Expansion Of Γδ Research Articles

Expansion and Polarization of Human γδT17 Cells in vitro from Peripheral Blood Mononuclear Cells.

γδ T cells play a critical role in homeostasis and diseases such as infectious diseases and tumors in both mice and humans. They can be categorized into two main functional subsets: IFN-γ-producing γδT1 cells and IL-17-producing γδT17 cells. While CD27 expression segregates these two subsets in mice, little is known about human γδT17 cell differentiation and expansion. Previous studies have identified γδT17 cells in human skin and mucosal tissues, including the oral cavity and colon. However, human γδ T cells from peripheral blood mononuclear cells (PBMCs) primarily produce IFN-γ. In this protocol, we describe a method for in vitro expansion and polarization of human γδT17 cells from PBMCs. Key Features • Expansion of γδ T cells from peripheral blood mononuclear cells. • Human IL-17A-producing γδ T-cell differentiation and expansion using IL-7 and anti-γδTCR. • Analysis of IL-17A production post γδ T-cell expansion. This protocol is used in: Science Advances (2022), DOI: 10.1126/sciadv.abm9120.

Characterization of expanded γδ T cells from patient with atypical X-linked severe combined immunodeficiency reveals preserved function and IL2RG-mediated signaling

Abnormally high γδ T cell numbers are common among atypical severe combined immunodeficiency (SCID) patients but detailed immunophenotyping and functional characterization of these expanded γδ T cells are limited. We have previously reported atypical SCID phenotype caused by hypomorphic IL2RG c.172C > T;p.(Pro58Ser) variant causing failure in IL2RG (CD132) plasma membrane targeting leading to impaired IL-2 responses in CD4 and CD8 T cells. Our aim was to investigate the phenotype and function of the index patient's abnormally large peripheral γδ T cell population.We found that in contrast to his αβ T cells, the patient's γδ T cells showed normal IL2RG cell surface expression and normal or enhanced IL2RG-mediated signaling. Vδ2+ population was proportionally increased with a preponderance of memory phenotypes and high overall tendency towards perforin expression. The patient's γδ T cells showed enhanced cytotoxicity towards A549 cancer cells. TCRvγ repertoire was versatile but sequencing of IL2RG revealed a novel c.534C>A; p.(Phe178Leu) somatic missense variant restricted to γδ T cells. Over time this variant became predominant in patient's γδ T cells and was enriched during in vitro expansion of patient's γδ T cells, indicating that the clones with this somatic variant may have growth or survival advantage. In silico analysis with DynaMut2 tool indicated that the overall effect of Phe178Leu substitution for IL2RG was predicted to be stabilizing and IL2RG-Pro58Ser/Phe178Leu to be more stable than IL2RG-Pro58Ser variant alone. Furthermore, IL2RG-Pro58Ser/Phe178Leu showed significantly increased cell surface expression when compared to IL2RG-Pro58Ser variant in stable HEK293 cell lines, suggesting that somatic IL2RG p.(Phe178Leu) variant may at least partially rescue the pathogenic effect of germline IL2RG p.(Pro58Ser) variant.In conclusion, our results indicate that expansion of γδ T cells associated with atypical SCID needs further studying and cannot exclusively be deemed as a homeostatic response to low numbers of conventional T cells. (Tuovinen et al., Journal of Clinical Immunology 2022)

Issue Information – Cover

Microbiology and ImmunologyVolume 67, Issue 5 p. i-ii COVER IMAGEFree Access Issue Information – Cover First published: 02 May 2023 https://doi.org/10.1111/1348-0421.12995AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Cover photograph: (1) The role of γδ T cells in skin is still unknown. Do they interact with sporozoite? Do they able to kill sporozoite or facilitate DC to recognize and phagocytize sporozoite? Lots to be answered in the future. (2) The responses of γδ T cells during liver-stage are mainly protective. Studies of attenuated sporozoite vaccination showed activation and expansion of γδ T cells. These γδ T cells produce large amount of IFN-γ and were able to protect subsequent challenge infection in human. γδ T cells are involved in activation and maturation of DC, and they may facilitate recruitment of inflammatory cells in the liver. γδ T cells also contribute to the activation and accumulation of CD8+ T cells directly or through DC. (3) Protective and pathogenic roles of γδ T cells during blood-stage have been proposed. Upon activation γδ T cells produce pro-inflammatory cytokines which enhance APC cells maturation. Also they are involved in accumulation and activation of myeloid cells by producing M-CSF. They can present antigens to naïve γδ T cells and initiate adaptive immune response. Moreover, γδ T cells facilitate antibody production and germinal center formation either directly contacting with B cells or through T follicular helper cells. In vitro studies of human γδ T cells showed capacity of antigen presentation, phagocytosis and cytotoxicity. However, these functions should be investigated in the tissue or in vivo. Microbiol Immunol: 67:239–247. Article link here Volume67, Issue5May 2023Pages i-ii RelatedInformation

Characterization of Expanded Gamma Delta T Cells from Atypical X-SCID Patient Reveals Preserved Function and IL2RG-Mediated Signaling

Abnormally high γδ T cell numbers among individuals with atypical SCID have been reported but detailed immunophenotyping and functional characterization of these expanded γδ T cells are limited. We have previously reported atypical SCID phenotype caused by hypomorphic IL2RG (NM_000206.3) c.172C > T;p.(Pro58Ser) variant. Here, we have further investigated the index patient’s abnormally large γδ T cell population in terms of function and phenotype by studying IL2RG cell surface expression, STAT tyrosine phosphorylation and blast formation in response to interleukin stimulation, immunophenotyping, TCRvγ sequencing, and target cell killing. In contrast to his ⍺β T cells, the patient’s γδ T cells showed normal IL2RG cell surface expression and normal or enhanced IL2RG-mediated signaling. Vδ2 + population was proportionally increased with a preponderance of memory phenotypes and high overall tendency towards perforin expression. The patient’s γδ T cells showed enhanced cytotoxicity towards A549 cancer cells. His TCRvγ repertoire was versatile but sequencing of IL2RG revealed a novel c.534C > A; p.(Phe178Leu) somatic missense variant restricted to γδ T cells. Over time this variant became predominant in γδ T cells, though initially present only in part of them. IL2RG-Pro58Ser/Phe178Leu variant showed higher cell surface expression compared to IL2RG-Pro58Ser variant in stable HEK293 cell lines, suggesting that somatic p.(Phe178Leu) variant may at least partially rescue the pathogenic effect of germline p.(Pro58Ser) variant. In conclusion, our report indicates that expansion of γδ T cells associated with atypical SCID needs further studying and cannot exclusively be deemed as a homeostatic response to low numbers of conventional T cells.

Isolation and expansion of murine γδ T cells from mouse splenocytes

Elucidation of the function of gamma delta T cells (γδ T cells) requires robust models that show how γδ T cells are commonly involved in inflammation, since very little is known about the factors that promote and control their development and function. There are few studies of murine γδ T cells primarily because these cells have proven difficult to isolate, expand and characterize. Here, we describe a simple method that utilizes key expansion elements to isolate and expand murine CD4−CD8−CD3+ γδ T cells typically found in secondary lymphoid tissues. Expansion of γδ T cells reached 150-fold by day 8 of culture, depended on exogenous IL-2, αCD3, and αCD28, and supported efficient and reproducible in vitro differentiation. These studies showed high production of cytokines IFNγ and Granzyme B, with the novel finding of IL-24 upregulation as well. Expression analysis of expanded γδ T cells, after treatment with IL-2, revealed high levels of Granzyme B, Granzyme D, and IFNγ. Lactate dehydrogenase (LDH) cytotoxicity assays showed that expanded γδ T cells were effective at inducing >90% cytolysis of murine MC38 colon cancer, E0771 breast cancer, and B16 melanoma cells at 10:1 effector to target ratios. These findings indicated that murine γδ T cells can be successfully isolated, expanded, and used to perform preclinical therapy studies.

TransCon IL-2 β/γ, a novel long-acting prodrug with sustained release of an IL-2Rβ/γ-selective IL-2 analog, demonstrates improved pharmacokinetics and profound expansion of cytotoxic immune cells in non-human primates.

2563 Background: Recombinant interleukin-2 (IL-2, aldesleukin) is an approved cancer immunotherapy but causes severe side effects including cytokine release syndrome (CRS) and vascular leak syndrome (VLS). This is believed to be due to activation of IL-2Rα+ endothelial cells and inflammatory eosinophils as well as high Cmax due to the short half-life requiring frequent high dose administrations. Also, potent activation of immunosuppressive IL-2Rα+ regulatory T cells (Tregs) may limit IL-2’s efficacy. TransCon IL-2 β/γ is a novel long-acting prodrug with sustained release of an IL-2Rβ/γ-selective IL-2 analog designed to optimally address these shortcomings. Methods: Naïve male and female cynomolgus monkeys received 2-3 doses of TransCon IL-2 β/γ intravenously Q2W. Blood samples were taken to assess plasma concentrations of IL-2 β/γ (active drug), serum cytokines, hematology and immunophenotyping. Histopathology was also performed. Results: Administration of TransCon IL-2 β/γ induced robust increases in absolute lymphocyte counts (ALC), with no/minor increases in eosinophil counts. The increase in ALC was primarily driven by potent activation and expansion of CD8+ T cells, NK cells and γδ T cells, relative to CD4+ T cells and Tregs. At doses with maximum pharmacodynamic (PD) responses, TransCon IL-2 β/γ induced on average > 20-fold ALC increases, > 19-fold CD8+ T cell increases, > 50-fold Effector Memory CD8+ T cell increases, > 18-fold NK cell increases and > 400-fold γδ T cell increases compared to pre-dose levels. These changes corresponded with a low Cmax and a long effective half-life ( > 30 hours) for IL-2 β/γ. A clear induction of soluble CD25 and chemokine MCP-1 was seen after dosing, while levels of IL-5, IL-6, IFN-γ and TNF-α remained low. Further, histopathology showed no signs of vascular damage, tissue necrosis, pulmonary edema or eosinophilia. Conclusions: In cynomolgus monkeys, TransCon IL-2 β/γ demonstrated remarkable PD responses in CD8+ T cells, NK cells and γδ T cells over Tregs and eosinophils. No signs of VLS or CRS were observed. These results are likely due to the selective IL-2Rβ/γ bias along with the slow and sustained release of IL-2 β/γ resulting in low Cmax and long circulatory half-life enabled by the TransCon prodrug technology. The massive expansion of γδ T cells, which are known to possess considerable anti-tumor activity, is to our knowledge a unique finding for TransCon IL-2 β/γ among IL-2 variants in clinical development. Altogether, the responses seen in monkeys are suggestive of a potentially substantial improvement in the therapeutic index beyond what has been achieved by aldesleukin or new IL-2 variants to date. TransCon IL-2 β/γ is currently being evaluated in a clinical Phase 1/2 trial as monotherapy and in combination with pembrolizumab (NCT05081609).

Optimization of methods for peripheral blood mononuclear cells isolation and expansion of human gamma delta T cells

Human Vγ9/Vδ2 T cells (γδ T cells) are immune surveillance cells both in innate and adaptive immunity and are a possible target for anticancer therapies, which can induce immune responses in a variety of cancers. Small non-peptide antigens such as zoledronate can do activation and expansion of T cells in vitro. It is evident that for adoptive cancer therapies, large numbers of functional cells are needed into cancer patients. Hence, optimization of methods needs to be carried out for the efficient expansion of these T cells. Standardization of peripheral blood mononuclear cells (PBMCs) isolation was devised. Cytokines (interleukin 2 (IL-2) and interleukin 15 (IL-15)) and zoledronate were also standardized for different concentrations. It was found that an increased number of PBMCs were recovered when washing was done at 1100 revolution per minute (rpm). Significantly high expansion fold was (2524 ± 787 expansion fold) achieved when stimulation of PBMCs was done with 1 μM of zoledronate and both cytokines IL-2 and IL-15 supported the expansion and survival of cells ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 17(3): 460-469 (2021) ©Biomedical Informatics (2021) 461 at the concentrations of 100 IU/ml and 10 ng/ml respectively. 14-day cultures showed highly pure (91.6 ± 5.1%) and live (96.5 ± 2.5%) expanded γδ T cells. This study aimed to standardize an easy to manipulate technique for the expansion of γδ T cells, giving a higher yield.

Characterization of Donor Variability for γδ T Cell ex vivo Expansion and Development of an Allogeneic γδ T Cell Immunotherapy.

Gamma delta (γδ) T cells recently emerged as an attractive candidate for cancer immunotherapy treatments due to their inherent cytotoxicity against both hematological and solid tumors. Moreover, γδ T cells provide a platform for the development of allogeneic cell therapies, as they can recognize antigens independent of MHC recognition and without the requirement for a chimeric antigen receptor. However, γδ T cell adoptive cell therapy depends on ex vivo expansion to manufacture sufficient cell product numbers, which remains challenging and limited by inter-donor variability. In the current study, we characterize the differences in expansion of γδ T cells from various donors that expand (EX) and donors that fail to expand, i.e., non-expanders (NE). Further, we demonstrate that IL-21 can be used to increase the expansion potential of NE. In order to reduce the risk of graft vs. host disease (GVHD) induced by an allogeneic T cell product, αβ T cell depletions must be considered due to the potential for HLA mismatch. Typically, αβ T cell depletions are performed at the end of expansion, prior to infusion. We show that γδ T cell cultures can be successfully αβ depleted on day 6 of expansion, providing a better environment for the γδ T cells to expand, and that the αβ T cell population remains below clinically acceptable standards for T cell-depleted allogeneic stem cell products. Finally, we assess the potential for a mixed donor γδ T cell therapy and characterize the effects of cryopreservation on γδ T cells. Collectively, these studies support the development of an improved allogeneic γδ T cell product and suggest the possibility of using mixed donor γδ T cell immunotherapies.

First-in-Human Phase I Trial of Adoptive Immunotherapy with Ex Vivo Expanded and Activated Γδ T Cells Following Haploidentical Bone Marrow Transplantation and Post-BMT Cyclophosphamide

INTRODUCTION: HAPLO BMT combined with cyclophosphamide infusion on days +3 and +4 following BMT (PTCy) for patients that lack an HLA-matched donor provides effective graft versus host disease (GVHD) prophylaxis but with a heightened risk of disease recurrence likely due to prolonged immunodeficiency. Multiple studies have shown that increases in circulating donor-derived gamma delta (γδ) T cells during the early post-transplant period is strongly associated with significant improvement in disease-free survival (DFS). We developed a clinical protocol to engineer this effect by infusing escalating doses of ex vivo expanded and activated donor γδ T cells (EAGD) during this period of immunodeficiency as prophylaxis against disease recurrence. As innate immune effectors γδ T cells immediately recognize and kill malignant cells in a broad-based non-MHC restricted manner and do not initiate GVHD. STUDY DESIGN AND METHODS: This single-center Phase I clinical trial of allogeneic haploidentical BMT + EAGD (NCT03533816), currently underway at the Kansas University Cancer Center, represents the first time BMT patients have received a large infusion of EAGD from a haploidentical donor during the early post-transplant time period. Subjects ≥18 years of age with AML, ALL, CML or MDS either in morphologic complete remission with high risk features, in first complete remission, or chronic phase (CML) eligible for BMT are enrolled in a 3 x 3 design with escalating EAGD doses from 1 x 106/kg to 3 x 106/kg to 1 x 107/kg. Subjects must have adequate organ function and a KPS ≥70. Any subjects with central nervous system neoplastic involvement, HIV infection, or a life expectancy <12 weeks are excluded. Haploidentical donors provide an initial non-mobilized leukapheresis product from which EAGD are manufactured and an unmanipulated bone marrow harvest as the source of hematopoietic stem cells. Closed system EAGD manufacturing is performed in the Miltenyi Prodigy® bioreactor by expansion of γδ T cells in conjunction with αβ T cell depletion. Specimens for composition, viability, sterility, identity, and purity are obtained prior to cryopreservation and results obtained prior to infusion. Subjects undergo standard of care conditioning therapy with fludarabine and cyclophosphamide followed by BMT and post-BMT cyclophosphamide (Cy). The EAGD product is thawed and infused directly within 5 days of neutrophil engraftment (ANC >500/mL for 3 consecutive days). Peripheral blood is collected at screening, pre and post EAGD infusion, then monthly thereafter through Day 100 with additional samples collected every 6 months through 1 year and annually thereafter. Biologic parameters include leukemia phenotype and genomics, multiparameter peripheral blood immunophenotyping, single-cell and serum Th1/Th2/Th17 cytokine analysis, and immunogenomics for both peripheral blood and the EAGD cell product. Primary endpoints include the following safety assessments; laboratory parameters, viral monitoring, physical exams, acute and chronic GVHD, as well as biopsy pathology. Relapse and overall survival will be measured as secondary endpoints. Descriptive statistics under each specific dose will be used to summarize baseline characteristics, safety variables and efficacy outcomes. Kaplan-Meier curves and summary statistics will be used to summarize time-to-event outcomes. STATUS: To date, 3 female subjects (age range, 44 - 54) with AML have been enrolled with 2 subjects receiving the EAGD infusion. To have no treatment-related adverse events have been recorded Disclosures Lamb: In8bio: Current Employment, Current equity holder in private company. Beelen:In8bio: Current Employment, Current equity holder in publicly-traded company. Youngblood:In8bio: Current Employment, Current equity holder in publicly-traded company. McGuirk:Pluristem Ltd: Research Funding; Allo Vir: Consultancy, Honoraria, Research Funding; Kite Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bellicum Pharmaceutical: Research Funding; Astellas: Research Funding; Fresenius Biotech: Research Funding; Novartis: Research Funding; Gamida Cell: Research Funding; Juno Therapeutics: Consultancy, Honoraria, Research Funding.

Putting mice on a keto diet

Infectious Disease Our immune responses to infections are influenced by extrinsic factors, including weather, social interactions, and diet. Goldberg et al. found that feeding mice a high-fat, low-carbohydrate ketogenic diet confers protection in the context of lethal influenza infection. By characterizing the immune response in the lungs, the authors identified that a ketogenic diet promoted the expansion of γδ T cells in the lung. Experiments with mice lacking γδ T cells confirmed the functional importance of these cells in conferring protection. The γδ T cells appear to improve barrier function in the lungs by modifying differentiation and function of the airway epithelial cells. Sci. Immunol. 4 , eaav2026 (2019).

Ketogenic diet activates protective γδ T cell responses against influenza virus infection

Abstract Influenza A virus (IAV) infection-associated morbidity and mortality are a key global healthcare concern, necessitating the identification of novel therapies capable of reducing the severity of IAV infections. In this study, we show that the consumption of a low-carbohydrate, high-fat ketogenic diet (KD) protects mice from lethal IAV infection and disease. KD feeding resulted in an expansion of γδ T cells in the lung that improved barrier functions, thereby enhancing anti-viral resistance. Expansion of these protective γδ T cells required metabolic adaptation to a ketogenic diet, as neither feeding mice a high-fat high-carbohydrate diet nor providing chemical ketone body substrate that bypasses hepatic ketogenesis protected against infection. Therefore, KD mediated immune-metabolic integration represents a viable avenue towards preventing or alleviating influenza disease.

γδ T Cell-Mediated Immunity to Cytomegalovirus Infection.

γδ T lymphocytes are unconventional immune cells, which have both innate- and adaptive-like features allowing them to respond to a wide spectrum of pathogens. For many years, we and others have reported on the role of these cells in the immune response to human cytomegalovirus in transplant patients, pregnant women, neonates, immunodeficient children, and healthy people. Indeed, and as described for CD8+ T cells, CMV infection leaves a specific imprint on the γδ T cell compartment: (i) driving a long-lasting expansion of oligoclonal γδ T cells in the blood of seropositive individuals, (ii) inducing their differentiation into effector/memory cells expressing a TEMRA phenotype, and (iii) enhancing their antiviral effector functions (i.e., cytotoxicity and IFNγ production). Recently, two studies using murine CMV (MCMV) have corroborated and extended these observations. In particular, they have illustrated the ability of adoptively transferred MCMV-induced γδ T cells to protect immune-deficient mice against virus-induced death. In vivo, expansion of γδ T cells is associated with the clearance of CMV infection as well as with reduced cancer occurrence or leukemia relapse risk in kidney transplant patients and allogeneic stem cell recipients, respectively. Taken together, all these studies show that γδ T cells are important immune effectors against CMV and cancer, which are life-threatening diseases affecting transplant recipients. The ability of CMV-induced γδ T cells to act independently of other immune cells opens the door to the development of novel cellular immunotherapies that could be particularly beneficial for immunocompromised transplant recipients.

Recombinase Activating Gene 1 Deficiencies Without Omenn Syndrome May Also Present With Eosinophilia and Bone Marrow Fibrosis.

BackgroundSevere combined immunodeficiency (SCID) syndromes are a heterogenous group of diseases characterized by impairment in both cellular and humoral immunity with a range of genetic disorders. Complete recombinase activating gene (RAG) deficiency is associated with classical T-B-NK+ SCID which is the most common phenotype of Turkish SCID patients. There is a broad spectrum of hypomorfic RAG mutations including Omenn syndrome, leaky or atypical SCID with expansion of γδ T cells, autoimmunity and cytomegalovirus (CMV) infections.MethodsTwenty-one (44%) patients had RAG1 deficiency of all 44 SCID patients followed up by pediatric immunology department. A retrospective analysis was conducted on the medical records of all SCID patients with RAG1 deficiency.ResultsEight patients were classified as T-B-NK+ SCID, five patients as T+B-NK+ SCID (three of these were Omenn phenotype), and eight patients as T+B+NK+ SCID phenotype. Mean age of the whole study group, mean age at onset of symptoms and mean age at diagnosis were 87.7 ± 73.8 (12 - 256), 4.4 ± 8.2 (1 - 36) and 29.1 ± 56.8 (1 - 244) months, respectively. Consanguinity was present in 11 (52%) of 21 patients. Autoimmunity was found in six patients (28%). Ten patients (47%) had CMV infection, four (19%) had Epstein-Barr virus (EBV) infections and three (14%) had Bacillus Calmette-Guerin (BCG) infections. Seven patients who had refractory cytopenia (two pancytopenia and five bicytopenia) underwent bone marrow biopsy, three of whom had bone marrow fibrosis. Future evaluations must be considered about bone marrow fibrosis in RAG1 deficiency patients. Eosinophilia was observed in 10 patients, seven of whom did not have Omenn phenotype.ConclusionNon-Omenn phenotype RAG1 deficiencies can also present with eosinophilia. This report is presented to emphasize that RAG1 mutations may lead to diverse clinical phenotypes.

Control of murine cytomegalovirus infection by γδ T cells.

Infections with cytomegalovirus (CMV) can cause severe disease in immunosuppressed patients and infected newborns. Innate as well as cellular and humoral adaptive immune effector functions contribute to the control of CMV in immunocompetent individuals. None of the innate or adaptive immune functions are essential for virus control, however. Expansion of γδ T cells has been observed during human CMV (HCMV) infection in the fetus and in transplant patients with HCMV reactivation but the protective function of γδ T cells under these conditions remains unclear. Here we show for murine CMV (MCMV) infections that mice that lack CD8 and CD4 αβ-T cells as well as B lymphocytes can control a MCMV infection that is lethal in RAG-1-/- mice lacking any T- and B-cells. γδ T cells, isolated from infected mice can kill MCMV infected target cells in vitro and, importantly, provide long-term protection in infected RAG-1-/- mice after adoptive transfer. γδ T cells in MCMV infected hosts undergo a prominent and long-lasting phenotypic change most compatible with the view that the majority of the γδ T cell population persists in an effector/memory state even after resolution of the acute phase of the infection. A clonotypically focused Vγ1 and Vγ2 repertoire was observed at later stages of the infection in the organs where MCMV persists. These findings add γδ T cells as yet another protective component to the anti-CMV immune response. Our data provide clear evidence that γδ T cells can provide an effective control mechanism of acute CMV infections, particularly when conventional adaptive immune mechanisms are insufficient or absent, like in transplant patient or in the developing immune system in utero. The findings have implications in the stem cell transplant setting, as antigen recognition by γδ T cells is not MHC-restricted and dual reactivity against CMV and tumors has been described.

Chitinase-like proteins promote IL-17-mediated neutrophilia in a tradeoff between nematode killing and host damage.

Enzymatically inactive chitinase-like proteins (CLPs) such as BRP-39, Ym1 and Ym2 are established markers of immune activation and pathology, yet their functions are essentially unknown. We show that Ym1 and Ym2 induce neutrophil accumulation through expansion of interleukin 17 (IL-17)-producing γδ T cells. While BRP-39 did not influence neutrophilia, it was required for IL-17 production in γδ T cells, suggesting IL-17 regulation is an inherent feature of murine CLPs. Using a model of lung migrating nematode infection, we also found that IL-17 and neutrophilic inflammation induced by Ym1 limited parasite survival but at the cost of enhanced lung injury. These studies describe effector functions of CLPs consistent with innate host defense traits of the chitinase family.

Abstract 2793: Phenotype and expansion profile of peripheral γδ T cells in patients with gastrointestinal cancer

Abstract During the last decade, adoptive transfer of autologous γδ T cells has been paid much attention to as an alternative immunotherapy for several types of cancer. It has been reported that human γδ T cells have potent cytotoxic activity against many types of tumor cell lines or primary cultured cells in vitro. Clinical studies in patients with metastatic renal cell carcinoma and non-small cell lung carcinoma showed that infusion of autologous γδ T cells is feasible and well tolerated. One of the problems in this therapy, however, is the difficulty for culturing peripheral γδ T cells in patients with cancer. Compared with healthy individuals, stimulation with phosphoantigens or bisphosphonates results in less expansion of γδ T cells in some patients. The tendency can be remarkably seen especially in those who have lower number of γδ T cells in peripheral blood. It is still unclear whether it is a unique phenomenon caused by something of malignancy or just due to the culture methodology. In this study, we investigated the characteristics of peripheral Vγ9Vδ2 T cells in patients with gastrointestinal cancer including their phenotype, the percentage in PBMC, and the proliferation activity in response to synthetic phosphoantigen, 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP). PBMC were prepared from patients, and then the phenotype and the number of Vγ9Vδ2 T cells were measured by flow cytometry. To expand Vγ9Vδ2 T cells in vitro, PBMC were cultured in AIM-V medium supplemented with 100 IU/mL IL-2, 100μM 2M3B1PP, and 10% autologous plasma. The expansion of Vγ9Vδ2 T cells were also examined using flow cytometry. We found that the percentage of the Vγ9Vδ2 T cells in peripheral T cells varied a lot depending on the patients. While their percentage in healthy individual was approx. 1.5-9.0%, it exceeded 10% in some patients, and even over 20% in several patients. Phenotypic analysis revealed that almost all the Vγ9Vδ2 T cells in these particular patients were effector-memory type cells with CD45RA-/CD27-/CCR7- and they massively expanded by the stimulation with 2M3B1PP. These results suggest that Vγ9Vδ2 T cells in these patients have been constantly exposed and stimulated with some specific antigens. On the other hand, the percentage of Vγ9Vδ2 T cells in T cells of some patients was less than 1.0%, and in the conventional culture methods the cells' response to 2M3B1PP was too low for them to proliferate themselves. However, their proliferation rate markedly increased when PBMC were inoculated at the high density (i.e. 1.3 x 104 Vγ9Vδ2 T cells per cm2 or more). The high density culture for 14 days has made it possible to expand the number of Vγ9Vδ2 T cells to 100-1000 folds. We conclude that Vγ9Vδ2 T cells in patients with gastrointestinal cancer have the equivalent level of proliferation ability to healthy individual even though the initial percentage is low, and thus these cells could be a possible effector cell type in a novel immunotherapy for gastrointestinal cancer. Citation Format: Yasunobu Kobayashi, Mayuko Sakai, Izumi Fujita, Hiroshi Komine, Masanori Matsushita, Koichi Shimizu, Atsushi Aruga, Hirohito Kobayashi, Keishi Tanigawa. Phenotype and expansion profile of peripheral γδ T cells in patients with gastrointestinal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2793. doi:10.1158/1538-7445.AM2014-2793

OR21: Gamma-delta T CELL EXPANSION ASSOCIATES WITH LESSER RISK OF ALLOANTIBODY DEVELOPMENT IN PEDIATRIC HEART TRANSPLANTATION

Aim To determine whether the expansion of γδ T cells observed in pediatric heart allograft recipients associates with transplant outcomes. Methods We studied a population of 58 pediatric heart transplant recipients under the auspices of the Institutional Review Board. We performed Ab determinations by solid phase assays (SPA-IgG, “LSAB”). Flow cytometry staining was performed to determine the percentage of lymphocytic lineage markers: CD3, CD4, CD8, CD19, CD16& 56, CD45, TCRγδ, TCRαβ. Results Pediatric heart allograft recipients often require total or partial thymectomy at the time of their first heart operation, which leads to lifelong alterations in their T cell repertoire. While the prevailing population of circulating T cells in non-thymectomized children carries the TCRαβ TCR (96 ± 3%), we observed a significant expansion of TCRγδ T cells by Flow Cytometry in 37 of 58 patients studied. Within the population with high circulating fraction of gd T’s (8–20% of lymphocytes, n = 37), 23 patients (23/37, or 62%) developed anti-HLA antibodies detected by SPA-IgG (LSAB), while 14 did not. In patients with normal TCRγδ fractions ( Conclusion Thymectomy in pediatric heart recipients which results in the expansion of γδ T cells is associated with lesser risk of alloantibody development. The finding that patient with low γδ T cells are more likely to produce anti HLA Abs suggests that thymectomy results in a breakdown of immune regulation. Th2 impairment may result in insufficient cytokine production to fully support antibody production in these patients. Although mechanistic studies are needed to understand the causation relationships underpinning this phenomenon, the association alone suggests that monitoring of γδ T cell expansion may constitute a useful tool for Ab development prediction in pediatric heart transplant patients.

Synergistic effects of ethanol and isopentenyl pyrophosphate on expansion of γδ T cells in synovial fluid from patients with arthritis.

Low to moderate ethanol consumption has been associated with protective effects in autoimmune diseases such as rheumatoid arthritis, RA. An expansion of γδ T cells induced by isopentenyl pyrophosphate, IPP, likewise seems to have a protective role in arthritis. The aim of this project was to test the hypothesis that low doses of ethanol can enhance IPP-induced expansion of synovial fluid γδ T cells from patients with arthritis and may thereby potentially account for the beneficial effects of ethanol on symptoms of the arthritic process. Thus, mononuclear cells from synovial fluid (SF) from 15 patients with arthritis and from peripheral blood (PB) from 15 healthy donors were stimulated with low concentrations of ethanol and IPP for 7 days in vitro. IPP in combination with ethanol 0.015%, 2.5 mM, equivalent to the decrease per hour in blood ethanol concentration due to metabolism, gave a significantly higher fractional expansion of SF γδ T cells compared with IPP alone after 7 days (ratio 10.1+/−4.0, p<0.0008, n = 12) in patients with arthritis. Similar results were obtained for PB γδ T cells from healthy controls (ratio 2.0+/−0.4, p<0.011, n = 15). The augmented expansion of γδ T cells in SF is explained by a higher proliferation (p = 0.0034, n = 11) and an increased survival (p<0.005, n = 11) in SF cultures stimulated with IPP plus ethanol compared to IPP alone. The synergistic effects of IPP and ethanol indicate a possible allosteric effect of ethanol. Similar effects could be seen when stimulating PB with ethanol in presence of risedronate, which has the ability to increase endogenous levels of IPP. We conclude that expansion of γδ T cells by combinatorial drug effects, possibly in fixed-dose combination, FDC, of ethanol in the presence of IPP might give a protective role in diseases such as arthritis.

Frequency and clonality of peripheral γδ T cells in psoriasis patients receiving anti-tumour necrosis factor-α therapy.

Hepatosplenic γδ T cell lymphoma (HSTCL) has been observed in patients with Crohn's disease (CD) who received anti-tumour necrosis factor (TNF)-α agents and thiopurines, but only one case was reported in a psoriasis patient worldwide. This difference could be due to differences in either the nature of the inflammatory diseases or in the use of immunomodulators. We investigated the impact of anti-TNF-α agents on the level and repertoire of γδ T cells in peripheral blood from psoriasis patients. Forty-five men and 10 women who were treated with anti-TNF-α agents for psoriasis were monitored for a median 11 months for the level and clonality of γδ T cells via flow cytometry and polymerase chain reaction (PCR) analysis of T cell receptor gamma (TCR-γ) gene rearrangements. Seventeen men had a repeated analysis within 48 h of the infliximab infusion to reveal a possible expansion of γδ T cells, as observed previously in CD patients. Ten psoriasis patients who were never exposed to biologicals and 20 healthy individuals served as controls. In the majority of psoriasis patients, the level and clonal pattern of γδ T cells was remarkably stable during infliximab treatment. A single male patient repeatedly experienced a significant increase in the level of γδ T cells after infliximab infusions. A monoclonal γδ T cell repertoire in a polyclonal background tended to be more frequent in anti-TNF-α-treated patients than naive patients, suggesting that anti-TNF-α therapy may promote the clonal selection of γδ T cells in psoriasis patients.

Gamma delta T cells in non-immune patients during primary schistosomal infection.

The mevalonate pathway is critical for the survival of Schistosoma. γδ T cells, a small subset of peripheral blood (PB) T cells, recognize low molecular weight phosphorylated antigens in the mevalonate pathway, which drive their expansion to exert protective and immunoregulatory effects. To evaluate their role in schistosomiasis, we measured γδ T cells in the PB of non-immune travelers who contracted Schistosoma hematobium or Schistosoma mansoni in Africa. The maximal level of γδ T-cells following infection was 5.78 ± 2.19% of the total T cells, versus 3.72 ± 3.15% in 16 healthy controls [P = 0.09] with no difference between S. hematobium and S. mansoni in this regard. However, among the nine patients in the cohort who presented with acute schistosomiasis syndrome (AS), the level (3.5 ± 1.9%) was significantly lower than in those who did not (8.6 ± 6.4%, P < 0.05), both before and after therapy. Furthermore, γδ T cells increased significantly in response to praziquantel therapy. In a patient with marked expansion of γδ T cells, most expressed the Vδ2 gene segment, a hallmark of cells responding to cognate antigens in the mevalonate pathways of the parasite or the human host. These results suggest an immunoregulatory role of antigen responsive γδ T cells in the clinical manifestations of early schistosomal infection.