner and functionally straddle the innate and adaptive immunity interface. AML shows high expression of phosphoantigens and UL-16 binding proteins that activate the Vδ2 and Vδ1 subtypes of γδ T cells, respectively, leading to γδ T cell-mediated cytotoxicity. Insights from murine models and clinical data in humans show improved overall survival, leukemia-free survival, reduced risk of relapse, enhanced graft-versus-leukemia effect, and decreased graft-versus-host disease in patients with AML who have higher reconstitution of γδ T cells following allogeneic hematopoietic stem cell transplantation. Clinical trials leveraging γδ T cell biology have used unmodified and modified allogeneic cells as well as bispecific engagers and monoclonal antibodies. In this review, we discuss γδ T cells' biology, roles in cancer and AML, and mechanisms of immune escape and antileukemia effect; we also discuss recent clinical advances related to γδ T cells in the field of AML therapeutics., Competing Interests: Competing interests: AR and AA: None. AM: Reports research funding to the institution from Chimeric Therapeutics, LinBio Sciences, Celgene, Sanofi-Aventis, CytoMed Therapeutics, BioSight, IGM Biosciences, Electra Therapeutics, Astex Pharmaceuticals. Other: Cero Therapeutics., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background and Aims: We aimed to investigate the immune characteristics of intestinal CD8 + gamma delta T (CD8 + γδ T) cells in Crohn's disease (CD) and their correlation with disease activity., Methods: The study cohorts included 21 CD patients and 21 healthy individuals. CD8 + γδ T cells were isolated from human ileal mucosa for detection by flow cytometry. The activation or inhibition status of cells was detected by detecting the expression of activation marker HLA-DR and the immunosuppressive molecule PD-1 on cells. The cytotoxicity of cells was assessed by detecting the expression of cytotoxic molecules (Perforin, Granzyme B, and TRAIL) in cells. Ratios of investigated cells were calculated as prediction factors by receiver operating characteristic curve (ROC) analysis., Results: The study revealed a reduction in intestinal CD8 + γδT cells among active CD patients, with a more pronounced reduction observed in moderately active patients compared to mildly active patients. Moreover, active CD patients exhibited heightened activation levels in their intestinal CD8 + γδT cells, whereas the activation was comparatively weakened in moderately active patients compared with mildly active patients. Additionally, the cytotoxicity of intestinal CD8 + γδT cells was enhanced solely in mildly active patients, while it was impaired in moderately active patients compared with mildly active patients. Furthermore, HLA-DR + CD8 + γδT cell ratio, CD8 + γδT ratio, and CD8 + γδT count were identified as indicators in the diagnosis of active CD. Meanwhile, the ratios of Granzyme B + CD8 + γδT cell and Perforin + CD8 + γδT cell were identified as indicators that distinguish mildly moderately active CD cases., Conclusions: Intestinal CD8 + γδT was reduced in active CD patients, but their activation and cytotoxicity were enhanced. However, with increased disease activity, intestinal CD8 + γδ T cells became dysfunctional. CD-specific perturbations observed in various phenotypic markers in CD8 + γδ T cells can be used as indicators to assist in diagnosing CD patients., (© 2024. The Author(s).)

Our previous study indicated that cytotoxicity of intraepithelial lymphocytes is a poor prognostic factor in feline intestinal T-cell lymphoma (FITL), but the effect of cytotoxic lymphocytes on mucosal epithelium is still unknown. Thus, we investigated the association between cytotoxic lymphocytes and mucosal epithelium in 71 cases of feline intestinal T-cell lymphoma (FITL): epithelial injury, basement membrane injury, cleaved-caspase-3 positivity of epithelial cells, and the number and Ki67 positivity of intraepithelial lymphocytes in granzyme B (GRB)+ and GRB- FITLs were evaluated. Epithelial injury score and the number of intraepithelial lymphocytes in granzyme B (GRB)+ FITL were significantly higher than those of GRB- FITL (P<0.05, P<0.05), but no significant differences were found in the basement membrane injury score, the percentage of cleaved-caspase-3+ epithelial cells, and the percentage of Ki67+ intraepithelial lymphocytes. There was a significant correlation between the epithelial injury score and the number of intraepithelial lymphocytes (P<0.05), but no significant correlation was observed between the epithelial injury score and Ki67+ percentage of intraepithelial lymphocytes. Because epithelial cell cleaved-caspase-3 positivity was observed in FITL, regardless of GRB expression in lymphocytes, GRB-mediated apoptosis may not contribute to epithelial injury in FITL. The association between increased number of intraepithelial lymphocytes and epithelial injury suggests that intraepithelial lymphocytes infiltration may contribute to epithelial injury in FITL.

Intestinal intraepithelial lymphocytes (IELs) exhibit prompt innate-like responses to microenvironmental cues and require strict control of effector functions. Here we showed that Aiolos, an Ikaros zinc-finger family member encoded by Ikzf3, acted as a regulator of IEL activation. Ikzf3 -/- CD8αα + IELs had elevated expression of NK receptors, cytotoxic enzymes, cytokines and chemokines. Single-cell RNA sequencing of Ikzf3 -/- and Ikzf3 +/+ IELs showed an amplified effector machinery in Ikzf3 -/- CD8αα + IELs compared to Ikzf3 +/+ counterparts. Ikzf3 -/- CD8αα + IELs had increased responsiveness to interleukin-15, which explained a substantial part, but not all, of the observed phenotypes. Aiolos binding sites were close to those for the transcription factors STAT5 and RUNX, which promote interleukin-15 signaling and cytolytic programs, and Ikzf3 deficiency partially increased chromatin accessibility and histone acetylation in these regions. Ikzf3 deficiency in mice enhanced susceptibility to colitis, underscoring the relevance of Aiolos in regulating the effector function in IELs., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Intraepithelial lymphocytes (IELs), including αβ and γδ T cells (T-IELs), constantly survey and play a critical role in maintaining the gastrointestinal epithelium. We show that cytotoxic molecules important for defense against cancer were highly expressed by T-IELs in the small intestine. In contrast, abundance of colonic T-IELs was dependent on the microbiome and displayed higher expression of TCF-1/ TCF7 and a reduced effector and cytotoxic profile, including low expression of granzymes. Targeted deletion of TCF-1 in γδ T-IELs induced a distinct effector profile and reduced colon tumor formation in mice. In addition, TCF-1 expression was significantly reduced in γδ T-IELs present in human colorectal cancers (CRCs) compared with normal healthy colon, which strongly correlated with an enhanced γδ T-IEL effector phenotype and improved patient survival. Our work identifies TCF-1 as a colon-specific T-IEL transcriptional regulator that could inform new immunotherapy strategies to treat CRC.

Intraepithelial lymphocytes (IEL) expressing γδ T-cell receptors (γδTCR) play key roles in elimination of colon cancer. However, the precise mechanisms by which progressing cancer cells evade immunosurveillance by these innate T cells are unknown. Here, we investigated how loss of the Apc tumor suppressor in gut tissue could enable nascent cancer cells to escape immunosurveillance by cytotoxic γδIELs. In contrast with healthy intestinal or colonic tissue, we found that γδIELs were largely absent from the microenvironment of both mouse and human tumors, and that butyrophilin-like (BTNL) molecules, which can critically regulate γδIEL through direct γδTCR interactions, were also downregulated in tumors. We then demonstrated that β-catenin activation through loss of Apc rapidly suppressed expression of the mRNA encoding the HNF4A and HNF4G transcription factors, preventing their binding to promoter regions of Btnl genes. Reexpression of BTNL1 and BTNL6 in cancer cells increased γδIEL survival and activation in coculture assays but failed to augment their cancer-killing ability in vitro or their recruitment to orthotopic tumors. However, inhibition of β-catenin signaling via genetic deletion of Bcl9/Bcl9L in either Apc-deficient or mutant β-catenin mouse models restored Hnf4a, Hnf4g, and Btnl gene expression and γδ T-cell infiltration into tumors. These observations highlight an immune-evasion mechanism specific to WNT-driven colon cancer cells that disrupts γδIEL immunosurveillance and furthers cancer progression., (©2023 The Authors; Published by the American Association for Cancer Research.)

γδ T cells are important tissue-resident, innate T cells that are critical for tissue homeostasis. γδ cells are associated with positive prognosis in most tumors; however, little is known about their heterogeneity in human cancers. Here, we phenotyped innate and adaptive cells in human colorectal (CRC) and endometrial cancer. We found striking differences in γδ subsets and function in tumors compared to normal tissue, and in the γδ subsets present in tumor types. In CRC, an amphiregulin (AREG)-producing subset emerges, while endometrial cancer is infiltrated by cytotoxic cells. In humanized CRC models, tumors induced this AREG phenotype in Vδ1 cells after adoptive transfer. To exploit the beneficial roles of γδ cells for cell therapy, we developed an expansion method that enhanced cytotoxic function and boosted metabolic flexibility, while eliminating AREG production, achieving greater tumor infiltration and tumor clearance. This method has broad applications in cellular therapy as an 'off-the-shelf' treatment option., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The human gut microbiome constantly converts natural products derived from the host and diet into numerous bioactive metabolites 1-3 . Dietary fats are essential micronutrients that undergo lipolysis to release free fatty acids (FAs) for absorption in the small intestine 4 . Gut commensal bacteria modify some unsaturated FAs-for example, linoleic acid (LA)-into various intestinal FA isomers that regulate host metabolism and have anticarcinogenic properties 5 . However, little is known about how this diet-microorganism FA isomerization network affects the mucosal immune system of the host. Here we report that both dietary factors and microbial factors influence the level of gut LA isomers (conjugated LAs (CLAs)) and that CLAs in turn modulate a distinct population of CD4 + intraepithelial lymphocytes (IELs) that express CD8αα in the small intestine. Genetic abolition of FA isomerization pathways in individual gut symbionts significantly decreases the number of CD4 + CD8αα + IELs in gnotobiotic mice. Restoration of CLAs increases CD4 + CD8αα + IEL levels in the presence of the transcription factor hepatocyte nuclear factor 4γ (HNF4γ). Mechanistically, HNF4γ facilitates CD4 + CD8αα + IEL development by modulating interleukin-18 signalling. In mice, specific deletion of HNF4γ in T cells leads to early mortality from infection by intestinal pathogens. Our data reveal a new role for bacterial FA metabolic pathways in the control of host intraepithelial immunological homeostasis by modulating the relative number of CD4 + T cells that were CD4 + CD8αα + ., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Located in the frontline against the largest population of microbiota, the intestinal mucosa of mammals has evolved to become an effective immune system. γδ T cells, a unique T cell subpopulation, are rare in circulation blood and lymphoid tissues, but rich in the intestinal mucosa, particularly in the epithelium. Via rapid production of cytokines and growth factors, intestinal γδ T cells are key contributors to epithelial homeostasis and immune surveillance of infection. Intriguingly, recent studies have revealed that the intestinal γδ T cells may play novel exciting functions ranging from epithelial plasticity and remodeling in response to carbohydrate diets to the recovery of ischemic stroke. In this review article, we update regulatory molecules newly defined in lymphopoiesis of the intestinal γδ T cells and their novel functions locally in the intestinal mucosa, such as epithelial remodeling, and distantly in pathological setting, e.g., ischemic brain injury repair, psychosocial stress responses, and fracture repair. The challenges and potential revenues in intestinal γδ T cell studies are discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Xia, Zeng, Luo, Liu, Zeng and Cao.)

CD8αα+ intestinal intraepithelial lymphocytes (iIELs) are known for their unique role in keeping the integrity of the intestinal epithelial barrier, but factors affecting the development of these cells have not been thoroughly understood. Here, we found that the transcriptional regulator interferon regulatory factor-2 (IRF-2) plays a cell-intrinsic, indispensable role in establishing iIEL populations. CD8αα+, but not CD8αβ+, iIELs bearing TCRαβ or TCRγδ were severely reduced in numbers in mice lacking this factor (Irf2-/- mice). Moreover, the majority of residual CD8αα+TCRαβ+ iIELs in these mice was immature as judged from their Thy1.2high phenotype and inefficient T-bet expression. Thymic IEL precursors isolated from Irf2-/- mice failed to efficiently generate CD8αα+TCRαβ+ and TCRγδ+ IELs upon transfer in vivo and CD8αα+TCRαβ+ cells in response to IL-15 in vitro. Double mutant mice lacking both interleukin-15 (IL-15) and IRF-2 showed an even more severe iIEL defect than in mice lacking IL-15 alone. Upon increasing agonistic TCR signal strength through OT-II TCR transgenesis, CD8αα+TCRαβ+ iIELs became more abundant but remained immature on the Irf2-/- background. Our current observations, thus, revealed the unique bimodal role that IRF-2 plays in promoting not only generation of IEL progenitors in the thymus but also maturation of iIELs in the periphery in IL-15-dependent and -independent manners., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

γδT intraepithelial lymphocyte represents up to 60% of the small intestine intraepithelial compartment. They are highly migrating cells and constantly interact with the epithelial cell layer and lamina propria cells. This migratory phenotype is related to the homeostasis of the small intestine, the control of bacterial and parasitic infections, and the epithelial shedding induced by LPS. Here, we demonstrate that Myo1f participates in the adhesion and migration of intraepithelial lymphocytes. Using long-tailed class I myosins KO mice, we identified the requirement of Myo1f for their migration to the small intestine intraepithelial compartment. The absence of Myo1f affects intraepithelial lymphocytes' homing due to reduced CCR9 and α4β7 surface expression. In vitro , we confirm that adhesion to integrin ligands and CCL25-dependent and independent migration of intraepithelial lymphocytes are Myo1f-dependent. Mechanistically, Myo1f deficiency prevents correct chemokine receptor and integrin polarization, leading to reduced tyrosine phosphorylation which could impact in signal transduction. Overall, we demonstrate that Myo1f has an essential role in the adhesion and migration in γδT intraepithelial lymphocytes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Martínez-Vargas, Sánchez-Bello, Miguel-Rodríguez, Hernández-Cázares, Santos-Argumedo and Talamás-Rohana.)

The aryl-hydrocarbon receptor (AHR) is a ligand-activated transcription factor that buoys intestinal immune responses. AHR induces its own negative regulator, the AHR repressor (AHRR). Here, we show that AHRR is vital to sustaining intestinal intraepithelial lymphocytes (IELs). AHRR deficiency reduced IEL representation in a cell-intrinsic fashion. Single-cell RNA sequencing revealed an oxidative stress profile in Ahrr -/- IELs. AHRR deficiency unleashed AHR-induced expression of CYP1A1, a monooxygenase that generates reactive oxygen species, increasing redox imbalance, lipid peroxidation, and ferroptosis in Ahrr -/- IELs. Dietary supplementation with selenium or vitamin E to restore redox homeostasis rescued Ahrr -/- IELs. Loss of IELs in Ahrr -/- mice caused susceptibility to Clostridium difficile infection and dextran sodium-sulfate-induced colitis. Inflamed tissue of inflammatory bowel disease patients showed reduced Ahrr expression that may contribute to disease. We conclude that AHR signaling must be tightly regulated to prevent oxidative stress and ferroptosis of IELs and to preserve intestinal immune responses., Competing Interests: Declaration of interests M. Colonna receives research support from Pfizer., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Under non-pathological conditions, human γδ T cells represent a small fraction of CD3 + T cells in peripheral blood (1-10%). They constitute a unique subset of T lymphocytes that recognize stress ligands or non-peptide antigens through MHC-independent presentation. Major human γδ T cell subsets, Vδ1 and Vδ2, expand in response to microbial infection or malignancy, but possess distinct tissue localization, antigen recognition, and effector responses. We hypothesized that differences at the gene, phenotypic, and functional level would provide evidence that γδ T cell subpopulations belong to distinct lineages. Comparisons between each subset and the identification of the molecular determinants that underpin their differences has been hampered by experimental challenges in obtaining sufficient numbers of purified cells. By utilizing a stringent FACS-based isolation method, we compared highly purified human Vδ1 and Vδ2 cells in terms of phenotype, gene expression profile, and functional responses. We found distinct genetic and phenotypic signatures that define functional differences in γδ T cell populations. Differences in TCR components, repertoire, and responses to calcium-dependent pathways suggest that Vδ1 and Vδ2 T cells are different lineages. These findings will facilitate further investigation into the ligand specificity and unique role of Vδ1 and Vδ2 cells in early immune responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sanz, Mann, Ryan, Bosque, Pennington, Hackstein and Soriano-Sarabia.)

Psoriasis vulgaris (PV) is an autoinflammatory dermatosis of unknown etiology. Current evidence suggests a pathogenic role of γδT cells, but the growing complexity of this population has made the offending subset difficult to pinpoint. The work on γδTCR int and γδTCR hi subsets, which express intermediate and high levels of γδTCR at their surface, respectively, is particularly scarce, leaving their inner workings in PV essentially unresolved. We have shown here that the γδTCR int /γδTCR hi cell composition and their transcriptom are related to the differential miRNA expression by performing a targeted miRNA and mRNA quantification (RT-qPCR) in multiplexed, flow-sorted γδ blood T cells from healthy controls ( n = 14) and patients with PV ( n = 13). A significant loss of miR-20a in bulk γδT cells (~fourfold decrease, PV vs. controls) largely mirrored increasing Vδ1 - Vδ2 - and γδ int Vδ1 - Vδ2 - cell densities in the bloodstream, culminating in a relative excess of γδ int Vδ1 - Vδ2 - cells for PV. Transcripts encoding DNA-binding factors ( ZBTB16 ), cytokine receptors ( IL18R1 ), and cell adhesion molecules ( SELPLG ) were depleted in the process, closely tracking miR-20a availability in bulk γδ T-cell RNA. Compared to controls, PV was also associated with enhanced miR-92b expression (~13-fold) in bulk γδT cells that lacked association with the γδT cell composition. The miR-29a and let-7c expressions remained unaltered in case-control comparisons. Overall, our data expand the current landscape of the peripheral γδT cell composition, underlining changes in its mRNA/miRNA transcriptional circuits that may inform PV pathogenesis.

Many studies have demonstrated a general decline and dysregulation in immune functions with age. It is not clear, however, how the aging affects the immune surveillance of the female reproductive tract (FRT) by γδ T cells, a unique population of T lymphocytes that was shown to regulate homeostasis of epithelial barriers. First, we analyzed γδ T cell presence in FRT in young (2 months) and old (18 months) wild-type (WT) C57BL/6 mice. We did not detect any changes in γδ T cell number nor distribution in the vaginas between the age groups, while in uteri, there was a twofold increase in γδ T cell number in aged mice. To check if γδ T lymphocytes regulate a metabolic and immune status of aging vaginal tissue, we compared the expression of 84 aging-associated genes in young and old WT and γδ T-cell-deficient ( Tcrd -/- ) mice. We discovered that only the Ltf (lactotransferrin) gene was downregulated in old Tcrd -/- mice. In both mouse strains, we found similar age-dependent changes in cytokine production upon vaginal inflammation due to Toll-like receptor 9 (TLR9) stimulation with CpG. With age in the vaginas, IL-1 α and IL-17A levels increased while IL-6, IL-10, MCP-1, and IFN γ levels were diminished in response to CpG. Similar trends were observed in uteri. Interestingly, under the inflammatory state, the lack of γδ T cells in young individuals enhanced MCP-1 production in the vagina and decreased MCP-1 level in the uterus in old females. Our gene expression data point to an antimicrobial role of γδ T lymphocytes. The profile of secreted inflammatory cytokines shifted during aging toward the proinflammatory type, and γδ T cells played a modest fine-tuning role in immunoregulation in aged FRT. We believe this work expands our understanding of γδ T cell functions and the inflammaging in the murine reproductive epithelia., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 Katarzyna Skulska et al.)

Gamma-delta (γδ) T cells contribute to the pathology of many immune-related diseases; however, no ex vivo assays to study their activities are currently available. Here, we established a methodology to characterize human allergen-reactive γδ T cells in peripheral blood using an activation-induced marker assay targeting upregulated 4-1BB and CD69 expression. Broad and reproducible ex vivo allergen-reactive γδ T cell responses were detected in donors sensitized to mouse, cockroach, house dust mite, and timothy grass, but the response did not differ from that in non-allergic participants. The reactivity to 4 different allergen extracts was readily detected in 54.2%-100% of allergic subjects in a donor- and allergen-specific pattern and was abrogated by T cell receptor (TCR) blocking. Analysis of CD40L upregulation and intracellular cytokine staining revealed a T helper type 1 (Th1)-polarized response against mouse and cockroach extract stimulation. These results support the existence of allergen-reactive γδ T cells and their potential use in rebalancing dysregulated Th2 responses in allergic diseases., Competing Interests: The authors have declared no conflict of interest., (© 2022 The Author(s).)

Mycobacterium leprae is a kind of disease-causing bacteria and results in leprosy in human. Gamma delta (γδ) T cell is a T-cell subset that is presented in both human dermis and epidermis. These cells bridge innate and adaptive immune responses and play critical roles in regulating anti-microbial defense, wound healing, and skin inflammation. Here, we investigated skin resident γδ T cells in patients with leprosy. Our data showed that γδ T cells significantly accumulated in skin lesions of leprosy patients with tuberculoid (TT) form. IL-23 can predominantly stimulate dermal γδ T cells to produce interleukin 17 (IL-17), a cytokine which may lead to disease protection. These γδ T cells expressed a specific set of surface molecules, and majority of these cells were Vδ1 + . Also, IL-23 can stimulate the expansion of dermal γδ T cells expansion. Moreover, our results revealed that the transcription factor RORγt was responsible for IL-17A expression in leprosy lesion. Therefore, these data indicated that IL-23-responsive dermal γδ T cells were the major resource of IL-17A production in the skin and could be a potential target in the treatment of leprosy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Shi, Ma, Ma, Lu, Zhu and Yang.)

Description of immune landscapes in malignant microenvironment is critical to the improvement of therapeutic strategies for various tumors. Acute myeloid leukemia (AML) remains a severe life-threatening malignancy and often confronts treatment dilemma in clinic. Although γδ T cells exhibit independent and potent cytotoxicity against leukemic cells in vitro and in the mouse models, efficacy of γδ T cell-based immunotherapy on AML patients has seemed unsatisfying so far. How the anti-AML capacity of γδ T cells is suppressed in vivo remains elusive. Herein, we found an aberrant γδ T cells subset expressing CD25 + CD127 low Vδ2 + in the bone marrows of patients with newly diagnosed AML. The emergence of this subset was significantly associated with disease status and risk stratification as well as with the abnormally increased bone morphogenetic protein 2 (BMP2). Mechanistically, BMP2 could directly induce CD25 + CD127 low Vδ2 + γδ T cells (named as Reg-Vδ2) in vitro . The immunosuppressive features of Reg-Vδ2 cells were identified by combining immunophenotypical and functional data. Furthermore, inhibition of BMP2 pathway significantly blocked the emergence of Reg-Vδ2 cells and enhanced the anti-AML immunity in humanized mice. These findings not only provide a novel insight into the mechanisms of immunosuppression in the context of leukemia, but also suggest potential targets for the treatment of AML and other hematopoietic malignancies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liang, Dong, Yue, Gao, Wu, Liu, Chang, Hao, Hu, Zhao, Jiang, Huang and Liu.)

Gut intraepithelial lymphocytes (IELs) are thought to calibrate glucagon-like peptide 1 (GLP-1) bioavailability, thereby regulating systemic glucose and lipid metabolism. Here, we show that the gut IEL GLP-1 receptor (GLP-1R) is not required for enteroendocrine L cell GLP-1 secretion and glucose homeostasis nor for the metabolic benefits of GLP-1R agonists (GLP-1RAs). Instead, the gut IEL GLP-1R is essential for the full effects of GLP-1RAs on gut microbiota. Moreover, independent of glucose control or weight loss, the anti-inflammatory actions of GLP-1RAs require the gut IEL GLP-1R to selectively restrain local and systemic T cell-induced, but not lipopolysaccharide-induced, inflammation. Such effects are mediated by the suppression of gut IEL effector functions linked to the dampening of proximal T cell receptor signaling in a protein-kinase-A-dependent manner. These data reposition key roles of the L cell-gut IEL GLP-1R axis, revealing mechanisms linking GLP-1R activation in gut IELs to modulation of microbiota composition and control of intestinal and systemic inflammation., Competing Interests: Declaration of interests D.J.D. has served as a speaker or consultant for Altimmune, Amgen, AMT Inc., Eli Lilly Inc., Kallyope Inc., Merck Inc., Novo Nordisk Inc., and Pfizer Inc. R.J.S. serves as a paid consultant for Novo Nordisk, Scohia, Fractyl, and ShouTi. R.J.S. has equity positions in Calibrate and Rewind Health., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Intestinal intraepithelial lymphocytes (IELs) play a crucial role in host defence against pathogens in the intestinal mucosa. The development of intestinal IELs is distinct from peripheral T lymphocytes and remains elusive. Fas-associated protein with death domain (FADD) is important for T cell development in the thymus. Here we describe a novel function of FADD in the IEL development. FADD (S191A), a mouse FADD mutant at Ser191 to Ala mimicking constitutively unphosphorylated FADD, promoted a rapid expansion of TCRαβ + IELs, not TCRγδ + IELs. Mechanism investigation indicated that the dephosphorylation of FADD was required for cell activation mainly in TCRαβ + CD8 + T cells. Consistently, FADD (S191A) as dephosphorylated FADD led to a high NF-κB activation in the TCR-dependent cell expansion. In addition, The FADD (S191A)-induced abnormal IEL populations resulted in the increased incidence and severity of colitis in mice. In summary, FADD signalling is involved in the intestinal IEL development and might be a regulator for intestinal mucosal homeostasis., (© 2022 John Wiley & Sons Ltd.)

Loss of Paneth cells and their antimicrobial granules compromises the intestinal epithelial barrier and is associated with Crohn's disease, a major type of inflammatory bowel disease 1-7 . Non-classical lymphoid cells, broadly referred to as intraepithelial lymphocytes (IELs), intercalate the intestinal epithelium 8,9 . This anatomical position has implicated them as first-line defenders in resistance to infections, but their role in inflammatory disease pathogenesis requires clarification. The identification of mediators that coordinate crosstalk between specific IEL and epithelial subsets could provide insight into intestinal barrier mechanisms in health and disease. Here we show that the subset of IELs that express γ and δ T cell receptor subunits (γδ IELs) promotes the viability of Paneth cells deficient in the Crohn's disease susceptibility gene ATG16L1. Using an ex vivo lymphocyte-epithelium co-culture system, we identified apoptosis inhibitor 5 (API5) as a Paneth cell-protective factor secreted by γδ IELs. In the Atg16l1-mutant mouse model, viral infection induced a loss of Paneth cells and enhanced susceptibility to intestinal injury by inhibiting the secretion of API5 from γδ IELs. Therapeutic administration of recombinant API5 protected Paneth cells in vivo in mice and ex vivo in human organoids with the ATG16L1 risk allele. Thus, we identify API5 as a protective γδ IEL effector that masks genetic susceptibility to Paneth cell death., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Intestinal intraepithelial lymphocytes (IELs) are distributed along the length of the intestine and are considered the frontline of immune surveillance. The precise molecular mechanisms, especially epigenetic regulation, of their development and function are poorly understood. The trimethylation of histone 3 at lysine 27 (H3K27Me3) is a kind of histone modifications and associated with gene repression. Kdm6b is an epigenetic enzyme responsible for the demethylation of H3K27Me3 and thus promotes gene expression. Here we identified Kdm6b as an important intracellular regulator of small intestinal IELs. Mice genetically deficient for Kdm6b showed greatly reduced numbers of TCRαβ + CD8αα + IELs. In the absence of Kdm6b, TCRαβ + CD8αα + IELs exhibited increased apoptosis, disturbed maturation and a compromised capability to lyse target cells. Both IL-15 and Kdm6b-mediated demethylation of histone 3 at lysine 27 are responsible for the maturation of TCRαβ + CD8αα + IELs through upregulating the expression of Gzmb and Fasl. In addition, Kdm6b also regulates the expression of the gut-homing molecule CCR9 by controlling H3K27Me3 level at its promoter. However, Kdm6b is dispensable for the reactivity of thymic precursors of TCRαβ + CD8αα + IELs (IELPs) to IL-15 and TGF-β. In conclusion, we showed that Kdm6b plays critical roles in the maturation and cytotoxic function of small intestinal TCRαβ + CD8αα + IELs., (© 2021. The Author(s).)