Vδ2 T-cell Research Articles

Application of γδ T cells with different memory phenotypes in clinical diagnosis of active pulmonary tuberculosis

ObjectiveTo investigate the application and clinical significance of different memory phenotypes of γδ T-cell subsets in the clinical diagnosis of pulmonary tuberculosis. MethodsIn total, 42 patients with tuberculosis (TB) according to the diagnostic criteria for tuberculosis (WS288-2017) who were treated at the Infectious Diseases Hospital affiliated with Soochow University from February 2023 to July 2023 were enrolled. Additionally, 16 patients with latent TB infection (LTBI) and 20 healthy controls (HCs) were included. Flow cytometry was used to measure the expression levels of γδ T cells, Vδ1 T-cell subsets/Vδ2 T-cell subsets, naive (CD45RA+CD27+) cells, central memory (CD45RA-CD27+) cells, effector memory (CD45RA-CD27-) cells, and terminally differentiated (CD45RA+CD27-) cells in the peripheral blood. The expression levels at different stages of TB infection were compared. ResultsThere were no significant differences in peripheral blood γδ T cells or Vδ1 T cells among the HC, LTBI and TB groups. The proportion of CD45RA-CD27+Vδ2 T cells in TB patients was significantly lower than that in HCs and LTBI patients, but the proportion of CD45RA+CD27-Vδ2 T cells was greater. ROC curve analysis revealed that CD45RA-CD27+Vδ2 T cells (AUC), CD45RA+CD27-Vδ2 T cells (AUC), and the combination of both (AUC) were effective in differentiating TB patients from LTBI patients. ConclusionThe proportions of CD45RA-CD27+Vδ2 T cells and CD45RA+CD27-Vδ2 T cells are potential biomarkers for the diagnosis of active TB infection and are helpful for distinguishing active TB infection from LTBI.

Vδ2 T-cell engagers bivalent for Vδ2-TCR binding provide anti-tumor immunity and support robust Vγ9Vδ2 T-cell expansion.

Vγ9Vδ2 T-cells are antitumor immune effector cells that can detect metabolic dysregulation in cancer cells through phosphoantigen-induced conformational changes in the butyrophilin (BTN) 2A1/3A1 complex. In order to clinically exploit the anticancer properties of Vγ9Vδ2 T-cells, various approaches have been studied including phosphoantigen stimulation, agonistic BTN3A-specific antibodies, adoptive transfer of expanded Vγ9Vδ2 T-cells, and more recently bispecific antibodies. While Vγ9Vδ2 T-cells constitute a sizeable population, typically making up ~1-10% of the total T cell population, lower numbers have been observed with increasing age and in the context of disease. We evaluated whether bivalent single domain antibodies (VHHs) that link Vδ2-TCR specific VHHs with different affinities could support Vγ9Vδ2 T-cell expansion and could be incorporated in a bispecific engager format when additionally linked to a tumor antigen specific VHH. Bivalent VHHs that link a high and low affinity Vδ2-TCR specific VHH can support Vγ9Vδ2 T-cell expansion. The majority of Vγ9Vδ2 T-cells that expanded following exposure to these bivalent VHHs had an effector or central memory phenotype and expressed relatively low levels of PD-1. Bispecific engagers that incorporated the bivalent Vδ2-TCR specific VHH as well as a tumor antigen specific VHH triggered antitumor effector functions and supported expansion of Vγ9Vδ2 T-cells in vitro and in an in vivo model in NOG-hIL-15 mice. By enhancing the number of Vγ9Vδ2 T-cells available to exert antitumor effector functions, these novel Vδ2-bivalent bispecific T cell engagers may promote the overall efficacy of bispecific Vγ9Vδ2 T-cell engagement, particularly in patients with relatively low levels of Vγ9Vδ2 T-cells.

LncRNA SNHG16 Inhibits Intracellular M. tuberculosis Growth Involving Cathelicidin Pathway, Autophagy, and Effector Cytokines Production.

Long noncoding small nucleolar RNA (LncRNA) host gene 16 (SNHG16) is associated with certain diseases, including cancers. However, its role and mechanism in Mycobacterium tuberculosis (Mtb) infection remain unclear. Here, we demonstrated that SNHG16 expression levels were suppressed in peripheral blood mononuclear cells (PBMCs) and CD14+ monocytes of tuberculosis (TB) patients. SNHG16 was up-regulated by acute Mtb infection of PBMCs from healthy control (HC) subjects. Such TB suppression of SNHG16 was consistent with an immunosuppressive-like state driven by IL-10 signaling as seen in TB patients. Notably, SNHG16 limited Mtb growth in macrophages/monocytes through autophagy and vitamin D receptor (VDR)-dependent cathelicidin (CAMP) antimicrobial pathways. Concurrently, SNHG16 was highly expressed in lymphocytes, including CD8+ and Vγ2 Vδ2 T-cell subsets in HCs. SNHG16 overexpression in lymphocytes allowed them to control Mtb infection in macrophages, and SNHG16 epigenetically increased the expression of anti-Mtb effector cytokines in lymphocytes by developing more accessible chromatin states in gene loci encoding IFN-γ, TNF-α, and Granzyme B. Furthermore, the adoptive transfer of SNHG16-overexpressing human PBMCs into Mtb-infected SCID mice conferred protective immunity against Mtb infection. Thus, SNHG16 drove the induction of pleiotropic effector functions that inhibited intracellular Mtb growth in vitro and in vivo, serving as an immunotherapy target in TB.

Arming Vδ2 T Cells with Chimeric Antigen Receptors to Combat Cancer.

Immunotherapy has emerged as a promising approach in the field of cancer treatment, with chimeric antigen receptor (CAR) T-cell therapy demonstrating remarkable success. However, challenges such as tumor antigen heterogeneity, immune evasion, and the limited persistence of CAR-T cells have prompted the exploration of alternative cell types for CAR-based strategies. Gamma delta T cells, a unique subset of lymphocytes with inherent tumor recognition capabilities and versatile immune functions, have garnered increasing attention in recent years. In this review, we present how arming Vδ2-T cells might be the basis for next-generation immunotherapies against solid tumors. Following a comprehensive overview of γδ T-cell biology and innovative CAR engineering strategies, we discuss the clinical potential of Vδ2 CAR-T cells in overcoming the current limitations of immunotherapy in solid tumors. Although the applications of Vδ2 CAR-T cells in cancer research are relatively in their infancy and many challenges are yet to be identified, Vδ2 CAR-T cells represent a promising breakthrough in cancer immunotherapy.

Abstract 5254: The “off-the-shelf ”CD19-CAR-Vδ1T cells demonstrated an enhanced therapeutic effect on B-cell malignancy models

Abstract Background: The commercialization of autologous chimeric antigen receptor T cell (CAR-T) is hindered by the expensive and time-consuming manufacturing process, presenting major challenges. The development of allogeneic products is proposed as an alternative strategy to overcome these obstacles. Vδ1T cells, possessing active homing capability, are known to infiltrate tumor tissues and have been linked with favorable patient prognosis due to their direct killing effect via innate immunity and their sensitive T cell receptors that can broadly recognize tumor antigens. Unlike conventional αβ T cells, Vδ1 TCRs are not restricted by major histocompatibility complexes (MHCs), thus preventing the development of graft-versus-host disease (GVHD). Therefore, Vδ1T cells were hypothesized to be a superior candidate for universal CAR-T cells. Nevertheless, the small portion of Vδ1T cells (less than 1%) in peripheral blood posts a significant challenge for CMC and production. Here we present the successful development of CD19-CAR-Vδ1T cells, which demonstrate enhanced therapeutic efficacy on B-cell malignancy models. Methods: High-purity CD19-CAR-Vδ1T cells were prepared using PersonGen's proprietary platform technology. Multiple CAR structures were tested for cytotoxicity, and the fourth-generation CAR-transduced Vδ1 cells designated as UTAA09 exhibited sustained killing of CD19+ target cells. UTAA09 underwent in-depth research regarding its anti-tumor efficacy and safety. Results: The Vδ1T cell purity exceeded 95%, while the residual TCRαβ count remained under 1%. The CAR transduction efficiency remained stable across batches, with medium CAR+~30%, and up to 2×1011 UTAA09 cells were harvested in compliance with GMP standards. The presence of secreted IL-2 significantly increased the persistence of UTAA09 in vitro, and UTAA09 showed comparable anti-tumor activity and expansion rates to CD19-CAR-αβT cells. Notably, no exhaustion phenotype was detected for UTAA09 after multiple antigen stimulation processes. Moreover, UTAA09 lacked detectable IL-17A, indicating a non-promoting characteristic in tumor growth. In the xenograft model, significant inhibition of tumor growth was observed in all mice, of which 40% achieved long-term disease-free survival (p<0.01) . On days 5, 12, and 19 after the administration of UTAA09, Vδ1T cells underwent significant expansion in all 5 mice tested. Subsequent analysis confirmed that the majority of these expanded Vδ1T cells were CAR+ cells, indicating the specific and long-lasting expansion of CD19-CAR+ Vδ1T cells in vivo. Importantly, none of the mice experienced significant weight loss, demonstrating the safety and outstanding in vivo effectiveness of UTAA09. Conclusion: UTAA09 shows potential as an off-the-shelf allogeneic cellular therapy for the treatment of B-cell malignancies and autoimmune diseases derived from B cells. Citation Format: Dan Chen, Fengtao You, Cheng Chen, Shufen Xiang, Ping Zhang, Hai Wu, Zhong Zheng, Min Wang, Xingbing Wang, Huimin Meng, Nan Yang, Bozhen Zhang, Lin Yang. The “off-the-shelf ”CD19-CAR-Vδ1T cells demonstrated an enhanced therapeutic effect on B-cell malignancy models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5254.

Vdelta1 T cells are more resistant than Vdelta2 T cells to the immunosuppressive properties of galectin-3.

Ovarian carcinomas have the highest lethality amongst gynecological tumors. A problem after primary resection is the recurrence of epithelial ovarian carcinomas which is often associated with chemotherapy resistance. To improve the clinical outcome, it is of high interest to consider alternative therapy strategies. Due to their pronounced plasticity, γδ T cells are attractive for T-cell-based immunotherapy. However, tumors might escape by the release of lectin galectin-3, which impairs γδ T-cell function. Hence, we tested the effect of galectin-3 on the different γδ T-cell subsets. After coculture between ovarian tumor cells and Vδ1 or Vδ2 T cells enhanced levels of galectin-3 were released. This protein did not affect the cytotoxicity of both γδ T-cell subsets, but differentially influenced the proliferation of the two γδ T-cell subsets. While increased galectin-3 levels and recombinant galectin-3 inhibited the proliferation of Vδ2 T cells, Vδ1 T cells were unaffected. In contrast to Vδ1 T cells, the Vδ2 T cells strongly upregulated the galectin-3 binding partner α3β1-integrin after their activation correlating with the immunosuppressive properties of galectin-3. In addition, galectin-3 reduced the effector memory compartment of zoledronate-activated Vδ2 T cells. Therefore, our data suggest that an activation of Vδ1 T-cell proliferation as part of a T-cell-based immunotherapy can be of advantage.

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.

Prognostic Immune Effector Signature in Adult Acute Lymphoblastic Leukemia Patients Is Dominated by γδ T Cells.

The success of immunotherapy has highlighted the critical role of the immune microenvironment in acute lymphoblastic leukemia (ALL); however, the immune landscape in ALL remains incompletely understood and most studies have focused on conventional T cells or NK cells. This study investigated the prognostic impact of circulating γδ T-cell alterations using high-dimensional analysis in a cohort of newly diagnosed adult ALL patients (10 B-ALL; 9 Philadelphia+ ALL; 9 T-ALL). Our analysis revealed common alterations in CD8+ T cells and γδ T cells of relapsed patients, including accumulation of early stage differentiation and increased expression of BTLA and CD73. We demonstrated that the circulating γδ T-cell signature was the most discriminating between relapsed and disease-free groups. In addition, Vδ2 T-cell alterations strongly discriminated patients by relapse status. Taken together, these data highlight the role of ɣδ T cells in adult ALL patients, among whom Vδ2 T cells may be a pivotal contributor to T-cell immunity in ALL. Our findings provide a strong rationale for further monitoring and potentiating Vδ2 T cells in ALL, including in the autologous setting.

Non-Classical HLA Class 1b and Hepatocellular Carcinoma.

A number of studies are underway to gain a better understanding of the role of immunity in the pathogenesis of hepatocellular carcinoma and to identify subgroups of individuals who may benefit the most from systemic therapy according to the etiology of their tumor. Human leukocyte antigens play a key role in antigen presentation to T cells. This is fundamental to the host's defense against pathogens and tumor cells. In addition, HLA-specific interactions with innate lymphoid cell receptors, such those present on natural killer cells and innate lymphoid cell type 2, have been shown to be important activators of immune function in the context of several liver diseases. More recent studies have highlighted the key role of members of the non-classical HLA-Ib and the transcript adjacent to the HLA-F locus, FAT10, in hepatocarcinoma. The present review analyzes the major contribution of these molecules to hepatic viral infection and hepatocellular prognosis. Particular attention has been paid to the association of natural killer and Vδ2 T-cell activation, mediated by specific HLA class Ib molecules, with risk assessment and novel treatment strategies to improve immunotherapy in HCC.

OPTIMIZED PROTOCOLS OF EXPANSION OF PERIPHERAL BLOOD-DERIVED δT-CELLS FOR THE ESTABLISHMENT OF AN OFF-THE-SHELF ADVANCED CELL THERAPY AGAINST CANCER

The use of autologous T cells expressing chimeric antigen receptors (CAR) has revolutionized the treatment of hematological malignancies. However, the use of allogeneic CAR-T cells offers many advantages over autologous therapies, including the immediate availability of cryopreserved batches for infusion and the use of healthy donor cells since it is possible to obtain high amounts of fully functional cells. Moreover, this strategy also allows for the scaling up of the production of CAR-T cells resulting in cost reduction. Cells that do not express TCR αβ, such as gamma delta (γδ) T, are an alternative source to allow widespread allogeneic therapy possible. Their ability to recognize targets in an MHC-independent manner and the expression of innate immunity receptors increases the potential of using these cells in immunotherapies. Aiming to improve the effectiveness of γδ T-cell-based immunotherapies against tumors, we tested various optimization strategies. The most widely used protocol for selectively expanding Vδ2T cells relies on zoledronic acid (ZOL) stimulation of PBMCs in combination with IL-2 or/and IL-15 cytokines. Here, we tested different concentrations of IL-2 (100 and 1,000 U/mL) and IL-15 (12.5 and 100 ng/mL) with 5 μM zoledronic acid. In addition, we compared cell expansion in three defined cell culture media, OpTmizer T Cell Expansion SFM, X-vivo10, and TexMACS (supplemented with 2% AB serum). A total of twenty-four different combinations of IL-2, IL-15, and media were tested in the present study.After twelve days of culture, cells were counted and labelled with anti-CD3, anti-CD8, anti-CD4, anti-Vδ2, anti-PD1, anti-CD69, and anti-NKG2D antibodies for flow cytometry analysis. Our results demonstrated that although TexMACS medium is capable of supporting T-cell activation and expansion, it was not optimal for maximal expansion of Vδ2T cells (fold expansion 5.99 ± 1.61). However, the fold expansion was similar in OpTmizer and X-vivo10 media (fold expansion 21.38 ± 5.13 and 18.37 ± 5.42, respectively). Among the medium supplementation strategies, cultivation in OpTmizer combined with 100 U/mL IL-2 + 12.5 ng/mL IL-15 led to highest expansion index (32.4-fold). As for the activation markers of Vδ2T cells, the frequency of NKG2D+ cells was similar for all conditions tested (above 85%). However, the percentage of CD69+ cells was higher in cells cultured in TexMACS medium compared to OpTmizer and X-vivo10 media (81.6±5.3, 69±9.9, 60.2±7.2, respectively). It is known that the Programmed Cell Death-1 receptor (PD-1) plays an important role in regulating immune cell function. We examined the expression of PD-1 after twelve days of expansion. We observed lower levels of PD-1 in cells expanded in X-vivo10 medium supplemented with 1,000 U/mL IL-2 + 100 ng/mL IL-15, whereas levels of expression were up-regulated significantly when cells were cultivated in the same conditions without IL-15 (28.7% versus 70.6%). Cells expanded in either OpTimizer or TexMACS demonstrated a similar frequency to PD-1 positive cells (41±13% and 49.7%±9.5). In conclusion, we reported an optimized protocol to improve the expansion and the quality of Vδ2 T cells. These findings set the groundwork for the establishment of an off-the-shelf advanced cellular immunotherapy using this alternative population of T cells.

The Proportion of Vδ1T Cells in Peripheral Blood Correlated with Prognosis of Sepsis.

Sepsis is a serious condition with a high mortality rate, and septic patients often have organ dysfunction, low tissue perfusion and hypoxia, lactic acidosis, oliguria, or functional brain changes. To observe the number and the function of Vδ1T cells in peripheral blood of septic patients, to analyze the clinical significance of detecting Vδ1T cells, and to clarify the correlation of their presence with the prognosis of sepsis. The basic data of the septic patients were recorded at admission. The immunosuppressive function-related molecules on the surface of Vδ1T cells were detected, and the immunosuppressive function of Vδ1T cells was also evaluated. Compared with the healthy controls, the proportion of Vδ1T cells in the blood of septic patients significantly decreased (P<0.01). The proportion of Vδ1T cells in septic patients correlated with the patients' condition (P<0.05). The expression of glucocorticoid-induced tumor necrosis factor receptor (GITR), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) on the surface of Vδ1T cells in the blood of septic patients significantly increased (P<0.01). The increase of Vδ1T cells in septic patients had inhibitory effects on T cell proliferation and interferon (IFN)-γ secretion. These findings implied that the immunosuppression of Vδ1Tcells in the peripheral blood of septic patients was significantly higher than that of the healthy controls (P<0.01). Changes in Vδ1T cells in septic patients were closely related to the patient's condition and prognosis.

T-Cell-Based Cellular Immunotherapy of Multiple Myeloma: Current Developments.

Simple SummaryOver the past two decades, there has been significant progress in the treatment of multiple myeloma. Starting with the approval of bortezomib and lenalidomide, followed by newer agents in the same classes, monoclonal antibodies, and most recently idecabtagene vicleucel and ciltacabtagene autoleucel, which are genetically engineered autologous T-cell-based therapies, our view of this disease has changed from incurable to controllable and potentially curable. In addition to multiple myeloma and B-cell lymphomas, T-cell-based therapies are also actively investigated in various types of hematological and non-hematological malignancies and are considered one of the most impactful evolutions in cancer therapeutics. This review aims to summarize existing data regarding the efficacy, toxicity, and management of unique adverse events in T-cell-based therapies that are both clinically available and under investigation. We will also address undergoing efforts to improve the survival outcomes of multiple myeloma patients through this treatment modality.T-cell-based cellular therapy was first approved in lymphoid malignancies (B-cell acute lymphoblastic leukemia and large B-cell lymphoma) and expanding its investigation and application both in hematological and non-hematological malignancies. Two anti-BCMA (B cell maturation antigen) CAR (Chimeric Antigen Receptor) T-cell therapies have been recently approved for relapsed and refractory multiple myeloma with excellent efficacy even in the heavily pre-treated patient population. This new therapeutic approach significantly changes our practice; however, there is still room for further investigation to optimize antigen receptor engineering, cell harvest/selection, treatment sequence, etc. They are also associated with unique adverse events, especially CRS (cytokine release syndrome) and ICANS (immune effector cell-associated neurotoxicity syndrome), which are not seen with other anti-myeloma therapies and require expertise for management and prevention. Other T-cell based therapies such as TCR (T Cell Receptor) engineered T-cells and non-genetically engineered adoptive T-cell transfers (Vγ9 Vδ2 T-cells and Marrow infiltrating lymphocytes) are also actively studied and worth attention. They can potentially overcome therapeutic challenges after the failure of CAR T-cell therapy through different mechanisms of action. This review aims to provide readers clinical data of T-cell-based therapies for multiple myeloma, management of unique toxicities and ongoing investigation in both clinical and pre-clinical settings.

Vδ2 T cells are associated with favorable clinical outcomes in patients with bladder cancer and their tumor reactivity can be boosted by BCG and zoledronate treatments

Background Bladder cancer is an important public health concern due to its prevalence, high risk of recurrence and associated cost of management. Although BCG instillation for urothelial cancer treatment is...

Phenotypic Changes of Peripheral γδ T Cell and Its Subsets in Patients With Coronary Artery Disease.

Coronary atherosclerotic heart disease (CAD) is a chronic inflammatory cardiovascular disease with high morbidity and mortality. Growing data indicate that many immune cells are involved in the development of atherosclerosis. However, the immunological roles of γδ T cells in the initiation and progression of CAD are not fully understood. Here, we used flow cytometry to determine phenotypical changes of γδ T cells and their subpopulations in peripheral blood samples collected from 37 CAD patients. The Pearson correlation coefficient was used to analyze the relationship between the clinical parameter (serum LDL-C level) and the changes of immunophenotypes of γδ T cells. Our results demonstrated that the frequencies and absolute numbers of total γδ T cells and Vδ2+ T cells were significantly decreased in CAD patients when compared to healthy individuals. However, the proportion of Vδ1+ T cells was much lower in CAD patients than that of healthy individuals. Most importantly, a significant alteration of the Vδ1/Vδ2 ratio was found in CAD patients. In addition, a series of surface markers that are associated with costimulatory signals (CD28, CD40L, CD80, CD86), activation levels (CD69, CD25, HLA-DR), activating NK cell receptors (NKp30, NKp46, NKG2D) and inhibitory receptors (PD-1, CTLA-4, PD-1, Tim-3) were determined and then analyzed in the total γδ T cells, Vδ2+T cells and Vδ2-T cells of CAD patients and healthy individuals. The data demonstrated that immunological activities of total γδ T cells, Vδ2+T cells, and Vδ2-T cells of CAD patients were much lower than those in healthy individuals. Moreover, we found that there were positive correlations between the serum LDL-C levels and frequencies of CD3+γδ+ T cells, CD69+Vδ2+T cells, NKG2D+Vδ2+T cells, and NKp46+Vδ2+T cells. By contrast, there was an inverse correlation between the levels of serum LDL-C and the frequencies of CD69+Vδ2-T cells and NKp46+Vδ2-T cells. Accordingly, these findings could help us to better understand the roles of γδ T cells in the CAD, and shed light on the development of novel diagnostic techniques and therapeutic strategies by targeting γδ T cells for CAD patients.

Gamma Delta T-Cell Based Cancer Immunotherapy: Past-Present-Future.

γδ T-cells directly recognize and kill transformed cells independently of HLA-antigen presentation, which makes them a highly promising effector cell compartment for cancer immunotherapy. Novel γδ T-cell-based immunotherapies, primarily focusing on the two major γδ T-cell subtypes that infiltrate tumors (i.e. Vδ1 and Vδ2), are being developed. The Vδ1 T-cell subset is enriched in tissues and contains both effector T-cells as well as regulatory T-cells with tumor-promoting potential. Vδ2 T-cells, in contrast, are enriched in circulation and consist of a large, relatively homogeneous, pro-inflammatory effector T-cell subset. Healthy individuals typically harbor in the order of 50-500 million Vγ9Vδ2 T-cells in the peripheral blood alone (1-10% of the total CD3+ T-cell population), which can rapidly expand upon stimulation. The Vγ9Vδ2 T-cell receptor senses intracellular phosphorylated metabolites, which accumulate in cancer cells as a result of mevalonate pathway dysregulation or upon pharmaceutical intervention. Early clinical studies investigating the therapeutic potential of Vγ9Vδ2 T-cells were based on either ex vivo expansion and adoptive transfer or their systemic activation with aminobisphosphonates or synthetic phosphoantigens, either alone or combined with low dose IL-2. Immune-related adverse events (irAE) were generally \\mild, but the clinical efficacy of these approaches provided overall limited benefit. In recent years, critical advances have renewed the excitement for the potential of Vγ9Vδ2 T-cells in cancer immunotherapy. Here, we review γδ T-cell-based therapeutic strategies and discuss the prospects of those currently evaluated in clinical studies in cancer patients as well as future therapies that might arise from current promising pre-clinical results.

Abstract 2820: Innate immune system in a bag: A novel immunotherapy comprising NK cells, Vδ1 and Vδ2 T-cells

Abstract The recent successes of chimeric antigen receptor (CAR) T cells in the treatment of hematological malignancies have led to an explosion of adoptive cell therapies for cancer. Currently approved therapies are still limited to patient-autologous αβ T cells. Although successful in some settings, this approach comes with challenges including associated toxicities, high production costs and the requirement to gene edit cells to avoid graft vs host disease in an allogeneic setting. In contrast to αβ T cells, innate immune cells such as Natural Killer (NK) cells and gamma delta (γδ) T cells offer opportunities to develop MHC unrestricted, fully allogeneic, off-the-shelf immunotherapies without the need for gene editing. Whilst NK cells offer great cytotoxic potential, control of MHC dysregulation and stress sensing receptors, γδ T cells on the other hand combine the power of a TCR with a variety of natural cytotoxicity receptors and the capability to orchestrate adaptive immune responses even in an HLA-mismatched context. Lately, Vδ1 T-cells have gained significant attention in the field of cancer immunotherapy based on their evolutionary conserved role in cancer immunosurveillance. Multiple groups are developing protocols to grow clinical grade innate immune cells at scale for the treatment of cancer. Each protocol is designed to grow one specific effector cell type of adequate quality. Despite their unique potential, each cell type faces its own challenges for clinical use. A treatment combining a variety of innate cells could be desirable to mitigate for these limitations. We have developed a first in class, scalable expansion process to simultaneously grow NK cells, Vδ1 and Vδ2 T-cells from a healthy donor leukapheresis in a single culture vessel. This 12-day process induces proliferation of all three cell types, resulting in a viable and cryo-preservable drug product where all cells express markers associated with great cytotoxicity, such as CD56 and NKp30. Named after the high expression of CD56, GDX056 shows great efficacy against haematological and solid tumor cells in-vitro. Integrating a viral transduction step into the expansion process allows us to generate a CAR modified tri-cellular product which shows unprecedented cytotoxicity against target cells at E:T ratios as low as 1:100. In an in vivo NALM-6 leukemia model all cell types show engraftment but different body distribution and persistence over time. Whilst efficacious, GDX056 also serves as a great research tool to study each cell type side-by-side. In summary, we have developed an easily manufacturable, first of its kind cellular product combining all major MHC unrestricted cell types. Whilst Vδ1 T cells and NK cells demonstrate anti-cancer activities, Vδ2 T cells are strong mediators of anti-bacterial biology, hence GDX056 potentially offers a unique combinatorial immunotherapy to be considered in scenarios where patients are prone to infections. Citation Format: André Simoes, Mihil Patel, Amy Lane, Sam Illingworth, Sarah Edwards, Istvan Kovacs, Michael Koslowski, Oliver Nussbaumer. Innate immune system in a bag: A novel immunotherapy comprising NK cells, Vδ1 and Vδ2 T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2820.

Abstract 2851: Moving on: Embracing γδ T-cell biology to create truly next generation immunotherapy concepts

Abstract Chimeric antigen receptor (CAR) modification of αβ T cells has revolutionized the field of oncology, driving the focus of attention to the immune system to target and fight malignancies. Currently available αβ T-cell therapies come with many challenges, including alloreactivity, off-target toxicities, cytokine release syndrome, limited survival of infused cells and the necessity to gene edit cells to overcome graft vs. host disease. Whilst some of these limitations can be overcome with complex and costly gene-engineering approaches, utilizing innate immune cells which are inherently non-HMC restricted and able to orchestrate wider immune responses might provide an alternative strategy avoiding many of these obstacles. Many groups developed protocols to grow and modify natural killer cells and invariant natural killer T-cells whilst others, including ours, focused on the development of Vδ1 γδ T-cell-based immunotherapies. Currently, these cell types are evaluated in the clinic in both non-engineered and engineered versions. Most of these engineering principles have been directly translated from the αβ CAR-T field without taking into consideration the biology of innate immune cells: they utilize αβ T-cell specific co-stimulatory molecules and signaling cassettes as well as armoring strategies developed for αβ T-cells. Vδ1 T-cells are tissue resident lymphocytes, which for most of their lifetime remain in epithelia rich tissues. They consistently survey tissues and monitor malignant transformation using a variety of natural cytotoxicity receptors, NK like receptors and the γδ TCR. In contrast to αβ T-cells, Vδ1 T-cells do not follow the ‘two-signal theory’: they must integrate multiple signals from an array of receptors in order to discriminate between healthy and malignant cells. We thus propose that CAR strategies exclusively utilizing CD3ζ activation domains are not using the full potential of innate immune cells for cellular immunotherapy. Another distinguishing feature of Vδ1 T-cells is the absence of traditional autocrine cytokine feedback loops seen in αβ T-cells. Our results demonstrate that traditional cytokine armoring strategies utilizing forced secretion of soluble or membrane-tethered IL-15 are detrimental to the biology of Vδ1 T-cells. By dissecting the IL-15 receptor biology we are now able to create cells that not only survive and grow in the absence of exogenous IL-15 but become more sensitive to endogenous levels. Applying γδ T-cell biology and an adapted understanding of the IL-15 pathway, we have now created novel engineering strategies to tailor specificity, potency, and proliferation of Vδ1 T-cells even in the presence of CAR whilst maintaining the cells’ inherent ability to discriminate between healthy and malignant cells. Preclinical evaluation of these concepts is ongoing with the aim to develop next generation tailored Vδ1T-cell immunotherapies. Citation Format: Jyothi Kumaran, Rajeev Karattil, André Simoes, Rebecca Alade, Mihil Patel, Liz Wood, Gonzalo Mercado Vico, Amy Lane, Sara Tamagno, Sarah Edwards, Andrea Venuso, Sam Illingworth, Alice Brown, Michael Koslowski, Istvan Kovacs, Oliver Nussbaumer. Moving on: Embracing γδ T-cell biology to create truly next generation immunotherapy concepts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2851.

A Simple and Robust Single-Step Method for CAR-Vδ1 γδT Cell Expansion and Transduction for Cancer Immunotherapy.

The γδT cell subset of peripheral lymphocytes exhibits potent cancer antigen recognition independent of classical peptide MHC complexes, making it an attractive candidate for allogeneic cancer adoptive immunotherapy. The Vδ1-T cell receptor (TCR)-expressing subset of peripheral γδT cells has remained enigmatic compared to its more prevalent Vγ9Vδ2-TCR and αβ-TCR-expressing counterparts. It took until 2021 before a first patient was dosed with an allogeneic adoptive Vδ1 cell product despite pre-clinical promise for oncology indications stretching back to the 1980s. A contributing factor to the paucity of clinical progress with Vδ1 cells is the lack of robust, consistent and GMP-compatible expansion protocols. Herein we describe a reproducible one-step, clinically translatable protocol for Vδ1-γδT cell expansion from peripheral blood mononuclear cells (PBMCs), that is further compatible with high-efficiency gene engineering for immunotherapy purposes. Briefly, αβTCR- and CD56-depleted PBMC stimulation with known-in-the-art T cell stimulators, anti-CD3 mAb (clone: OKT-3) and IL-15, leads to robust Vδ1 cell expansion of high purity and innate-like anti-tumor efficacy. These Vδ1 cells can be virally transduced to express chimeric antigen receptors (CARs) using standard techniques, and the CAR-Vδ1 exhibit antigen-specific persistence, cytotoxicity and produce IFN-γ. Practicable, GMP-compatible engineered Vδ1 cell expansion methods will be crucial to the wide-spread clinical testing of these cells for oncology indications.

OP37 Bacterial suppression of intestinal fungi via activation of human gut Vδ2+ T-cells

Abstract Background The human gut is colonized by diverse microbes including fungi such as Candida albicans, which has been implicated in the pathogenesis of Crohn’s disease (CD). Intestinal C. albicans typically exists as commensal yeast, but can also form tissue-invasive filaments that secrete Candidalysin toxin to disrupt epithelial barriers. Commensal gut bacteria produce molecules that induce host immunity, including phosphoantigen HMB-PP which stimulates Vδ2+ T-cell homing to intestine and mucosal expression of IL-22 to promote barrier integrity. We aimed to identify mechanisms of gut barrier defence against fungal invasion in health and CD. Methods Colonic biopsies / resected tissue were stimulated or not with HMB-PP in the presence or absence of anti-IL-22 blocking antibody and analysed by flow-cytometry / microscopy to determine extent of fungal outgrowth. Fungal isolates were identified using standard morphological and biochemical criteria before assessment of filamentation, cell wall composition, and NETosis induction. Results Mucosal Vδ2+ T-cell activation with bacterial phosphoantigen HMB-PP, in the presence of epithelial damage-associated cytokine IL-15, potently suppressed growth of endogenous fungi in human colonic organ cultures. Vδ2-mediated fungal suppression required IL-22 and was sufficient to restrict the growth of multiple fungal species, including a range of emerging pathogenic moulds as well as archetypal pathobiont C. albicans. In Vδ2-deficient CD patients, mucosal biopsy stimulation with HMB-PP failed to control fungal growth. CD-derived C. albicans strains also displayed rapid filamentation ex vivo and higher levels of NOD2 ligand chitin than were observed in isolates from healthy controls. Comparing hypoxic with oxygenated cultures that mimic inflamed intestine, pSILAC proteomic analysis of a representative CD-derived C. albicans strain confirmed features of invasive growth (e.g. DAO1, IHD1), whereas a healthy control isolate exhibited metabolic hallmarks of symbiosis (Jen1p, SCH9). These divergent characteristics directly impacted on host anti-fungal immune responses, since the CD-derived strain rapidly induced neutrophil extracellular traps in vitro, whereas the healthy control isolate did not. Conclusion These data suggest that commensal bacteria can activate host Vδ2 T-cells to suppress fungal invasion of the gut epithelium via an IL22-dependent mechanism that is deficient in CD patients.

Immune tumoral microenvironment in gliomas: focus on CD3+ T cells, Vδ1+ T cells, and microglia/macrophages.

Gliomas are histologically defined as low-grade gliomas (LGG) and high-grade gliomas (HGG). The most common type of HGG is the glioblastoma (GBM). We aimed to determine the immunological characteristics of CD3 T-cells, Vδ1 T-cells, and microglia/macrophages infiltrating brain gliomas. We collected 24 formalin-fixed paraffin-embedded samples issued from 19 cases of GBM and 5 cases of LGG. An immunohistochemical analysis was performed using anti-CD3, anti-Vδ1, and anti-iba-1 antibodies. Labelling indexes (LI) of CD3 and Vδ1 were evaluated quantitatively, and other CD3, Vδ1, and iba-1 staining characteristics were evaluated qualitatively. The median age of patients was 49years in GBM and 52years in LGG. The sex ratio was 1.4 and GBM predominated in males (p = 0.05). In GBM, the medians of CD3-LI and Vδ1-LI were 30 and 3.5 respectively. CD3-LI inversely correlated with survival in GBM cases (r = - 0.543; p = 0.016). CD3 staining intensity correlated with CD3-LI (p < 0.0001) and with the survival in GBM cases (p = 0.003). Compared to LGG, the CD3-LI, the intensity of intra-tumoral Vδ1 staining, and the amount of iba-1 were higher in GBM (p = 0.042; p = 0.014; and p = 0.001 respectively). The iba-1 organization was more amoeboid in older patients and more branched in younger patients (p = 0.028) and tended to be more amoeboid in cases with high iba-1 amount (p = 0.09). Our results suggest that a high level of CD3-LI and a strong intra-tumoral infiltration of Vδ1 T-cells as well as a high involvement of TAM can be considered potential markers of poor prognosis of GBM. However, this requires further studies on more balanced GBM-LGG sample, including an expanded panel of biomarkers.