Unraveling the role of γδ T cells in the pathogenesis of an oncogenic avian herpesvirus.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that causes deadly lymphomas in chickens. In chickens, up to 50% of all peripheral T cells are gamma delta (γδ) T cells. Until now, their role in MDV pathogenesis and tumor formation remains poorly understood. To investigate the role of γδ T cells in MDV pathogenesis, we infected recently generated γδ T cell knockout chickens with very virulent MDV. Strikingly, disease and tumor incidence were highly increased in the absence of γδ T cells, indicating that γδ T cells play an important role in the immune response against MDV. In the absence of γδ T cells, virus replication was drastically increased in the thymus and spleen, which are potential sites of T cell transformation. Taken together, our data provide the first evidence that γδ T cells play an important role in the pathogenesis and tumor formation of this highly oncogenic herpesvirus.IMPORTANCEGamma delta (γδ) T cells are the most abundant T cells in chickens, but their role in fighting pathogens remains poorly understood. Marek's disease virus (MDV) is an important veterinary pathogen, that causes one of the most frequent cancers in animals and is used as a model for virus-induced tumor formation. Our study revealed that γδ T cells play a crucial role in combating MDV, as disease and tumor incidence drastically increased in the absence of these cells. γδ T cells restricted virus replication in the key lymphoid organs, thereby decreasing the likelihood of causing tumors and disease. This study provides novel insights into the role of γδ T cells in the pathogenesis of this highly oncogenic virus.

Key players of immunosuppression in epithelial malignancies: Tumor-infiltrating myeloid cells and γδ T cells.

BACKGROUND: The tumor microenvironment of solid tumors governs the differentiation of otherwise non-immunosuppressive macrophages and gamma delta (γδ) T cells into strong immunosuppressors while promoting suppressive abilities of known immunosuppressors such as myeloid-derived suppressor cells (MDSCs) upon infiltration into the tumor beds. RECENT FINDINGS: In epithelial malignancies, tumor-associated macrophages (TAMs), precursor monocytic MDSCs (M-MDSCs), and gamma delta (γδ) T cells often acquire strong immunosuppressive abilities that dampen spontaneous immune responses by tumor-infiltrating T cells and B lymphocytes against cancer. Both M-MDSCs and γδ T cells have been associated with worse prognosis for multiple epithelial cancers. CONCLUSION: Here we discuss recent discoveries on how tumor-associated macrophages and precursor M-MDSCs as well as tumor associated-γδ T cells acquire immunosuppressive abilities in the tumor beds, promote cancer metastasis, and perspectives on how possible novel interventions could restore the effective adaptive immune responses in epithelial cancers.

Functional and biological implications of clonotypic diversity among human donor-unrestricted T cells.

T cells express a T-cell receptor (TCR) heterodimer that is the product of germline rearrangement and junctional editing resulting in immense clonotypic diversity. The generation of diverse TCR repertoires enables the recognition of pathogen-derived peptide antigens presented by polymorphic major histocompatibility complex (MHC) molecules. However, T cells also recognize nonpeptide antigens through nearly monomorphic antigen-presenting systems, such as cluster of differentiation 1 (CD1), MHC-related protein 1 (MR1) and butyrophilins (BTNs). This potential for shared immune responses across genetically diverse populations led to their designation as donor-unrestricted T cells (DURTs). As might be expected, some CD1-, MR1- and BTN-restricted T cells express a TCR that is conserved across unrelated individuals. However, several recent studies have reported unexpected diversity among DURT TCRs, and increasing evidence suggests that this diversity has functional consequences. Recent reports also challenge the dogma that immune cells are either innate or adaptive and suggest that DURT TCRs may act in both capacities. Here, we review this evidence and propose an expanded view of the role for clonotypic diversity among DURTs in humans, including new perspectives on how DURT TCRs may integrate their adaptive and innate immune functions.

Directing the migration of serum-free, ex vivo-expanded Vγ9Vδ2 T cells.

Vγ9Vδ2 T cells represent a promising cancer therapy platform because the implementation of allogenic, off-the-shelf product candidates is possible. However, intravenous administration of human Vγ9Vδ2 T cells manufactured under good manufacturing practice (GMP)-compliant, serum-free conditions are not tested easily in most mouse models, mainly because they lack the ability to migrate from the blood to tissues or tumors. We demonstrate that these T cells do not migrate from the circulation to the mouse bone marrow (BM), the site of many malignancies. Thus, there is a need to better characterize human γδ T-cell migration in vivo and develop strategies to direct these cells to in vivo sites of therapeutic interest. To better understand the migration of these cells and possibly influence their migration, NSG mice were conditioned with agents to clear BM cellular compartments, i.e., busulfan or total body irradiation (TBI), or promote T-cell migration to inflamed BM, i.e., incomplete Freund's adjuvant (IFA), prior to administering γδ T cells. Conditioning with TBI, unlike busulfan or IFA, increases the percentage and number of γδ T cells accumulating in the mouse BM, and cells in the peripheral blood (PB) and BM display identical surface protein profiles. To better understand the mechanism by which cells migrate to the BM, mice were conditioned with TBI and administered γδ T cells or tracker-stained red blood cells. The mechanism by which γδ T cells enter the BM after radiation is passive migration from the circulation, not homing. We tested if these ex vivo-expanded cells can migrate based on chemokine expression patterns and showed that it is possible to initiate homing by utilizing highly expressed chemokine receptors on the expanded γδ T cells. γδ T cells highly express CCR2, which provides chemokine attraction to C-C motif chemokine ligand 2 (CCL2)-expressing cells. IFNγ-primed mesenchymal stromal cells (MSCs) (γMSCs) express CCL2, and we developed in vitro and in vivo models to test γδ T-cell homing to CCL2-expressing cells. Using an established neuroblastoma NSG mouse model, we show that intratumorally-injected γMSCs increase the homing of γδ T cells to this tumor. These studies provide insight into the migration of serum-free, ex vivo-expanded Vγ9Vδ2 T cells in NSG mice, which is critical to understanding the fundamental properties of these cells.

Broad-spectrum defenders: γδ T cells take on a multitude of immune challenges.

A recent article in Proceedings of the National Academy of Sciences investigated γδ T cell antigen specificity in mice and humans, in which the authors show that γδ T cell antigen specificity is not constrained to one epitope. Rather, γδ T cells recognize a broad range of diverse antigens containing similar chemical structures or properties. In this News and Views, the importance of γδ T cell antigen polyspecificity during immune responses is highlighted.

Unraveling the secrets of γδ T cells with single-cell biology.

Recent technological advancements have enabled us to study the molecular features of cellular states at the single-cell level, providing unprecedented resolution for comprehending the identity and function of a cell. By applying these techniques across multiple time frames, tissues, and diseases, we can delve deeper into the mechanisms governing the development and functions of cell lineages. In this review, I focus on γδ T cells, which are a unique and functionally nonredundant T cell lineage categorized under the umbrella of unconventional T cells. I discuss how single-cell biology is providing unique insights into their development and functions. Furthermore, I explore how single-cell methods can be used to answer several key questions about their biology. These investigations will be essential to fully understand their translational potential, including their role in cytotoxicity and tissue repair in cancer and regeneration.

OMIP-099: 31-color spectral flow cytometry panel to investigate the steady-state phenotype of human T cells.

We have developed a 31-color panel to define the steady-state phenotype of T cells in human peripheral blood (Table 1). The panel presented here was optimized using cryopreserved peripheral blood mononuclear cells (PBMC). The markers included in this panel were chosen in order to characterize the steady-state phenotype of T cells and includes markers (CD45RA, CD45RO, CCR7, CD95) to distinguish the main subsets (e.g., naïve, TEM , TCM , TEMRA , TSCM etc.) of CD4, CD8, and γδ T cells. This panel also includes markers for the identification of differentiation status (CD27, CD28), activation/antigen experience status (CD11a, CD49d, CD38, HLA-DR, CD56, and CD39), co-inhibitory marker expression (PD-1, TIM-3), and CD4 T helper subsets (CXCR3, CXCR5, CCR4, CCR6, Foxp3, CD25, and CD127). This optimized panel provides a broad assessment of the steady-state phenotype of human T cells.

Surface protein and functional analyses identify CD4+CD39+ TCR αß+ and activated TCR Vδ1+ cells with distinct pro-inflammatory functions in Crohn's disease lesions.

Crohn's disease (CD) is a chronic immune-mediated disorder of the gastrointestinal tract. Extensive screening studies have revealed the accumulation of immune cell subsets with unique plasticity and immunoregulatory properties in patients with CD. We performed phenotypic and functional studies on inflamed and non-inflamed bioptic tissue to investigate the presence of distinct T cells in the intestinal mucosa of CD patients. We analysed hundreds of surface molecules expressed on cells isolated from the intestinal tissue of CD patients using anti-CD45 mAbs-based barcoding. A gene ontology enrichment analysis showed that proteins that regulate the activation of T cells were the most enriched group. We, therefore, designed T-cell focused multicolour flow-cytometry panels and performed clustering analysis which revealed an accumulation of activated TEM CD4+CD39+ T cells producing IL-17 and IL-21 and increased frequency of terminally differentiated TCR Vδ1+ cells producing TNF-α and IFN-γ in inflamed tissue of CD patients. The different functional capacities of CD4+ and TCR Vδ1+ cells in CD lesions indicate their non-overlapping contribution to inflammation. The abnormally high number of terminally differentiated TCR Vδ1+ cells suggests that they are continuously activated in inflamed tissue, making them a potential target for novel therapies.

Anti-PD1 does not improve pyroptosis induced by γδ T cells but promotes tumor regression in a pleural mesothelioma mouse model.

Introduction: Mesothelioma is an aggressive tumor in the pleural cavity that is difficult to treat. Diagnosis is usually late with minimal treatment options available for the patients and with unfavorable outcomes. However, recent advances in immunotherapy using γδ T cells may have potential against mesothelioma, given its ample tumoricidal and tumor-migratory properties could allow its infiltration to the widespread tumor mass. Thus, we hypothesize that Vδ2 T cells can perform cytotoxic activities against mesothelioma especially when combined with immune checkpoint blocker against PD-1. Methods: Human Vδ2 T cells were expanded from peripheral blood mononuclear cells using Tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino) ethylidene-1,1-bisphosphonate (PTA) plus IL-2 for 13 days, before used to test for cytotoxicity against mesothelioma cell lines. Mesothelioma-bearing mice was established by Intrapleural administration of mesothelioma cell lines to test for the efficacy of Vδ2 T cells plus anti-PD-1 antibody combination treatment. Pyroptosis was evaluated by cell morphology, western blot analysis, and ELISA experiments. Flow cytometry was used to examine expression of BTN2A1, BTN3A1, PD-L1, PD-L2 on mesothelioma cell lines. Immunofluorescence staining was performed to detect Vδ2 T cells post adoptive transfer and characteristics of pyroptosis in ex vivo mesothelioma tissue sections. Results: Indeed, our data demonstrated that Vδ2 T cells killing mesothelioma can be enhanced by anti-PD-1 antibody in vitro, especially for high PD-1 expressing cells, and in vivo in the intrapleural mesothelioma mice model established by us. Adoptive transfer of Vδ2 T cells into these mice leads to tumor regression by 30-40% compared to control. Immunofluorescence of the tumor section confirmed infiltration of Vδ2 T cells into the tumor, especially to cells with BTN2A1 expression (a Vδ2 T cell activating molecule) despite PD-L1 co-localization. Interestingly, these cells co-expressed cleaved gasdermin D, suggesting that pyroptosis was induced by Vδ2 T cells. This was verified by Vδ2 T/mesothelioma co-culture experiments demonstrating membrane ballooning morphology, increased cleaved caspase-3 and gasdermin E, and upregulated IL-1ß and IL-18. Discussion: Vδ2 T cells plus anti-PD1 exhibited cytotoxicity against mesothelioma in vivo. However, we found no advantage for anti-PD-1 against PD-1 high expressing Vδ2 T cells in promoting pyroptosis. Taken together, our work demonstrated that Vδ2 T cells combined with anti-PD-1 antibody can be developed as a potential combination immunotherapy for mesothelioma.

Linked Th17 and Calgranulin Responses in Maternal-cord Blood Dyads of Preterm Gestations with Histologic Chorioamnionitis.

Introduction: Maternal-fetal immune crosstalk mechanisms are increasingly identified in the pathogenesis of gestational disorders, including histologic chorioamnionitis (HCA). Although an inflammatory Th17 immune phenotype has been described in preterm neonates with HCA, the associated maternal Th17 response is relatively unknown. To refine our understanding of Th17 biology in this context, we examined Th17 responses in maternal-cord blood dyads of preterm gestations. Materials and methods: Paired maternal and cord blood (CB) samples were prospectively collected from preterm gestations (23-34 weeks) with HCA or controls. Th17-linked cell frequencies and plasma calgranulin (S100A8, S100A12) levels were determined by flow cytometry and enzyme-linked immunoassay, respectively. Results: Analyses of 47 maternal-cord blood pairs showed striking parallel increases in Th17 cell frequencies as well as plasma calgranulin levels in the presence of fetal inflammation. Cord blood S100A12 levels were directly correlated with Th17 cell frequencies. In CB cultures, rh-S100A12 promoted in vitro propagation of Th17-type CD4+ cells. Conclusions: Maternal and CB Th17-linked responses are dually amplified in gestations with HCA, supporting a biological role for maternal-fetal interactions in this disorder. In addition to advancing current knowledge of neonatal Th17 mechanisms, these data shed new light on their association with maternal inflammation.

A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma.

Chimeric antigen receptor T cell (CAR-T) therapy has been applied in the treatment of B-cell lymphoma; however, CAR-T manufacturing requires virus- or non-virus-based genetic modification, which causes high manufacturing costs and potential safety concerns. Antibody-cell conjugation (ACC) technology, which originated from bio-orthogonal click chemistry, provides an efficient approach for arming immune cells with cancer-targeting antibodies without genetic modification. Here, we applied ACC technology in Vγ9Vδ2 T (γδ2 T) cells to generate a novel off-the-shelf CD20-targeting cell therapy ACE1831 (rituximab-conjugated γδ2 T cells) against relapsed/refractory B-cell lymphoma. ACE1831 exhibited superior cytotoxicity against B-cell lymphoma cells and rituximab-resistant cells compared to γδ2 T cells without rituximab conjugation. The in vivo xenograft study demonstrated that ACE1831 treatment strongly suppressed the aggressive proliferation of B-cell lymphoma and prolonged the survival of tumor-bearing mice with no observed toxicity. Mass spectrometry analysis indicated that cell activation receptors including the TCR complex, integrins and cytokine receptors were conjugated with rituximab. Intriguingly, the antigen recognition of the ACC-linked antibody/receptor complex stimulated NFAT activation and contributed to ACE1831-mediated cytotoxicity against CD20-expressing cancer cells. This study elucidates the role of the ACC-linked antibody/receptor complex in cytotoxicity and supports the potential of ACE1831 as an off-the-shelf γδ2 cell therapy against relapsed/refractory B-cell lymphoma.

Peripheral T Cell Populations are Differentially Affected in Familial Mediterranean Fever, Chronic Granulomatous Disease, and Gout.

Both innate errors of immunity, such as familial Mediterranean fever (FMF) and chronic granulomatous disease (CGD), and the common inflammatory disease gout are characterized by episodes of sterile inflammatory attacks in the absence of an infection. While these disorders encompass distinct pathologies due to differentially affected metabolic pathways and inflammasome activation mechanisms, their common features are the excessive production of interleukin (IL)-1ß and innate immune cell hyperreactivity. On the other hand, the role of T cells and innate-like lymphocytes such as gamma delta (γδ) T cells in these pathologies is ill-defined. In order to widen our understanding of T cell involvement in CGD, FMF and gout pathology, we developed multicolour immunophenotyping panels for flow cytometry to characterize γδ T cells as well as CD4 and CD8 T cell populations in terms of their cytokine production, activation status, memory or naive phenotypes, exhaustion status, homing receptor expression, and cytotoxic activity. Our study is the first deep immunophenotyping analysis of T cell populations in CGD, FMF, and gout patients. We found that CGD affects the frequencies and activation status of T cells, while gout impairs the cytokine production capacity of Vδ2 T cells. FMF was characterized by decreased percentages of regulatory T cells in circulation and attenuated IFN-γ production capacity by Vδ2 T cells. Autoinflammatory syndromes and congenital defects of phagocyte differentially affect T cell compartments. Future studies are warranted to assess whether these phenotypical changes are relevant for disease pathology.

Tissue-resident and innate-like T cells in patients with advanced chronic liver disease.

Chronic liver disease results from the orchestrated interplay of components of innate and adaptive immunity in response to liver tissue damage. Recruitment, positioning, and activation of immune cells can contribute to hepatic cell death, inflammation, and fibrogenesis. With disease progression and increasing portal pressure, repeated translocation of bacterial components from the intestinal lumen through the epithelial and vascular barriers leads to persistent mucosal, hepatic, and systemic inflammation which contributes to tissue damage, immune dysfunction, and microbial infection. It is increasingly recognised that innate-like and adaptive T-cell subsets located in the liver, mucosal surfaces, and body cavities play a critical role in the progression of advanced liver disease and inflammatory complications of cirrhosis. Mucosal-associated invariant T cells, natural killer T cells, γδ T cells, and tissue-resident memory T cells in the gut, liver, and ascitic fluid share certain characteristic features, which include that they recognise microbial products, tissue alarmins, cytokines, and stress ligands in tissues, and perform effector functions in chronic liver disease. This review highlights recent advances in the comprehension of human tissue-resident and unconventional T-cell populations and discusses the mechanisms by which they contribute to inflammation, fibrosis, immunosuppression, and antimicrobial surveillance in patients with cirrhosis. Understanding the complex interactions of immune cells in different compartments and their contribution to disease progression will provide further insights for effective diagnostic interventions and novel immunomodulatory strategies in patients with advanced chronic liver disease.

IL-17a-producing γδT cells and NKG2D signaling mediate bacterial endotoxin-induced neonatal lung injury: implications for bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease in preterm birth survivors characterized by inflammation, impaired alveolarization and dysmorphic vasculature. Activated IL-17A+ lymphocytes are key drivers of inflammation in preterm infants. We have shown that in immature mice chronic airway exposure to lipopolysaccharide (LPS) induces pulmonary inflammation, increased IL-17a expression, and hypoalveolarization, a BPD-like phenotype. The source of IL-17a and contribution to lung pathology is unknown. The natural-killer group 2, member D (NKG2D) receptor mediates activation and IL-17a production in γδ T cells by binding to stress molecules. LPS induces NKG2D ligand expression, including Rae-1 and MULT1. We hypothesized that IL-17a+ γδ T cells and NKG2D signaling mediate neonatal LPS-induced lung injury. Immature C57BL/6J (wild type), Nkg2d-/- or Tcrd-/- (lacking γδ T cells) mice were inoculated with 3ug/10ul of LPS from E. coli O26:B6 or 10ul of PBS intranasally on day of life 3, 5, 7, and 10. Selected mice were treated with neutralizing antibodies against IL-17a, or NKG2D intraperitoneally. Lung immune cells were assessed by flow cytometry and gene expression was analyzed by qPCR. Alveolar growth was assessed by lung morphometry. We established that anti-IL-17a antibody treatment attenuated LPS-induced hypoalveolarization. We found that LPS induced the fraction of IL-17a+NKG2D+ γδ T cells, a major source of IL-17a in the neonatal lung. LPS also induced lung mRNA expression of NKG2D, Rae-1, MULT1, and the DNA damage regulator p53. Anti-NKG2D treatment attenuated the effect of LPS on γδ T cell IL-17a expression, immune cell infiltration and hypoalveolarization. LPS-induced hypoalveolarization was also attenuated in Nkg2d-/- and Tcrd-/- mice. In tracheal aspirates of preterm infants IL-17A and its upstream regulator IL-23 were higher in infants who later developed BPD. Also, human ligands of NKG2D, MICA and MICB were present in the aspirates and MICA correlated with median FiO2. Our novel findings demonstrate a central role for activated IL-17a+ γδ T cells and NKG2D signaling in neonatal LPS-induced lung injury. Future studies will determine the role of NKG2D ligands and effectors, other NKG2D+ cells in early-life endotoxin-induced lung injury and inflammation with a long-term goal to understand how inflammation contributes to BPD pathogenesis.

Aging unconventionally: γδ T cells, iNKT cells, and MAIT cells in aging.

Unconventional T cells include γδ T cells, invariant Natural Killer T cells (iNKT) cells and Mucosal Associated Invariant T (MAIT) cells, which are distinguished from conventional T cells by their recognition of non-peptide ligands presented by non-polymorphic antigen presenting molecules and rapid effector functions that are pre-programmed during their development. Here we review current knowledge of the effect of age on unconventional T cells, from early life to old age, in both mice and humans. We then discuss the role of unconventional T cells in age-associated diseases and infections, highlighting the similarities between members of the unconventional T cell family in the context of aging.

Infiltrated IL-17A-producing gamma delta T cells play a protective role in sepsis-induced liver injury and are regulated by CCR6 and gut commensal microbes.

Introduction: Sepsis is a common but serious disease in intensive care units, which may induce multiple organ dysfunctions such as liver injury. Previous studies have demonstrated that gamma delta (γδ) T cells play a protective role in sepsis. However, the function and mechanism of γδ T cells in sepsis-induced liver injury have not been fully elucidated. IL-17A-producing γδ T cells are a newly identified cell subtype. Methods: We utilized IL-17A-deficient mice to investigate the role of IL-17A-producing γδ T cells in sepsis using the cecum ligation and puncture (CLP) model. Results: Our findings suggested that these cells were the major source of IL-17A and protected against sepsis-induced liver injury. Flow cytometry analysis revealed that these γδ T cells expressed Vγ4 TCR and migrated into liver from peripheral post CLP, in a CCR6-dependent manner. When CLP mice were treated with anti-CCR6 antibody to block CCR6-CCL20 axis, the recruitment of Vγ4+ γδ T cells was abolished, indicating a CCR6-dependent manner of migration. Interestingly, pseudo germ-free CLP mice treated with antibiotics showed that hepatic IL-17A+ γδ T cells were regulated by gut commensal microbes. E. coli alone were able to restore the protective effect in pseudo germ-free mice by rescuing hepatic IL-17A+ γδ T cell population. Conclusion: Our research has shown that Vγ4+ IL-17A+ γδ T cells infiltrating into the liver play a crucial role in protecting against sepsis-induced liver injury. This protection was contingent upon the recruitment of CCR6 and regulated by gut commensal microbes.

MR1 deficiency enhances IL-17-mediated allergic contact dermatitis.

Major histocompatibility complex (MHC) class Ib molecules present antigens to subsets of T cells primarily involved in host defense against pathogenic microbes and influence the development of immune-mediated diseases. The MHC class Ib molecule MHC-related protein 1 (MR1) functions as a platform to select MR1-restricted T cells, including mucosal-associated invariant T (MAIT) cells in the thymus, and presents ligands to them in the periphery. MAIT cells constitute an innate-like T-cell subset that recognizes microbial vitamin B2 metabolites and plays a defensive role against microbes. In this study, we investigated the function of MR1 in allergic contact dermatitis (ACD) by examining wild-type (WT) and MR1-deficient (MR1-/-) mice in which ACD was induced with 2,4-dinitrofluorobenzene (DNFB). MR1-/- mice exhibited exaggerated ACD lesions compared with WT mice. More neutrophils were recruited in the lesions in MR1-/- mice than in WT mice. WT mice contained fewer MAIT cells in their skin lesions following elicitation with DNFB, and MR1-/- mice lacking MAIT cells exhibited a significant increase in IL-17-producing αß and γδ T cells in the skin. Collectively, MR1-/- mice displayed exacerbated ACD from an early phase with an enhanced type 3 immune response, although the precise mechanism of this enhancement remains elusive.

Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers.

Adoptive cell therapy (ACT) utilizing γδ T cells is becoming a promising option for the treatment of cancer, because it offers an off-the-shelf allogeneic product that is safe, potent, and clinically effective. Approaches to engineer or enhance immune-competent cells for ACT, like expression of chimeric antigen receptors (CARs) or combination treatments with bispecific T cell engagers, have improved the specificity and cytotoxic potential of ACTs and have shown great promise in preclinical and clinical settings. Here, we test whether electroporation of γδ T cells with CAR or secreted bispecific T cell engager (sBite) mRNA is an effective approach to improve the cytotoxicity of γδ T cells. Using a CD19-specific CAR, approximately 60% of γδ T cells are modified after mRNA electroporation and these cells show potent anticancer activity in vitro and in vivo against two CD19-positive cancer cell lines. In addition, expression and secretion of a CD19 sBite enhances γδ T cell cytotoxicity, both in vitro and in vivo, and promotes killing of target cells by modified and unmodified γδ T cells. Taken together, we show that transient transfection of γδ T cells with CAR or sBite mRNA by electroporation can be an effective treatment platform as a cancer therapeutic.

γδ T cells recruitment and local proliferation in brain parenchyma benefit anti-neuroinflammation after cerebral microbleeds.

Background: Cerebral microbleeds (CMBs) are an early sign of many neurological disorders and accompanied by local neuroinflammation and brain damage. As important regulators of immune response and neuroinflammation, the biological behavior and role of γδ T cells after CMBs remain largely unknown. Methods: We made a spot injury of microvessel in the somatosensory cortex to mimic the model of CMBs by two-photon laser and in vivo tracked dynamical behaviors of γδ T cells induced by CMBs using TCR-δGFP transgenic mice. Biological features of γδ T cells in the peri-CMBs parenchyma were decoded by flow cytometry and Raman spectra. In wildtype and γδ T cell-deficient mice, neuroinflammation and neurite degeneration in the peri-CMBs cortex were studied by RNAseq, immunostaining and in vivo imaging respectively. Results: After CMBs, γδ T cells in the dural vessels were tracked to cross the meningeal structure and invade the brain parenchyma in a few days, where the division process of γδ T cells were captured. Parenchymal γδ T cells were highly expressed by CXCR6 and CCR6, similar to meningeal γδ T cells, positive for IL-17A and Ki67 (more than 98%), and they contained abundant substances for energy metabolism and nucleic acid synthesis. In γδ T cell-deficient mice, cortical samples showed the upregulation of neuroinflammatory signaling pathways, enhanced glial response and M1 microglial polarization, and earlier neuronal degeneration in the peri-CMBs brain parenchyma compared with wildtype mice. Conclusion: CMBs induce the accumulation and local proliferation of γδ T cells in the brain parenchyma, and γδ T cells exert anti-neuroinflammatory and neuroprotective effects at the early stage of CMBs.