BACKGROUND:Chronic hepatitis B virus (HBV) infection is characterised by immune dysfunction. While conventional T cell responses have been extensively studied, the role of γδ T cells, innate-like cytotoxic lymphocytes enriched in the liver, remains incompletely understood. OBJECTIVE:To characterise γδ T cell subsets in HBV infection and assess their association with viral control and antibody-dependent cellular cytotoxicity (ADCC). DESIGN:Peripheral blood from patients with chronic (n=83) and acute (n=16) HBV infection, healthy controls (n=31), and cord-blood donors (n=3) was analysed using multiparameter flow cytometry, single-cell RNA sequencing and in vitro ADCC assays. RESULTS:A distinct CD16+ γδ T cell subset inversely correlated with hepatitis B core-related antigen (HBcrAg), a surrogate of intrahepatic viral replication. CD16+ γδ T cells displayed a cytotoxic signature, whereas CD16⁻ cells showed inflammatory, non-cytotoxic profiles. On hepatitis B surface antigen-specific antibody stimulation, CD16+ γδ T cells mounted potent ADCC responses, mainly mediated by Vδ2+ cells expressing the activating receptor CD226, while Vδ1+ cells preferentially expressed the inhibitory receptor TIGIT. Cytotoxic CD16+ Vδ2+ γδ T cells were present in both blood and liver. CD16+ γδ T cells were expanded and highly functional in acute HBV but reduced and partially impaired in chronic infection. Neonatal cord-blood-derived γδ T cells lacked CD16 expression and displayed limited ADCC potential. CONCLUSIONS:CD16+ γδ T cells mediate antibody-dependent antiviral immunity in HBV infection. Their inverse association with HBcrAg links γδ T cell-mediated ADCC to viral control and highlights this pathway as a target for HBV cure strategies.

γδ T cells are one of the first T cell subsets developing in early ontogeny and show various effector functions in immune homeostasis and response in the young and the old. However, their maturation trajectories from infancy to children, adults and elderly have not been systematically defined. Here, we generated a single-cell transcriptome atlas of 106,711 γδ T cells from 223 individuals spanning infancy to old age. Our analysis reveals that γδ T cell aging is non-linear, characterized by pronounced childhood transitions followed by relative stability throughout adulthood despite marked inter-individual variability. In childhood, changes from developmental and mitochondrial programs toward cytotoxicity and inflammaging were evident. This includes maturation trajectories from GZMK⁺ intermediates to GZMB+Perforin+ effectors at both RNA and protein levels. Taken together, our study delineates the aging trajectories of human γδ T cells, establishes γδ T cells as a cellular paradigm of non-linear immune aging, and provides a comprehensive resource for investigating γδ T cell biology across the human lifespan. ### Competing Interest Statement The authors have declared no competing interest. German Research Foundation (DFG), EXC 2155 (RESIST), Project ID 390874280, FOR2799, Project ID RA3077/1-2 Hannover Medical School (MHH), HiLF (internal project funding)

Cytomegalovirus (CMV) reactivation is a frequent complication after allogeneic hematopoietic stem cell transplantation (aHSCT) and critically shapes immune reconstitution. However, the clonal and functional dynamics of T cell responses to CMV remain insufficiently defined. In this study, we combined longitudinal single-cell RNA sequencing with paired T cell receptor sequencing to track γδ and αβ T cell clones at clonal resolution across five post-transplant time points in patients with and without CMV reactivation. This integrative approach revealed marked, patient-specific expansion of non-Vγ9Vδ2 γδ T cell clones, particularly within Vδ1⁺ and Vδ3⁺ subsets, which was associated with differentiation toward cytotoxic and antiviral effector states characterized by IFN-γ and TNF-α expression. Conventional CD8⁺ αβ T cells showed comparatively modest clonal dynamics during CMV reactivation in this cohort. Longitudinal tracking of individual clonotypes demonstrated heterogeneous but recurrent trajectories, with expanding γδ T cell clones frequently acquiring antiviral and cytotoxic phenotypes following CMV reactivation. These findings highlight the adaptive-like behavior and functional plasticity of non-Vγ9Vδ2 γδ T cells and provide a high-resolution framework for studying antiviral immune responses during immune reconstitution.

Background Although most studies of anticancer T-cell immunity focus on αβ T cells, γδ T cells are attracting increasing attention due to their involvement in antitumor immune responses in various cancer entities, including melanoma. While immune checkpoint blockade (ICB) using the antagonistic programmed cell death protein 1 (PD-1) antibodies nivolumab and pembrolizumab significantly improved the survival of patients with melanoma with distant metastasis, prognosis remains poor. PD-1 is not only expressed by αβ T cells but also by γδ T cells, making this numerically minor population of unconventional T cells, whose role in melanoma is still elusive, a target of ICB.Methods Here, we present a detailed γδ T-cell profiling study in late-stage melanoma at single-cell level using mass and polychromatic flow cytometry, T-cell receptor repertoire analyses and immunohistochemistry.Results Our analyses link high frequencies of peripheral Vδ1 T cells before the start of anti-PD-1 therapy to a significantly reduced overall survival. In these patients, the Vδ1 compartment is dominated by a late-differentiated senescent-like phenotype that is presumably unresponsive to therapy. This phenotype is less prevalent at the tumor site and analysis of RNA sequencing data revealed that the abundance of Vδ1 T cells within the tumor was positively associated with survival.Conclusions Our study suggests that Vδ1 T cells are associated with clinical outcomes, with a responsive subset expanding under ICB in patients where such a response remains possible. The observed clinical effects may be supported by the infiltration of these cells into the tumor, where they contribute to cancer immunosurveillance.

Colorectal cancer (CRC) remains a significant global health concern. Improving the efficacy of immunotherapy, particularly for microsatellite-stable tumors, requires a better understanding of the unconventional T-cell populations that regulate intestinal immunity. γδ T cells are uniquely positioned at the epithelial barrier and function as rapid sentinels, recognizing stress signals independently of classical antigen presentation. In a healthy colon, γδ intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) form separate compartments influenced by tissue-specific butyrophilin-like (BTNL) interactions, microbiota-derived signals, and cytokine environments. These signals imprint divergent effector programs, ranging from IFN-γ-producing, cytotoxic responses to IL-17-driven tissue repair and inflammation. In CRC, however, these subsets exhibit remarkable plasticity. In mouse models, for example, Vγ1+ and Vγ7+ IELs mediate potent antitumor immunity, whereas Vγ4+ and Vγ6+ LPLs can acquire IL-17-dependent pro-tumor functions. In contrast, human data depict a different balance. Across multiple cohorts, tumor-infiltrating γδ T cells, predominantly the Vδ1+ and Vδ2+ subsets, exhibit robust cytotoxic and IFN-γ-associated phenotypes. Meanwhile, the existence of bona fide IL-17-producing γδ T cells remains highly controversial. Higher γδ T-cell abundance correlates with better outcomes, even in tumors with defective HLA class I expression, where γδ T cells can mediate the therapeutic effects of PD-1 blockade. Emerging findings reveal subset heterogeneity, context-dependent functional states, and a crucial role for NK-receptor-mediated recognition, particularly via NKG2D. Together, these insights position γδ T cells as pivotal yet understudied regulators of CRC progression and immunotherapy responsiveness. Understanding their subset-specific biology could lead to next-generation γδ-based therapies tailored to the unique immunogenic constraints of colorectal cancer.

The perinatal period represents a unique immunological window of opportunity, in which γδ T cells play a central role. New methods, including single-cell and high-resolution TCR repertoire analyses, have transformed our understanding of how γδ T cells develop, diversify, and function from fetal life into adulthood. Early γδ T cell waves display TCRs shaped by low TdT activity, biased V(D)J recombination, and favored V-J and V-D pairings that give rise to public, near germline-encoded TCRs with preprogrammed effector functions. These fetal-derived populations contribute to rapid pathogen responses and unique pathogen-driven expansions during congenital infections, revealing fundamental differences between perinatal and adult γδ T cell immunity. However, it is less clear how pre- and perinatal γδ T cells persist and influence individual immune responses later in life. This review synthesizes current insights into ontogeny, effector programming, and repertoire dynamics of γδ T cells in early life. We focus on deciphering the evident changes in the γδ T cell compartment during gestation, birth, and early life in humans.

Resolving T cell clonality at single-cell spatial resolution remains a major challenge. Here, we developed an in situ hybridization panel comprising probes for immune and tissue cell types alongside comprehensive coverage of TRAV, TRBV, TRGV, and TRDV gene segments, enabling unbiased spatial mapping of T cell clones in situ. As a proof of principle, we applied this approach to human kidney biopsies from patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis and controls. Combinatorial T cell receptor variable (TRV) gene expression allowed identification of T cell clones and their spatial organization. Confined clusters of clonally related αβ T cells were found in proximity to increased numbers of antigen-presenting cells, consistent with local immune activation, while γδ T cells occupied distinct peripheral niches. Although paired αβ chain detection is currently limited, this method establishes a scalable framework for spatially resolved clonotype analysis and provides a broadly applicable tool to investigate tissue-level immune organization across diseases.

OBJECTIVE:Cerebral ischemia remains a major cause of disability, and the contribution of the hyperacute immune response is increasingly recognized. The aim of this study was to investigate the local inflammatory response in the affected brain of stroke patients with large vessel occlusion during the hyperacute phase of stroke. METHODS:To decipher the role of myeloid immune cells in stroke-induced inflammation, we performed an unsupervised multiomics analysis of innate immune pathways in ischemic blood obtained directly from the occluded middle cerebral artery in 54 patients undergoing mechanical recanalization. Paired nonischemic arterial blood served as an internal control. RESULTS:Stroke triggered a local hyperacute activation of classical monocytes and neutrophils in the ischemic cerebral vasculature, with interleukin (IL)-1β emerging as a key mediator of inflammation. Elevated plasma adenosine triphosphate levels and inflammasome priming in intravascular monocytes were associated with IL-1β production within the occluded middle cerebral artery within 4.5 hours of stroke onset. IL-1β release coincided with increased neutrophil-attracting chemokines (C-X-C motif chemokine ligand 1 [CXCL1], IL-8). Locally activated neutrophils formed neutrophil extracellular traps (NETs) within the ischemic vasculature, a hallmark of thromboinflammation. Postmortem analyses revealed NET deposition within ischemic brain parenchyma. INTERPRETATION:These findings indicate increased IL-1β expression and enhanced NET formation within the cerebral circulation in stroke caused by large vessel occlusion, suggesting that these mechanisms might contribute to early stroke pathophysiology and represent potential targets for immunomodulatory strategies. ANN NEUROL 2026.

Efficient production of milligram quantities of properly folded, functional eukaryotic proteins remains a significant challenge for structural and biochemical research, especially for membrane proteins and complex glycoproteins. The Drosophila Schneider 2 (S2) expression system provides a scalable, cost-effective alternative to baculovirus and mammalian platforms. However, its broader use is hampered by lengthy cell line development and variable expression levels. In this study, we present an optimized S2 expression workflow that reduces the time required to create stable cell lines from approximately 4 weeks to about 2 weeks without compromising protein yield. To further enhance expression, especially for low-expressing constructs, we used fluorescence-activated cell sorting (FACS) to select for high-expressing oligoclonal populations. Using a soluble human leucocyte antigen class II (HLA) and the transmembrane protein hCD81 as model proteins, FACS enrichment increased yields by ∼124-fold for the soluble protein and by ∼4.6-fold for the membrane protein. Protein functionality was confirmed through receptor-binding assays, SEC-MALS, and high-resolution crystallography for HLA, and ligand-binding assays for hCD81. Overall, this faster, scalable workflow enables rapid, high-yield production of structurally intact, functionally active soluble and membrane proteins from small culture volumes, establishing S2 cells as a fast and reliable platform for eukaryotic protein expression.

γδ T cells are one of the first T cell subsets developing in early ontogeny and show various effector functions in immune homeostasis and response in the young and the old. However, their maturation trajectories from infancy to childhood, adulthood, and old age have not been systematically defined. Here, we generated a single-cell transcriptome atlas of 106,711 γδ T cells from 223 individuals spanning infancy to old age. Our analysis reveals that γδ T cell aging is non-linear, characterized by pronounced childhood transitions followed by relative stability throughout adulthood despite marked inter-individual variability. In childhood, changes from developmental and mitochondrial programs toward cytotoxicity and inflammaging were evident. This includes maturation trajectories from GZMK⁺ intermediates to GZMB+Perforin+ effectors at both RNA and protein levels. Taken together, our study delineates the aging trajectories of human γδ T cells, establishes γδ T cells as a cellular paradigm of non-linear immune aging, and provides a comprehensive resource for investigating γδ T cell biology across the human lifespan.

Fungal skin infections significantly contribute to the global human disease burden, yet our understanding of cutaneous immunity against dermatophytes remains limited. Previously, we developed a model of epicutaneous infection with Microsporum canis in C57BL/6 mice, which highlighted the critical role of IL-17RA signaling in antidermatophyte defenses. In this study, we expanded our investigation to the human pathogen Nannizzia gypsea and demonstrated that skin yBTCRint and CD8/CD4 double-negative (3TCR+ T cells are the principal producers of IL-17A during dermatophytosis. These IL-17A+ T cells exhibited an activated/memory phenotype, including a subset of proliferating tissue-resident cells. Notably, restriction of lymphocyte trafficking after fingolimod administration in infected mice did not lead to increased susceptibility, indicating that local antifungal defenses are independent of T-cell priming in lymph nodes. In addition, Rag1-/- mice lacking T and B lymphocytes effectively controlled infection and exhibited increased IL-17A production by innate lymphoid cells. Furthermore, Rag2-/-Il2rg-/- mice, devoid of T, B, and innate lymphoid cells, were highly susceptible to dermatophytosis compared with Rag2-/-or wild-type mice, demonstrating that innate lymphoid cells are sufficient to antifungal defenses in T-cell-deficient mice. In conclusion, our study underscores the coordinated interplay between skin yBT, a(3T, and innate lymphoid cell subsets in controlling primary N gypsea dermatophytosis.

γδ T cells, long regarded as unconventional relatives of αβ T cells, have emerged as pivotal players in immunity, with unique biology and therapeutic promise. Recent advances in single-cell multiomics, refined mouse models, and human cohort studies have deepened insights into their TCR-ligand interactions, developmental pathways, and context-dependent functions. This mini-review synthesizes current understanding from structural studies of γδ TCR recognition and developmental regulation-including inborn errors of immunity-to adaptive-like clonal expansions shaped by infection, aging, and environmental cues. It also highlights their dual roles in cancer, where subsets can exert potent cytotoxicity or promote tumor progression, and discusses strategies to optimize their antitumor potential through checkpoint blockade, metabolic modulation, and engineered receptors. Beyond immunity to malignancy, γδ T cells contribute to tissue homeostasis, repair, and regulation of inflammatory processes in diverse organs, influencing outcomes in neuroinflammation, autoimmunity, and fibrotic diseases. Together, these perspectives form the foundation of a special issue in the European Journal of Immunology (https://onlinelibrary.wiley.com/doi/toc/10.1002/(ISSN)1521-4141.T-cells) dedicated to advancing the understanding of γδ T cell biology and clinical potential.

Oral squamous cell carcinoma (OSCC) is a highly aggressive malignancy, with a low 5-y survival rate and frequent local recurrence or metastasis. This study explores the role of γδT cells in the development and progression of OSCC. γδT cells, which exhibit innate and adaptive immune characteristics, are known for their dual role in cancer, acting as anti- and protumor agents depending on the context. Using a murine model of OSCC induced by the carcinogen 4-nitroquinoline-1-oxide (4NQO), which adequately mimics the progression of human OSCC, we investigated the impact of γδT cells on tumor growth and the tumor microenvironment. We first characterized the γδT cells of the tongue epithelium, the primary site for cancer development in this model. The results indicate that γδT cells are predominantly of the Vγ6+ subset, expanding postnatally in a microbiota-dependent manner. Upon 4NQO administration, depletion of γδT cells did not significantly alter the kinetics of OSCC progression but did result in a reduction in tumor size and number, suggesting a role in promoting tumor growth. Interestingly, the absence of IL-17, a key cytokine produced by the Vγ6+ subset, also resulted in reduced tumor volume without affecting disease progression, corroborating the protumor role of these cells in OSCC. Further analysis revealed that IL-17-producing γδT cells facilitate angiogenesis within the tumor microenvironment by promoting the expression of angiogenic factors. Of note, while 4NQO treatment increased the oral microbial load and altered its composition, IL-17 deficiency did not affect the oral microbiota, indicating that the effects of IL-17-producing γδT cells on OSCC are independent of microbial changes. This study highlights the pathologic role of IL-17-producing γδT cells in OSCC, particularly in promoting tumor growth through angiogenesis. This underscores the importance of γδT cells in OSCC and the need for further research into therapeutic strategies targeting these cells.

Alloreactive T cells mediate graft-versus-leukemia (GvL) reactions and acute graft-versus-host disease (aGvHD) in AML patients following allogeneic hematopoietic stem cell transplantation. To investigate biomarkers that identify alloreactive T cells associated with either beneficial GvL or detrimental aGvHD, we collected graft samples and two post-transplant follow-up blood samples (day 30 and day 100) of ten AML patients undergoing hematopoietic stem cell transplantation and profiled over 777,000 CD45+ leukocytes in total by combinatorial barcoding-based mega-scale single-cell RNA sequencing. Using immune receptor sequences as intrinsic clonal barcodes, we observed that especially CD8+ graft-derived T cells persisted and displayed enhanced proliferation, clonal expansion, and likely alloreactivity. Notably, patient-derived peripheral leukocytes that survived the conditioning, as identified by sex-chromosome-related genes, were primarily CD4+ T helper cells. MDGA1 expression on T cells and NK cells emerged as a novel biomarker potentially associated with aGvHD. Additionally, we observed a significant deficiency of ADGRG1 expression, a marker of alloreactive cytotoxic T cells, by αβ and γδ T cells from relapsed patients. In conclusion, mega-scale single-cell monitoring of graft and hematopoietic immune cell reconstitution allowed us to demonstrate that MDGA1 and ADGRG1 may function as complementary biomarkers expressed by distinct circulating T cells that are associated with divergent outcomes in AML patients, enabling precise risk stratification of alloHSCT outcomes and presenting potential therapeutic targets.

Intraepithelial lymphocytes expressing the γδ T cell receptor (γδ IELs) provide immunosurveillance of the intestinal barrier but are reduced in patients with active Crohn’s disease (CD). Here, we report an underappreciated role for γδ IELs in maintaining immunological tolerance during the onset and progression of CD-like ileitis using TNF ΔARE/+ mice. We found that TNF-induced down-regulation of epithelial hepatocyte nuclear factor 4-gamma/butyrophilin is followed by a loss of ileal Vγ7 IELs and impaired barrier surveillance before the histological onset of disease. A reduction of immunoregulatory CD39 + γδ IELs coincided with the influx of immature, peripheral CD39 neg γδ T cells into the epithelium, leading to an expansion of induced IELs, whereas an earlier depletion of γδ IELs correlated with accelerated onset of ileal inflammation. Our findings identify multiple layers of γδ IEL dysregulation before ileitis development, indicating that the loss of steady-state immunoregulatory γδ IELs may contribute to the initiation of ileal CD.

Conventional dendritic cells (cDCs) are important antigen presenting cells which link innate and adaptive immunity by transferring antigenic information from peripheral organs to T cells in lymph nodes (LNs). However, despite their central function in the induction of adaptive immune responses, the kinetics and molecular regulation of the cDC life cycle and migration remain poorly understood. Using a variety of in vivo techniques, we examine the kinetics of cDC turnover in the intestine and address the molecular changes throughout the various stages of the cDC life cycle - from tissue entry and differentiation to CCR7 upregulation and subsequent migration into draining LNs. Our data demonstrate that the life cycle of gut cDCs is highly dynamic, characterised by continuous alterations in transcriptome, protein expression and proliferation rates. These progressive changes culminate in cDC homeostatic activation and migration resulting in a resource-intensive daily turnover of up to a quarter of intestinal cDCs and an almost complete daily replacement of the migratory cDC compartment in the mesenteric LN. This high turnover rate ensures that the mesenteric LN maintains an accurate reflection of the intestinal immunological state, supporting rapid adaptation to emerging immune challenges.

γδ T cells are unconventional T cells that group into different subsets based on the usage of variable γδ T cell receptor (TCR) gene segments, body location, and functionality. γδ T cells that secrete the proinflammatory cytokine interleukin 17 (IL-17) predominantly express a Vγ4 + or Vγ6 + γδ TCR. The biology and the importance of the γδ TCR of IL-17-producing γδ T cells are not well understood. Here, we investigated the IL-17 production capability of γδ T cells in mice deficient for Vγ4 + and Vγ6 + γδ TCRs using flow cytometry, TCR-seq, and single-cell transcriptomics. Our data show that Vγ1 T cells only partially compensate for the loss of IL-17 + Vγ4 and Vγ6 T cell subsets in lymphoid and nonlymphoid tissues. They develop pre- and postnatally and were predominantly detectable in their physiological body habitats. Collectively, the data underscore the nonredundant roles of Vγ4⁺ and Vγ6⁺ subsets in IL-17-mediated immunity.

The physiological significance of thymic positive selection and its reliance on a single stromal cell type, cortical thymic epithelial cells, remain incompletely understood. The lysosomal cysteine protease cathepsin L (CTSL) has been implicated in generating major histocompatibility complex class II-bound peptides in cortical thymic epithelial cells for efficient CD4+ T cell differentiation. Here, we addressed the extent and nature of the CD4+ T cell repertoire changes associated with CTSL deficiency. In the absence of CTSL, a highly selective loss of T cell receptors resulted in a markedly reduced repertoire diversity. However, a similarly large proportion of nominally 'CTSL-independent' T cell receptors were retained. Clones representative of the second category experienced weaker positive selection signals in the absence of CTSL, which were sufficient for further maturation yet imprinted aberrant responsiveness to agonist stimulation and impaired homeostatic behavior. Together, these findings demonstrate that CTSL is crucial for both shaping full repertoire diversity and optimizing CD4+ T cell functionality.