Γδ T Cells Research Articles

Evaluating Tumor-Intrinsic and Patient-Specific Mechanisms of Resistance to Teclistamab in Anti-BCMA Exposed and Naïve Multiple Myeloma

Background: Teclistamab (Tec) is a CD3 x BCMA bispecific antibody granted accelerated FDA approval for treating relapsed/refractory (RR) multiple myeloma (MM) based off the results of the MajesTEC-1 trial (Usmani S, et al. Lancet 2021, Moreau P, et al. N Engl J Med 2022). However, patients with prior exposure to anti-BCMA therapies were excluded from this trial and there is no published data evaluating Tec efficacy in this setting. Additionally, little is known about tumor-intrinsic and patient-specific immunologic predictors of response to Tec. Methods: We performed an IRB-approved analysis of clinical outcomes for 52 commercially treated Tec patients treated as of 07/28/2023 at our center. Results were correlated to pre-treatment BCMA expression measured by immunohistochemistry (IHC). We also profiled peripheral blood T-cells from pre-treatment peripheral blood mononuclear cell (PBMC) samples from a sub cohort of patients via high-dimensional spectral cytometry using a 37-color panel including lineage, exhaustion, and activation markers. Results: Our commercial cohort was older (median age 70 vs 64), more heavily pretreated (median prior lines of therapy 7 vs 5) and had higher incidence of both high-risk cytogenetic abnormalities (33% vs 26%) and extramedullary disease (35% vs 17%) than the MajesTEC-1 population. Additionally, 52% had prior exposure to anti-BCMA therapies, including belantamab mafodotin (31%), anti-BCMA CAR T-cell therapies (37%), and anti-BCMA bispecific antibody therapies (4%), with some having exposure to multiple prior anti-BCMA therapies (17%). Among response evaluable patients (90%), with a median follow-up time of 100 days, the overall response rate (ORR) to Tec was 64% (36% ≥VGPR). Progression free survival (PFS) in patients without prior BCMA therapy was higher than in the anti-BCMA exposed population (median PFS NR vs 102 days, p = 0.032, Figure Panel A). However, the anti-BCMA therapy exposed population was more heavily pretreated (8 vs 5 median prior lines of therapy) and had more patients with high-risk cytogenetic abnormalities (46% vs 19%) and extramedullary disease (42% vs 27%) than the anti-BCMA therapy naïve cohort. No major differences in BCMA expression were noted between responding and non-responding patients or between anti-BCMA exposed and anti-BCMA naïve patients. However, two patients had absent BCMA expression following prior treatment with other BCMA targeting agents and did not respond to Tec. Cytokine release syndrome (CRS) during step-up dosing was strongly associated with Tec efficacy (median PFS NR vs 27 days, p <0.0001), with a 100% ORR for patients with CRS. Immune profiling experiments (Figure Panel B) revealed that Tec responders had peripheral blood enriched for both CD8 + effector memory T-cells (TEM, >6-fold increase, p = 0.0499) and CD8 + effector memory T-cells re-expressing CD45RA (TEMRA, >5-fold increase, p = 0.012) when compared to Tec non-responders. Tec non-responders had peripheral blood enriched for TIGIT + regulatory T-cells (Tregs, 3-fold increase, p = 0.03) when compared to Tec responders. No differences in PD-1, CTLA-4, LAG-3 or TIM-3 expression were observed in Tec responders vs non-responders. Patients with recent relapse from non-BCMA targeting bispecific antibodies (including products targeting FcRH5 or GPRC5D) had peripheral blood enriched for γδ T-cells with high TIGIT expression. Conclusion: Tec is an effective therapy in RRMM with a slightly lower efficacy observed in patients with prior anti-BCMA therapy exposure. PFS reductions among these anti-BCMA exposed patients may be partly due to independent disease associated risk factors. A pre-Tec peripheral blood T-cell population enriched with highly cytotoxic effector T-cells was associated with response to therapy, while suppressive TIGIT +Tregs were associated with nonresponse, suggesting a potential therapeutic role for TIGIT blockade or CD25 + cell depletion to enhance the therapeutic efficacy of bispecific antibodies. Clinical and translational data from additional patients will be presented at the meeting.

Protective role of IL-17-producing γδ T cells in a laser-induced choroidal neovascularization mouse model

BackgroundVision loss in patients with wet/exudative age-related macular degeneration (AMD) is associated with choroidal neovascularization (CNV), and AMD is the leading cause of irreversible vision impairment in older adults. Interleukin-17A (IL-17A) is a component of the microenvironment associated with some autoimmune diseases. Previous studies have indicated that wet AMD patients have elevated serum IL-17A levels. However, the effect of IL-17A on AMD progression needs to be better understood. We aimed to investigate the role of IL-17A in a laser-induced CNV mouse model.MethodsWe established a laser-induced CNV mouse model in wild-type (WT) and IL-17A-deficient mice and then evaluated the disease severity of these mice by using fluorescence angiography. We performed enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS) to analyze the levels of IL-17A and to investigate the immune cell populations in the eyes of WT and IL-17A-deficient mice. We used ARPE-19 cells to clarify the effect of IL-17A under oxidative stress.ResultsIn the laser-induced CNV model, the CNV lesions were larger in IL-17A-deficient mice than in WT mice. The numbers of γδ T cells, CD3+CD4+RORγt+ T cells, Treg cells, and neutrophils were decreased and the number of macrophages was increased in the eyes of IL-17A-deficient mice compared with WT mice. In WT mice, IL-17A-producing γδ T-cell numbers increased in a time-dependent manner from day 7 to 28 after laser injury. IL-6 levels increased and IL-10, IL-24, IL-17F, and GM-CSF levels decreased in the eyes of IL-17A-deficient mice after laser injury. In vitro, IL-17A inhibited apoptosis and induced the expression of the antioxidant protein HO-1 in ARPE-19 cells under oxidative stress conditions. IL-17A facilitated the repair of oxidative stress-induced barrier dysfunction in ARPE-19 cells.ConclusionsOur findings provide new insight into the protective effect of IL-17A in a laser-induced CNV model and reveal a novel regulatory role of IL-17A-producing γδ T cells in the ocular microenvironment in wet AMD.

Characteristics of circulating immune cells in HBV-related acute-on-chronic liver failure following artificial liver treatment

Background and aimLiver failure, which is predominantly caused by hepatitis B (HBV) can be improved by an artificial liver support system (ALSS). This study investigated the phenotypic heterogeneity of immunocytes in patients with HBV-related acute-on-chronic liver failure (HBV-ACLF) before and after ALSS therapy.MethodsA total of 22 patients with HBV-ACLF who received ALSS therapy were included in the study. Patients with Grade I according to the ACLF Research Consortium score were considered to have improved. Demographic and laboratory data were collected and analyzed during hospitalization. Immunological features of peripheral blood in the patients before and after ALSS were detected by mass cytometry analyses.ResultsIn total, 12 patients improved and 10 patients did not. According to the immunological features data after ALSS, the proportion of circulating monocytes was significantly higher in non-improved patients, but there were fewer γδT cells compared with those in improved patients. Characterization of 37 cell clusters revealed that the frequency of effector CD8+ T (P = 0.003), CD4+ TCM (P = 0.033), CD4+ TEM (P = 0.039), and inhibitory natural killer (NK) cells (P = 0.029) decreased in HBV-ACLF patients after ALSS therapy. Sub group analyses after treatment showed that the improved patients had higher proportions of CD4+ TCM (P = 0.010), CD4+ TEM (P = 0.021), and γδT cells (P = 0.003) and a lower proportion of monocytes (P = 0.012) compared with the non-improved patients.ConclusionsChanges in effector CD8+ T cells, effector and memory CD4+ T cells, and inhibitory NK cells are associated with ALSS treatment of HBV-ACLF. Moreover, monocytes and γδT cells exhibited the main differences when patients obtained different prognoses. The phenotypic heterogeneity of lymphocytes and monocytes may contribute to the prognosis of ALSS and future immunotherapy strategies.

Investigation of the shared gene signatures and molecular mechanisms between obstructive sleep apnea syndrome and asthma

BackgroundObstructive sleep apnea syndrome (OSAS) is highly related with asthma from the epidemiology to pathogenesis, while the underlying mechanism is still unclear. Herein, we aimed to reveal the shared gene signatures and molecular mechanisms underlying the coexistence of OSAS and asthma and verified relating pathway in mouse models. We downloaded GSE75097 of OSAS and GSE165934 of asthma from GEO database and performed differential expression analysis and functional enrichment analysis to screen differentially expressed genes (DEGs) and potential pathogenic pathway. PPI network was constructed with the STRING database. Hub genes were identified with cytoHubba and immune infiltration analysis was performed with cibersort for further verification. Potential drugs were screened with Comparative Toxicogenomics Database and miRNA-gene network was constructed. Besides, to test the pulmonary function and inflammatory cytokine, mouse models with OSAS and asthma were constructed, followed by validating the involvement of NOD1/NOD2-RIPK2-NF-κB-MCPIP-1 pathway in associated diseases. ResultsIn total, 104 DEGs were identified, in which PLAUR, RIPK2, PELI1, ZC3H12A, and TNFAIP8 are the hub genes, while NOD-like receptor signaling pathway and apoptosis signaling pathway were the potential influential pathways. Increased γδT cells and neutrophils were detected in asthma patients through immune infiltration analysis. Significant difference was detected among genders in OSAS, and acetaminophen is a potential drug in the comorbidity by screening the drugs in the Comparative Toxicogenomics Database. Mice with OSAS and asthma presented with worse pulmonary function and higher levels of inflammatory cytokines. The relative proteins, including NOD1, NOD2, RIPK2, NF-κB, and MCPIP-1, were up-regulated in mice with the OSAS and asthma. ConclusionsThis research firstly elucidates NOD1/NOD2-RIPK2-NF-κB-MCPIP-1 pathway as the shared pathway in the development of OSAS and asthma through bioinformatics and experimental methods. There is an interactive deterioration model between OSAS and asthma. This study may provide some potential biomarkers in the future research of the underlying pathogenesis and treatment of comorbidity of OSAS and asthma.

Examining the impact of immunosuppressive drugs on antibody-dependent cellular cytotoxicity (ADCC) of human peripheral blood natural killer (NK) cells and gamma delta (γδ) T cells

BackgroundHuman natural killer (NK) cells and gamma delta (γδ) T cells may impact outcomes of solid organ transplantation (SOT) such as lung transplantation (LTx) following the differential engagement of an array of activating and inhibitory receptors. Amongst these, CD16 may be particularly important due to its capacity to bind IgG to trigger antibody-dependent cellular cytotoxicity (ADCC) and the production of proinflammatory cytokines. While the use of immunosuppressive drugs (ISDs) is an integral component of SOT practice, their relative impact on various immune cells, especially γδT cells and CD16-induced functional responses, is still unclear. MethodsThe ADCC responses of peripheral blood NK cells and γδT cells from both healthy blood donors and adult lung transplant recipients (LTRs) were assessed by flow cytometry. Specifically, the degranulation response, as reflected in the expression of CD107a, and the capacity of both NK cells and γδT cells to produce IFN-γ and TNF-α was assessed following rituximab (RTX)-induced activation. Additionally, the effect of cyclosporine A (CsA), tacrolimus (TAC), prednisolone (Prdl) and azathioprine (AZA) at the concentration of 1 ng/ml, 10 ng/ml, 100 ng/ml, and 1000 ng/ml on these responses was also compared in both cell types. ResultsFlow cytometric analyses of CD16 expresion showed that its expression on γδT cells was both at lower levels and more variable than that on peripheral blood NK cells. Nevertheless functional analyses showed that despite these differences, γδT cells like NK cells can be readily activated by engagement with RTX to degranulate and produce cytokines such as IFNg and TNF-a. RTX-induced degranulation by either NK cells or γδT cells from healthy donors was not impacted by co-culture with individual ISDs. However, CsA and TAC but not Prdl and AZA did inhibit the production of IFN-γ and TNF-α by both cell types. Flow cytometric analyses of RTX-induced activation of NK cells and γδT cells from LTRs suggested their capacity to degranulate was not markedly impacted by transplantation with similar levels of cells expressing CD107 pre- and post-LTx. However an impairment in the ability of NK cells to produce cytokines was observed in samples obtained post LTx whereas γδT cell cytokine responses were not significantly impacted. ConclusionsIn conclusion, the findings show that despite differences in the expression levels of CD16, γδT cells like NK cells can be readily activated by engagement with RTX and that in vitro exposure to CsA and TAC (calcineurin inhibitors) had a measurable effect on cytokine production but not degranulation by both NK cells and gdT cells from healthy donors. Finally the observation that in PBMC obtained from LTx recipients, NK cells but not γδT cells exhibited impaired cytokine reponses suggests that transplantation or chronic exposure to ISDs differentially impacts their potential to respond to the introduction of an allograft and/or transplant-associated infections.

Sex-Related Effect of Aging in Gingival Gamma-Delta T Cells

Aging affects the number and function of gamma-delta (γδ) T cells in a tissue-specific manner, modifying the risk for inflammatory disease. These aging-related γδT-cell variations in gingival tissues that could increase the risk for inflammation and periodontal disease remain unknown. Here we sought to identify quantitative and qualitative variations in gingival γδT cells associated with aging that could have an impact in oral immunoinflammatory responses. For this, gingival tissues from young (4 mo) and aged (24 mo) male and female mice were collected and analyzed by flow cytometry. Cell suspensions were stimulated and stained with eFluor450 (cell viability), anti-CD45 (hematopoietic cells), anti-CD3 (lymphocytes), anti-TCRγδ (γδT cells), anti–IL-15rα (cell proliferation), and anti–Notch-3 (senescence marker). Detection of intracellular cytokines IL-17A and interferon γ (IFNγ) was performed. Gingival expression of specific γ- and δ-chains and cytokines was evaluated by quantitative reverse transcription polymerase chain reaction. A significantly higher number of IL-17A–producing γδT cells and IL-17A expression levels were observed in gingival tissues from aged females but not males. Similarly, the number of gingival Notch-3+ γδT cells increased with aging only in females. IL-15rα was not detected in gingival γδT cells. Chains γ1, 2, 4, 5, 6, and 7 as well as δ1, 2, 4, and 6 were detected. Detection levels of all γ chains except γ1 as well as δ1 and δ2 changed with aging in males, females, or both. Interestingly, number of IL-17A–producing conventional T cells similarly increased with aging only in females. Both sexes showed increased IFNγ+ conventional T-cell numbers with aging; however, it reached significance only in females. In conclusion, the number of gingival IL-17A–producing γδT cells and IL-17A expression increase naturally with aging specifically in females. This sexual dimorphism in gingival γδT and conventional Th17 cell numbers and phenotypes suggests distinct aging-related mechanisms of periodontitis in males and females.

High-dimensional immune profiling using mass cytometry reveals IL-17A-producing γδ T cells as biomarkers in patients with T-cell-activated idiopathic severe aplastic anemia

Severe aplastic anemia (SAA) is a bone marrow failure syndrome characterized by activated T cells. Features of T-cell activation in the pathophysiology of SAA remain unknown. To understand T cell activation states, we investigated the atlas of peripheral immune cells and the secreted cytokine network with single cell mass cytometry analysis. We found decreased γδ T-cell frequencies in all patients with SAA, together with a significantly increased proportion of interleukin (IL)-17A-producing cell subsets. Cytokine network analysis of immune cells showed significant positive relationship between IL and 17A production from immune cells and disease severity of severe aplastic anemia. On separating SAA into two distinct subgroups based on T-cell activation stage, the proportion of γδ T cells tended to decrease in the T-cell-activated SAA group compared with non-T-cell-activated group. And the proportion of IL-17A-producing γδ T cells (γδT17) within γδ T cells was newly found to be significantly higher in the T-cell-activated SAA group, implying that IL-17A production by γδ T cells was associated with T-cell activation. Overall, our study revealed a role of γδT17 cells in mediating autoreactive T-cell activation in SAA and provided a novel diagnostic indicator for monitoring autoreactive T-cell activation status during the progression of aplastic anemia in the clinic.

Mammary γδ T cells promote IL-17A-mediated immunity against Staphylococcus aureus-induced mastitis in a microbiota-dependent manner

Mastitis, a common disease for female during lactation period that could cause a health risk for human or huge economic losses for animals, is mainly caused by S.aureus invasion. Here, we found that neutrophil recruitment via IL-17A-mediated signaling was required for host defense against S.aureus-induced mastitis in a mouse model. The rapid accumulation and activation of Vγ4+ γδ Tcells in the early stage of infection triggered the IL-17A-mediated immune response. Interestingly, the accumulation and influence of γδT17 cells in host defense against S.aureus-induced mastitis in a commensal microbiota-dependent manner. Overall, this study, focusing on γδT17 cells, clarified innate immune response mechanisms against S.aureus-induced mastitis, and provided a specific response to target for future immunotherapies. Meanwhile, a link between commensal microbiota community and host defense to S.aureus mammary gland infection may unveil potential therapeutic strategies to combat these intractable infections.

Ligand-based targeting of c-kit using engineered γδ T cells as a strategy for treating acute myeloid leukemia.

The application of immunotherapies such as chimeric antigen receptor (CAR) T therapy or bi-specific T cell engager (BiTE) therapy to manage myeloid malignancies has proven more challenging than for B-cell malignancies. This is attributed to a shortage of leukemia-specific cell-surface antigens that distinguish healthy from malignant myeloid populations, and the inability to manage myeloid depletion unlike B-cell aplasia. Therefore, the development of targeted therapeutics for myeloid malignancies, such as acute myeloid leukemia (AML), requires new approaches. Herein, we developed a ligand-based CAR and secreted bi-specific T cell engager (sBite) to target c-kit using its cognate ligand, stem cell factor (SCF). c-kit is highly expressed on AML blasts and correlates with resistance to chemotherapy and poor prognosis, making it an ideal candidate for which to develop targeted therapeutics. We utilize γδ T cells as a cytotoxic alternative to αβ T cells and a transient transfection system as both a safety precaution and switch to remove alloreactive modified cells that may hinder successful transplant. Additionally, the use of γδ T cells permits its use as an allogeneic, off-the-shelf therapeutic. To this end, we show mSCF CAR- and hSCF sBite-modified γδ T cells are proficient in killing c-kit+ AML cell lines and sca-1+ murine bone marrow cells in vitro. In vivo, hSCF sBite-modified γδ T cells moderately extend survival of NSG mice engrafted with disseminated AML, but therapeutic efficacy is limited by lack of γδ T-cell homing to murine bone marrow. Together, these data demonstrate preclinical efficacy and support further investigation of SCF-based γδ T-cell therapeutics for the treatment of myeloid malignancies.

NIMG-16. THE PROGNOSTIC VALUE AND BIOLOGICAL SIGNIFICANCE OF MRI CE-T1-BASED RADIOMICS MODELS IN CNS5-IDENTIFIED GBM

Abstract BACKGROUND Following the publication of the 2021 WHO Classification of CNS Tumors (Fifth Edition) (CNS5), prognostic markers to guide the individualized treatment of patients with glioblastoma (GBM) (CNS5) need to be explored. Radiomics is a non-invasive, reproducible, and cost-effective method that has been used in the prognostic assessment of multiple solid tumors. The prognostic value and biological significance of MRI T1 - weighted enhancement (CE-T1) - based radiomics models in GBM (CNS5) has not been reported. METHODS The tumor region of MRI CE-T1 phase was segmented, and radiomics features were extracted. The consistency analysis, univariate Cox regression and two machine learning algorithms were used for feature reduction. Then, the radiomics prognostic model was established to calculated the radiomics score (RS). The independent external validation was independently verified using the bicenter radiotherapy cohorts. The enrichment analyses of the differential genes between RS-high and -low groups were implemented to explore the biological significance. RESULTS A prognostic model composed of six radiomics features were built. A high RS (HR=3.718, 95%CI: 2.222−6.220, P< 0.001) was an independent risk factor for overall survival (OS). The result was externally validated by two cohorts. Through biological significance exploration, RS was found to be significantly correlated with DNA repair (P=0.009) and glycolysis (P=0.001) pathway enrichment scores. RS was associated with γδT cell infiltration and the expression of LAG3. CONCLUSION The MRI CE-T1 based radiomics prognostic models constructed can predict GBM (CNS5) prognosis noninvasively and effectively, which is relevant to DNA repair, and may guide the screening of radiosensitive populations.

Gamma-delta T cells: their atherogenic actions and therapeutic potential in atherosclerosis

Abstract Introduction Atherosclerosis, a chronic inflammatory disease of the arteries, is a major contributor to ischemic attacks in the heart. Immune cells are studied in atherosclerosis and recognized as important players in plaque instability; however, multipotent γδ-T cells are rarely studied. Here, we aim to investigate their role in chronic arterial inflammation and the therapeutic potential of targeting them in preventing plaque instability. Methods Depletion and repletion strategies were adopted to examine the critical role of bone marrow derived γδ-T cells in atherosclerosis. Their direct atherogenicity mediated by interferon- γ (IFN-γ) and perforin (pfp) was investigated in mixed chimeric mice with specific protein deficiency restricted to bone marrow derived γδ-T cells. Anti -mouse TCR γ/δ antibody was employed to examine the master regulatory role of γδ-T cells in plaque stability. Mice with established atherosclerosis were treated with small molecule CXCR3-antagonist, AMG487 to retard γδ-T cell-modulated plaque instability. Results We confirmed that bone marrow derived γδ-T cells promote atherosclerosis and plaque stability. They directly contribute to lesion inflammation and cell death via IFN-γ and pfp leading to expanding vulnerable plaques. In addition, γδ-T cells express innate activating receptors, NKG2D and DNAM-1, for their activation in lesion and activated γδ-T cells recruited proinflammatory and cytotoxic immune cells via chemoattractants Rantes and MIP-1α, suggesting their pivotal roles in lesion progression. AMG487 treatment inhibited γδ-T cells and other immune cells and reduced atherosclerotic lesions with stable plaque characteristics - less cytotoxicity, more vascular smooth cells and thicker caps. Conclusion We have shown that bone marrow-derived CD27-positive γδ-T cells are most important immune cells in atherosclerosis. They contribute lesion cytotoxicity and inflammation by direct effector functions and influencing other atherogenic cells, and targeting them with AMG487 may be beneficial to reduce generation of vulnerable plaques and subsequent rupture so that it can significantly reduce MI.

Tyrosine Kinase Inhibition Activates Intratumoral γδ T Cells in Gastrointestinal Stromal Tumor.

γδ T cells are a rare but potent subset of T cells with pleiotropic functions. They commonly reside within tumors but the response of γδ T cells to tyrosine kinase inhibition is unknown. To address this, we studied a genetically engineered mouse model of gastrointestinal stromal tumor (GIST) driven by oncogenic Kit signaling that responds to the Kit inhibitor imatinib. At baseline, γδ T cells were antitumoral, as blockade of either γδ T-cell receptor or IL17A increased tumor weight and decreased antitumor immunity. However, imatinib therapy further stimulated intratumoral γδ T cells, as determined by flow cytometry and single-cell RNA sequencing (scRNA-seq). Imatinib expanded a highly activated γδ T-cell subset with increased IL17A production and higher expression of immune checkpoints and cytolytic effector molecules. Consistent with the mouse model, γδ T cells produced IL17A in fresh human GIST specimens, and imatinib treatment increased γδ T-cell gene signatures, as measured by bulk tumor RNA-seq. Furthermore, tumor γδ T cells correlated with survival in patients with GIST. Our findings highlight the interplay between tumor cell oncogene signaling and antitumor immune responses and identify γδ T cells as targets for immunotherapy in GIST.

Combining Allogeneic Gamma Delta T Cells with a Phosphoantigen Prodrug Promotes Apoptosis of Acute Myeloid Leukemia Stem and Progenitor Cells

Despite recent therapeutic advances, patients with relapsed/refractory acute myeloid leukemia (r/r AML) have limited treatment options and poor prognosis. Leukemic stem cells (LSCs) are considered as relapse-initiating cells and the “culprit” of treatment resistance. Allogeneic and autologous gamma delta (γδ) T cell-based therapies have been explored for a long time and proven to be safe in patients through multiple clinical trials. However, quite a few AML blasts obtained from r/r AML patients show resistance to γδ T cell-mediated cytotoxicity. Novel strategies targeted LSCs may improve efficacy of γδ T cell-mediated r/r AML therapy. Given that γδ T cells exert antitumor capacity via multiple pathways, to identify the way matters most in the resistance of γδ T cell-mediated cytotoxicity, we examined the median fluorescence intensity (MFI) of the ligands expressed in tumor cells by flow cytometry, including butyrophilin 3A (BTN3A), butyrophilin 2A1 (BTN2A1), the human MHC class I chain-related genes (MICA and MICB), the unique-long 16 binding proteins (ULBP1, ULBP2/5/6, ULBP3), Fas, the receptor of TNF-related apoptosis-inducing ligand (TRAIL) DR5, CD112 and CD155. The cytotoxicity activity of γδ T cells was measured by flow cytometry-based killing assay or bright-lite luciferase assay system after coculturing with AML cell lines and blasts at an appropriate effector to target (E:T) ratio. Then we employed a Support Vector Machine model to predict the killing efficiency and identified that BTN3A and BTN2A1 play the predominant role in γδ T cell-mediated cytotoxicity based on the Shapley additive explanation. BTN3A and BTN2A1 are the key ligands in the γδ TCR-phosphoantigen (pAg) recognition activity. PAgs, which are produced in tumor cells function as “molecular glues” to promote heteromeric association of BTN3A1 and BTN2A1 (Yuan L et al., BioRxiv 2022). To promote the γδ T cell-mediated cytotoxicity targeting r/r AML, here we synthesized a pAg prodrug, which show great plasma stability and membrane permeability. We anticipate this small molecule would lead pAg accumulation in tumor cells and cause increased γδ T cell activation. To identify the killing efficacy of pAg prodrug-induced γδ T cell activation, we select KG-1α (a human leukemia stem cell-like cell) as target cell. Zoledronate (ZOL), which is employed to stimulate γδ T cells in multiple γδ T cell-based clinical trials, is used in our killing assay as compared group. As expected, combining γδ T cells with pAg prodrug or ZOL both significantly improved the percent of specific killing when the E:T ratio is 1:1. The pAg prodrug showed more potent activity than ZOL group with EC50 value of 0.1 nM vs 17.93μM. And the specific killing percent of pAg prodrug group is 83.26±2.1 % (1 nM), comparing with negative control 15.17±3.4 % (only γδT cells). Futhermore, at relatively low E:T ratio (1:4), γδ T cells with pAg prodrug remain excellent cytotoxicity when targeting MV411 cell and OCI-AML3 cell, which are widely used in r/r AML research. After coculturing with primary leukemia stem and progenitor cells (CD34 +) obtained from r/r AML patients for 6~8 h, the apoptosis percentage of CD34 + leukemia cells was 42.34% comparing with negative control (only γδT cells) 2.53%. Then we evaluated the in vivo efficacy of combining γδT cells and pAg prodrug in NSG mice bearing MV411 cells. Compared with only γδT cells-treated group, combination with pAg prodrug presented better tumor clearing. To elucidate the pathway of pAg prodrug-mediated killing, we tested mitochondria apoptosis related events. After treated by γδ T cells with pAg prodrug, KG-1α cells showed loss of mitochondrial outer membrane potential and increasingly actived caspase 3/7. Meanwhile, pAg prodrug directly activates effector γδ T cells with increased secretion of CD107a and IFN-γ. Considering the possibility of toxicity towards normal hematopoietic stem and progenitor cells, we conducted colony-forming units (CFUs) assay. After treatment with γδT cells and pAg prodrug, the number of CFUs of CD34 + cells from cord blood was not effected compared with the CD34 + cells only group. Overall, we explored the predominant factor in γδ T cell-mediated killing and synthesized a pAg prodrug to improve the killing efficacy. We demonstrated that combining γδ T cells with pAg prodrug has effectively promoted apoptosis of AML stem and progenitor cells sparing normal hematopoietic stem and progenitor cells.

RAB22A As a Predictor of Exosome Secretion in the Progression and Relapse of Multiple Myeloma

Background: Multiple myeloma（MM）is an incurable malignant plasma cell disease in the blood system. After remission, patients will still have multiple recurrences, and the remission time after each recurrence will gradually become shorter. RAB22A is a member of RAS oncogene family, and there is no relevant research in MM at present. In this study, we explored the role of RAB22A in exosome secretion, Epithelial-mesenchymal transition（EMT）and immune regulation. Methods: We obtained MM samples from GEO data sets (GSE5900, GSE4581 and GSE146649), and analyzed the difference of expression of RAB22A. We downloaded the “IOBR” package, and used the “PCA” and “ssGSEA” algorithms to calculate the EMT scores and exosome scores of the high/low RAB22A expression groups. The “CIBERSORT” package was used to analyze the infiltration of immune cells. RT-PCR and Western-blot were used to verify the expression level of RAB22A in normal people and MM patients. After regulating the expression of RAB22A, the exosomes of MSC were collected for immunofluorescence staining and CCK-8 experiment. Results: The expression level of RAB22A in SMM and MM patients was significantly higher than that in normal people and MGUS patients, and the expression level of RAB22A in relapse MM patients was significantly higher than that in newly diagnosed patients. We calculated the EMT score and exosomes score by “IOBR” package. The results showed that the EMT score and exosomes score of high RAB22A group were significantly higher than those of low RAB22A group, and the exosomes score of MSC in recurrent patients was significantly higher than that of newly diagnosed patients. In addition, the infiltration levels of Monocyte, NK cells resting, Eosinophils, T cells regulatory (Treg) and T cells CD4 memory activated were positively correlated with RAB22A, while plasma cells and T cells gamma delta were negatively correlated with RAB22A. The expression levels of m6A genes (METTL14, VIRMA, RBM15 and FMR1) in the high RAB22A group were significantly higher than those in the low RAB22A group. We constructed a nomogram based on m6A genes significantly related to RAB22A to predict the risk value of high RAB22A expression, and the calibration curve showed that the nomogram had good prediction ability. We screened sensitive drugs according to the difference of IC50 values between high and low RAB22A groups, and found that cisplatin, Doxorubicin and bcl-2 inhibitors had higher sensitivity in low RAB22A group. Finally, we verified the above results in the samples of our center. The results showed that the expression level of RAB22A in MM patients was significantly higher than that of donors. After down-regulating the expression of RAB22A in MM-MSC, the secretion of exosomes decreased（Figure 1). Down-regulation of RAB22A reduced the proliferation of MM cells, and the addition of exosomes of MM-MSC partially reversed this effect. Conclusion: RAB22A is a potential therapeutic target to improve the prognosis of MM, which is closely related to exosome secretion, EMT and immune cell infiltration.

Engineering Off-the-Shelf Gamma Delta CAR T Cells for the Treatment of Acute Myeloid Leukemia

Chimeric antigen receptor T cell therapy (CART) therapy has shown remarkable success in the treatment of B cell acute lymphoblastic leukemias (B-ALL) and lymphomas. However, CART therapies for acute myeloid leukemia (AML), where 5-year survival rates are significantly lower than for B-ALL, are only in their infancy. CD33-CART have potent activity against AML in preclinical models and a first-in-child/first-in-human phase 1/2 CD33-CART clinical trial for AML is ongoing in the Pediatric Oncology Branch of the National Cancer Institute (NCT03971799). Nonetheless, published outcomes suggest a modest efficacy of approximately 50% (Shahzad et al., Front Immunol 2023), highlighting the critical need to develop new strategies to improve CART accessibility and a more robust anti-AML response. We hypothesized that off-the-shelf gamma delta (γδ) CD33 CART cells could potentially overcome current challenges for the treatment of AML. γδ lineage T cells are unconventional lymphocytes whose functions are not restricted to MHC-mediated antigen presentation; they are primed for immediate responses, including tumor killing. Furthermore, allogeneicγδ T cells have the potential to induce robust anti-tumor cytotoxicity without causing graft versus host disease (GVHD). Here, we generated γδ CAR T cells from healthy donor elutriated lymphocytes by activation with zoledronic acid and IL-2 for 7-14 days. Within 9 days post stimulation, the vast majority of lymphocytes were Vδ2+ and 30-40% were successfully transduced with a lentiviral CD33 CAR construct harboring the 4-1BB costimulatory domain. Importantly, and unlike conventional alpha beta (ab) T lymphocytes, >98% of these γδ CD33CAR T cells expressed IFNγ under basal conditions. This characteristic likely accounted for the efficient in vitro killing of AML cell lines by untransduced γδ T lymphocytes under conditions of high effector/target (E/T) ratios. While untransduced γδ T cells did not exhibit cytotoxicity following repeat AML stimulations, γδ CD33CAR T lymphocytes exhibited proficient in vitro cytotoxicity, with killing rates that were more rapid than those initiated by ab CD33 CART (Figure 1). These characteristics were associated with a prolonged metabolic activity of γδT cells; γδ CD33 CART expressed high levels of the GLUT1 glucose transporter for >14 days post activation whereas GLUT1 levels on ab CD33 CART returned to resting within 10 days. High GLUT1 levels were linked to efficient killing under conditions of basal glucose levels. Most notably, γδ CD33CAR T lymphocytes achieved high in vivo cytotoxicity, assessed using bioluminescent AML cell line xenografts in humanized NSG mice. Together, these data highlight the feasibility of generating allogeneic γδ CD33CART with a strong anti-AML cytotoxic response.

Single-Cell Analyses Unveil a Potential Mechanism Promoting Graft-Versus-Leukemia Response in Haploidentical Transplantation Using Peripheral Stem Cells Plus Cord Blood Grafts through Early Immune Reconstruction of CD8+ Infg+ T Cells

Abstract Background Haploidentical donor stem cell transplantation (haplo-SCT) by using G-CSF-primed peripheral stem cells combined with bone marrow grafts (PBSC+BM) represents a widely employed strategy for acute myeloid leukemia (AML). While leukemic relapse is one of the main reasons for the failure of SCT. Surprisingly, our clinical data found that haploidentical peripheral stem cells combined with cord blood (haplo-PBSC+cord) transplantation had significantly lower incidence of relapse than haplo-PBSC+BM transplantation. Nonetheless, the underlying mechanism needs to be explored. Method From 2021 to 2022, bone marrow samples at 1 and 3 months post-transplantation from 4 AML patients who underwent haplo-PBSC+cord transplantation and 3 AML patients who received haplo-PBSC+BM transplantation were obtained. We used single-cell RNA-sequence (scRNA-seq) technology for analysis of hematopoietic reconstitution disparity between the PBSC+cord and PBSC+BM groups. Results Significant disparities manifest in the initial stages of hematopoietic reconstitution between the PBSC+cord and the PBSC+BM groups. Notably, at 1 month and 3 months post-transplantation, the PBSC+cord group demonstrated conspicuous reconstitution of lymphoid and erythroid cells, with CD8+ T cells exhibiting the most pronounced effect. Differential gene analysis revealed that T cells in the PBSC+cord group display elevated expression of graft-versus-leukemia (GVL)-related cytokines, including IFN-γ, CCL3, and IL1B, when compared to the PBSC+BM group. Furthermore, noteworthy differences in T cell subsets were observed between the two groups. Within 1 month and 3 months post-transplantation, the PBSC+cord group experienced up-regulation of CD8+ effector memory T cells (TEM), characterized by heightened expression of the IFNG gene. Gene Set Variation Analysis (GSVA) unveiled significant enrichment of CD8+ IFNG+ TEM in T cell activation, cytotoxicity, cytokine secretion, and other pathways, suggesting its potential GVL effect. On the contrary, the PBSC+BM group demonstrated a higher proportion of CD4+ T cells, γδT cells, and MAIT cells at 1 month and 3 months post-transplantation. Among CD8+ T cells, the PBSC+BM group showcased a more robust reconstruction of CD8+ TIGIT+ T cell subsets, which highly express exhaustion-related genes like TIGIT, LAG3, PDCD1 (PD-1), HAVCR2 (TIM-3), and exhibited increased apoptosis-related pathway activity. Conclusion Our investigation identified a potential mechanism promoting GVL response in the immune reconstitution of haplo-PBSC+cord transplantation.

A Rare Case of Primary Cutaneous Gamma-Delta T-cell Lymphoma with Aberrant B-cell Marker Expression.

Primary cutaneous gamma-delta T-cell lymphoma (PCGDTL) is a rare and diagnostically challenging primary skin lymphoma. We present a case of a 78-year-old otherwise healthy man who developed nonhealing nodules on his right posterior calf. Initial biopsy showed a dense, atypical, lymphoid infiltrate with gamma-delta and cytotoxic T-cell immunophenotypes. The diagnosis of PCGDTL was rendered; however, concurrent flow cytometry revealed expression of aberrant B-cell markers, including CD19 and cytoplasmic CD79a. Subsequent immunohistochemical studies corroborated this result. We report the extremely rare phenomenon of aberrant B-cell marker expression in PCGDTL, the first formally reported case to our knowledge.

Dominant CD4+ T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV

In people with HIV (PWH), the post-antiretroviral therapy (ART) window is critical for immune restoration and HIV reservoir stabilization. We employ deep immune profiling and Tcell receptor (TCR) sequencing and examine proliferation to assess how ART impacts Tcell homeostasis. In PWH on long-term ART, lymphocyte frequencies and phenotypes are mostly stable. By contrast, broad phenotypic changes in natural killer (NK) cells, γδ Tcells, B cells, and CD4+ and CD8+ Tcells are observed in the post-ART window. Whereas CD8+ Tcells mostly restore, memory CD4+ T subsets and cytolytic NK cells show incomplete restoration 1.4 years post ART. Surprisingly, the hierarchies and frequencies of dominant CD4 TCR clonotypes (0.1%-11% of all CD4+ Tcells) remain stable post ART, suggesting that clonal homeostasis can be independent of homeostatic processes regulating CD4+ Tcell absolute number, phenotypes, and function. The slow restoration of host immunity post ART also has implications for the design of ART interruption studies.

Enhancement of exercise expanded gamma-delta T-cells anti-leukemic activity with TIGIT blockade

Enhancement of exercise expanded gamma-delta T-cells anti-leukemic activity with TIGIT blockade

Cell-free multi-omics analysis reveals potential biomarkers in gastrointestinal cancer patients’ blood

During cancer progression, tumorigenic and immune signals are spread through circulating molecules, such as cell-free DNA (cfDNA) and cell-free RNA (cfRNA) in the blood. So far, they have not been comprehensively investigated in gastrointestinal cancers. Here, we profile 4 categories of cell-free omics data from patients with colorectal cancer and patients with stomach adenocarcinoma and then assay 15 types of genomic, epigenomic, and transcriptomic variations. We find that multi-omics data are more appropriate for detection of cancer genes compared with single-omics data. In particular, cfRNAs are more sensitive and informative than cfDNAs in terms of detection rate, enriched functional pathways, etc. Moreover, we identify several peripheral immune signatures that are suppressed in patients with cancer. Specifically, we establish a γδ-T cell score and a cancer-associated-fibroblast (CAF) score, providing insights into clinical statuses like cancer stage and survival. Overall, we reveal a cell-free multi-molecular landscape that is useful for blood monitoring in personalized cancer treatment.