T Cells Research Articles

Effect of cyclophosphamide combined with glucocorticoid therapy on idiopathic membranous nephropathy: A multicenter open-label randomized controlled trial.

We aimed to evaluate the effect of cyclophosphamide combined with glucocorticoid therapy on idiopathic membranous nephropathy through a multicenter open-label randomized controlled trial. 92 patients with idiopathic membranous nephropathy admitted from March 2020 to September 2022 were included and assigned to a control group (n=46) and a research group (n=46) using a random number table. The control group was given glucocorticoid, and the research group was given cyclophosphamide combined with glucocorticoid. Clinical efficacy, renal function-related indicators (serum creatinine, blood urea nitrogen and albumin, and 24-hour urine protein quantification), inflammatory factors (interleukin (IL)-6, IL-18, transforming growth factor-β, and tumor necrosis factor-α), immune function-related indicators (anti-phospholipase A2 receptor antibody, and T-lymphocyte subsets), oxidative stress-related indicators (heme oxygenase-1, superoxide dismutase, malondialdehyde, and nitric oxide), blood lipid-related indicators (total cholesterol, triacylglycerol, and low-density lipoprotein), and adverse reactions were compared. The overall remission rate of the research group was higher than that of the control group (93.48 vs. 78.26%, p<0.05). After treatment, the research group had lower levels of 24-hour urine protein quantification, serum creatinine, blood urea nitrogen, IL-6, IL-18, transforming growth factor-β, tumor necrosis factor-α, heme oxygenase-1, malondialdehyde, anti-phospholipase A2 receptor antibody, CD8+, total cholesterol, triacylglycerol and low-density lipoprotein, higher levels of albumin, superoxide dismutase, nitric oxide, and CD4+ and a higher CD4+/CD8+ ratio than the control group (p<0.05). Cyclophosphamide combined with glucocorticoid therapy is effective for improving the overall remission rate and can suppress inflammatory responses and oxidative stress in patients with idiopathic membranous nephropathy.

Persistence of lung structural and functional alterations at one year post-COVID-19 is associated with increased serum PD-L2 levels and altered CD4/CD8 ratio.

Persistent respiratory symptoms and lung abnormalities post-COVID-19 are public health problems. This study evaluated biomarkers to stratify high-risk patients to the development or persistence of post-COVID-19 interstitial lung disease. One hundred eighteen patients discharged with residual lung abnormalities compatible with interstitial lung disease (COVID-ILD patients) after a severe COVID-19 were followed for 1 year (post-COVID-ILD patients). Physical examination, pulmonary function tests, and chest high-resolution computed tomography (HRCT) were performed. Soluble forms (s) of PD-L1, PD-L2, TIM-3, and GAL-9 were evaluated in serum and cell culture supernatant, as well as T-cells subsets and the transmembrane expression of PD-L1 and PD-L2 on the cell surface. Eighty percent of the post-COVID-ILD patients normalized their lung function at 1-year follow-up, 8% presented COVID-independent ILD, and 12% still showed functional and HRCT alterations. PD-L2 levels were heterogeneous during acute COVID-19 (aCOVID); patients who increased (at least 30%) their sPD-L2 levels at 1 year post-COVID-19 and exhibited altered CD4/CD8 ratio showed persistence of chest tomographic and functional alterations. By contrast, patients who decreased sPD-L2 displayed a complete lung recovery. sPD-L1, sTIM-3, and sGAL-9 increased significantly during aCOVID and decreased in all patients after 1-year follow-up. Increased sPD-L2 and an altered CD4/CD8 ratio after 12 months of aCOVID are associated with the persistence of lung lesions, suggesting that they may contribute to lung damage post-COVID-19.

Impact of LAG-3/FGL1 pathway on immune evasive contexture and clinical outcomes in advanced urothelial carcinoma

BackgroundAnti-programmed death-1 (PD-1)/anti-PD-ligand-1 (PD-L1) pathway inhibition is a standard regimen for advanced urothelial carcinoma (UC); however, its limited efficacy has been reflected in reported medium response rates. This study explored...

Impact of sub chronic administration of deltamethrin on autoimmune activity in rat

Deltamethrin (DLM) is a newer kind of insecticide that is used on pets, livestock, and crops, as well as to combat malaria vectors and household pests. It belongs to the synthetic pyrethroid group and is being promoted as an alternative to organophosphate chemicals due to its persistent and destructive effects. The current study aimed to evaluate the impact of sub-chronic oral exposure to DLM on autoimmune activity in rats. Three groups of male albino rats (15 rats/group) including the control group, the ethanol-treated group (1 ml/rat), and the DLM-treated group (5 mg/kg b.w). Samples of blood were taken from all groups at 4-, 8- and 12-week intervals for the determination of hematological, cytokines, and immunological parameters. T lymphocyte subsets and Treg lymphocytes were determined in serum using flow cytometric acquisition. The results revealed that DLM significantly increased TNF-α, IL-33, IL-6, IL-17, IgG, IgM, WBCs, differential count, and platelets while decreasing Hb concentration and RBCs. Additionally, DLM decreased the number of T-cell subsets (CD3, CD4, CD5, and CD8) and Treg lymphocytes. All of these impacts became more severe over time. It is possible to conclude that the sub-chronic oral exposure to DLM disturbed autoimmune activity through the disturbances in immunological indices, CDs subset Treg lymphocytes.

Guanylate binding protein 5 is an immune-related biomarker of oral squamous cell carcinoma: A retrospective prognostic study with bioinformatic analysis.

Cancer utilizes immunosuppressive mechanisms to create a tumor microenvironment favorable for its progression. The purpose of this study is to histologically characterize the immunological properties of the tumor microenvironment of oral squamous cell carcinoma (OSCC) and identify key molecules involved in the immunological microenvironment and patient prognosis. First, overlapping differentially expressed genes (DEGs) were screened from OSCC transcriptome data in public databases. Correlation analysis of DEGs with known immune-related genes identified genes involved in the immune microenvironment of OSCC. Next, stromal patterns of tumor were classified and immunohistochemical staining was performed for immune cell markers (CD3, CD4, Foxp3, CD8, CD20, CD68, and CD163), programmed death-ligand 1 (PD-L1), and guanylate binding protein 5 (GBP5) in resected specimens obtained from 110 patients with OSCC who underwent resection. Correlations between each factor and their prognostic impact were analyzed. Among the novel OSCC-specific immune-related genes screened (including ADAMDEC1, CXCL9, CXCL13, DPT, GBP5, IDO1, and PLA2G7), GBP5 was selected as the target gene. Histopathologic analysis showed that multiple T-cell subsets and CD20-positive cells were less common in the advanced stages, whereas CD163-positive cells were more common in advanced stages. The immature type in the stromal pattern category was associated with less immune cell infiltration, lower expression of PD-L1 in immune cells, lower expression of GBP5 in the stroma, and shorter overall survival and recurrence-free survival. Expression of GBP5 in the tumor and stroma correlated with immune cell infiltration of tumors and PD-L1 expression in tumor and immune cells. Patients with low tumor GBP5 expression and high stromal expression had significantly longer overall survival and recurrence-free survival. The stromal pattern category may reflect both invasive and immunomodulatory potentials of cancer-associated fibroblasts in OSCC. GBP5 has been suggested as a potential biomarker to predict the prognosis and therapeutic efficacy of immune checkpoint inhibitors.

Live Rotavirus Vaccination Appears Low-risk in Infants Born to Mothers With Inflammatory Bowel Disease on Biologics

Biologic therapies in the context of inflammatory bowel disease and pregnancy lead to improved maternal and fetal outcomes. Placental transfer results in detectable drug concentrations in infants. Rotavirus infection results in diarrheal related hospitalizations; however, the live oral vaccine is not currently recommended in biologic exposed infants. The aim of this study was to assess the effect of maternal biologic therapies on the infant immune system and safety of live rotavirus vaccination in biologic-exposed infants. Biologic-exposed infants underwent standardized clinical assessments, drug concentration, and immune function testing (complete blood count, differential, immunoglobulin levels, extended B-cell and T-cell subset enumeration, Recent Thymic Emigrants, regulatory T-cell numbers, mitogen stimulation assays, and review of T-cell Receptor Excision Circles in the newborn screen). Rotavirus vaccine-specific adverse effects following immunizations up to 42 days post the last dose of the vaccine series were recorded. There were 57 infants born to 52 mothers with inflammatory bowel disease exposed to infliximab (n= 21), adalimumab (n= 19), vedolizumab (n= 10), and ustekinumab (n= 7) in the third trimester for a median of 39 weeks (interquartile range, 38-39 weeks) at delivery. Immunologic assessments validated for age were normal in all infants despite median infliximab concentrations of 6.1 ug/mL (range, 0.4-28.8 ug/mL), adalimumab concentrations of 1.7ug/mL (range, 0.7-7.9 ug/mL), ustekinumab concentrations of 0.6 ug/mL (range, 0-1.1), and undetectable for vedolizumab at 10.7 weeks (interquartile range, 9.4-12.4) of age. The live oral rotavirus vaccine series was provided to 50 infants with the first dose given at a median of 13weeks of age. No adverse effects following immunization were reported. Immune function testing was normal, and administration of live rotavirus vaccination appeared low-risk in biologic-exposed infants irrespective of circulating drug levels.

Comparative proteomic profiling of the ovine and human PBMC inflammatory response

Understanding the cellular and molecular mechanisms of inflammation requires robust animal models. Sheep are commonly used in immune-related studies, yet the validity of sheep as animal models for immune and inflammatory diseases remains to be established. This cross-species comparative study analyzed the in vitro inflammatory response of ovine (oPBMCs) and human PBMCs (hPBMCs) using mass spectrometry, profiling the proteome of the secretome and whole cell lysate. Of the entire cell lysate proteome (oPBMCs: 4217, hPBMCs: 4574 proteins) 47.8% and in the secretome proteome (oPBMCs: 1913, hPBMCs: 1375 proteins) 32.8% were orthologous between species, among them 32 orthologous CD antigens, indicating the presence of six immune cell subsets. Following inflammatory stimulation, 71 proteins in oPBMCs and 176 in hPBMCs showed differential abundance, with only 7 overlapping. Network and Gene Ontology analyses identified 16 shared inflammatory-related terms and 17 canonical pathways with similar activation/inhibition patterns in both species, demonstrating significant conservation in specific immune and inflammatory responses. However, ovine PMBCs also contained a unique WC1+γδ T-cell subset, not detected in hPBMCs. Furthermore, differences in the activation/inhibition trends of seven canonical pathways and the sets of DAPs between sheep and humans, emphasize the need to consider interspecies differences in translational studies and inflammation research.

Vanin-2 is expressed in peripheral blood T cells and upregulated in patients with systemic lupus erythematosus.

Members of the vanin gene family include VNN1, VNN2, and VNN3 in humans. Although the functions of vanins have been widely examined in myeloid cells, their expression and functions have not been clarified in T lymphocytes. This study aimed to elucidate the significance of Vanin-2 (VNN2) on human peripheral blood T lymphocytes and study its expression in systemic lupus erythematosus (SLE). The differential expression of Vanins was analyzed by bioinformatics. VNN2 expressions in peripheral blood T-cell subsets were analyzed by single-cell RNA sequencing data and flow cytometry. Changes of VNN2 expression before and after T-cell activation were further clarified by western blot. The function of VNN2+ cells was studied by granzyme B (GZMB) and perforin detection. Changes in VNN2+ proportions in T-cell subsets of patients with SLE were further analyzed. In the present study, only VNN2 among vanins showed distinguishable expression in T cells. VNN2+ percentages were higher in CD8+ T cells those in CD4+ T cells. VNN2+ T cells were with a higher memory T-cell composition. VNN2 expression was significantly increased after T-cell stimulation. VNN2+ T cells had higher levels of GZMB and perforin secretion than VNN2- T cells. Clinically, VNN2+ percentages in T cells of patients with SLE were upregulated. Together, these data suggested that VNN2 is expressed in peripheral blood T cells characterized more GZMB and perforin secretion, and increased VNN2+ T cells in patients with SLE could reflect altered T-cell functions in vivo.

Enhancement of Tumor Antigen-specific T-cell Responses by Immune Checkpoint Blockade in Human Papillomavirus-related Head and Neck Squamous Cell Carcinoma.

Human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is clinically and immunologically distinct from HPV-negative HNSCC. Herein, we investigated the presence of tumor antigens HPV E6/E7 and wild-type p53-specific T-cell responses, and the impact of immune checkpoint blockade in patients with HPV-positive HNSCC. Peripheral blood mononuclear cells (PBMCs) from patients with HPV-positive HNSCC were stimulated with HPV E6/E7 or wild-type p53-derived peptide mixture and evaluated using the interferon-γ enzyme-linked immunosorbent spot assay. Flow cytometry was performed to analyze the proportion of T-cell subsets and T cells expressing immune checkpoint molecules. HPV E6/E7-specific T cells were detected in 22 (95.7%) of 23 patients, whereas wild-type p53-specific T cells were detected in 3 (15.0%) of 20 patients. Seven (43.8%) of 16 patients exhibited wild-type p53-specific T-cell responses, as determined using whole proteins instead of peptides. Immune checkpoint blockade enhanced wild-type p53-specific T-cell responses in 9 (45.0%) of 20 patients. Flow cytometric analysis of PBMCs revealed that responders exhibiting enhanced wild-type p53-specific T-cell responses following immune checkpoint blockade had a significantly higher proportion of Ki-67+CD4+ T cells, Ki-67+CD8+ T cells, regulatory T cells, PD-1+CD4+ T cells, and TIM-3+CD4+ T cells than non-responders. Our findings indicate that tumor antigen-specific T cells are present in the peripheral blood of patients with HPV-positive HNSCC. Blockade of checkpoint pathways can enhance T-cell responses in certain patients, probably via activated T cells, Tregs, and/or exhausted CD4+ T cells.

Bone Marrow-Suppressive Treatment in Children Is Associated with Diminished IFN-γ Response from T Cells upon Polyclonal and Varicella Zoster Virus Peptide Stimulation.

Severe haematological diseases and lymphoid malignancies require bone marrow (BM)-suppressive treatments. Knowledge regarding the impact of BM-suppressive treatments on children's memory T cells is very limited. Memory T cells play a crucial role in defending against herpesviruses, which is particularly relevant in paediatric cancer care. We studied 53 children in total; 34 with cancer and 2 with severe haematological disorders, with some receiving BM-suppressive treatment with or without allogeneic-haematopoietic stem cell transplantation (allo-HSCT), alongside 17 healthy controls. We focused on peripheral blood proportions of memory T-cell subsets using flow cytometry and analysed cytokine-secreting T cells with a four-parameter FluoroSpot assay in response to T-cell mitogen and varicella zoster virus (VZV) peptides. Patients on BM-suppressive treatment showed increased clusters of differentiation (CD)4+ and CD8+ effector memory (TEM)/terminally differentiated effector (TEFF) T cells compared to the healthy controls. They also exhibited, amongst other things, when compared to the healthy controls, a reduced total number of cytokine-secreting cells, by means of interferon (IFN)-γ, interleukin (IL)-17A, IL-10, and IL-22, following mitogen activation. A diminished IFN-γ response among the children with BM-suppressive treatment was observed upon VZV-peptide stimulation, compared to the healthy children. Collectively, the findings herein indicate that the children who are undergoing or have finished BM-suppressive treatment display qualitative differences in their T-cell memory compartment, potentially increasing their susceptibility to severe viral infections and impacting their immunotherapy, which relies on the functional ability of autologous T cells.

Cytomegalovirus infection is associated with thymic dysfunction and chronic graft-versus-host disease after pediatric hematopoietic stem cell transplantation

Pediatric hematopoietic stem cell transplantation (HSCT) is challenged by chronic graft-versus-host disease (cGvHD) significantly affecting survival and long-term morbidity, but underlying mechanisms including the impact of post-HSCT CMV infection are sparsely studied.We first investigated the impact of CMV infection for development of cGvHD in 322 children undergoing standard myeloablative HSCT between 2000 and 2018. Clinically significant CMV infection (n = 61) was an independent risk factor for chronic GvHD in a multivariable Cox regression analysis (HR = 2.17, 95% CI = 1.18–3.97, P = 0.013). We next explored the underlying mechanisms in a subcohort of 39 children. CMV infection was followed by reduced concentration of recent thymic emigrants (17.5 vs. 51.9 × 106/L, P = 0.048) and naïve CD4+ and CD8+ T cells at 6 months post-HSCT (all P < 0.05). Furthermore, CD25highFOXP3+ Tregs tended to be lower in patients with CMV infection (2.9 vs. 9.6 × 106/L, P = 0.055), including Tregs expressing the naivety markers CD45RA and Helios. CD8+ T-cell numbers rose after CMV infection and was dominated by exhausted PD1-expressing cells (66% vs. 39%, P = 0.023).These findings indicate that post-HSCT CMV infection is a main risk factor for development of chronic GvHD after pediatric HSCT and suggest that this effect is caused by reduced thymic function with a persistently impaired production of naïve and regulatory T cells in combination with increased peripheral T-cell exhaustion.

Quantification and Profiling of Early and Late Differentiation Stage T Cells in Mantle Cell Lymphoma Reveals Immunotherapeutic Targets in Subsets of Patients.

With the aim to advance the understanding of immune regulation in MCL and to identify targetable T-cell subsets, we set out to combine image analysis and spatial omic technology focused on both early and late differentiation stages of T cells. MCL patient tissue (n = 102) was explored using image analysis and GeoMx spatial omics profiling of 69 proteins and 1812 mRNAs. Tumor cells, T helper (TH) cells and cytotoxic (TC) cells of early (CD57-) and late (CD57+) differentiation stage were analyzed. An image analysis workflow was developed based on fine-tuned Cellpose models for cell segmentation and classification. TC and CD57+ subsets of T cells were enriched in tumor-rich compared to tumor-sparse regions. Tumor-sparse regions had a higher expression of several key immune suppressive proteins, tentatively controlling T-cell expansion in regions close to the tumor. We revealed that T cells in late differentiation stages (CD57+) are enriched among MCL infiltrating T cells and are predictive of an increased expression of immune suppressive markers. CD47, IDO1 and CTLA-4 were identified as potential targets for patients with T-cell-rich MCL TIME, while GITR might be a feasible target for MCL patients with sparse T-cell infiltration. In subgroups of patients with a high degree of CD57+ TC-cell infiltration, several immune checkpoint inhibitors, including TIGIT, PD-L1 and LAG3 were increased, emphasizing the immune-suppressive features of this highly differentiated T-cell subset not previously described in MCL.

Recent Advances in Immune-Based Therapies for Acute Myeloid Leukemia.

Despite advancements, acute myeloid leukemia (AML) remains unconquered by current therapies. Evidence of immune evasion during AML progression, such as HLA loss and T-cell exhaustion, suggests that antileukemic immune responses contribute to disease control and could be harnessed by immunotherapy. In this review, we discuss a spectrum of AML immunotherapy targets, encompassing cancer cell-intrinsic and surface antigens as well as targeting in the leukemic milieu, and how they can be tailored for personalized approaches. These targets are overviewed across major immunotherapy modalities applied to AML: immune checkpoint inhibitors, antibody-drug conjugates, therapeutic vaccines, bispecific/trispecific antibodies, and chimeric antigen receptor (CAR)-T and CAR-NK cells. Significance: Immune therapies in AML treatment show evolving promise. Ongoing research aims to customize approaches for varied patient profiles and clinical scenarios. This review covers immune surveillance mechanisms, therapy options like checkpoint inhibitors, antibodies, CAR-T/NK cells, and vaccines, as well as resistance mechanisms and microenvironment considerations.

Abstract PO-030: Single cell spatial analysis of large B cell lymphoma identifies biomarkers correlated with genetic subtypes and clincal outcomes

Abstract Single cell and multiplex imaging modalities have opened new insights into the organization and architecture of the immune microenvironment of cancer. Our group was one of the first to demonstrate these methods in the analysis of Hodgkin [1,2] and non-Hodgkin lymphoma [3]. Most recently, we demonstrated that spatial biomarkers outperform gene-expression biomarkers in predicting relapse after autologous transplant [4]. Here we present previously unpublished data demonstrating single cell multiplexed spatial analysis, using the imaging mass cytometry platform, in a cohort of 426 patients with large cell lymphoma. Gene expression and mutational analysis are available for a majority of cases allowing us to correlate spatial signature with genetic classes such as LymphGen [5], and deconvolution methods such as EcoTyper [6]. Using this cohort, we identify spatial biomarkers that improve prediction of clinical outcomes beyond cell of origin or genetic subgroup and identify novel targets for clinical development. Finally, we will present our approaches to move our discovery level spatial profiling into clinical diagnostic space through application of automated multiplex immunofluorescence imaging and a machine learning based image analysis pipeline. [1] Aoki T, et al. Single-Cell Transcriptome Analysis Reveals Disease-Defining T-cell Subsets in the Tumor Microenvironment of Classic Hodgkin Lymphoma. Cancer Discov. 2020 doi: 10.1158/2159-8290.CD-19-0680. [2] Xu, A. et al. Single cell spatial analysis and biomarker discovery in Hodgkin lymphoma. Biorxiv, 2023. https://doi.org/10.1101/2023.05.24.542195. [3] Colombo AR, et al. Single-cell spatial analysis of tumor immune architecture in diffuse large B-cell lymphoma. Blood Adv. 2022 doi: 10.1182/bloodadvances.2022007493. [4] Aoki T, et al. Spatially Resolved Tumor Microenvironment Predicts Treatment Outcomes in Relapsed/Refractory Hodgkin Lymphoma. J Clin Oncol. 2024 doi: 10.1200/JCO.23.01115. [5] Wright GW, et al. A Probabilistic Classification Tool for Genetic Subtypes of Diffuse Large B Cell Lymphoma with Therapeutic Implications. Cancer Cell. 2020 doi: 10.1016/j.ccell.2020.03.015. [6] Steen CB, et al. The landscape of tumor cell states and ecosystems in diffuse large B cell lymphoma. Cancer Cell. 2021 doi: 10.1016/j.ccell.2021.08.011. Citation Format: Alexander Xu, Joseph Lownik, Anton Villamejor, Simeon Mahov, Ying Li, Yidan Xu-Monette, Ken Young, Akil Merchant. Single cell spatial analysis of large B cell lymphoma identifies biomarkers correlated with genetic subtypes and clincal outcomes [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-030.

IMMU-05. TARGETING THE DYSREGULATED DNA METHYLOME DURING CAR T CELL MANUFACTURING IMPROVES THEIR FITNESS IN IMMUNOCOMPETENT BRAIN TUMOR MODELS

Abstract BACKGROUND Epigenetic reprogramming and T-cell exhaustion are key factors impacting the effectiveness of chimeric antigen receptor (CAR) T-cell immunotherapies in brain tumors. These processes are regulated by DNA methylation, an epigenetic modification that is crucial for gene regulation. Here, we investigated whether targeting the dysregulated DNA methylome in engineered immune cells can improve their anti-tumor efficacy. METHODS We optimized a transduction protocol that incorporates adding DNA methyltransferase inhibitors (DNMTi) early during the manufacturing process of murine CAR T-cells targeting B7-H3. We then used standard in vitro assays and immunocompetent glioma models to evaluate whether DNMTi treatment improved CAR T-cell effector functions by regulating activation, memory differentiation, and cytokine responses in adoptive T-cells. RESULTS In repeated-stimulation assays, CAR T-cells generated in the presence of DNMTi exhibited significantly higher levels of expansion and persistence compared to CAR T-cells without DNMTi exposure. Notably, CAR T-cells manufactured in the presence of DNMTi persisted up to 12 repeat stimulations, in contrast to a maximum of 7 stimulations for CAR T-cells generated without DNMTi treatment. Moreover, DNMTi-treated CAR T-cells expanded up to 55-fold higher than T-cells generated without DNMTi treatment upon repeated-stimulation with B7-H3-positive glioma cells. This was also associated with remarkably enhanced secretion of immune-stimulatory cytokines that persisted across repetitive stimulations. Furthermore, DNMTi-treated CAR T-cells exhibited greater cytotoxicity at lower effector-to-target ratios compared to untreated CAR T-cells, with sustained differential cytotoxicity even after the 4th stimulation. Preliminary in vivo data from immunocompetent glioma models further support the potential of DNMTi pre-treatment, showing improved T-cell persistence and reduced dose requirements. Mechanistic studies suggest that DNMT inhibition enhances B7-H3 CAR T-cell anti-tumor activity by regulating memory differentiation of CAR T-cells early in the production phase. CONCLUSION This study proposes that DNMTi pre-treatment may offer a promising approach for optimizing CAR T-cell manufacturing and enhancing performance in brain tumors.

IMMU-06. CAR DESIGN SHAPES THE BRAIN TUMOR IMMUNE MICROENVIRONMENT: IMPACT ON ACTIVITY AND PERSISTENCE OF ANTI-GLIOMA ADOPTIVE T CELL IMMUNOTHERAPY

Abstract BACKGROUND B7-H3 CAR T-cells effectively eradicate brain tumors in xenograft models and have proven safe in clinical trials, yet no objective responses were observed in patients. The limited efficacy is partly attributed to the immunosuppressive tumor microenvironment (TME), a characteristic not fully recapitulated in investigations using xenograft models. In this study, we set to optimize B7-H3 CAR structure and examine the effects of CAR design on the brain TME using an immunocompetent glioma model. METHODS We generated a panel of fully murine B7-H3 CARs with variations in transmembrane, co-stimulatory, and activation domains. This enabled us to comprehensively investigate their effector functions within the intact immune system. High-dimensional flow cytometry and single-cell RNA sequencing were then used to investigate changes in the brain TME following CAR T-cell therapy. RESULTS While five out of six B7-H3 CARs with single co-stimulatory domains exhibited potent functionality in vitro, optimizing co-stimulation and signaling failed to enhance their anti-glioma efficacy in vivo. To further enhance therapeutic effectiveness and persistence, we incorporated 4-1BB and CD28 co-stimulation through transgenic expression of 4-1BBL on CD28-based CAR T-cells. Although this design significantly improved anti-glioma efficacy in vitro, it conferred no additional benefits in vivo. Analysis of the TME revealed that CAR T-cell therapy influenced the immune composition of the brain TME. Successful anti-tumor responses were dictated by the recruitment, activation, and spatial localization of unique inflammatory ‘immune hubs’ containing specific subsets of macrophages and endogenous T-cells. Interestingly, depletion of brain macrophages using CSF1R inhibitor markedly inhibited CAR T-cell anti-tumor activity. CONCLUSION Our study highlights the critical role of CAR structural design and its modulation of the TME in mediating the efficacy of CAR T-cell therapy in brain tumors. Yet, more specific targeting and/or reprogramming of the TME is warranted for the successful design of effective adoptive immunotherapies for high-grade glioma.

Co-stimulation of CD28/CD40 signaling molecule potentiates CAR-T cell efficacy and stemness

CD19 chimeric antigen receptor T (CD19CAR-T) cells have achieved promising outcomes in relapsed/refractory B-cell malignancies. However, recurrences occur due to the loss of CAR-T cell persistence. We developed dual T/B-cell co-stimulatory molecules (CD28 and CD40) in CAR-T cells to enhance intense tumoricidal activity and persistence. CD19.28.40z CAR-T cells promoted pNF-κB and pRelB downstream signaling while diminishing NFAT signaling upon antigen exposure. CD19.28.40z CAR-T cells demonstrated greater proliferation which translated into effective anti-tumor cytotoxicity in long-term co-culture assay. Repetitive weekly antigen stimulation unveiled continuous CAR-T cell expansion while preserving central memory T-cell subset and lower expression of exhaustion phenotypes. The intrinsic genes underlying CD19.28.40z CAR-T cell responses were compared to conventional CARs and demonstrated the up-regulated genes associated with T-cell proliferation and memory as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway. Enrichment of genes toward T-cell stemness, particularly SELL, IL-7r, TCF7, and KLF2, was observed. Effective and continuing anti-tumor cytotoxicity in vivo was exhibited in both B-ALL and B-NHL xenograft models while demonstrating persistent T-cell memory signatures. The functional enhancement of CD37.28.40z CAR-T cell activities against CD37+ tumor cells was further validated. The modification of dual T/B-cell signaling molecules remarkably maximized the efficacy of CAR-T cell therapy.

Effects of Tongluo Jiedu prescription on immune function and oxidative stress in patients with oral cancer

BACKGROUND Oral cancer, which is caused by mucous membrane variation, represents a prevalent malignant tumor in the oral and maxillofacial region, posing a significant threat to patients’ lives and safety. While surgical intervention stands as a cornerstone treatment for oral cancer patients, it carries the risk of incomplete treatment or high rates of postoperative recurrence. Hence, a multifaceted approach incorporating diverse treatment modalities is essential to enhance patient prognosis. AIM To analyze the application effect of Tongluo Jiedu prescription as adjuvant therapy and its influence on patient prognosis in patients with oral cancer. METHODS Eighty oral cancer patients in our hospital were selected and divided into the observation group and control group by a random number table. The control group was treated with continuous arterial infusion chemotherapy of cisplatin and 5-fluorouracil. The observation group was additionally given Tongluo Jiadu prescription. The inflammatory stress level, peripheral blood T-cell subsets, and immune function of the two groups were subsequently observed. SPSS 21.0 was used for data analysis. RESULTS The observation group demonstrated lower levels of interleukin-6 and C-reactive protein, and a higher level of tumor necrosis factor in comparison to the control group. After treatment, the immune function in the observation group was significantly better than in the control group. CONCLUSION Tongluo Jiedu prescription can improve the immune function and oxidative stress level of patients with oral cancer and accelerate the recovery process.

Heat shock protein 90α reduces CD8+ T cell exhaustion in acute lung injury induced by lipopolysaccharide

CD8+ T-cell exhaustion is a promising prognostic indicator of sepsis-induced acute respiratory distress syndrome (ARDS). Patients with sepsis-related ARDS had reduced levels of HSP90AA1. However, whether the changes in CD8+ T cells were related to HSP90α, encoded by the HSP90AA1 gene, was unclear. This study aimed to examine the regulatory mechanism of HSP90α and its impact on CD8+ T-cell exhaustion in lipopolysaccharide (LPS)-induced acute lung injury (ALI). In this study, by conducting a mouse model of ALI, we found that one week after LPS-induced ALI, CD8+ T cells showed exhaustion characteristics. At this time, proliferation and cytokine release in CD8+ T cells were reduced. The inhibitory costimulatory factors PD-1 and Tim-3, on the other hand, were enhanced. Meanwhile, the expression of HSP90α and STAT1 decreased significantly. The in vitro studies showed that HSP90α stimulation or inhibition affected the CD8+ T-cell exhaustion phenotype. Interference with STAT1 reduced the expression of HSP90α and impaired its regulation of CD8+ T cells. The Co-Immunoprecipitation results indicated that HSP90α can directly or indirectly bind to TOX to regulate TOX expression and downstream signal transduction. In summary, by inhibiting TOX-mediated exhaustion signaling pathways, HSP90α inhibited CD8+ T-cell exhaustion in ALI. The participation of STAT1 in the regulation of HSP90α was required.

PDL1 targeting by miR-138-5p amplifies anti-tumor immunity and Jurkat cells survival in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) has constituted over 80% of the lung cancer population with a poor prognosis. Over the past decade, immunotherapy has been constructed in the enlargement of immune checkpoint inhibitors as a promising approach for NSCLC treatment. Evading the immune system using the PD-1/PD-L1 axis is an intelligent way for cancers, and T cells cannot respond fully and confront cancer. Recently, the miR-138 was reported as a PD-L1 regulator in NSCLC. However, its inhibitory impact on T-cell exhaustion has not been characterized. The present study aims to impair PD-L1 (B7-H1) expression in Adenocarcinoma cell lines using miR-138-5p and determines how it prevents Jurak cell exhaustion. To gain the purpose, first, 18 highly significant dysregulated miRNAs containing hsa-miR-138 and CD274-mRNA network were detected in NSCLC based on bioinformatics analysis. Moreover, our study revealed a high level of miR-138-5p could make significant changes like PDL1 downregulation, proliferation, and mortality rate in A549/Calu6 cells. We also simulate cancer environmental conditions by culturing Jurak cells and NSCLC cell lines under the influence of stimulator cytokines to show how miR-138-5p survives Jurak cells by targeting PD-L1/PD-1pathway.