T-cell Dysfunction Research Articles

State of the Field: Cytotoxic Immune Cell Responses in C. neoformans and C. deneoformans Infection.

Cryptococcus neoformans is an environmental pathogen that causes life-threatening disease in immunocompromised persons. The majority of immunological studies have centered on CD4+ T-cell dysfunction and associated cytokine signaling pathways, optimization of phagocytic cell function against fungal cells, and identification of robust antigens for vaccine development. However, a growing body of literature exists regarding cytotoxic cells, specifically CD8+ T-cells, Natural Killer cells, gamma/delta T-cells, NK T-cells, and Cytotoxic CD4+ T-cells, and their role in the innate and adaptive immune response during C. neoformans and C. deneoformans infection. In this review, we (1) provide a comprehensive report of data gathered from mouse and human studies on cytotoxic cell function and phenotype, (2) discuss harmonious and conflicting results on cellular responses in mice models and human infection, (3) identify gaps of knowledge in the field ripe for exploration, and (4) highlight how innovative immunological tools could enhance the study of cytotoxic cells and their potential immunomodulation during cryptococcosis.

Development of a Competitive Nutrient-Based T-Cell Immunotherapy Designed to Block the Adaptive Warburg Effect in Acute Myeloid Leukemia.

Background: T-cell-based adoptive cell therapies have emerged at the forefront of cancer immunotherapies; however, failed long-term survival and inevitable exhaustion of transplanted T lymphocytes in vivo limits clinical efficacy. Leukemia blasts possess enhanced glycolysis (Warburg effect), exploiting their microenvironment to deprive nutrients (e.g., glucose) from T cells, leading to T-cell dysfunction and leukemia progression. Methods: Thus, we explored whether genetic reprogramming of T-cell metabolism could improve their survival and empower T cells with a competitive glucose-uptake advantage against blasts and inhibit their uncontrolled proliferation. Results: Here, we discovered that high-glucose concentration reduced the T-cell expression of glucose transporter GLUT1 (SLC2A1) and TFAM (mitochondrion transcription factor A), an essential transcriptional regulator of mitochondrial biogenesis, leading to their impaired expansion ex vivo. To overcome the glucose-induced genetic deficiency in metabolism, we engineered T cells with lentiviral overexpression of SLC2A1 and/or TFAM transgene. Multi-omics analyses revealed that metabolic reprogramming promoted T-cell proliferation by increasing IL-2 release and reducing exhaustion. Moreover, the engineered T cells competitively deprived glucose from allogenic blasts and lessened leukemia burden in vitro. Conclusions: Our findings propose a novel T-cell immunotherapy that utilizes a dual strategy of starving blasts and cytotoxicity for preventing uncontrolled leukemia proliferation.

Predictors of interferon-gamma release assay results and their association with COVID-19 infection outcomes.

An 'indeterminate' interferon-gamma release assay (IGRA) result used in the diagnosis of latent TB infection (LTBI) is most commonly due to an inadequate control (or 'mitogen') response, which may reflect underlying T-cell dysfunction. We performed a single-centre, retrospective study on COVID-19 patients admitted to a tertiary referral hospital who had IGRA testing conducted over a 5-month period. The primary outcomes included predictors of indeterminate IGRA results and associations with COVID-19 outcomes. A total of 181 patients were included for analysis. Approximately one-third of patients hospitalised with COVID-19 with IGRA testing performed (60/181) had an indeterminate result. The likelihood of an indeterminate IGRA was increased in patients with a history of solid organ transplant and a higher severity of COVID-19 at the time of testing. An indeterminate IGRA was associated with a higher risk of severe COVID-19 and a higher risk of admission to the ICU during admission to the hospital. No difference in mortality between the two subgroups was found. Our study demonstrated that COVID-19 patients on immunosuppression had a high likelihood of an indeterminate IGRA result, which was associated with markers of disease severity and immunosuppression. In this cohort, an indeterminate result was associated with worse COVID-19 outcomes.

Different growth patterns in two siblings with Schimke immuno-osseous-dysplasia.

Schimke immuno-osseous-dysplasia (SIOD) is an autosomal recessive systemic disease due to pathogenic variants in SMARCAL1. Manifestations include nephrotic syndrome (NS), kidney failure, T-cell dysfunction, vaso-occlusive disease, and disproportionate short stature, a general feature of this disease. Here, we present a markedly different growth pattern in two brothers with SIOD sharing the same homozygous R561C missense variant. The index patient presented at the age of 11years with NS and severely disproportionate short stature, followed by kidney failure at the age of 16, and severely reduced adult height (z-score - 8.0). In contrast, the younger brother showed normal growth until the age of 8years. Mild proteinuria was noted at the age of 4.5, followed by NS at 9.5years, kidney failure at 11years, progressive disproportionate stature, and reduced adult height (z-score - 4.5). Both brothers had comparable disproportion in adulthood (sitting height index z-score - 0.88 and - 1.44, respectively).

Validation, Diagnostic, and Monitoring Roles of Multiomic Studies in Putative Immunotoxiepigenetic Models of Immune-Mediated Inflammatory Diseases

Abstract Immune-mediated inflammatory diseases (IMIDs) are mediated by immune dysfunction. Putative models of T-cell-mediated immune dysfunction in IMIDs exist and are yet to be validated by multiomics studies. Prompt diagnosis and monitoring are crucial for IMID management. The aim of this review was to discuss the putative T-cell dysfunction model of IMIDs and the role of multiomics studies for validation of the disease models as well as its diagnostic and monitoring potential in IMIDs. Online searches on databases such as Google Scholar, PubMed, Biomed Central, and SciELO were carried out. An attempt was made to review articles with keywords such as genomics, proteomics, transcriptomics, metabolomics, T cell immunopathogenesis/dysfunction, immune dysfunction, and IMIDs. The putative T-cell dysfunction model of IMIDs and its validation with multiomics studies were discussed. The diagnostic and monitoring roles of multiomic studies for management of IMIDs were also discussed. The review concluded that multiomic studies have validating potential for the putative T-cell dysfunction model of IMIDs as well as diagnostic and monitoring roles in IMIDs.

Abstract A050: MDSC-derived transmembrane TNF is a central regulator of stromal inflammation and T-cell dysfunction in pancreatic cancer

Abstract INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy characterized by stromal inflammation and T-cell dysfunction. Neutrophilic/granulocytic myeloid-derived suppressor cells (gMDSC) infiltrate early in PDAC development and contribute to a highly immunosuppressive tumor microenvironment (TME). We have previously shown that gMDSC-derived tumor necrosis factor (TNF) is a novel regulator of therapeutic resistance in the PDAC TME, and pharmacological targeting of transmembrane (tm)TNF-TNFR2 signaling can reprogram the TME to improve chemosensitivity. Here we dissect the functional consequences of gMDSC-derived tmTnf on cancer-associated fibroblast (CAF) skewness and CD8+ T-cell function. METHODS: To assess global effects of tmTNF in orthotopically and subcutaneously injected KPC models of PDAC, we compared T-cell/CAF phenotypes in littermate vs transgenic mice overexpressing exclusively tmTnf (tmTnfOE)—in which site-directed mutagenesis of TNF cleavage sites renders tmTNF uncleavable, resulting in systemically overexpressed tmTNF signaling. To investigate effects of gMDSC-derived tmTnf in vitro, J774M cells that phenocopy gMDSCs were engineered to overexpress exclusively tmTnf (J-tmTnfOE) or wild-type Tnf (J-TnfWT) following CRISPR/cas9 silencing of endogenous Tnf (J-TnfKO). These cells were co-cultured with dual reporter CAFs (Il6GFP=inflammatory iCAF; Acta2dsRed=myofibroblastic myCAF) and CD8+ T-cells followed by cell trace violet proliferation assay, flow cytometry phenotyping, and IFNγ ELISA. RESULTS: Compared to littermate controls, flank and orthotopic KPC tumor-bearing tmTnfOE mice displayed accelerated tumor kinetics and volume, respectively, as well as increase in stromal fibrosis and collagen deposition (Trichrome/Sirius Red). Flow cytometric phenotyping revealed increased proportion of iCAF (PDPN+Ly6C+) vs myCAF (PDPN+Ly6C-) in tmTNFOE vs littermate mice. Moreover, intratumoral gMDSCs isolated from tmTNFOE mice augmented CAF-Il6GFP:Acta2dsRed ratios ex vivo. CD8+ T-cell phenotyping revealed higher proportions of CD39+Ly108- dysfunctional and KLRG1+CD127- short-lived effector T-cells (SLEC), with concurrent reduction in CD127+KLRG1- memory progenitor effector T-cells (MPEC), in tmTnfOE compared with littermate tumors. Using confocal microscopy in vitro, we confirmed that transmembrane sequestration of Tnf was more pronounced in J-tmTNFOE vs J-TnfWT, with complete loss of subcellular Tnf in J-TnfKO cells. To define the precise contribution of gMDSC-tmTNF on CAF/T-cell skewness, co-culture of J-tmTnfOE with dual-reporter CAFs resulted in striking induction in CAF-Il6GFP compared with J-TnfKO-CAF co-cultures. Moreover, J-tmTnfOE significantly restrained CD8+ T-cell proliferation, MPEC differentiation, and IFN-γ release relative to J-TnfKO. CONCLUSIONS: gMDSC-tmTnf is a central regulator of inflammatory CAF polarization and CD8+ T-cell dysfunction in PDAC. Selective pharmacologic or genetic strategies to disrupt gMDSC-tmTNF derived signaling in the TME may improve chemoimmunotherapy sensitivity in PDAC. Citation Format: Andrew M Adams, Haleh Amirian, Christine I Rafie, Karthik Rajkumar, Erin Dickey, Harper M Marsh, Manan Patel, Siddharth Mehra, Nagaraj Nagathihalli, Nipun Merchant, Yoon S Yap, Luisa Cimmino, Erietta Stelekati, Anna Bianchi, Jashodeep Datta. MDSC-derived transmembrane TNF is a central regulator of stromal inflammation and T-cell dysfunction in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A050.

Targeting GProtein-Coupled Receptors in Immuno-Oncological Therapies.

The advent of cancer immunotherapy based on PD-1 and CTLA-4 immune checkpoint blockade (ICB) has revolutionized cancer treatment. However, many cancers do not respond to ICB, highlighting the urgent need for additional approaches to achieve durable cancer remission. The large family of G protein-coupled receptors (GPCRs) is the target of more than 30% of all approved drugs, but GPCRs have been underexploited in cancer immunotherapy. In this review, we discuss the central role of GPCRs in immune cell migration and function and describe how single-cell transcriptomic studies are illuminating the complexity of the human tumor immune GPCRome. These receptors include multiple GPCRs expressed in CD8 Tcells that are activated by inflammatory mediators, protons, neurotransmitters, and metabolites that accumulate in the tumor microenvironment, thereby promoting Tcell dysfunction. We also discuss new opportunities to target GPCRs as a multimodal approach to enhance the response to ICB for a myriad of human malignancies.

Tumor-associated neutrophils upregulate Nectin2 expression, creating the immunosuppressive microenvironment in pancreatic ductal adenocarcinoma

BackgroundTumor-associated neutrophils (TANs) constitute an abundant component among tumor-infiltrating immune cells and have recently emerged as a critical player in pancreatic ductal adenocarcinoma (PDAC) progression. This study aimed to elucidate the pro-tumor mechanisms of TAN and identify a novel target for effective immunotherapy against PDAC.MethodsMicroarray and cytokine array analyses were performed to identify the mechanisms underlying the function of TANs. Human and mouse TANs were obtained from differentiated HL-60 cells and orthotopically transplanted PDAC tumors, respectively. The interactions of TANs with cancer and cytotoxic T-cells were evaluated through in vitro co-culture and in vivo orthotopic or subcutaneous models. Single-cell transcriptomes from patients with PDAC were analyzed to validate the cellular findings.ResultsIncreased neutrophil infiltration in the tumor microenvironment was associated with poor survival in patients with PDAC. TANs secreted abundant amounts of chemokine ligand 5 (CCL5), subsequently enhancing cancer cell migration and invasion. TANs subpopulations negatively correlated with cytotoxic CD8+ T-cell infiltration in PDAC and promoted T-cell dysfunction. TANs upregulated the membranous expression of Nectin2, which contributed to CD8+ T-cell exhaustion. Blocking Nectin2 improved CD8+ T-cell function and suppressed tumor progression in the mouse model. Single-cell analysis of human PDAC revealed two immunosuppressive TANs phenotypes: Nectin2+ TANs and OLR1+ TANs. Endoplasmic reticulum stress regulated the protumor activities in TANs.ConclusionsTANs enhance PDAC progression by secreting CCL5 and upregulating Nectin2. Targeting the immune checkpoint Nectin2 could represent a novel strategy to enhance immunotherapy efficacy in PDAC.

Breakdown and Repair of Peripheral Immune Tolerance in Type 1 Diabetes.

Failures in peripheral immune tolerance mechanisms create a permissive environment for autoimmune diabetes initiation and disease progression. Biomarker analyses provide tools that allow recognition of this loss of tolerance, reflecting a serial acquisition of pathogenic characteristics causally linked to islet β-cell dysfunction and death. Autoimmune effector cell activation and expansion, ineffective immune regulation, and tissue response to injury during active disease each represent challenges to homeostasis; however, they also represent targets for therapeutic intervention, with the potential for restoration of tolerance. Limited success in recent clinical trials demonstrates that tolerance in type 1 diabetes (T1D) is achievable, but currently occurs in few subjects and is not durable in most. Combining therapeutic agents to rebuild multiple immune components to restore tolerance, particularly addressing both effector and regulatory T-cell dysfunction, is needed.

H3K9 lactylation in malignant cells facilitates CD8+ T cell dysfunction and poor immunotherapy response

Histone lysine lactylation (Kla) is a post-translational modification, and its role in tumor immune escape remains unclear. Here, we find that increased histone lactylation is associated with poor response to immunotherapy in head and neck squamous cell carcinoma (HNSCC). H3K9la is identified as a specific modification site in HNSCC. Using cleavage under targets and tagmentation analyses, interleukin-11 (IL-11) is identified as a downstream regulatory gene of H3K9la. IL-11 transcriptionally activates immune checkpoint genes through JAK2/STAT3 signaling in CD8+ Tcells. Additionally, IL-11 overexpression promotes tumor progression and CD8+ Tcell dysfunction invivo. Moreover, IL11 knockdown reverses lactate-induced CD8+ Tcell exhaustion, and cholesterol-modified siIL11 restores CD8+ Tcell killing activity and enhances immunotherapy efficacy. Clinically, H3K9la positively correlates with IL-11 expression and unfavorable immunotherapy responses in patients. This study reveals the crucial role of histone lactylation in immune escape, providing insights into immunotherapy strategies for HNSCC.

Age-associated contraction of tumor-specific T cells impairs antitumor immunity.

Progressive decline of the adaptive immune system with increasing age coincides with a sharp increase in cancer incidence. In this study, we set out to understand whether deficits in antitumor immunity with advanced age promote tumor progression and/or drive resistance to immunotherapy. We found that multiple syngeneic cancers grew more rapidly in aged versus young adult mice, driven by dysfunctional CD8+ T-cell responses. By systematically mapping immune cell profiles within tumors, we identified loss of tumor antigen-specific CD8+ T cells as a primary feature accelerating the growth of tumors in aged mice and driving resistance to immunotherapy. When antigen-specific T cells from young adult mice were administered to aged mice, tumor outgrowth was delayed and the aged animals became sensitive to PD-1 blockade. These studies reveal how age-associated CD8+ T-cell dysfunction may license tumorigenesis in elderly patients and have important implications for the use of aged mice as pre-clinical models of aging and cancer.

Spatial transcriptomics reveals profound subclonal heterogeneity and T-cell dysfunction in extramedullary myeloma

AbstractExtramedullary disease (EMD) is a high-risk feature of multiple myeloma (MM) and remains a poor prognostic factor, even in the era of novel immunotherapies. Here, we applied spatial transcriptomics (RNA tomography for spatially resolved transcriptomics [tomo-seq] [n = 2] and 10x Visium [n = 12]) and single-cell RNA sequencing (n = 3) to a set of 14 EMD biopsies to dissect the 3-dimensional architecture of tumor cells and their microenvironment. Overall, infiltrating immune and stromal cells showed both intrapatient and interpatient variations, with no uniform distribution over the lesion. We observed substantial heterogeneity at the copy number level within plasma cells, including the emergence of new subclones in circumscribed areas of the tumor, which is consistent with genomic instability. We further identified the spatial expression differences between GPRC5D and TNFRSF17, 2 important antigens for bispecific antibody therapy. EMD masses were infiltrated by various immune cells, including T cells. Notably, exhausted TIM3+/PD-1+ T cells diffusely colocalized with MM cells, whereas functional and activated CD8+ T cells showed a focal infiltration pattern along with M1 macrophages in tumor-free regions. This segregation of fit and exhausted T cells was resolved in the case of response to T-cell–engaging bispecific antibodies. MM and microenvironment cells were embedded in a complex network that influenced immune activation and angiogenesis, and oxidative phosphorylation represented the major metabolic program within EMD lesions. In summary, spatial transcriptomics has revealed a multicellular ecosystem in EMD with checkpoint inhibition and dual targeting as potential new therapeutic avenues.

Low-level HIV-1 viremia affects T-cell activation and senescence in long-term treated adults in the INSTI era

BackgroundAround 10% of people with HIV (PWH) exhibit a low-level viremia (LLV) under antiretroviral therapy (ART). However, its origin and clinical significance are largely unknown, particularly at viremias between 50 and 200 copies/mL and under modern ART based on integrase strand transfer inhibitors (INSTIs). Our aim was to characterize their poor immune response against HIV in comparison to individuals with suppressed viremia (SV) and non-HIV controls (NHC).MethodsTransversal observational study in 81 matched participants: 27 PWH with LLV, 27 PWH with SV, and 27 NHC. Activation (CD25, HLA-DR, and CD38) and senescence [CD57, PD1, and HAVCR2 (TIM3)] were characterized in peripheral T-cell subsets by spectral flow cytometry. 45 soluble biomarkers of systemic inflammation were evaluated by immunoassays. Differences in cell frequencies and plasma biomarkers among groups were evaluated by a generalized additive model for location, scale, and shape (GAMLSS) and generalized linear model (GLM) respectively, adjusted by age, sex at birth, and ART regimen.ResultsThe median age was 53 years and 77.8% were male. Compared to NHC, PWH showed a lower CD4+/CD8+ ratio and increased activation, senescence, and inflammation, highlighting IL-13 in LLV. In addition, LLV showed a downtrend in the frequency of CD8+ naive and effector memory (EM) type 1 compared to SV, along with higher activation and senescence in CD4+ and CD8+ EM and terminally differentiated effector memory RA+ (TEMRA) subpopulations. No significant differences in systemic inflammation were observed between PWH groups.ConclusionLLV between 50 and 200 copies/mL leads to reduced cytotoxic activity and T-cell dysfunction that could affect cytokine production, being unable to control and eliminate infected cells. The increase in senescence markers suggests a progressive loss of immunological memory and a reduction in the proliferative capacity of immune cells. This accelerated immune aging could lead to an increased risk of developing future comorbidities. These findings strongly advocate for heightened surveillance of these PWH to promptly identify potential future complications.

Both Th1 and Th2 CD4 + T-Cell Lineage Infiltrations Decrease in Post-hematopoietic Stem Cell Transplantation Colon Adenoma.

As long-term survival improves after allogeneic hematopoietic stem cell transplantation (HSCT), the risk for secondary solid cancers, including colon cancer, also increases. However, the pathogenesis of secondary solid cancers in post-HSCT patients remains unclear. This study aimed to investigate the involvement of local immunity in colon carcinogenesis in post-HSCT patients by assessing the infiltrating T cells in colon adenomas as premalignant lesions of colon cancer in adenoma-carcinoma sequence. Colon adenoma samples obtained from 19 post-HSCT patients and 57 non-HSCT participants were analyzed via immunohistochemistry. Double staining of CD4/T-bet, CD4/GATA3, and CD4/FoxP3 was performed for evaluation of helper T-cell lineages (Th1, Th2, and regulatory T cells, respectively) and CD8 staining for CD8+ T cells. There were no significant between-group differences in the number of infiltrating CD4+ T cells and CD8+ T cells in adenomas. However, the number of both CD4+/T-bet+ and CD4+/GATA3+ T cells was significantly lower in the post-HSCT adenomas than in the non-HSCT adenomas (P = 0.0171 and 0.0009, respectively), whereas no significant differences were found in the number of CD4+/FoxP3+ cells. Although the number of infiltrating CD4+ and CD8+ T cells, and even Treg cell counts, is sufficiently recovered post-HSCT, CD4+ T-cell dysfunction due to suppressed activation and differentiation in colon adenomas might be involved in colon carcinogenesis in post-HSCT patients. Elucidating the pathogenesis will contribute to the development of effective screening and prevention programs for secondary colon cancer in post-HSCT patients.

Abstract We077: Mechanisms of T-lymphocyte Dysfunction in Dilated Cardiomyopathy Patients

Preclinical studies have shown that T-cells infiltrate the heart during heart failure (HF), promoting remodeling. However, it remains unclear whether activated T-cells infiltrate the human failing hearts also. Importantly, cellular mechanisms that are involved in T-cell activation during human HF are unknown. To address this question, we analyzed published single-cell RNA sequencing data from patients with dilated cardiomyopathy (DCM) and compared it with control hearts. Our analysis focused on characterizing the gene expression of cardiac T-cells to uncover potential mechanisms underlying their role in human HF. We found that patients with DCM had significantly more cardiac CD3+ T-cells (%CD45+ cells; 35.5±5.9 vs 22.0±0.8 p < 0.01), with gene signatures indicative of higher TCR dependent antigenic activation, proliferation, and exhaustion. Although there were no differences in the proportion of CD4+ helper and CD8+ cytotoxic T-cells between groups, patients with DCM had a higher proportion of activated (%CD45+ cells, 6.6±0.8 vs 3.1±0.3 p = 0.03) and proliferative CD4+ T-cells (%CD45+ cells, 8.0±0.8 vs 3.1±0.3 p < 0.01). Additionally, genes involved in TNF and TGFβ signaling were significantly enriched in cardiac T-cells derived from patients with DCM (normalized enrichment scores = 2.35, p adj < 0.01, and 1.72, p adj < 0.01, respectively),consistent with preclinical studies published by us and others. Recently, we also showed that dysfunctional T-cells exhibit increased estrogenic signaling with activation of Estrogen Receptor (ER)α in mice. Thus, we also looked at estrogen signaling in T-cells infiltrated into the DCM hearts and found that, indeed, cardiac T-cells in DCM patients exhibit significant upregulation of genes involved in estrogen signaling (normalized enrichment score = 1.69, p adj < 0.01). This comprehensive analysis, for the first time, shows that T-cells are antigenically activated, actively infiltrate the failing human hearts and exhibit increased estrogenic signaling during DCM, providing novel insights and potential avenues for targeted immunotherapies.

PD-L1+ macrophage and tumor cell abundance and proximity to T cells in the pretreatment large B-cell lymphoma microenvironment impact CD19 CAR-T cell immunotherapy efficacy.

CD19-targeted chimeric antigen receptor T-cell (CAR-T) immunotherapy has transformed the management of relapsed/refractory large B-cell lymphoma (LBCL), yet durable remissions are observed in less than half of treated patients. The tumor microenvironment (TME) is a key and understudied factor impacting CD19 CAR-T therapy outcomes. Using NanoString nCounter transcriptome profiling (n = 24) and multiplex immunohistochemistry (mIHC, n = 15), we studied the TME in pretreatment biopsies from patients with LBCL undergoing CD19 CAR-T therapy. Patients who achieved complete response (CR) after CAR-T therapy demonstrated higher expression of genes associated with T-cell trafficking and function, whereas those who did not achieve CR had higher expression of genes associated with macrophages and T-cell dysfunction. Distinct patterns of immune infiltration and fibrosis in the TME were associated with CAR-T therapy outcomes, and these findings were corroborated using artificial intelligence-assisted image analyses. Patients who achieved CR had a lower proportion of the biopsy occupied by an interspersed immune infiltrate and a higher proportion of hypocellular/fibrotic regions. Furthermore, mIHC revealed lower density of CD4+ T cells and higher densities of both macrophages and tumor cells expressing PD-L1 in non-CR patients. Spatial analysis revealed that PD-1+ T cells were in close proximity to PD-L1+ macrophages or PD-L1+ tumor cells in patients who did not compared to those who did achieve CR after CAR-T therapy. These findings suggest that morphologic patterns in the TME and engagement of the PD-1/PD-L1 axis in pretreatment biopsies may impact CD19 CAR-T immunotherapy response in patients with LBCL.

AB042. Combined anti-PD-L1 and anti-VEGFR2 therapy promotes the antitumor immune response in glioblastoma multiforme by reprogramming tumor microenvironment.

Inhibitors of programmed cell death ligand 1 (PD-L1) and vascular endothelial growth factor receptor 2 (VEGFR2) are commonly used in the clinic, but they are beneficial for only a minority of glioblastoma multiforme (GBM) patients. GBM has significant immunosuppressive properties, and there are many immunosuppressive cells and dysfunctional effector T-cell in the tumor microenvironment (TME), which is one of the important reasons for the failure of clinical treatment of GBM. P21-activated kinase 4 (PAK4) is a threonine protein kinase, and as a pivotal immune suppressor in the TME. PAK4 knockdown attenuates vascular abnormalities and promotes T-cell infiltration. Using RNA sequencing (RNA-seq) technology, western blotting, and immunofluorescence, we identified changes in genes expression following VEGFR2 knockdown. The impact of anti-PD-L1 and anti-VEGFR2 on GBM cells apoptosis was assessed using coculture assays, western blotting, and flow cytometry. Additionally, the therapeutic efficacy of anti-PD-L1 and anti-VEGFR2 therapy was evaluated through in vivo experiments, immunohistochemistry, and immunofluorescence. Our studies revealed that VEGFR2 binds and phosphorylates signal transducer and activator of transcription 3 (p-STAT3), thereby regulating the expression of PAK4. Anti-PD-L1 and anti-VEGFR2 therapy can increase the secretion of interferon-gamma (IFN-γ), granzyme B, and perforin by immune cells and promoting the cytotoxic effects of cytotoxic cluster of differentiation 8 (CD8)+ T cells, and overexpression of PAK4 could reverse this effect. We also demonstrated that combination therapy with anti-PD-L1 and anti-VEGFR2 agents prevents tumor growth in an intracranial tumor model. Our results support that anti-VEGFR2 therapy can downregulate PAK4, reprogram the TME by increasing CD8+ T cells infiltration and activation, and enhance the therapeutic effect of anti-PD-L1 therapy on GBM cells.

Targeting myeloid-derived suppressor cells promotes antiparasitic T-cell immunity and enhances the efficacy of PD-1 blockade

Immune exhaustion corresponds to a loss of effector function of T cells that associates with cancer or chronic infection. Here, our objective was to decipher the mechanisms involved in the immune suppression of myeloid-derived suppressor cells (MDSCs) and to explore the potential to target these cells for immunotherapy to enhance checkpoint blockade efficacy in a chronic parasite infection. We demonstrated that programmed cell-death-1 (PD-1) expression was significantly upregulated and associated with T-cell dysfunction in advanced alveolar echinococcosis (AE) patients and in Echinococcus multilocularis-infected mice. PD-1 blockade ex vivo failed to reverse AE patients’ peripheral blood T-cell dysfunction. PD-1/PD-L1 blockade or PD-1 deficiency had no significant effects on metacestode in mouse model. This was due to the inhibitory capacities of immunosuppressive granulocytic MDSCs (G-MDSCs), especially in the liver surrounding the parasite pseudotumor. MDSCs suppressed T-cell function in vitro in an indoleamine 2, 3 dioxygenase 1 (IDO1)-dependent manner. Although depleting MDSCs alone restored T-cell effector functions and led to some limitation of disease progression in E. multilocularis-infected mice, combination with PD-1 blockade was better to induce antiparasitic efficacy. Our findings provide preclinical evidence in support of targeting MDSC or combining such an approach with checkpoint blockade in patients with advanced AE.

T-cell dysfunction in CLL is mediated through expression of Siglec-10 ligands CD24 and CD52 on CLL cells

AbstractAutologous T-cell–based therapies, such as chimeric antigen receptor (CAR) T-cell therapy, exhibit low success rates in chronic lymphocytic leukemia (CLL) and correlate with a dysfunctional T-cell phenotype observed in patients. Despite various proposed mechanisms of T-cell dysfunction in CLL, the specific CLL-derived factors responsible remain unidentified. This study aimed to investigate the mechanisms through which CLL cells suppress CAR T-cell activation and function. We found that CLL-derived T cells get activated, albeit in a delayed fashion, and specifically that restimulation of CAR T cells in the presence of CLL cells causes impaired cytokine production and reduced proliferation. Notably, coculture of T cells with CD40-activated CLL cells did not lead to T-cell dysfunction, and this required direct cell contact between the CD40-stimulated CLL cells and T cells. Inhibition of kinases involved in the CD40 signaling cascade revealed that the Spare Respiratory Capacity (SRC) kinase inhibitor dasatinib prevented rescue of T-cell function independent of CD40-mediated increased levels of costimulatory and adhesion ligands on CLL cells. Transcriptome profiling of CD40-stimulated CLL cells with or without dasatinib identified widespread differential gene expression. Selecting for surface receptor genes revealed CD40-mediated downregulation of the Sialic acid-binding Ig-like lectin 10 (Siglec-10) ligands CD24 and CD52, which was prevented by dasatinib, suggesting a role for these ligands in functional T-cell suppression in CLL. Indeed, blocking CD24 and/or CD52 markedly reduced CAR T-cell dysfunction upon coculture with resting CLL cells. These results demonstrated that T cells derived from CLL patients can be reinvigorated by manipulating CLL–T-cell interactions. Targeting CD24- and CD52-mediated CLL–T-cell interaction could be a promising therapeutic strategy to enhance T-cell function in CLL.

Combination of oncolytic Maraba virus with immune checkpoint blockade overcomes therapy resistance in an immunologically cold model of advanced melanoma with dysfunctional T-cell receptor signalling

BackgroundOver the past decade, cancer immunotherapies have revolutionized the treatment of melanoma; however, responses vary across patient populations. Recently, baseline tumor size has been identified as an independent prognostic factor...