Vδ2 Subset Research Articles

Dynamics of Peripheral Lymphocyte Subsets from Birth until Old Age

The immune system is inexperienced before birth and tends to be tolerogenic, rather than immunogenic. After birth, the adaptive immune system develops while facing microbial challenges, but it can become impaired as old age progresses and persistent inflammation can lead to chronic morbidity, disability and frailty. To investigate the potential contributions of lymphocyte subsets to immunity from birth until old age, we enumerated circulating innate and conventional lymphocytes and measured serum cytokine levels in 10 cord blood samples and in peripheral blood from 10 healthy term neonates, 23 healthy school-age children, 25 young adults and 11 older subjects. Flow cytometric analysis revealed that B cell frequencies increase during childhood and gradually decrease into adulthood, whereas natural killer cell frequencies increase throughout life. T cell frequencies remained relatively constant throughout life, as did their expression of CD4 and CD8. However, all four innate T cell populations studied—invariant natural killer T cells, mucosa-associated invariant T cells and the Vδ1 and the Vδ2 subsets of γδ T cells—were extremely rare in cord blood and in peripheral blood of neonates, but they expanded after birth reaching highest levels in adulthood. Analysis of serum cytokine levels revealed that proinflammatory and T helper type 1 (Th1) cytokine levels increase in adulthood, whereas Th2 and Th17 cytokine levels remain relatively constant. These changes in lymphocyte numbers and cytokine levels across the lifetime are likely to affect immunocompetence, leaving newborn and elderly people susceptible to infection, cancer and immune-mediated disease.

The Alteration of Circulating Invariant Natural Killer T, γδT, and Natural Killer Cells after Ischemic Stroke in Relation to Clinical Outcomes: A Prospective Case-Control Study.

The adaptive response occurs only after 7-10 days of antigen presentation. Nevertheless, the autoreactive T cells infiltrate the stroke lesion within the first 48 h. Thus, we hypothesized that the unconventional lymphocytes as invariant natural killer T cells (iNKT) and γδT cells that share immediate innate and delayed adaptive response features are involved in acute stroke pathophysiology. We assessed prospectively the quantity of circulating iNKT cells, γδT cells, and NK cells with flow cytometry in 52 subjects within three months after stroke, and we compared the results with those obtained in age-, sex-, and vascular risk factor-matched controls. We studied lymphocyte parameters regarding clinical outcomes, infarct volume, stroke-associated infection (SAI), and burden risk factors. The reduced number of circulating γδT cells and decreased percentage of the Vδ2 subset in the acute phase of stroke correlated with worse neurological status in the recovery phase. In subjects treated with thrombolysis and those who developed SAI, a lower percentage of γδT cells in the 90-day follow-up was observed. An increased percentage of iNKT cells in the acute and subacute phases of stroke was observed, and it was related to the worse clinical status. The circulating NK cells do not change temporarily or affect the outcomes after stroke. It seems that γδT cells play a long-lasting role in ischemic stroke, mainly related to the Vδ2 subset. The role of iNKT cells appears to be detrimental, especially in the acute and subacute phases of stroke. The effect of circulating NK cells on the outcome after stroke seems negligible.

Dysregulation of Gamma Delta T-Cells, CD8+ T Cells, CD4+ T Cells, and Natural Killer Cells in Brain Pathology: Understanding HIV-Induced Impairment and Exploring Targeted GSH Therapy.

This review comprehensively explores the dysregulation of Gamma Delta T-cells, CD8+ T Cells, and Natural Killer T Cells in the context of Human Immunodeficiency Virus (HIV) infection and its implications for brain pathology. It encompasses an overview of the HIV disease process, immune cell dysregulation, association with neurological diseases, and the critical role of Glutathione (GSH) in T-cell function. The alterations in Gamma Delta T-cells during chronic infection, the intricate dynamics of Vδ1 and Vδ2 subsets, and the potential of Vγ9Vδ2 T cells in inhibiting HIV replication are discussed. Additionally, the review addresses the exhaustion, impaired cytotoxicity, and premature senescence of CD8+ T cells, as well as the dysregulation of Natural Killer Cells (NKCs) and their impact on overall immune system activity. Furthermore, it examines the role of Gamma Delta (γδ) T-cells in brain injuries, infections, and tumors and highlights the therapeutic implications of elevated GSH levels in promoting a T helper 1 (Th1) immune response. However, HIV-infected patients with decreased GSH exhibit a T helper 2 (Th2) bias, compromising protection against intracellular pathogens. Finally, the review discusses studies in murine models demonstrating the impact of GSH levels on immune responses and underscores the therapeutic potential of targeting GSH to enhance immunity in HIV patients. Overall, this review provides valuable insights into the complex interplay between immune dysregulation, GSH levels, and HIV-associated brain pathology, offering insights into potential therapeutic avenues for mitigating immune compromise and neurological impairments in HIV patients.

C5a enhances inflammation and chemotaxis of γδ T cells in malignant pleural effusion

BackgroundThe inhibitory effect of γδT17 cells on the formation of murine malignant pleural effusions (MPE) has been established. However, there is limited understanding regarding the phenotypic characterization of γδ T cells in MPE patients and their recruitment to the pleural cavity. MethodsWe quantified γδ T cell prevalence in pleural effusions and corresponding peripheral blood from malignant and benign patients using immunohistochemistry and flow cytometry. The expression of effector memory phenotype, stimulatory/inhibitory/chemokine receptors and cytokines on γδ T cells in MPE was analyzed using multicolor flow cytometry. The infiltration of γδ T cells in MPE was assessed through immunofluorescence, ELISA, flow cytometry and transwell migration assay. ResultsWe observed a significant infiltration of γδ T cells in MPE, surpassing the levels found in blood and benign pleural effusion. γδ T cells in MPE exhibited heightened expression of CD56 and an effector memory phenotype, while displaying lower levels of PD-1. Furthermore, γδ T cells in MPE showed higher levels of cytokines (IFN-γ, IL-17A and IL-22) and chemokine receptors (CCR2, CCR5 and CCR6). CCR2 expression was notably higher in the Vδ2 subtype compared to Vδ1 cells. Moreover, the complement C5a enhanced cytokine release by γδ T cells, upregulated CCR2 expression in Vδ2 subsets, and stimulated the production of chemokines (CCL2, CCL7 and CCL20) in MPE. In vitro utilizing CCR2 neutralising and C5aR antagonist significantly reduced the recruitment of γδ T cells. Conclusionsγδ T cells infiltrate MPE by overexpressing CCR2 and exhibit hightened inflammation, which is further augmented by C5a.

Vδ1+ and Vδ2+ gamma delta T cell subsets exert distinct functional profiles with aviremic HIV infection and aging

Abstract Populations of gamma delta T cells exert different effector function profiles (inflammatory (Th1/Th17) vs. immunoregulatory (Th2/Treg)) in response to non-antigen specific signals in a microenvironment. The nature of gamma delta T cell lineage commitment versus functional capacity at the single cell level is unclear. Also, gamma delta T cells are implicated as drivers of the chronic inflammation found with normal aging and ART-suppressed HIV infection; however, the precise subsets involved in this phenomenon are currently unknown. To address these questions, we optimized a 31-color Intracellular Cytokine Staining (ICS) panel enabling measurement of 10 cytokines, as well as Ki67, granzyme B, and CD107a for use with PBMC from participants of a HIV and Aging cohort. Multi-variate analysis (opt-SNE) of the collected dataset revealed striking differences in the overall functional potential of the V delta 1 (Vδ1) and V delta 2 (Vδ2) subsets, with many Vδ1+ cells not producing any of the cytokines measured. Distinct differences of both the Vδ1+ and Vδ2+ subsets were found between the HIV infected and age-stratified subject groups. Individual gamma delta T cells with a ‘Th0’ phenotype, producing both Th1 and Th2 lineage cytokines, were present in both Vδ1+ and Vδ2+ subsets. Also, HIV+ subjects lacked Vδ2+ cells expressing CD107a, indicating a specific defect in the cytotoxic function of this subset with HIV infection. Further investigation of gamma delta T cell functions and phenotypes could provide new insight into the biology of this unique T cell population and reveal novel therapeutic targets. Supported by a grant from NIH (R01AG060890-01)

γδ T cells are effectors of immunotherapy in cancers with HLA class I defects

DNA mismatch repair-deficient (MMR-d) cancers present an abundance of neoantigens that is thought to explain their exceptional responsiveness to immune checkpoint blockade (ICB)1,2. Here, in contrast to other cancer types3–5, we observed that 20 out of 21 (95%) MMR-d cancers with genomic inactivation of β2-microglobulin (encoded by B2M) retained responsiveness to ICB, suggesting the involvement of immune effector cells other than CD8+ T cells in this context. We next identified a strong association between B2M inactivation and increased infiltration by γδ T cells in MMR-d cancers. These γδ T cells mainly comprised the Vδ1 and Vδ3 subsets, and expressed high levels of PD-1, other activation markers, including cytotoxic molecules, and a broad repertoire of killer-cell immunoglobulin-like receptors. In vitro, PD-1+ γδ T cells that were isolated from MMR-d colon cancers exhibited enhanced reactivity to human leukocyte antigen (HLA)-class-I-negative MMR-d colon cancer cell lines and B2M-knockout patient-derived tumour organoids compared with antigen-presentation-proficient cells. By comparing paired tumour samples from patients with MMR-d colon cancer that were obtained before and after dual PD-1 and CTLA-4 blockade, we found that immune checkpoint blockade substantially increased the frequency of γδ T cells in B2M-deficient cancers. Taken together, these data indicate that γδ T cells contribute to the response to immune checkpoint blockade in patients with HLA-class-I-negative MMR-d colon cancers, and underline the potential of γδ T cells in cancer immunotherapy.

EBV latent membrane protein 1 augments γδ T cell cytotoxicity against nasopharyngeal carcinoma by induction of butyrophilin molecules.

Nasopharyngeal carcinoma (NPC) is a diverse cancer with no well-defined tumor antigen, associated with oncogenic Epstein-Barr Virus (EBV), and with usually late-stage diagnosis and survival <40%. Current radiotherapy and chemotherapy have low effectiveness and cause adverse effects, which calls for the need of new therapy. In this regard, adoptive immunotherapy using γδ T cells has potential, but needs to be coupled with butyrophilin 2A1 and 3A1 protein expression to achieve tumoricidal effect. Methods: Human γδ T cells were expanded (with Zol or PTA) and used for cytotoxicity assay against NPC cells, which were treated with the EBV EBNA1-targeting peptide (L2)P4. Effect of (L2)P4 on BTN2A1/BTN3A1 expression in NPC cells was examined by flow cytometry and Western blot. An NPC-bearing NSG mice model was established to test the effectiveness of P4 and adoptive γδ T cells. Immunofluorescence was performed on NPC tissue sections to examine the presence of γδ T cells and expression of BTN2A1 and BTN3A1. EBV gene expression post-(L2)P4 treatment was assessed by qRT-PCR, and the relationship of LMP1, NLRC5 and BTN2A1/BTN3A1 was examined by transfection, reporter assay, Western blot, and inhibition experiments. Results: Zol- or PTA-expanded the Vδ2 subset of γδ T cells that exerted killing against certain NPC cells. (L2)P4 reactivates latent EBV, which increased BTN2A1 and BTN3A1 expression and conferred higher susceptibility towards Vδ2 T cells cytotoxicity in vitro, as well as enhanced tumor regression in vivo by adoptive transfer of Vδ2 T cells. Mechanistically, (L2)P4 induced EBV LMP1, leading to IFN-γ/p-JNK and NLRC5 activation, and subsequently stimulated the expression of BTN2A1 and BTN3A1. Conclusions: This study demonstrated the effectiveness of using the EBV-targeting probe (L2)P4 and adoptive γδ T cells as a promising combinatorial immunotherapy against NPC. The identification of the LMP1-IFN-γ/p-JNK-NLRC5-BTN2A1/BTN3A1 axis may lead to new insight and therapeutic targets against NPC and other EBV+ tumors.

CNSC-13. TUMOR INFILTRATION OF GAMMA-DELTA T CELLS IN GLIOBLASTOMA

Abstract Gamma-delta (γδ) T cells are innate immunity effector lymphocytes with known prominent anti-tumor reactivity against aggressive glioblastoma (GBM). However, therapeutic approaches have had limited success due to the protective blood-brain-barrier and the immunosuppressive GBM tumor microenvironment. In this study, we determined Vδ1 a Vδ2 γδ T cell populations in peripheral blood and paired tumor tissue samples in patients (n=40) following the resection and throughtout the therapy follow-up. Tumor samples were processed using enzymatic kits and gentleMACSTM Dissociator (Miltenyi Biotec Inc.) and tumor-infiltrating γδ T lymphocytes (TILs) were analyzed by flow cytometry.We found infiltration of both intratumoral CD3+ γδ T cell subsets in 68% tumor samples. We detected Vδ1 γδ T cells in the range 0-0.8% (median 0.26%). Majority of GBM patients presented the Vδ2 subset among TILs in the range 0-13.8% (median 1.5%). Functional studies showed prominent cytotoxicity of magnetically sorted Vδ1 a Vδ2 γδ T cells against GBM cell lines and more importantly against primary tumors. Detailed phenotypic profiling and single-cell sequencing of Vδ2 γδ T cells is currently underway. Next, we identified the EphA2 receptor as one of the targets for tumor-reactive Vδ1 γδ T cells. Specifically, we found that blocking of EphA2 expression resulted in significant inhibition of GBM killing mediated by Vδ1 γδ T cells. Furthermore, Luminex xMAP technology identified significantly elevated levels of stress ligand MICA and check-point inhibitor ligands PD-L1 (B7-H1, CD274) and B7-H3 (CD276) and galectin-9 in patients‘ plasma samples at diagnosis compared to age-matched controls.The patient’s clinical course and therapeutic protocols will be discussed.This study was supported by Ministry of Health, Czech Republic (grant NV19-05-00410 to AK) by Ministry of Health, Czech Republic-conceptual development of research organization (FNBr, 65269705). All rights reserved.

Tissue- and age-specific adaptations of human γδT cells in early life

Abstract γδT cells are unconventional T lymphocytes which seed peripheral tissues in early life. They provide frontline anti-pathogen, anti-cancer and homeostatic responses, rapidly initiated in response to signals of cellular stress independent of MHC-recognition. However, the role of γδT cells in humans has been difficult to assess given their purported tissue residency. Using tissues obtained from organ donors, we conducted flow cytometry, TCR-sequencing, and stimulation assays on γδT cells isolated from blood, spleen, lung, intestines and lymph nodes (LN) to create an ‘atlas’ across the human body throughout life. γδT cells were enriched in the blood, spleen, lung, and jejunum in early life, whereas LN proportions were maintained at low levels. Vδ1 and Vδ2 γδT cell subsets also exhibited tissue- and age-specific phenotypes: Vδ1 cells predominated in all organs in infancy except the lung. They were ‘naïve-like’, acquiring a mature phenotype with age, whereas Vδ2 possessed an effector memory signature, even in donors a few days old. Functionally, Vδ2 cells produced IFNγ, TNFα, GzmB and perforin at high levels in childhood. By contrast, Vδ1 cells favored Areg production during infancy but acquired a cytotoxic profile into adulthood. TCR analysis revealed clonal expansion in mucosal organs, whereas lymphoid sites had increased diversity in early life. Despite this site-specific clonal expansion, V-chain usage was stably distributed across organs and did not show the tissue-specific biases seen in mice. Our analyses reveal that γδT cells may play an outsized role in pediatric tissue immune responses and identify potentially divergent roles of specific subsets that contribute to tissue immunity during the most vulnerable years of life.

Off-the-shelf Vδ1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma

BackgroundGlypican-3 (GPC-3) is an oncofetal protein that is highly expressed in various solid tumors, but rarely expressed in healthy adult tissues and represents a rational target of particular relevance in...

Characteristics of Human Peripheral Blood γδ T Cells Expanded With Zoledronate.

This study aimed to investigate the characteristics of human peripheral blood γδ T cells, which were expanded ex vivo in the presence of zoledronate (ZOL). Human peripheral blood cells were cultured with IL-2 and IL-15 in the presence or absence of ZOL, which was added as a phospho-antigen, and their phenotypes were assessed by flow cytometry. Expanded γδ T cells were transduced with CD19 CAR vector, and the cytotoxicity was evaluated in vitro and in vivo by flow cytometry. Ex vivo expansion did not hamper the expression of activating receptors. Interestingly, ZOL promoted the expression of CD226 (DNAM-1), TRAIL, and FAS-L in the Vδ1 subset of γδ T cells. Expanded γδ T cells containing CD19 CAR+ γδ T cells removed B cell lymphoma cells effectively in vivo. γδ T cells could be a promising immunotherapeutic for cancer.

MAIT and Vδ2 Unconventional T Cells Predict Favorable Outcome after Allogeneic HCT and Are Supported By a Diverse Intestinal Microbiome

MAIT and Vδ2 Unconventional T Cells Predict Favorable Outcome after Allogeneic HCT and Are Supported By a Diverse Intestinal Microbiome

Activated na\xefve \u03b3\u03b4 T cells accelerate deep molecular response to BCR-ABL inhibitors in patients with chronic myeloid leukemia

Tyrosine kinase inhibitors (TKIs) that target BCR-ABL are the frontline treatments in chronic myeloid leukemia (CML). Growing evidence has shown that TKIs also enhance immunity. Since gamma-delta T (γδT) cells possess the potent anticancer capability, here we investigated the potential involvement of γδT cells in TKI treatments for CML. We characterized γδT cells isolated from chronic-phase CML patients before and during TKI treatments. γδT expression increased significantly in CML patients who achieved major molecular response (MMR) and deep molecular response (DMR). Their Vδ2 subset of γδT also expanded, and increased expression of activating molecules, namely IFN-γ, perforin, and CD107a, as well as γδT cytotoxicity. Mechanistically, TKIs augmented the efflux of isopentenyl pyrophosphate (IPP) from CML cells, which stimulated IFN-γ production and γδT expansion. Notably, the size of the IFN-γ+ naïve γδT population in TKI-treated CML patients was strongly correlated with their rates to reach DMR and with the duration on DMR. Statistical analysis suggests that a cutoff of 7.5% IFN-γ+ naïve subpopulation of γδT in CML patients could serve as a determinant for MR4.0 sustainability. Our results highlight γδT cells as a positive regulator for TKI responses in CML patients.

Tissue distribution of γδ T cell subsets in oesophageal adenocarcinoma

The global obesity epidemic is contributing to increased prevalence of diseases fuelled by chronic inflammation, including cancer. Oesophageal adenocarcinoma (OAC) is an obesity-associated malignancy with increasing prevalence, dismal prognosis, and severely dysregulated immune processes. We previously reported that αβ T cells migrate to omentum and liver in OAC and contribute to inflammation in these tissues. Here, we assessed the tissue distribution and phenotype of gamma/delta (γδ) T cells in the blood, omentum, liver and tumour of OAC patients.Our data show that the Vδ1 and Vδ3 subsets of γδ T cells are most prevalent in omentum and liver of OAC patients. Furthermore, γδ T cells are predominantly pro-inflammatory in these tissues, and co-express IFN-γ and IL-17. Moreover, γδ T cells exhibit cytotoxic capabilities in OAC omentum and liver.This study provides the first indication that γδ T cells contribute to obesity-associated inflammation in OAC and might be exploited therapeutically.

Allogeneic Vδ1 gamma delta T cells engineered with glypican-3 (GPC3)-specific CAR expressing soluble IL-15 have enhanced antitumor efficacy against hepatocellular carcinoma in preclinical models.

e14511 Background: Autologous αβ chimeric antigen receptor (CAR) T cell therapy has shown promising clinical results in hematologic malignancies but limited success in solid tumors. Allogeneic αβ T cell therapy may overcome several challenges faced by autologous therapy but carries the risk of graft-versus-host disease (GvHD) and does not readily recognize multiple tumor-associated antigens. Gamma delta (γδ) T cells are highly cytolytic effectors that can recognize and kill tumor cells in an MHC-unrestricted manner without causing GvHD. The Vδ1 subset is preferentially localized in peripheral tissue and is critical for tumor immunosurveillance. Engineering Vδ1 T cells with CARs can further enhance antitumor activity and represents an attractive and safe approach to treating solid tumors. However, their clinical use has been hindered by the limited number of circulating Vδ1 T cells. Here, we describe the development of the first allogeneic Vδ1 T cells that have been expanded from healthy donor PBMCs and genetically modified to secrete IL-15 (sIL15) and express a CAR targeting glypican-3 (GPC3), a rational target for hepatocellular carcinoma (HCC). Methods: Vδ1 T cells in healthy donor PBMCs were activated by a Vδ1-specific monoclonal antibody and transduced with 41BBζ or 41BBζ-sIL15 GPC3-CARs prior to cell expansion, αβ T cell depletion and cryopreservation. In vitro characterization included: 1) co-culture assays with GPC3-expressing HCC targets HepG2 and PLC/PRF/5, 2) phenotypic analysis by flow cytometry, and 3) cytokine production by multiplexed immunoassay. For in vivo assessment of tumor control, immunodeficient NSG mice were subcutaneously injected with HepG2 cells and treated with a single dose of 41BBζ or 41BBζ-sIL15 GPC3-CAR Vδ1 T cells. Additionally, tissues were harvested 7 days post transfer and analyzed by flow cytometry for Vδ1 T cell tissue homing and proliferation, or at end of study and analyzed for GvHD by immunohistochemistry. Results: Vδ1 T cells expanded over 10,000-fold and routinely reached &gt;80% purity. Expanded Vδ1 T cells showed a primarily naïve-like phenotype (CD45RA+CD27+) with minimal exhaustion receptor expression and displayed robust proliferation, cytokine production, and cytotoxic activity against HCC cell lines expressing low and high GPC3 levels in vitro. In a HepG2 mouse model, GPC3-CAR Vδ1 T cells primarily accumulated and proliferated in the tumor, and a single dose was able to efficiently control tumor burden without causing GvHD. Importantly, 41BBζ-sIL15 GPC3-CAR Vδ1 cells displayed enhanced tumor-specific proliferation that resulted in better tumor control without any toxicity. Conclusions: Our results show that expanded Vδ1 T cells engineered with GPC3-CAR and sIL-15 represent a promising platform for safe and effective off-the-shelf treatment of HCC.

Functional characterization of gamma-delta (γδ) T cells in allergy

Abstract Despite a growing consensus on the involvement of γδ T cells in allergy and other human immunological disorders, the detailed mechanisms remain hypothetical due to lack of investigative tools. Herein, we sought to develop functional assays to study the role of γδ T cells in allergy. Using an Activation-Induced Marker (AIM) assay, based on the upregulation of 4-1BB (CD137) and CD69, we were able to detect ex vivo allergen-specific responses from γδ T cells to multiple allergen extracts in human PBMCs, including mouse, cockroach (CR), house dust mite (HDM) and timothy grass (TG) allergens, which were reproducible and observed in multiple allergic cohorts. The magnitude of allergen-reactive γδ T cells differed between allergic and non-allergic cohorts with HDM and TG allergy, but not mouse or CR allergy. Further characterization showed that the γδ T cells reactive to mouse allergen were mostly the Vδ2 subset and from effector memory (Tem and Temra) compartments. Interestingly, in mouse-specific allergic donors we found differential polarized responses for γδ and ab T cells, with a Th1 and Th2 pattern, respectively. Using a complementary AIM assay for CD40L (CD154) combined with intracellular staining (ICS), we observed that Th1 polarization (INFg and TNFa) in responses to mouse extract was also common in non-allergic donors. Conversely, In the case of CR responses, Th1 polarization of Vδ2 T cells was only observed in the allergic cohort. Overall, these results suggest that allergen-specific Vδ2 T cells are potential to skew dysregulated cytokine responses in immunotherapy. In conclusion, we developed new functional assays that serve as tools for in-depth characterization and deciphering the roles of γδ T cells in allergy.

Rapid activation and expansion of peripheral gammadelta (γδ) T cells in SARS-CoV-2-infected nonhuman primates following aerosol exposure

Abstract Infection with SARS-CoV-2, the novel coronavirus responsible for the current COVID-19 pandemic, results in a wide range of pathologies from asymptomatic/mild outcome to fulminant pneumonia and progression to acute respiratory distress syndrome (ARDS). The cellular mechanisms underlying this spectrum of disease remain unknown. We investigated the dynamic changes in frequency and activation status of peripheral innate lymphocyte subsets in two different species of nonhuman primates. Cohorts of Rhesus macaques (n=8) and African Green monkeys (n=8) were infected with SARS-CoV-2 either by small particle or mucosal (IN/IT) route. Flow cytometric analysis of PBMCs revealed a rapid and significant increase in circulating γδ T cell frequencies and activation at d1 postexposure across all animals inoculated by aerosol, which was correlative with peak viral loads in lung lavage and Vδ2 T cell frequencies. Further, elevated GranzymeB expression in both Vδ1 and Vδ2 subsets of γδ T cells was suggestive of increased cytotoxic function in early infection. In contrast, mucosally-infected animals showed little change in circulating γδ T cell frequencies from d1 through d7 post-infection, nor were there remarkable differences in early MAIT and NK cell frequencies. Collectively, these data suggest γδ T cells may be early responders to deep lung SARS-CoV-2 infection and the kinetics and quality of immune response are dependent upon route of exposure despite resulting in similar peak viral loads from infection. Further understanding of γδ T cell functions within the pulmonary compartment during SARS-CoV-2-driven acute inflammation may reveal tractable therapeutic approaches to further mitigate ARDS-like responses in COVID-19 patients.

Dysregulation of IL-17/IL-22 Effector Functions in Blood and Gut Mucosal Gamma Delta T Cells Correlates With Increase in Circulating Leaky Gut and Inflammatory Markers During cART-Treated Chronic SIV Infection in Macaques.

HIV-associated inflammation has been implicated in the premature aging and increased risk of age-associated comorbidities in cART-treated individuals. However, the immune mechanisms underlying the chronic inflammatory state of cART-suppressed HIV infection remain unclear. Here, we investigated the role of γδT cells, a group of innate IL-17 producing T lymphocytes, in the development of systemic inflammation and leaky gut phenotype during cART-suppressed SIV infection of macaques. Plasma levels of inflammatory mediators, intestinal epithelial barrier disruption (IEBD) and microbial translocation (MT) biomarkers, and Th1/Th17-type cytokine functions were longitudinally assessed in blood and gut mucosa of SIV-infected, cART-suppressed macaques. Among the various gut mucosal IL-17/IL-22-producing T lymphocyte subsets including Th17, γδT, CD161+ CD8+ T, and MAIT cells, a specific decline in the Vδ2 subset of γδT cells and impaired IL-17/IL-22 production in γδT cells significantly correlated with the subsequent increase in plasma IEBD/MT markers (IFABP, LPS-binding protein, and sCD14) and pro-inflammatory cytokines (IL-6, IL-1β, IP10, etc.) despite continued viral suppression during long-term cART. Further, the plasma inflammatory cytokine signature during long-term cART was distinct from acute SIV infection and resembled the inflammatory cytokine profile of uninfected aging (inflammaging) macaques. Overall, our data suggest that during cART-suppressed chronic SIV infection, dysregulation of IL-17/IL-22 cytokine effector functions and decline of Vδ2 γδT cell subsets may contribute to gut epithelial barrier disruption and development of a distinct plasma inflammatory signature characteristic of inflammaging. Our results advance the current understanding of the impact of chronic HIV/SIV infection on γδT cell functions and demonstrate that in the setting of long-term cART, the loss of epithelial barrier-protective functions of Vδ2 T cells and ensuing IEBD/MT occurs before the hallmark expansion of Vδ1 subsets and skewed Vδ2/Vδ1 ratio. Thus, our work suggests that novel therapeutic approaches toward restoring IL-17/IL-22 cytokine functions of intestinal Vδ2 T cells may be beneficial in preserving gut epithelial barrier function and reducing chronic inflammation in HIV-infected individuals.

Evidence that tissue resident human enthesis γδT-cells can produce IL-17A independently of IL-23R transcript expression

ObjectivesMurine models of interleukin (IL)-23-driven spondyloarthritis (SpA) have demonstrated entheseal accumulation of γδT-cells which were responsible for the majority of local IL-17A production. However, IL-23 blockers are ineffective in axial...

Single-cell RNA sequencing unveils the shared and the distinct cytotoxic hallmarks of human TCRVδ1 and TCRVδ2 γδ T lymphocytes

γδ T lymphocytes represent ∼1% of human peripheral blood mononuclear cells and even more cells in most tissues of vertebrates. Although they have important anticancer functions, most current single-cell RNA sequencing (scRNA-seq) studies do not identify γδ T lymphocytes because their transcriptomes at the single-cell level are unknown. Here we show that high-resolution clustering of large scRNA-seq datasets and a combination of gene signatures allow the specific detection of human γδ T lymphocytes and identification of their T cell receptor (TCR)Vδ1 and TCRVδ2 subsets in large datasets from complex cell mixtures. In t-distributed stochastic neighbor embedding plots from blood and tumor samples, the few γδ T lymphocytes appear collectively embedded between cytotoxic CD8 T and NK cells. Their TCRVδ1 and TCRVδ2 subsets form close yet distinct subclusters, respectively neighboring NK and CD8 T cells because of expression of shared and distinct cytotoxic maturation genes. Similar pseudotime maturation trajectories of TCRVδ1 and TCRVδ2 γδ T lymphocytes were discovered, unveiling in both subsets an unattended pool of terminally differentiated effector memory cells with preserved proliferative capacity, a finding confirmed by in vitro proliferation assays. Overall, the single-cell transcriptomes of thousands of individual γδ T lymphocytes from different CMV+ and CMV- donors reflect cytotoxic maturation stages driven by the immunological history of donors. This landmark study establishes the rationale for identification, subtyping, and deep characterization of human γδ T lymphocytes in further scRNA-seq studies of complex tissues in physiological and disease conditions.