Γδ T Cells Research Articles

Abstract 1523: The predictive potential of gamma-delta T cells and germinal B cells in cancer therapy for enhanced clinical outcomes

Abstract Introduction: Immune Checkpoint Blockade (ICB) therapy elicits clinical responses in a subset of patients in multiple cancer types, yet predicting response remains an open problem. While CD8-T cells remains to be focus, emerging evidence suggests role of other immune cell subtypes in favorable outcome of patients. This study presents the abundance of gamma-delta T (gdt) cell subpopulations in responders and non-responders. The analysis further contrasts differential abundances under immunotherapy and non-immunotherapy regimens across diverse cancers. Methods: We performed joint analysis of data from 250 non-immunotherapy cohorts and 17 ICB cohorts. To infer differential abundance in each cohort, we first identified genes robustly upregulated in tumors of patients with favorable outcome, and then unbiasedly evaluated upregulated genes for enrichment for immune cell populations. The topmost differential abundant subpopulations were validated by de novo assembly and single cell analyses. Results: Our analysis revealed similarities in the immune microenvironment across responders, irrespective of immunotherapy or non-immunotherapy modalities, strongly suggesting shared factors influencing response. Notably, gdt cells emerged as consistent predictors of better prognosis, being associated with favorable responses to ICB therapy and improved long-term survival in non-immunotherapy-treated cancer patients. Two independent validation approaches reinforce these findings. First, taking advantage of the fact that gamma and delta receptors are unique to gdt cells, we obtained precise estimates of gdt cells in tumors from their RNA-seq using de novo assembly. The assessment recapitulated our finding that favorable prognosis of tumors with of gdt cell levels. Second, analysis of several single-cell RNA-sequencing cohorts showed that patients with favorable prognoses consistently exhibit high gdt cell levels within tumors. Conclusion: The study underscores a strikingly consistent responder immune microenvironment, whether under immunotherapy or non-immunotherapy setting, suggesting a shared response and resistance mechanisms. The likelihood of cross-resistance has implications when clinicians decide whether immunotherapy should be administered before or after the traditional therapies for patients. Our study also elucidates the role of gdt cells as potent biomarkers for ICB therapy and their broader significance in enhancing patient survival across various cancer types. Ultimately, these insights may refine patient selection and inform tailored treatment strategies, could elevating clinical outcomes in the realm of precision oncology. Citation Format: Aaron Segura, Mikaela Dicome, Li Song, Xiaole Shirley Liu, Avinash Sahu. The predictive potential of gamma-delta T cells and germinal B cells in cancer therapy for enhanced clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1523.

Abstract 5335: Immune cell population variations in the livers of commonly used mouse strains

Abstract Previous studies have shown that the distribution of major immune cell populations differ across mouse strains in peripheral blood and hematopoietic organs at baseline. Additionally, variations in immune responses exist between mouse strains in Leishmania spp. and Melanoma models, but these studies in the context of liver diseases including Non Alcoholic Fatty Liver Disease (NAFLD) and Hepatocellular Carcinoma (HCC) have not been carried out. With the effort to state if these variations exist within the liver, we performed comprehensive spectral flow cytometry analysis on the liver and spleens of three female mouse strains commonly used in cancer research (BALB/c, C57BL/6, and FVBN). NAFLD was induced by providing mice with a methionine choline deficient diet. To promote HCC, a single genotype (myc/p53+/-) was induced by administering transposon-based vectors to overexpress Myc-Luc and CRISPR/Cas-9 vectors to knock down p53 via hydrodynamic tail-vein injection. To confirm vector integration and monitor tumor growth (luciferase activity), in vivo imaging was performed at different time points. As a result, we confirm throughout this study that differences exist within immune cell populations of the liver and spleen at baseline. Moreover, we depict differences in the shift of immune cell populations across mouse strains in the pathologic states included. In NAFLD, there is an increase of dendritic cells (DCs), macrophages, NK cells, ILC1s, T regulatory cells (Tregs), TH1, and TH2, and a decrease of CD4+ T cells, B cells, γδ-T cells, and NKT cells in the liver across all mouse strains, when compared to controls. However, the population of neutrophils in BALB/c decreases, and the population of CD8+ T cells increases in C57BL/6 mice, which differs from the patterns of change seen in the other mouse strains. Moreover, in spleens, NAFLD promotes an increase of TH1 and NK cells, and a decrease of TH2 and NKT Cells across the mouse strains included in the study, whereas the patterns of change differ between mouse strains among the remaining immune cell types. When evaluating liver immune cell population shifts on our HCC model, BALB/c mice showed a decrease in macrophages and ILC1s, C57BL/6 mice showed a decrease in γδ-T cells, prominent shifts across Neutrophils, TH1, and NKT cells, and null changes in TH2 and CD4+ T cell shifts, and FVBN mice had a less prominent decrease of B cells, which differed from the shifts observed in the mouse strains used for comparison. Immune cell populations in the spleens of our HCC mouse model also presented with differences across the mouse strains studied. In conclusion, we highlight that key differences in immune profiles exist across commonly used mouse strains for cancer research in the presence of liver pathology including HCC, suggesting that mouse strains could be possible confounders in the study of immune oncology of HCC. Citation Format: Francisco J. Rodriguez-Matos, Patrick Huang, Rajiv Trehan, Benjamin Ruf, Chi Ma, Tim F. Greten. Immune cell population variations in the livers of commonly used mouse strains [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5335.

Abstract 5229: Enhancing solid tumor treatment with engineered blood-derived Vδ1 platform: Safe and effective targeting of mesothelin-expressing tumors with Vδ1 γδT cells

Abstract Mesothelin (MSLN) is a solid tumor-associated antigen that has been thoroughly studied and is expressed in several tumor indications that have significant unmet therapeutic needs. Vδ1-enriched γδT cells differentiate themselves from other MSLN-targeted modalities by mitigating the challenges of targeting tumors with heterogeneous MSLN expression through their inherent capability to detect malignant cells while sparing normal cells. The ability to effectively combat cancer cells that express mesothelin is significantly increased by the expression of a chimeric antigen receptor (CAR). Novel armoring is being evaluated to improve engineered Vδ1 persistence in the solid tumor microenvironment. Cytokine armoring, through novel engineering, enhances sensitivity to cytokines and recovers cell fitness that is impaired by 'traditional' cytokine armoring options. Extensive in vitro and in vivo safety and efficacy studies were carried out to validate the product's potency. These studies aimed to determine cytotoxicity, expansion, and persistence. MSLN CAR cytokine-armored Vδ1 cells exhibited high post-thaw viability and survival. Furthermore, cytokine-armored Vδ1 cells displayed elevated innate receptor expression and gene-engineered cells maintained their baseline innate phenotype, indicating that they can maintain their ‘innate’ effectiveness over time. In vitro studies demonstrated broad anti-tumor activity against multiple tumor cell lines expressing various levels of mesothelin. Additionally, in long-term cytotoxicity assays, cytokine-armored cells demonstrated improved tumor-clearance capabilities. The effector cells also exhibited an enhanced innate cytotoxic phenotype that equips the cells with the capability to persist and proliferate in response to soluble cytokines and upon cytokine trans-presentation by adjacent cells. In vitro studies further revealed that there is a distinction in the killing activity between tumor and normal cells, which is important for ensuring that the product is safe for use in patients. An orthotopic lung tumor-bearing mouse model was developed to confirm their anti-tumor effectiveness in vivo. The model's outcomes demonstrated that MSLN CAR cytokine-armored Vδ1 cells exhibit higher in vivo efficacy in comparison to their unarmored counterparts at low effector-to-target ratios. With no significant healthy tissue damage found, the histopathology report confirmed an excellent safety profile. Terminal biodistribution studies demonstrated that armored drug candidates preferentially home to and persist in tumor-bearing tissues. Overall, these findings imply that the product might be highly effective in treating tumors that express mesothelin. This strategy could greatly advance our knowledge of how Vδ1-enriched γδT cells function as a platform against solid tumors. Citation Format: Nandhu Mohan Sobhana, Andre Simoes, Sarah Edwards, Sweta Mishra, Gary Gu, George Papadopoulos, Stefan Kiesgen, Rebecca Alade, njing Li, Andrea Venuso, Jyothi Kumaran, Kristy Waddington, Tabassum Huseni, Rajeev Karattil, Lilly Reynolds, Maria Sharif, David Wotherspoon, Corina Tudor, Istvan Kovacs, Mei Rosa Ng, Yana Wang, Lan Cao. Enhancing solid tumor treatment with engineered blood-derived Vδ1 platform: Safe and effective targeting of mesothelin-expressing tumors with Vδ1 γδT cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5229.

Abstract 5486: Spatial profiling reveals unique immune-transcriptomic features of tertiary lymphoid structures in melanoma

Abstract Background:Previous studies have shown that intratumoral immune aggregates called tertiary lymphoid structures (TLS) are associated with exceptional response to immune checkpoint blockade (ICB) in several cancer types. However, the specific mechanisms through which TLS contribute to ICB-response remain poorly characterized in part due to technical limitations with isolating TLS. Here, we use the NanoString GeoMx digital spatial profiler to interrogate TLS with deep spatial and transcriptional resolution and identify cellular and molecular features of response-associated TLS in melanoma. Methods:Twelve neoadjuvant ICB-treated high-risk resectable melanoma patients [NCT02519322] with formalin-fixed paraffin embedded (FFPE) surgical resections were included in this study. Major pathologic response (MPR) defined as <10% viable tumor at time of surgery was used to evaluate response (R – responder; NR – non-responder). TLS were sampled (via H&E) using 1mm core punches and assembled into a tissue microarray (TMA). The TLS TMA was probed using the human whole transcriptome atlas (huWTA) and the human T-cell receptor (huTCR) atlas. Melanoma (S100B) and immune markers (CD45 and CD20) were used to guide region-of-interest (ROI) selection of classical TLS. Differential gene expression was calculated using a linear mixed model and corrected with Benjamini-Hochberg. Immune deconvolution was performed using CIBERSORTx with FDR cutoff of < 0.25. Results:Fifty percent (6/12) of patients included in this study demonstrated MPR following treatment with neoadjuvant ICB. Principal component analysis of huWTA probes revealed distinct transcriptional states of R TLS vs NR TLS, with PC1 strongly correlating with response status (r=0.88, p<0.0001). Differential immune analysis showed that R TLS harbored more CD8+ T-cells (p<0.001), NK cells (p=0.004), γδ T-cells (p=0.017) and memory B-cells (p=0.026) and preferentially upregulated inflammatory genes such as pattern recognition receptors (TLR8 and KLRC2) and immune-stimulatory markers (CD27 and CD103). Immune-repertoire analysis revealed a trend towards increased TCR diversity in R TLS (p = 0.3) and distinct TCR signatures in R TLS underscored by elevated expression of TRDJ4 (p<0.001), TRGJ1/2(p<0.001), TRGV4(p<0.001) and TRAV1-2(p=0.018) genes amongst others. Conclusions:These findings demonstrate that, although melanomas from both R and NR patients contain TLS, the immune-phenotype of R TLS is more cytotoxic and characterized by a more diverse TLS-resident T-cell population. These findings present new insights into the therapeutic underpinnings of TLS in cancer and will inform future investigations evaluating their formation/function in solid tumors. Citation Format: Manoj Chelvanambi, Brenda Melendez, Elise F. Nassif, Rossana N. Lazcano, Matthew J. Lastrapes, Bharat S. Bhushan, Sarah B. Johnson, Khalida Wani, Davis R. Ingram, Y David Seo, Beth A. Helmink, Michael G. White, Russell G. Witt, Laurence P. Diggs, Golnaz Morad, Monika Zelazowska, Somnath Paul, Florentia Dimitriou, Ashish V. Damania, Matthew C. Wong, Neeta Somaiah, Nadim J. Ajami, Emily Z. Keung, Wolf H. Fridman, James P. Allison, Padmanee Sharma, Kevin M. McBride, Tina Cascone, Christina L. Roland, Alexander J. Lazar, Jennifer A. Wargo. Spatial profiling reveals unique immune-transcriptomic features of tertiary lymphoid structures in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5486.

Abstract 3927: Metabolic inhibition of BATF2 dampens type-I interferon-mediated immune sensing of cancer

Abstract Background: The type-I interferon (IFN-I) system appeared before adaptive immunity during evolution as the primary defense system for metazoans. Initiating cancers frequently disable this first line of defense before establishing T-cell exhaustion programs. However, the molecules linking IFN-I, danger signals, and unique metabolic conditions in the tumor microenvironment (TME) remain to be discovered. Methods: We performed centered log-ratio transformation on the cluster composition data using the R-package ALDEx2 to compare the clusters in single-cell data sets. IFN-I signatures were quantified using qPCR and ELISA. ChIP was performed to examine the H3K27me3 epigenetic marker on the promoters of BATF2 and IFN-I genes in bone marrow-derived macrophage (BMDM). For the cigarette smoke carcinogen-induced head and neck squamous cell carcinoma (HNSCC) model, experimental mice were fed with water containing 50 μg/mL of 4-NQO for 16 weeks and then switched to regular feeding water. We started monitoring 10 weeks post-treatment for the number, location, and diameter of the lesions. TME analysis was performed using single-cell RNA-Seq and multispectral imaging. Results: We analyzed three large independent cohorts of patients and found that BATF2 strongly correlated with the immunogenicity of HNSCC, with a stronger correlation coefficient than STING. We generated Batf2−/− BMDM and found that Batf2 deficiency rendered cells insensitive to STING agonists. However, the dsRNA-induced IFN-I activation was not affected by Batf2 deletion. BATF2 interacted with STING, promoting its phosphorylation and oligomerization. Glutamine is enriched in the TME. We found that glutamine inhibited the BATF2-STING pathway by increasing a repressive epigenetic marker, H3K27me3, on the promoters of BATF2 and IFNB1. The expression of BATF2 in cancer cells resulted in IFN-I- and γδ T-cell-dependent tumor rejection. Single-cell immune profiling showed that Batf2-expressing tumors contained significantly expanded Cxcl10+ myeloid cells and γδ T-cells. Utilizing the 4-NQO-induced HNSCC model, we found that BATF2 limited suppressive myeloid cells and expanded effector cells in the TME, serving as a potent tumor suppressor. Conclusion: BATF2 is a glutamine-responsive tumor suppressor that limits tumor initiation by engaging the STING-γδ T-cell pathway. Acknowledgments: This work was supported by NIH grants R01 DE026728, U01 DE029255, R01 DE031951, and U01 DE033330 Citation Format: Wang Gong, Hülya Taner, Yuesong Wu, Zaiye Li, Wanqing Cheng, Christopher Donnelly, Felipe Nör, Yumin He, Zackary Fitzsimonds, Jianqian Li, Haitao Wen, Steven Chinn, Yuying Xie, James Moon, Yu Leo Lei. Metabolic inhibition of BATF2 dampens type-I interferon-mediated immune sensing of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3927.

Abstract 6722: A CD33 antigen targeted Gamma Delta T-cell engager in combination with zoledronate promotes Vg9Vd2+ T cell proliferation and cytotoxicity against acute myeloid leukemia

Abstract Vγ9Vδ2+ T cell (GDT) targeted immunotherapy is of interest to harness its MHC-independent cytotoxic potential to promote anti-tumor immunity. However, it remains unclear whether GDT (1-5% of total T cells) are present at sufficient numbers to be therapeutically relevant. Accumulation of intracellular phosphoantigens promotes heterodimerization of butyrophilin (BTN) 2A1 and BTN3A1 on the cell surface, leading to TCR-mediated activation of GDT. To date, clinical trials in cancer patients have failed to demonstrate monotherapy activity using a variety of GDT activating agents, including aminobisphosphonates such as zoledronic acid (ZA), Vδ2+ targeted T cell engagers and BTN3A1 targeted antibodies. These data leave fundamental questions regarding the therapeutic potential of GDT unanswered, including whether they are present in sufficient numbers, whether the agents in question are providing adequate GDT activation, and whether GDT are being directed to tumor cell targets. The studies herein were thus designed to further understand the role of phosphoantigens, butyrophilins and antigen presenting cells (APCs) in activating GDT. Specifically, APC mediated GDT activation was studied in an in vitro co-culture system in the presence and absence of ZA or a CD33-directed GDT engager incorporating a heterodimer of BTN2A1 and BTN3A1 to dissect the contribution of phosphoantigens, butyrophilins and other costimulatory molecules on GDT proliferation and activation. To this end, while BTN2A1/3A1-Fc-CD33scFv treatment alone in T and monocyte-derived dendritic cell (mDC) co-cultures or in unfractionated PBMCs increased the frequency of GDT by 2 to 10-fold after 7-12 days, the combination with low dose ZA (0.5 to 2 μM) synergistically increased GDT proliferation by 40 to 100-fold. These results indicate that GDT proliferation relies both on the abundance of BTN2A1/3A1 heterodimers and other costimulatory signals activated by ZA. Synergistic effects of ZA and BTN2A1/3A1-Fc-CD33scFv in GDT proliferation was similarly observed in human PBMC-engrafted NSG-IL-15tg mice 12 days after treatment. Importantly, the addition of BTN2A1/3A1-Fc-CD33scFv to T (or PBMC) and CD33+ AML co-cultures enhanced both GDT proliferation and anti-leukemic activity while sparing healthy CD33+ immune cells. Collectively, these results demonstrate that GDT proliferation and effector function is dependent both upon the abundance of butyrophilin heterodimers, and other cell surface molecules which are modulated by intracellular phosphoantigen accumulation. Citation Format: Anne Y. Lai, Chunyan Wang, Dana C. Baiu, Kelsey Smith, Arpita Patel, Kinsley Evans, Noah Murr, Derek Franklin, Mahmud Hussain, Joseph Pate, Nathan Oien, Vishruti Makani, Abhinav Shukla, Keith Wilson, George Fromm, Taylor H. Schreiber, Jenny E. Gumperz, Suresh De Silva. A CD33 antigen targeted Gamma Delta T-cell engager in combination with zoledronate promotes Vg9Vd2+ T cell proliferation and cytotoxicity against acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6722.

Abstract 813: Primary cutaneous gamma-delta T-Cell lymphoma in the United States: A nationwide analysis of demographics and survival outcomes among the Hispanic population

Abstract BACKGROUND: Primary Cutaneous Gamma-Delta T-Cell Lymphoma (PCGDTCL) is a rare subtype of cutaneous T-cell lymphoma (CTCL) that, despite comprising <1% of cutaneous lymphomas, has gained recognition in recent years as an entity separate from other rare CTCLs. (LeukemiaPMID 35732829) A 5 year survival of 11% demonstrates a low response to current available therapies. (BloodPMID 30635287, Clin Hematol Int.PMID 35950208) Due to lack of consistency in behavior between CTCLs, there exists a need for studies of demographic, clinical, and survival disparities, including between Hispanics (HI) vs. non-Hispanics (NH). METHODS: Data were analyzed on PCGDTCL patients in the United States reported to the National Cancer Database (NCDB) between 2004-2019. Demographic and treatment characteristics were compared between ethnic groups. Kaplan-Meier and Cox regression analyses were used to compare OS between populations. Multivariate analysis and propensity score matching was performed with adjustment for age, stage, co-morbidity score, and insurance status, type of facility and great circle distance. RESULTS: Among 132 PCGDTCL patients studied (5% HI, 92% NH), the HI group was younger at diagnosis than NH (49.5y vs. 60y) (p=0.384); The majority of HI (50%) was diagnosed between 2010-2012 vs 2017-2019 for NH (45%) (p=0.673). Most of HI and NH were white (67% vs 79%). Private insurance was the most prevalent type in HI (67%) vs government sponsored for NH (49%). The most non-Insured group was NH (3%) vs HI (0%). The most prevalent bracket for Census Median Income (2008-2012) was $63,000+ for both HI (50%) and NH (31%). 17% HI vs 13%. NH had median income <$38,000. For Charlson-Deyo Score (comorbidities score), HI had 0% =/> 2 score, vs NH 6%. NCI-designated comprehensive cancer centers were the most prevalent type of facility providing care for HI (100%) and NH (56%) Survival probability (OS) at 2, 5 and 10y (HI vs NH) were (80% vs 48%), (80% vs 44%), and (80% vs 26%), respectively. Median survival time (MS) was not reached for HI vs 1.9 years for NH. Overall survival (OS) difference favored HI (p=0.13) On multivariate analysis, there was no independent variable associated with better or worse OS. Interestingly, the propensity matched analysis demonstrated significant MS difference between HI vs NH (median not reached vs. 1.35 years). CONCLUSION: There was no significant difference in OS between groups; however, the weighed MS favored the HI cohort, likely in part due to the difference in numbers between cohorts. The drastically low survival in all demographics is consistent with prior studies. These results demonstrate an unmet need for more comprehensive studies with the potential inclusion of correlatives looking at intrinsic biologic characteristics that may offer insight into druggable targets, aiming for improvement in survival outcomes for this rare lymphoma. Citation Format: Katherine Thiel, Rosa White, Qianqian Liu, Joel Michalek, Adolfo Enrique Diaz Duque. Primary cutaneous gamma-delta T-Cell lymphoma in the United States: A nationwide analysis of demographics and survival outcomes among the Hispanic population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 813.

Abstract 5228: Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile

Abstract Acute myeloid leukemia (AML) is an aggressive hematological malignancy that, despite advances in treatment, has an overall 5-year survival rate of less than 30%. There is an urgent unmet need for new and effective therapies for AML. Chimeric antigen receptor (CAR)-modified cell therapies are emerging as promising options. However, the selection of suitable antigens is challenging, due to the heterogeneous expression of potential antigen targets in AML. Most antigens, such as CD123, are expressed on both leukemic cells and normal hematopoietic stem and progenitor cells. In addition, conventional autologous or allogeneic T cell therapies present many limitations, such as cytokine release syndrome (CRS) and graft-vs-host disease (GvHD). We therefore evaluated CD123-targeting CAR designs in vδ1-enriched γδT cells, an innate immune cell platform that is inherently non-MHC restricted, has low risks of CRS and GvHD, and is capable of discriminating between malignant and healthy cells in preclinical models. We engineered vδ1-enriched γδT cells from peripheral blood of healthy individuals to express various CD123-targeting CAR constructs. The CD123-targeting CAR-engineered vδ1-enriched γδT cells expressed high levels of natural cytotoxicity receptors, suggesting capability to target innate receptor ligands on AML cell surface. CD123-targeting CAR-expressing vδ1-enriched γδT cells demonstrated enhanced cytotoxicity in vitro against multiple AML cell lines, in both short-term co-culture and repeated antigen stimulation assays. CD123-targeting CAR-expressing vδ1-enriched γδT cells also showed enhanced efficacy and survival in vivo against mouse models of human AML. Interestingly, attenuation of CAR activity via novel designs preserved the inherent selectivity of vδ1-enriched γδT cells against AML cells relative to normal hematopoietic stem or progenitor cells, which also express CD123. Together, our results demonstrated through rational CAR designing and engineering in vδ1-enriched γδT cells, an allogeneic cell therapy targeting CD123 can be generated with selective cytotoxicity against AML cells while sparing normal hematopoietic stem cells. Such product designs have the potential to be a transformative therapeutic option for a broad population of AML patients. Citation Format: Mei Rosa Ng, Sarah L. Hayward, Di Zhang, Gary Gu, Bri Paulhus, Donjeta Gjuka, Collin Crowley, Sophie McLaughlin, Anya Lublinsky, Angel Mathew, Evans Berreondo Giron, Doanh Mai, Madisen Lee, Megha Wal, Rebecca Alade, Andre Simoes, Robin Lytle, James Gavin, Shira Cramer, Zhong-hua Yan, Taylor Hickman, Rashmi Choudhary, Cori Abikoff, Yana Wang, Istvan Kovacs, Nandhu M. Sobhana, Lan Cao. Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5228.

Abstract 5255: A B7-H3-CAR-modified Vδ1 T cells showed potentanti-solid tumor potential

Abstract Background: CAR-αβT cells have made limited advancement in treating solid tumors. Vδ1 T cells, a subtype of γδT cells, are rare in human peripheral blood, but they are the main tissue-resident immune cells found in epithelial and mucosal tissues. This suggests that Vδ1 T cells might be a potential modality for treating solid tumors. To explore this possibility, we hypothesized that Vδ1 T cells modified with B7-H3 CAR (B7-H3-CAR-Vδ1 T) could have great potential of anti-solid tumor activity, making them a promising candidate for further development as a universal cellular therapy product. Methods: A proprietary manufacturing procedure to produce Vδ1 T cells from the leukopak of healthy donors has been established under GMP compliance. The immune checkpoint molecule B7-H3 was selected as the target. The allogeneic B7-H3-CAR-Vδ1 T cells carrying IL-2 (UTAA06), which displayed the most prolonged anti-tumor activity, were chosen for comprehensive functional validation. Single-cell sequencing and mass spectrometry flow cytometry were utilized to characterize the biology of UTAA06. In vivo effectiveness of UTAA06 was confirmed in animal models. Results: A clinical-grade manufacturing process for Vδ1 T cells is developed. This process results in a purity of over 95% of Vδ1 T cells, with an expansion of more than 20,000-fold, and a CAR lentiviral transduction efficiency of over 50%.žSingle-cell sequencing and mass cytometry analysis both demonstrated that the Vδ1 T cells produced by our approach exhibited high expression levels of natural cytotoxic receptors (including NKG2D, DNAM1, and NKp series receptors), chemokine receptors (such as CXCR3 and CXCR6), anti-tumor related molecules (such as CD27 and CD137), and low expression levels of inhibitory molecules (such as PD1 and LAG3).ž UTAA06 cells did not produce IL17A, which is a signal protein with pro-tumor activity. Meanwhile, UTAA06 demonstrated significant and specific inhibitory effects on the growth of various solid tumor cell lines.žUTAA06 cells were found to significantly suppress tumor cell growth in solid tumor mouse models. The complete remission (CR) for each tumor model was 83.3% of neuroblastoma (SK-N-AS, N=6), 66.7% of pancreatic cancer (SW1990, N=6), 100% of lung cancer (A549, N=6), and 70% of colorectal cancer (HCT-15, N=10) respectively. In addition, a potent capacity of UTAA06 to penetrate tumor tissues with a favorable safety profile in mice was observed. Conclusion: UTAA06 exhibited extraordinary anticancer properties both in vitro and in vivo against solid tumors. Additionally, the outstanding manufacturing process of UTAA06 will facilitate the clinical application of this product. Citation Format: Licui Jiang, Changsong Qi, Fengtao You, Shufen Xiang, Ping Zhang, Min Wang, Hai Wu, Kunmin Yan, Jiequn Huang, Huimin Meng, Nan Yang, Shirley Zhang, Lin Shen, Lin Yang. A B7-H3-CAR-modified Vδ1 T cells showed potentanti-solid tumor potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5255.

Advancements in γδT cell engineering: paving the way for enhanced cancer immunotherapy.

Comprising only 1-10% of the circulating T cell population, γδT cells play a pivotal role in cancer immunotherapy due to their unique amalgamation of innate and adaptive immune features. These cells can secrete cytokines, including interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), and can directly eliminate tumor cells through mechanisms like Fas/FasL and antibody-dependent cell-mediated cytotoxicity (ADCC). Unlike conventional αβT cells, γδT cells can target a wide variety of cancer cells independently of major histocompatibility complex (MHC) presentation and function as antigen-presenting cells (APCs). Their ability of recognizing antigens in a non-MHC restricted manner makes them an ideal candidate for allogeneic immunotherapy. Additionally, γδT cells exhibit specific tissue tropism, and rapid responsiveness upon reaching cellular targets, indicating a high level of cellular precision and adaptability. Despite these capabilities, the therapeutic potential of γδT cells has been hindered by some limitations, including their restricted abundance, unsatisfactory expansion, limited persistence, and complex biology and plasticity. To address these issues, gene-engineering strategies like the use of chimeric antigen receptor (CAR) T therapy, T cell receptor (TCR) gene transfer, and the combination with γδT cell engagers are being explored. This review will outline the progress in various engineering strategies, discuss their implications and challenges that lie ahead, and the future directions for engineered γδT cells in both monotherapy and combination immunotherapy.

Aberrant phenotypes of circulating γδ-T cells may be involved in the onset of systemic lupus erythematosus.

Human gamma-delta T cells (γδ-T cells) play crucial roles in both innate and adaptive immune responses. However, much less is known about the immune status of γδT cells in systemic lupus erythematosus (SLE) patients. The objective of this study was to explore potential relationships between the frequency of γδ-T-cell subpopulations and disease activity, autoantibody titres and renal involvement in patients with SLE. Circulating γδ-T cells and their subsets (Vδ1+ T cells, Vδ2+ T cells and γδ-T-cell subpopulations defined by expression of surface receptors, including NKG2D, NKp30, NKp46 and PD-1), were identified via flow cytometry. Sixty active SLE patients were selected, including 41 new-onset and 19 relapsing cases. One hundred healthy controls (HCs) were enrolled as the control group. Percentages of these cell subsets in SLE patients and HCs and their relationships with disease activity were analysed. Twenty-two of the 41 new-onset SLE patients were assessed before and after treatment. Changes in the frequencies of these cell subsets and their relationships with renal involvement were also analysed. Compared with that in HCs, the percentage of total γδ-T cells among CD3+ T cells in SLE patients was significantly lower. An imbalance in the proportions of Vδ1+ and Vδ2+ T cells among γδ-T cells was observed. The proportion of Vδ1+ T cells among γδ-T cells was significantly greater in SLE patients than in HCs, while the proportion of Vδ2+ T cells was significantly lower. Expression levels of PD-1, NKG2D, NKp30 and NKp46 in Vδ1+ T cells and Vδ2+ T cells from SLE patients were generally significantly increased, except for expression of NKG2D in Vδ2+ T cells. Moreover, Vδ2+ T cells, Vδ1+ T cells and Vδ1+PD-1+ T cells were associated with disease activity, and an increase in Vδ2+ T-cell frequency and a decrease in PD-1 expression by γδ-T cells might be associated with effective treatment. Interestingly, our results indicated that Vδ2+ T cells and their Vδ2+NKp30+ T-cell subpopulation might be associated with renal involvement in SLE. A broad range of anomalies in the proportions of γδ-T-cell subsets and γδ-T cells in SLE patients may be involved in the pathogenesis of SLE. There is a strong association between Vδ2+ T cells and their Vδ2+NKp30+ T-cell subpopulation and LN occurrence. Our results indicate that γδ-T cells and their subpopulations might be key players in disease immunopathology and renal involvement in SLE.

Engineering a Dual Specificity γδ T-Cell Receptor for Cancer Immunotherapy.

γδ T-cells provide immune surveillance against cancer, straddling both innate and adaptive immunity. G115 is a clonal γδ T-cell receptor (TCR) of the Vγ9Vδ2 subtype which can confer responsiveness to phosphoantigens (PAgs) when genetically introduced into conventional αβ T-cells. Cancer immunotherapy using γδ TCR-engineered T-cells is currently under clinical evaluation. In this study, we sought to broaden the cancer specificity of the G115 γδ TCR by insertion of a tumour-binding peptide into the complementarity-determining region (CDR) three regions of the TCR δ2 chain. Peptides were selected from the foot and mouth disease virus A20 peptide which binds with high affinity and selectivity to αvβ6, an epithelial-selective integrin that is expressed by a range of solid tumours. Insertion of an A20-derived 12mer peptide achieved the best results, enabling the resulting G115 + A12 T-cells to kill both PAg and αvβ6-expressing tumour cells. Cytolytic activity of G115 + A12 T-cells against PAg-presenting K562 target cells was enhanced compared to G115 control cells, in keeping with the critical role of CDR3 δ2 length for optimal PAg recognition. Activation was accompanied by interferon (IFN)-γ release in the presence of either target antigen, providing a novel dual-specificity approach for cancer immunotherapy.

Langerhans cells and gamma delta Т-lymphocytes in the pathogenesis of psoriasis

Psoriasis is a frequently occurring inflammatory skin disease. Fundamental research into the pathogenesis of psoriasis has significantly expanded our understanding of skin immunology, which has enabled the introduction of innovative and highly effective therapies. Psoriasis is a largely T-lymphocyte-mediated disease in which activation of innate immune cells and pathogenic T cells leads to inflammation and hyperproliferation of keratinocytes. Langerhans cells like dendritic cells (DCs), which play an important role in the immune system, are mainly distributed in the epidermis. Since human and mouse skin DC subpopulations share common ontogenetic characteristics, we can further investigate the role of DCs in psoriatic inflammation in mice. γδT cells are an unconventional population of T-lymphocytes that play an indispensable role in host defense, immune surveillance, and immune system homeostasis. Although γδT cells constitute only a small fraction of the total T cell pool, emerging evidence suggests that aberrantly activated γδT cells may play a role in the pathogenesis of psoriasis. Dermal γδT cells are the major IL-17-producing cells in the skin that respond to IL-23 stimulation. In addition, γδT cells have characteristics of memory cells that mediate repeated episodes of psoriatic inflammation. This review discusses the mechanisms by which Langerhans cells and γδT cells participate in the pathogenesis of psoriasis.

The γδ T Cell Club: bringing a global scientific community together in the age of Zoom.

Immunologists are very social people-they love to meet other immunologists and talk about immunology (and immunologists). Constantly! γδ T-cell researchers are no exception. On the contrary, as there are not so many of them compared to, say, researchers working on dendritic cells, they especially crave frequent interactions with like-minded scientists. This is where the technological solutions being developed during the coronavirus disease 2019 (COVID-19) pandemic come into play that have, almost overnight, allowed researchers to hold meetings and lectures online. We here describe how we set up the virtual 'γδ T Cell Club', a monthly webinar series that aims to bring the field closer together, and present our musings about what we have learned from this experience, which we hope is useful for other researchers interested in connecting online.

Radiation Dermatitis: Radiation-Induced Effects on the Structural and Immunological Barrier Function of the Epidermis.

An important hallmark of radiation dermatitis is the impairment of the mitotic ability of the stem/progenitor cells in the basal cell layers due to radiation-induced DNA damage, leading to suppressed cell renewal in the epidermis. However, this mechanism alone does not adequately explain the complex pathogenesis of radiation-induced skin injury. In this review, we summarize the latest findings on the complex pathogenesis of radiation dermatitis and correlate these with the clinical features of radiation-induced skin reactions. The current studies show that skin exposure to ionizing radiation induces cellular senescence in the epidermal keratinocytes. As part of their epithelial stress response, these senescent keratinocytes secrete pro-inflammatory mediators, thereby triggering skin inflammation. Keratinocyte-derived cytokines and chemokines modulate intercellular communication with the immune cells, activating skin-resident and recruiting skin-infiltrating immune cells within the epidermis and dermis, thereby orchestrating the inflammatory response to radiation-induced tissue damage. The increased expression of specific chemoattractant chemokines leads to increased recruitment of neutrophils into the irradiated skin, where they release cytotoxic granules that are responsible for the exacerbation of an inflammatory state. Moreover, the importance of IL-17-expressing γδ-T cells to the radiation-induced hyperproliferation of keratinocytes was demonstrated, leading to reactive hyperplasia of the epidermis. Radiation-induced, reactive hyperproliferation of the keratinocytes disturbs the fine-tuned keratinization and cornification processes, leading to structural dysfunction of the epidermal barrier. In summary, in response to ionizing radiation, epidermal keratinocytes have important structural and immunoregulatory barrier functions in the skin, coordinating interacting immune responses to eliminate radiation-induced damage and to initiate the healing process.

Persistence of Contact Lens-Induced Corneal Parainflammation Following Lens Removal.

Contact lens wear induces corneal parainflammation involving increased immune cell numbers after 24hours' (CD11c+, Lyz2+, γδ-T cells) and six days' (Ly6G+ cells) wear. We investigated the time course of onset and resolution of these responses. LysMcre or C57BL/6J mice were fitted with a contact lens (four to 48hours). Contralateral eyes did not wear lenses. After lens removal, Lyz2+, MHC-II+ or Ly6G+ cells were examined by quantitative imaging. RT-qPCR determined cytokine gene expression. Lens wear for 24hours increased corneal Lyz2+ cells versus contralateral eyes approximately two-fold. Corneas remained free of visible pathology. The Lyz2+ response was not observed after four or 12 hours' wear, nor after 12 hours' wear plus 12 hours' no wear. Lens removal after 24hours' wear further increased Lyz2+ cells (∼48% after one day), which persisted for four days, returning to baseline by seven days. Lyz2+ cells in contralateral eyes remained at baseline. MHC-II+ cells showed a similar response but without increasing after lens removal. Lens wear for 48 hours showed reduced Lyz2+ cells versus 24hours' wear with one day discontinuation, correlating with reduced IL-1β and IL-18 gene expression. Lens wear for 24hours did not induce Ly6G+ responses six days after removal. Lens-induced corneal parainflammation involving Lyz2+ cells requires 24hours' wear but persists after lens discontinuation, requiring seven days for reversal. Lens wear for 48 hours may suppress initial Lyz2+ cell and cytokine responses. The significance of parainflammation during and after lens wear remains to be determined.

A radiomics model to predict γδ T-cell abundance and overall survival in head and neck squamous cell carcinoma.

γδ T cells are becoming increasingly popular because of their attractive potential for antitumor immunotherapy. However, the role and assessment of γδ T cells in head and neck squamous cell carcinoma (HNSCC) are not well understood. We aimed to explore the prognostic value of γδ T cell and predict its abundance using a radiomics model. Computer tomography images with corresponding gene expression data and clinicopathological data were obtained from online databases. After outlining the volumes of interest manually, the radiomic features were screened using maximum melevance minimum redundancy and recursive feature elimination algorithms. A radiomics model was developed to predict γδ T-cell abundance using gradient boosting machine. Kaplan-Meier survival curves and univariate and multivariate Cox regression analyses were used for the survival analysis. In this study, we confirmed that γδ T-cell abundance was an independent predictor of favorable overall survival (OS) in patients with HNSCC. Moreover, a radiomics model was built to predict the γδ T-cell abundance level (the areas under the operating characteristic curves of 0.847 and 0.798 in the training and validation sets, respectively). The calibration and decision curves analysis demonstrated the fitness of the model. The high radiomic score was an independent protective factor for OS. Our results indicated that γδ T-cell abundance was a promising prognostic predictor in HNSCC, and the radiomics model could discriminate its abundance levels and predict OS. The noninvasive radiomics model provided a potentially powerful prediction tool to aid clinical judgment and antitumor immunotherapy.

Interleukin-2-induced skin inflammation.

Recombinant human IL-2 has been used to treat inflammatory diseases and cancer; however, side effects like skin rashes limit the use of this therapeutic. To identify key molecules and cells inducing this side effect, we characterized IL-2-induced cutaneous immune reactions and investigated the relevance of CD25 (IL-2 receptor α) in the process. We injected IL-2 intradermally into WT mice and observed increases in immune cell subsets in the skin with preferential increases in frequencies of IL-4- and IL-13-producing group 2 innate lymphoid cells and IL-17-producing dermal γδ Tcells. This overall led to a shift toward type 2/type 17 immune responses. In addition, using a novel topical genetic deletion approach, we reduced CD25 on skin, specifically on all cutaneous cells, and found that IL-2-dependent effects were reduced, hinting that CD25 - at least partly - induces this skin inflammation. Reduction of CD25 specifically on skin Tregs further augmented IL-2-induced immune cell infiltration, hinting that CD25 on skin Tregs is crucial to restrain IL-2-induced inflammation. Overall, our data support that innate lymphoid immune cells are key cells inducing side effects during IL-2 therapy and underline the significance of CD25 in this process.

Toll-like receptor 2 deficiency promotes the generation of alloreactive γδT17 cells after cardiac transplantation in mice

Homograft rejection is the main cause of heart transplantation failure. The role of TLR2, a major member of the toll-like receptor (TLR) family, in transplantation rejection is has yet to be elucidated. In this study, we used a mouse model of acute cardiac transplantation rejection to investigate whether the TLR2 signalling pathway can regulate cardiac transplantation rejection by regulating alloreactive IL-17+γδT (γδT17) cells. We found that the expression of TLR2 on the surface of dendritic cells (DCs) and macrophages increased during acute transplantation rejection. In addition, our investigation revealed that γδT17 cells exert a significant influence on acute cardiac transplantation rejection. TLR2 gene knockout resulted in an increase in alloreactive γδT17 cells in the spleen and heart grafts of recipient mice compared with wild-type recipient mice and an increase in the mRNA expression of IL-17, IL-1β, CCR6, and CCL20 in the heart grafts. In an in vitro experiment, a mixed lymphocyte reaction was conducted to assess the impact of TLR2 deficiency on the generation of γδT17 cells, which further substantiated a significant increase compared to that in wild-type controls. Furthermore, the mixed lymphocyte reaction showed that TLR2 regulated the production of γδT17 cells by regulating the ability of DCs to secrete IL-1β. These results suggest that TLR2 signalling is important for regulating the generation of γδT17 cells after cardiac allograft transplantation.

A Bcl11bN797K variant isolated from an immunodeficient patient inhibits early thymocyte development in mice.

BCL11B is a transcription factor with six C2H2-type zinc-finger domains. Studies in mice have shown that Bcl11b plays essential roles in T cell development. Several germline heterozygous BCL11B variants have been identified in human patients with inborn errors of immunity (IEI) patients. Among these, two de novo mis-sense variants cause asparagine (N) to lysine (K) replacement in distinct zinc-finger domains, BCL11BN441K and BCL11BN807K. To elucidate the pathogenesis of the BCL11BN807K variant, we generated a mouse model of BCL11BN807K by inserting the corresponding mutation, Bcl11bN797K, into the mouse genome. In Bcl11b+/N797K mice, the proportion of immature CD4-CD8+ single-positive thymocytes was increased, and the development of invariant natural killer cells was severely inhibited in a T-cell-intrinsic manner. Under competitive conditions, γδT cell development was outcompeted by control cells. Bcl11bN797K/N797K mice died within one day of birth. Recipient mice reconstituted with Bcl11bN797K/N797K fetal liver cells nearly lacked CD4+CD8+ double-positive thymocytes, which was consistent with the lack of their emergence in culture from Bcl11bN797K/N797K fetal liver progenitors. Interestingly, Bcl11bN797K/N797K progenitors gave rise to aberrant c-Kit+ and CD44+ cells both in vivo and in vitro. The increase in the proportion of immature CD8 single-positive thymocytes in the Bcl11bN797K mutants is caused, in part, by the inefficient activation of the Cd4 gene due to the attenuated function of the two Cd4 enhancers via distinct mechanisms. Therefore, we conclude that immunodeficient patient-derived Bcl11bN797K mutant mice elucidated a novel role for Bcl11b in driving the appropriate transition of CD4-CD8- into CD4+CD8+ thymocytes.