Γδ T-cell Expansion Research Articles

Identification of clonally expanded γδ T-cell populations during CAR-Tcell therapy.

Anti-CD19 Chimeric Antigen Receptor (CAR)-Tcell therapies have shown promise for treating B cell malignancies, but the clinical outcome is influenced by both the CAR-T product and the patient's immune system. The role of γδ Tcells in the context of CAR-T cell therapy remains poorly understood. This study investigates the transcriptional heterogeneity, clonal expansion and dynamics of γδ Tcells in patients undergoing anti-CD19 CAR-T cell therapy. Longitudinal single cell multi-omics analysis was performed on γδ Tcells from four patients receiving anti-CD19 CAR-T cell therapy. Single cell RNA-seq, antibody-based protein profiling (AbSeq) and full-length TCRγδ sequences revealed clonally expanded populations displaying plasticity in Tcell differentiation, and temporal dynamics of large clones, suggesting ongoing expansion and differentiation. Clonally expanded γδ Tcells had heterogeneous gene expression profiles, occupying seven transcriptionally distinct clusters. Analysis of chemokine markers indicated cluster-specific homing tendencies of circulating γδ Tcells to peripheral tissues. We found unexpectedly high frequencies of Vδ1 and Vδ3 cells in the blood with distinct gene and protein expression profiles. This analysis provides insights into the dynamic and heterogeneous nature of γδ Tcells following anti-CD19 CAR-T cell therapy, contributing valuable information for optimizing CAR-T cell therapies in B cell malignancies.

Analysis of the components of Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) and its regulation of γδ T-cell function

BackgroundMycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) is a peptide antigen released from the mycobacterial cytoplasm into the supernatant of Mycobacterium tuberculosis (Mtb) attenuated H37Ra strain after autoclaving at 121 °C for 20 min. Mtb-HAg can specifically induce γδ T-cell proliferation in vitro. However, the exact composition of Mtb-HAg and the protein antigens that are responsible for its function are currently unknown.MethodsMtb-HAg extracted from the Mtb H37Ra strain was subjected to LC‒MS mass spectrometry. Twelve of the identified protein fractions were recombinantly expressed in Escherichia coli by genetic engineering technology using pET-28a as a plasmid and purified by Ni–NTA agarose resin to stimulate peripheral blood mononuclear cells (PBMCs) from different healthy individuals. The proliferation of γδ T cells and major γδ T-cell subset types as well as the production of TNF-α and IFN-γ were determined by flow cytometry. Their proliferating γδ T cells were isolated and purified using MACS separation columns, and Mtb H37Ra-infected THP-1 was co-cultured with isolated and purified γδ T cells to quantify Mycobacterium viability by counting CFUs.ResultsIn this study, Mtb-HAg from the attenuated Mtb H37Ra strain was analysed by LC‒MS mass spectrometry, and a total of 564 proteins were identified. Analysis of the identified protein fractions revealed that the major protein components included heat shock proteins and Mtb-specific antigenic proteins. Recombinant expression of 10 of these proteins in by Escherichia coli genetic engineering technology was used to successfully stimulate PBMCs from different healthy individuals, but 2 of the proteins, EsxJ and EsxA, were not expressed. Flow cytometry results showed that, compared with the IL-2 control, HspX, GroEL1, and GroES specifically induced γδ T-cell expansion, with Vγ2δ2 T cells as the main subset, and the secretion of the antimicrobial cytokines TNF-α and IFN-γ. In contrast, HtpG, DnaK, GroEL2, HbhA, Mpt63, EsxB, and EsxN were unable to promote γδ T-cell proliferation and the secretion of TNF-α and IFN-γ. None of the above recombinant proteins were able to induce the secretion of TNF-α and IFN-γ by αβ T cells. In addition, TNF-α, IFN-γ-producing γδ T cells inhibit the growth of intracellular Mtb.ConclusionActivated γδ T cells induced by Mtb-HAg components HspX, GroES, GroEL1 to produce TNF-α, IFN-γ modulate macrophages to inhibit intracellular Mtb growth. These data lay the foundation for subsequent studies on the mechanism by which Mtb-HAg induces γδ T-cell proliferation in vitro, as well as the development of preventive and therapeutic vaccines and rapid diagnostic reagents.

Ex vivo expansion and activation of Vγ9Vδ2 T cells by CELMoDs in combination with zoledronic acid.

As multiple myeloma (MM) progresses, immune effector cells decrease in number and function and become exhausted. This remains an insurmountable clinical issue that must be addressed by development of novel modalities to revitalize anti-MM immunity. Human Vγ9Vδ2 T (Vδ2+ γδ T) cells serve as the first line of defense against pathogens as well as tumors and can be expanded ex vivo from peripheral blood mononuclear cells (PBMCs) upon treatment with amino-bisphosphonates in combination with IL-2. Here, we demonstrated that next-generation immunomodulators called cereblon E3 ligase modulators (CELMoDs), as well as lenalidomide and pomalidomide, expanded Th1-like Vδ2+ γδ T cells from PBMCs in the presence of zoledronic acid (ZA). However, the expansion of Th1-like Vδ2+ γδ T cells by these immunomodulatory drugs was abolished under IL-2 blockade, although IL-2 production was induced in PBMCs. BTN3A1 triggers phosphoantigen presentation to γδ T-cell receptors and is required for γδ T-cell expansion and activation. ZA but not these immunomodulatory drugs upregulated BTN3A1 in monocytes. These results suggest that immunomodulatory drugs and ZA have cooperative roles in expansion of Th1-like Vδ2+ γδ T cells, and provide the important knowledge for clinical application of human Vδ2+ γδ T cells as effector cells.

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.

Research Progress of γδT Cells in Tumor Immunotherapy.

Background: γδT cells are special innate lymphoid cells, which are not restricted by major histocompatibility complex (MHC). γδT cells mainly exist in human epidermis and mucosal epithelium. They can secrete a variety of cytokines and chemokines involved in immune regulation, and produce effective cytotoxic responses to cancer cells. Purpose: To investigate the role of γδT cells in tumor immunotherapy, to understand its anti-tumor mechanism, and to explore the synergistic effect with other treatment modalities. This therapy is expected to become an important means of cancer treatment. Research Design: In this review presents a comprehensive analysis of the existing literature, focusing on the efficacy of γδT cells in a variety of tumor types. Results: The mechanism of γδT cells recognizing tumor antigens and killing tumor was clarified. The tumor immunotherapy based on γδT cells and its application in clinical practice were summarized. Conclusions: γδT cells have shown promising potential in tumor immunotherapy, but the therapeutic effect varies according to the type of tumor, and some patients have poor response. There are still some challenges in the treatment of this disease, such as non-standard expansion regimens and different responses of patients, indicating that the existing treatment methods are not complete. Future research should focus on perfecting γδT cell expansion protocols, gaining a deeper understanding of its anti-tumor mechanisms, and exploring synergies with other treatment modalities. This multifaceted study will promote the development of γδT cells in the field of cancer immunotherapy.

The dynamics of γδ T cell responses in nonhuman primates during SARS-CoV-2 infection

Although most SARS-CoV-2 infections are mild, some patients develop systemic inflammation and progress to acute respiratory distress syndrome (ARDS). However, the cellular mechanisms underlying this spectrum of disease remain unclear. γδT cells are T lymphocyte subsets that have key roles in systemic and mucosal immune responses during infection and inflammation. Here we show that peripheral γδT cells are rapidly activated following aerosol or intra-tracheal/intra-nasal (IT/IN) SARS-CoV-2 infection in nonhuman primates. Our results demonstrate a rapid expansion of Vδ1 γδT cells at day1 that correlate significantly with lung viral loads during the first week of infection. Furthermore, increase in levels of CCR6 and Granzyme B expression in Vδ1 T cells during viral clearance imply a role in innate-like epithelial barrier-protective and cytotoxic functions. Importantly, the early activation and mobilization of circulating HLA-DR+CXCR3+ γδT cells along with significant correlations of Vδ1 T cells with IL-1Ra and SCF levels in bronchoalveolar lavage suggest a novel role for Vδ1 T cells in regulating lung inflammation during aerosol SARS-CoV-2 infection. A deeper understanding of the immunoregulatory functions of MHC-unrestricted Vδ1 T cells in lungs during early SARS-CoV-2 infection is particularly important in the wake of emerging new variants with increased transmissibility and immune evasion potential.

A diversified role for γδT cells in vector-borne diseases.

Vector-borne diseases have high morbidity and mortality and are major health threats worldwide. γδT cells represent a small but essential subpopulation of T cells. They reside in most human tissues and exert important functions in both natural and adaptive immune responses. Emerging evidence have shown that the activation and expansion of γδT cells invoked by pathogens play a diversified role in the regulation of host-pathogen interactions and disease progression. A better understanding of such a role for γδT cells may contribute significantly to developing novel preventative and therapeutic strategies. Herein, we summarize recent exciting findings in the field, with a focus on the role of γδT cells in the infection of vector-borne pathogens.

Reviving human γδT cells from apoptosis induced by IL-12/18 via p-JNK inhibition.

γδT cells recognize and exert cytotoxicity against tumor cells independently of MHC restriction and have antigen presentation and regulatory functions to promote adaptive immune responses. They are considered as potential immune cells for cellular immunotherapy in cancer patients. However, it is challenging to ex vivo expand human γδT cells that have superb effector functions and long-term survival for adoptive cancer therapy. We found that IL-12/18 combination could drastically promote IFN-γ secretion and cytotoxicity in human γδT cells. However, the enhanced activation of human γδT cells is accompanied by increased apoptosis and elevated expressions of co-inhibitory receptors under the stimulation of IL-12/18. We further demonstrated that IL-12/18 induced apoptosis of human γδT cells was in a phosphoantigen or IFN-γ-independent manner. Transcriptomic analysis suggested that IL-12/18-induced apoptosis of human γδT cells was mediated by the activation of JNK pathway. p-JNK inhibitor (SP-600125) treatment effectively revived human γδT cells from the apoptosis induced by IL-12/18 and maintained their enhanced IFN-γ production and cytotoxicity against tumor cells. Our results provide a novel and feasible strategy for ex vivo expansion of cytokine-activated human γδT cells, which could promote the efficacy of γδT cell adoptive immunotherapy in cancer patients.

Properties and Roles of γδT Cells in Plasmodium yoelii nigeriensis NSM Infected C57BL/6 Mice.

BackgroundMany kinds of immune cells are involved in malaria infection. γδT cells represent a special type of immune cell between natural and adaptive immune cells that play critical roles in anti-parasite infection.MethodsIn this study, malaria infection model was constructed. Distribution of γδT cells in various immune organs and dynamic changes of γδT cells in the spleens of C57BL/6 mice after infection were detected by flow cytometry. And activation status of γδT cells was detected by flow cytometry. Then γδT cells in naive and infected mice were sorted and performed single-cell RNA sequencing (scRNA-seq). Finally, γδTCR KO mice model was constructed and the effect of γδT cell depletion on mouse T and B cell immunity against Plasmodium infection was explored.ResultsHere, splenic γδT cells were found to increase significantly on day 14 after Plasmodium yoelii nigeriensis NSM infection in C57BL/6 mice. Higher level of CD69, ICOS and PD-1, lower level of CD62L, and decreased IFN-γ producing after stimulation by PMA and ionomycin were found in γδT cells from infected mice, compared with naive mice. Moreover, 11 clusters were identified in γδT cells by scRNA-seq based t-SNE analysis. Cluster 4, 5, and 7 in γδT cells from infected mice were found the expression of numerous genes involved in immune response. In the same time, the GO enrichment analysis revealed that the marker genes in the infection group were involved in innate and adaptive immunity, pathway enrichment analysis identified the marker genes in the infected group shared many key signalling molecules with other cells or against pathogen infection. Furthermore, increased parasitaemia, decreased numbers of RBC and PLT, and increased numbers of WBC were found in the peripheral blood from γδTCR KO mice. Finally, lower IFN-γ and CD69 expressing CD4+ and CD8+ T cells, lower B cell percentage and numbers, and less CD69 expressing B cells were found in the spleen from γδTCR KO infected mice, and lower levels of IgG and IgM antibodies in the serum were also observed than WT mice.ConclusionsOverall, this study demonstrates the diversity of γδT cells in the spleen of Plasmodium yoelii nigeriensis NSM infected C57BL/6 mice at both the protein and RNA levels, and suggests that the expansion of γδT cells in cluster 4, 5 and 7 could promote both cellular and humoral immune responses.

Zoledronic acid conjugated calcium phosphate nanoparticles for applications in cancer immunotherapy

Zoledronic acid (Zol) is a potent bisphosphonate drug used in cancer immunotherapy due to its ability to activate γδT cells and bring about depletion of tumor-associated macrophages. Zoledronic acid pulsed monocytes are utilized for the activation and proliferation of γδT cells. For adoptive γδT cell transfer, isolated peripheral blood mononuclear cells (PBMC) are treated with Zol under in vitro conditions for γδT cell expansion. Herein, we optimized, zoledronate conjugated calcium phosphate nanoparticles (Zol-nCP) to improve in vitro γδT cell proliferation. PBMC treated with Zol-nCP conjugates resulted in 28.7% enhancement in γδT cell proliferation in comparison to free Zol. In addition, the treatment of RAW 264.7 macrophages with Zol-nCP resulted in 25% enhanced Zol mediated toxicity compared to the bare drug. This enhancement in toxicity to macrophages can be utilized for better depletion of tumor-associated macrophages compared to free Zol. The development of this nanoconjugate may have great potential in enhancing the clinical efficacy of bisphosphonate-mediated cancer immunotherapy.

Study on the Killing Effect of γδT Cells Activated by Rukangyin on Breast Cancer MDA-MB-231 Cells.

Objective To investigate the killing effect of rukangyin (RKY) activated γδT cells on breast cancer cells MDA-MB-231 and to provide a basis for Chinese medicine combined with immunotherapy for breast cancer. Methods Thus, study isolates peripheral blood mononuclear cells (PBMC) and uses CCK8 to select the optimal concentration of Rukang drink, ZOL (zoledronic acid), and PHA (phytoagglutinin) to activate γδT cells. There are 8 groups including the ① PBMC control group, ② RKY group, ③ ZOL group, ④ PHA group, ⑤RKY+ZOL group, ⑥RKY+PHA group, ⑦ZOL+PHA group, and ⑧ RKY+ZOL+PHA group. At 0 and 14 days of culture, cell viability and γδT cell expansion were detected by flow cytometry. The 8 groups of amplified γδT were cocultured with breast cancer MDA-MB-231 cells labeled with fluorescent dye CFSE at a ratio of 10 : 1 to determine the lethality of γδT cells on breast cancer MDA-MB-231 cells. Results The optimal concentrations of RKY, ZOL, and PHA to activate γδT cell proliferation were 4.5 mg/l, 3 μM, and 60 μg/ml, respectively. On day 0 of culture, the values (, %) of γδT cells in groups ① to ⑧ were 3.50 ± 0.72, 3.97 ± 0.45, 3.99 ± 0.15, 4.37 ± 0.24, 4.47 ± 0.97, 4.59 ± 1.35, 3.45 ± 0.40, and 3.89 ± 0.48, while when a comparison between groups was made, F = 1.093 and p = 0.412; there is no significant difference between groups. Besides, when being cultured for 14 days, the values (, %) of γδT cells in groups ① to ⑧ were 4.77 ± 0.78, 23.22 ± 2.73, 26.4 ± 0.92, 28.66 ± 1.43, 27.99 ± 1.10, 30.21 ± 1.91, 32.51 ± 0.74, and 33.21 ± 0.42. Then, based on the comparison between groups, F = 119.917 and p < 0.001, there are obvious statistical differences between groups. Furthermore, the expansion values of γδT cells were compared before and after culture for 0 and 14 days. The t values of group ① to group ⑧ were 2.072, 12.051, 41.641, 29.015, 27.777, 18.972, 59.836, and 79.622. Except for the PBMC control group (p = 0.107), there are significant statistical differences (p < 0.001). The number of γδT cell expansion at 14 days was the RKY+ZOL+PHA group>ZOL+PHA group>RKY+PHA group>PHA group>RKY+ZOL group>ZOL group>RKY group>PBMC control group. From group ① to group ⑧, the γδT cell expansion multiples were 1.14 ± 0.44, 5.25 ± 0.77, 5.70 ± 0.89, 6.05 ± 1.03, 6.21 ± 0.09, 6.76 ± 1.46, 7.52 ± 1.05, and 7.97 ± 1.55, respectively, while the comparison between groups was F = 17.772 and p < 0.001. As for the amplification factor, there was RKY+ZOL+PHA group>ZOL+PHA group>RKY+PHA group>RKY+ZOL group>PHA group>ZOL group>RKY group>PBMC control group. In the killing experiment, the killing rate (, %) of group ① to group ⑧ was 1.08 ± 0.03, 1.89 ± 0.14, 1.22 ± 0.11, 1.31 ± 0.09, 1.48 ± 0.10, 2.02 ± 0.21, 2.18 ± 0.27, and 2.37 ± 0.35, whereas the comparison between groups was F = 20.498 and p < 0.001. In terms of killing rate, there was RKY+ZOL+PHA group>ZOL+PHA group>RKY+PHA group>RKY group>RKY+ZOL group>PHA group>ZOL group>PBMC control group. Conclusion Rukangyin can increase the lethality of γδT cells against MDA-MB-231 cells by activating the proliferation of γδT cells, which provides a basis for Chinese medicine combined with immunotherapy for breast cancer.

P-005: Novel strategy of elotuzumab and zoledronic acid with Th1-like γδT cells against myeloma

<h3>Background</h3> We are now in the new era of immunotherapies against multiple myeloma (MM) such as anti-CD38 and anti-SLAMF7 therapeutic monoclonal antibodies (mAbs); however, MM progression falls into attenuation and exhaustion of effector cells via immune checkpoint axes such as PD-1/PD-L1 and TIGIT/CD155, leading to hamper the clinical efficacy of these therapeutic mAbs. <h3>Methods</h3> To overcome this issue, we have been studying the utilization of Vδ2 γδT cells in peripheral blood mononuclear cells, which can be expanded and induced as Th1-like phenotype ex vivo using aminobisphosphonates in combination with IL-2, as effectors for MM treatment. The present study was aimed to develop the novel therapeutic strategies of γδT cells in combination with anti-MM mAbs. <h3>Results</h3> The next generation IMiDs, cereblon E3 ligase modulators (CELMoDs) of iberdomide and CC-92480 have been developed. CELMoDs was able to expand and activate γδT cells at much lower dose compared to IMiDs of lenalidomide and pomalidomide, in combination with zoledronic acid (ZA) ex vivo. Since the expanded Th1-like γδT cells highly expressed CD16, FcγRIIIa without expression of checkpoint molecules such as PD-1, TIGIT, CTLA-4, and LAG-3, we next examined whether anti-MM mAbs, elotuzumab (ELO), daratumumab (DARA), and isatuximab (ISA), induced ADCC in the presence of the γδT cells. DARA and ISA did not induce cytotoxicity against CD38-expressing MM cells in the presence of the γδT cells; however, addition of ELO further increased cytotoxicity of the γδT cells against SLAMF7-expressing MM.1S and OPM-2 cells but not RPMI 8226 cells with marginal SLAMF7 expression, suggesting induction of ELO's ADCC with the γδT cells. We next examined the cytotoxic effect of the combinatory treatment on osteoclasts (OCs) because MM cells resides in the bone marrow and the interaction between MM cells and OCs forms vicious cycle of MM cell growth and bone destruction. Of note, OCs, which can be induced from monocytes using M-CSF and RANK ligand, highly expressed SLAMF7 along with elevation of NFATc1 transcription factor. Although the expanded γδT cells can target OCs, addition of ELO augmented the cytotoxic effect of γδT cells on OCs, suggesting the emergence of ELO's ADCC with Th1-like γδT cells towards OCs. As γδT cells recognize not only neoantigens but also phosphoantigens (pAgs), we treated MM cells and OCs with ZA to enhance the expression of pAgs, and γδT cells then incubated with the ZA-treated cells. ZA-stimulation augmented the cytotoxicity of γδT cells towards MM cells and OCs even in the presence of ELO. <h3>Conclusions</h3> Taken together, our findings demonstrated the effective usage of ELO and Th1-like γδT cells with ZA against MM cells and ambient cells in MM bone marrow. Further study is warranted on the effective revitalization and expansion of Th1-like γδT cells using CELMoDs.

Dichotomous and stable gamma delta T-cell number and function in healthy individuals

BackgroundGamma-delta (γδ) T lymphocytes are primed to potently respond to pathogens and transformed cells by recognizing a broad range of antigens. However, adoptive immunotherapy with γδT cells has exhibited mixed...

Efficient Ex Vivo Expansion of γδ T-Cells from AML Patients Requires Elimination of Circulating Leukemic Blasts

γδ T-cells play an important role in immune surveillance of acute myeloid leukemia (AML). The main circulating subtype expresses a Vγ9Vδ2 T-cell receptor and may be expanded <em>ex vivo</em> following aminobisphosphonate activation. While such protocols operate robustly in healthy donors, they are often inefficient using blood samples from patients with advanced malignancy.

Gamma/Delta T-Cells Enhance Carboplatin-induced Cytotoxicity Towards Advanced Bladder Cancer Cells.

Bladder cancer (BLCA, urothelial bladder cancer) is one of the most common malignancies with increasing incidence and mortality worldwide. Poor diagnosis and the limitation of treatment is still an unmet need in clinical practice. γδ T-Cells have been paid increasing attention because of their potent cytotoxicity against tumors. Herein, we investigated the cytolytic effect of γδ T-cells in combination with the chemotherapeutic drug, carboplatin, against BLCA cells. The standard protocol for the induction and expansion of peripheral blood mononuclear cell-derived γδ T-cells was a zoledronic acid/interleukin-2-based medium system for 2 weeks. The cytotoxicity of γδ T-cells with and without carboplatin against BLCA cells was examined. After incubation, T-cell receptor-positive γδ T-cells showed a natural killer cell-like phenotypic characteristic and dose-dependently increased cytotoxicity against BLCA cells. Interestingly, we found that in advanced BLCA cells, which were more resistant to carboplatin, the cell viability was significantly (p<0.05) reduced in the presence of γδ T-cells. Our findings showed that γδ T-cell therapy has potent benefit in cancer treatment.

Hypoxia regulates the differentiation and anti-tumor effector functions of γδT cells in oral cancer.

Hypoxia within the tumor microenvironment (TME) is a key factor contributing to immunosuppression in tumors, co-relating with poor treatment outcome and decreased overall survival in advanced oral cancer (OC) patients. Vδ2 is a dominant subset of gamma delta T cells (γδT cells) present in the peripheral blood which exhibits potent anti-tumor cytotoxicity and is evolving as a key player of anti-cancer cellular therapy. However, the fate of γδT cells in hypoxic oral tumors remains elusive. In the present study, we compared the effect of hypoxia (1% O2 ) and normoxia (21% O2 ) on the expansion, proliferation, activation status, cytokine secretion and cytotoxicity of γδT cells isolated from OC patients and healthy individuals. Hypoxia-exposed γδT cells exhibited reduced cytotoxicity against oral tumor cells. Our data demonstrated that hypoxia reduces the calcium efflux and the expression of degranulation marker CD107a in γδT cells, which explains the decreased anti-tumor cytotoxicity of γδT cells observed under hypoxia. Hypoxia-exposed γδT cells differentiated to γδT17 [γδ T cells that produce interleukin (IL)-17] cells, which corroborated our observations of increased γδT17 cells observed in the oral tumors. Co-culture of γδT cells with CD8 T cells in the presence of hypoxia showed that programmed cell death ligand 1 (PD-L1)high γδT cells brought about apoptosis of programmed cell death 1 (PD-1)high CD8 T cells which could be significantly reversed upon blocking PD-1. Thus, future immunotherapeutic treatment modality for oral cancer may use a combined approach of blocking the PD-1/PD-L1 signaling and targeting hypoxia-inducible factor 1α, which may help in reversing hypoxia-induced immunosuppression.

Cytokine Pre-activated γδT cells in Cancer Adoptive Immunotherapy

Abstract Adoptive cell therapy harnesses the power of immune system by stimulating and expanding immune cells to combat cancer. The application of tumour-specific αβT cell-based therapy may be hindered by limited tumour antigens. γδT cells are promising candidates for cellular immunotherapy because of their abundant IFN-γ or IL-17 production and MHC-independent cytotoxicity against a broad spectrum of tumors. Numerous attempts to use γδT cells in cancer immunotherapy have been made and resulted in variable efficacy and an overall good safety profile. Better expansion strategies are needed to improve the efficacy of the clinical trials using γδT cell-based immunotherapies. In the current project, we aim to explore the efficacy of cytokine pre-activated γδT cells in cancer immunotherapy. Pre-activated γδT cells by a combination of cytokines were characterised for their surface activation marker expression, cytokine production, and cytotoxicity against tumour cells. We found that IL-12/18, IL-12/15/18, IL-12/18/21 and IL-12/15/18/21 are the most effective cytokine combinations for in vitro expansion of γδT cells in terms of activation and cytokine production. IL-21 upregulated their cytotoxicity against tumour cells in murine systems. All four combinations showed upregulated cytotoxicity of human γδT cells. These findings were extended to in vivo studies using murine hepatocellular carcinoma model.

Activation of Human γδ T Cells: Modulation by Toll-Like Receptor 8 Ligands and Role of Monocytes.

Background: Human Vγ9Vδ2 γδ T cells can kill a variety of cancer cells and have attracted substantial interest for cancer immunotherapy. Toll-like receptor (TLR) ligands are promising adjuvants for cancer immunotherapy, but TLR7/8 ligand Resiquimod has been shown to inhibit CD4 T-cell activation in a monocyte-dependent manner. Therefore, we studied the modulation of human γδ T-cell activation by TLR7/8 ligands. Methods: Peripheral blood mononuclear cells (PBMC) or purified γδ T cells together with purified monocytes were stimulated with zoledronic acid or phosphoantigens in the absence or presence of various imidazoquinoline TLR7 or TLR8 agonists. Read-out systems included interferon-γ induction and cellular expansion of γδ T cells, as well as viability, cell surface antigen modulation, and IL-1β and TNF-α production of monocytes. Results: TLR8 ligand TL8-506 and TLR7/8 ligand Resiquimod (but not TLR7 ligands) rapidly induced IFN-γ expression in γδ T cells within PBMC, and co-stimulated phosphoantigen-induced IFN-γ expression in γδ T cells. On the other hand, TLR8 ligands potently suppressed γδ T-cell expansion in response to zoledronic acid and phosphoantigen. Purified monocytes secreted large amounts of IL-1β and TNF-α when stimulated with TLR8 ligands but simultaneously underwent substantial cell death after 24 h. Conclusions: TLR8 ligand-activated monocytes potently co-stimulate early γδ T-cell activation but failed to provide accessory cell function for in vitro expansion of γδ T cells.

DeSUMOylase SENP7-Mediated Epithelial Signaling Triggers Intestinal Inflammation via Expansion of Gamma-Delta T Cells

Inflammatory bowel disease (IBD) is a complex autoimmune disorder recently shown to be associated with SUMOylation, a post-translational modification mechanism. Here, we have identified a link between epithelial deSUMOylases and inflammation in IBD. DeSUMOylase SENP7 was seen to be upregulated specifically in intestinal epithelial cells in both human IBD and a mouse model. In steady state, but not IBD, SENP7 expression was negatively regulated by a direct interaction and ubiquitination by SIAH2. Upregulated SENP7 in inflamed tissue displayed a distinct interactome. These changes led to an expansion of localized proinflammatory γδ Tcells. Furthermore, invivo knockdown of SENP7 or depletion of γδ Tcells abrogated dextran sulfate sodium (DSS)-induced gut inflammation. Strong statistical correlations between upregulated SENP7 and high clinical disease indices were observed in IBD patients. Overall, our data reveal that epithelial SENP7 is necessary and sufficient for controlling gut inflammation, thus highlighting its importance as a potential drug target.

ESGCT 27th Annual Congress In collaboration with SETGyc Barcelona, Spain October 22–25, 2019 Abstracts

Xenotransplantation of transgenic porcine pancreatic islets offers a promising alternative source to circumvent current limitations posed by the scarcity of allogeneic donors. We have investigated the feasibility to generate tissue engineered SLA silenced islet cell clusters (ICC) to decrease xenogeneic immune responses Pancreatic islets single cell suspensions were generated by enzymatic digestion of porcine ICCs. Single cells were silenced for SLA expression by lentiviral vectors encoding for Nanoluciferase as reporter gene and for short hairpin RNAs targeting beta2-microglobulin or class II transactivator, respectively. SLA transcripts were evaluated by real-time PCR and protein levels by flow cytometry and fluorescence microscopy analyses. The effect of SLA silencing was evaluated in human T and NK cell cytotoxicity assays. SLA-silenced pancreatic beta-cells were then reassembled into ICCs in stirred bioreactors. SLA class I silencing reached a level of up to 84% and class II by up to 50% on pancreatic islet cell. Silencing SLA expression did not affect cell viability and the insulin-producing beta-cell phenotype. Xenogeneic T-cell immune responses (p < 0.05) as well as antibody-mediated cellular-dependent immune responses (p < 0.01) were significantly decreased. Silencing SLA class I expression did not increase susceptibility to NK-cell cytotoxicity. In stirred bioreactors, tissue engineered islets showing the typical 3D-structure and morphology of ICC were assembled from SLA-silenced pancreatic cell suspensions. These data shows the feasibility to generate low immunogenic porcine ICC from transgenic pigs after single cell engineering and post-transduction islet reassembling that might serve as a robust alternative to allogeneic pancreatic islet cell transplantation.