Limited T-cell Research Articles

Generation of ex vivo autologous hematopoietic stem cell-derived T lymphocytes for cancer immunotherapy

CD19CAR-T cell therapy demonstrated promising outcomes in relapsed/refractory B-cell malignancies. Nonetheless, the limited T-cell function and ineffective T-cell apheresis for therapeutic purposes are still concern in heavily pretreated patients. We investigated the feasibility of generating hematopoietic stem cell-derived T lymphocytes (HSC-T) for cancer immunotherapy. The patients’ autologous peripheral blood HSCs were enriched for CD34+ and CD3+ cells. The CD34+ cells were then cultured following three steps of lymphoid progenitor differentiation, T-cell differentiation, and T-cell maturation processes. HSC-T cells were successfully generated with robust fold expansion of 3735 times. After lymphoid progenitor differentiation, CD5+ and CD7+ cells remarkably increased (65–84 %) while CD34+ cells consequentially declined. The mature CD3+ cells were detected up to 40 % and 90 % on days 42 and 52, respectively. The majority of HSC-T population was naïve phenotype compared to CD3-T cells (73 % vs 34 %) and CD8:CD4 ratio was 2:1. The higher level of cytokine and cytotoxic granule secretion in HSC-T was observed after activation. HSC-T cells were assessed for clinical application and found that CD19CAR-transduced HSC-T cells demonstrated higher cytokine secretion and a trend of superior cytotoxicity against CD19+ target cells compared to control CAR-T cells. A chronic antigen stimulation assay revealed similar T-cell proliferation, stemness, and exhaustion phenotypes among CAR-T cell types. In conclusions, autologous HSC-T was feasible to generate with preserved T-cell efficacy. The HSC-T cells are potentially utilized as an alternative option for cellular immunotherapy.

The single cell transcriptional landscape of fibrosarcoma.

235 Background: Fibrosarcoma (FS) is a relatively uncommon subtype of soft tissue sarcoma, which predominantly occurs in the extremities and girdles of elderly individuals. FS are famous for its high recurrence and metastasis rate. However, the current treatment regimens have shown poor efficacy in treating these progressive lesions. Therefore, a deep understanding of the tumor microenvironment characteristics of fibrosarcoma is needed to develop more effective drugs.Therefore, a deeper understanding of the tumor microenvironment characteristics of fibrosarcoma is needed for further development of therapeutic drugs. Methods: We performed single-cell transcriptome sequencing for the first time on 6 patients bearing with FS. Bulk-seq data of soft tissue sarcoma was downloaded from public database including TCGA and GEO for prognostic analysis. What' s more, a cohort of 54 FS patients was retrospectively collected for protein detection. Results: The tumor microenvironment landscape was characterized by the presence of malignant fibroblast, diverse macrophages, limited T-cell presence, and high levels of vascular infiltration. The expression of siglec15 was significantly upregulated in the malignant cells. And the prognostic significance of siglec15 was confirmed by the TCGA public database. What’s more, the high expression of siglec15 protein was negatively related to overall survival of fibrosarcoma patients from our institution. We also explored the interactions among different cell subtypes, especially the interactions between malignant cells and endothelial cells, as well as macrophages and endothelial cells, which not only promote tumor progression but also inhibit T-cell infiltration and function. Conclusions: In total, our work has uncovered the low T cell infiltration landscape of FS. The primary reason of insufficient immune infiltration may be due to the blockage of T cell extravasation caused by the interaction between endothelial cells, malignant cells and macrophages.

FRACTION: protocol of a phase II study of Fedratinib and Nivolumab combination in patients with myelofibrosis and resistance or suboptimal response to JAK-inhibitor treatment of the German MPN study group (GSG-MPN)

Development of Janus-kinase (JAK) inhibitors has revolutionized the therapeutic landscape for patients with myeloproliferative neoplasia (MPN). Following approval of the first JAK1/2-inhibitor Ruxolitinib, symptoms of this inflammatory disease, characterized by splenomegaly, release of inflammatory cytokines and appearance of thrombosis, could be effectively reduced for the first time. However, JAK-inhibitor treatment is limited in several aspects: 1) duration of response: 3 years after initiation of therapy more than 50% of patients have discontinued JAK-inhibitor treatment due to lack of efficacy or resistance; 2) reduction of disease burden: while effective in reducing inflammation and constitutional symptoms, JAK-inhibitors fail to reduce the malignant clone in the majority of patients and therefore lack long-term efficacy. Early clinical trials for patients with myelofibrosis (MF) have tried to address these issues for patients with suboptimal response to Ruxolitinib therapy while combination therapies with Fedratinib are rare. Recent reports provided first evidence on how the JAK2-V617F mutated myeloid cells may influence T-cell responses. JAK2-V617F promoted the synthesis of PD-L1 in MPN cells leading to limited anti-neoplastic T-cell responses, metabolic changes in T-cells and eventually JAK2-V617F-driven immune-escape of MPN cells. These findings may facilitate the use of immunotherapeutic approaches for JAK-mutated clones. Immune checkpoints refer to a variety of inhibitory pathways that are crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage. The FRACTION study is a single arm, open label Phase II trial investigating the combination of Fedratinib with the PD-1 inhibitor Nivolumab in patients with myelofibrosis and suboptimal or lack of response to JAK-inhibitor therapy. Over a 12 months period the trial assesses longer term outcomes, particularly the effects on clinical outcomes, such as induction of clinical remissions, quality of life and improvement of anemia. No prospective clinical trial data exist for combinations of JAK- and immune-checkpoint-inhibitors in the planned MF study population and this study will provide new findings that may contribute to advancing the treatment landscape for MF patients with suboptimal responses and limited alternatives.

Recruitment of T-cells to the lung using an ex vivo co-culture paradigm

Precision cut lung slices (PCLS) bridge a gap between in vivo and in vitro studies by maintaining anatomical organization with structural integrity and intercellular signaling pathways. Applications of PCLS have included the modeling of inflammatory lung diseases, metabolism studies, and drug development. In the lungs, immune responses are carried out by a network of T- and B- cells, the latter of which are resident. The limited resident T-cell population of the lung diminishes accurate representations of pathogen response capacity in PCLS. Addressing this, we set out to increase pulmonary T-cell populations ex vivo. We hypothesized that thymus and bone marrow-derived T-cells would work synergistically to populate the lung in co-culture experiments. A murine organotypic lung co-culture model was developed and characterized for tissue health and T-cell recruitment over 3 days ex vivo using adult neurobasal media with 4 mM glucose + 2% B27 supplement. Lung slices were cultured independently, with bone marrow, thymus, or both. Immune colonization of the lung was assessed using immunohistochemistry for CD3+ T-cells and ACK2+ cells. Cells were counted in alveolar and airway spaces after 3 days of culture. Co-culture of lung slices with bone marrow did not increase CD3+ immunoreactive T-cells while thymus co-culture increased CD3+ T-cells by 76% in the alveolar space and by 39% in the airway, relative to lung alone. When lung slices were cultured with bone marrow plus thymus, CD3+ T-cells increased by 206% in the alveolar space and by 251% in the airway, relative to the lung alone. Co-culture with thymus increased ACK2+ cells by 48% in the airway, while lung culture with both thymus and bone marrow, ACK2+ cells increased by 35% in the alveolar space and 85% in the airway, relative to the lung alone. These results suggest that the increased T-cell population corresponding with thymus and bone marrow co-culture could be a result of cell-cell interaction or the secretion of growth factors. Cell secretions or growth factor release could stimulate thymic secretion of T-cells or could stimulate T-cell proliferation in the lung, suggesting that co-culture with thymus and bone marrow can elicit a T-cell response ex vivo. Future studies will center around differentiating what drives T-cell population to be increased in lung co-cultures and what T-cell subsets are being recruited in PCLS co-cultures while analyzing their capacity for response to pathogens ex vivo. Anschutz Foundation Pandemic Preparedness Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of autophagy-related protein 7 enhances anti-tumor immune response and improves efficacy of immune checkpoint blockade in microsatellite instability colorectal cancer

BackgroundThe efficacy of anti-PD-1 therapy is primarily hindered by the limited T-cell immune response rate and immune evasion capacity of tumor cells. Autophagy-related protein 7 (ATG7) plays an important role in autophagy and it has been linked to cancer. However, the role of ATG7 in the effect of immune checkpoint blockade (ICB) treatment on high microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) CRC is still poorly understood.MethodsIn this study, patients from the cancer genome altas (TCGA) COAD/READ cohorts were used to investigate the biological mechanism driving ATG7 development. Several assays were conducted including the colony formation, cell viability, qRT-PCR, western blot, immunofluorescence, flow cytometry, ELISA, immunohistochemistry staining and in vivo tumorigenicity tests.ResultsWe found that ATG7 plays a crucial role in MSI-H CRC. Its knockdown decreased tumor growth and caused an infiltration of CD8+ T effector cells in vivo. ATG7 inhibition restored surface major histocompatibility complex I (MHC-I) levels, causing improved antigen presentation and anti-tumor T cell response by activating reactive oxygen species (ROS)/NF-κB pathway. Meanwhile, ATG7 inhibition also suppressed cholesterol accumulation and augmentation of anti-tumor immune responses. Combining ATG7 inhibition and statins improved the therapeutic benefit of anti-PD-1 in MSI-H CRC. Importantly, CRC patients with high expression of both ATG7 and recombinant 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) experienced worse prognosis compared to those with low ATG7 and HMGCR expression.ConclusionsInhibition of ATG7 leads to upregulation of MHC-I expression, augments immune response and suppresses cholesterol accumulation. These findings demonstrate that ATG7 inhibition has therapeutic potential and application of statins can increase the sensitivity to immune checkpoint inhibitors.

IFN-Type-I Response and Systemic Immunity in Rectal Adenocarcinoma Patients Treated with Conventional or Hypofractionated Neoadjuvant Radiotherapy.

The IFN-type-I pathway is involved in radiotherapy (RT)-mediated immune responses. Large RT fractions have been suggested to potently induce this pathway. Neoadjuvant hypofractionated short-course (scRT) and conventional long-course (lcRT) RT applied for the treatment of locally advanced rectal adenocarcinoma patients provides a unique model to address the immuno-stimulatory properties of RT on a systemic level. We prospectively analyzed the IFNβ plasma levels and lymphocyte counts (LCs) of rectal adenocarcinoma patients before and after treatment with scRT (n = 22) and lcRT (n = 40). Flow cytometry was conducted to assess the effects on lymphocytic subpopulations in a subset of 20 patients. A statistically significant increase in the post-RT IFNβ plasma levels was noted in patients undergoing scRT (p = 0.004). Improved pathological tumor regression was associated with elevated post-RT IFNβ levels (p = 0.003). Although all patients experienced substantial lymphopenia after treatment, the post-RT LC of patients treated with scRT were significantly higher compared to lcRT (p = 0.001). Patients undergoing scRT displayed significantly lower percentages of regulatory CD4+/CD25+ T-cells after therapy (p = 0.02). scRT enables effective stimulation of the IFN-type-I pathway on a systemic level and confers decreased lymphocytic cytotoxicity and limited regulatory T-cell activation compared to lcRT, supporting its increasing role in immuno-RT trials.

Applications and Opportunities for Immune Cell CAR Engineering in Comparative Oncology.

Chimeric antigen receptor (CAR) T-adoptive cell therapy has transformed the treatment of human hematologic malignancies. However, its application for the treatment of solid tumors remains challenging. An exciting avenue for advancing this field lies in the use of pet dogs, in which cancers that recapitulate the biology, immunological features, and clinical course of human malignancies arise spontaneously. Moreover, their large size, outbred genetic background, shared environment with humans, and immunocompetency make dogs ideal for investigating and optimizing CAR therapies before human trials. Here, we will outline how challenges in early clinical trials in patients with canine lymphoma, including issues related to autologous CAR T-cell manufacturing, limited CAR T-cell persistence, and tumor antigen escape, mirrored challenges observed in human CAR T trials. We will then highlight emerging adoptive cell therapy strategies currently under investigation in dogs with hematological and solid cancers, which will provide crucial safety and efficacy data on novel CAR T regimens that can be used to support clinical trials. By drawing from ongoing studies, we will illustrate how canine patients with spontaneous cancer may serve as compelling screening platforms to establish innovative CAR therapy approaches and identify predictive biomarkers of response, with a specific emphasis on solid tumors. With increased funding for canine immunotherapy studies, multi-institutional investigations are poised to generate highly impactful clinical data that should translate into more effective human trials, ultimately benefiting both human and canine cancer patients.

Evaluation of the effectiveness and safety of sequential vaccination with inactivated SARS-CoV-2 vaccine and Ad5-nCoV booster in pediatric liver transplant recipients.

Amidst the COVID-19 pandemic, uncertainty persists among caregivers regarding the vaccination of pediatric liver transplant recipients (PLTRs). This study evaluates the immunogenicity and safety of COVID-19 vaccination in this vulnerable population. A cohort of 30 PLTRs underwent sequential vaccinations with an inactivated SARS-CoV-2 vaccine followed by an Ad5-nCoV booster. We collected and analyzed blood samples pre-vaccination and four weeks post-vaccination to quantify antibody and IGRA (IFN-γ Release Assay) levels. We also documented any adverse reactions occurring within seven days post-vaccination and monitored participants for infections over six months post-vaccination, culminating in a comprehensive statistical analysis. The Ad5-nCoV booster substantially elevated IgG (T1: 18.01, 20%; T2: 66.61, 55%) and nAb (T1: 119.29, 8%; T2: 3799.75, 80%) levels, as well as T-cell responses, in comparison to the initial dose. The first dose was associated with some common adverse reactions, such as injection site pain (13.3%) and fever (16.6%), but a low rate of systemic reactions (16.0%). There was no significant difference in Omicron infection rates or RTPCR conversion times between vaccinated and unvaccinated groups. Notably, following Omicron infection, vaccinated individuals exhibited significantly higher SARS-CoV-2 IgG and nAb titers (average IgG: 231.21 vs. 62.09 S/CO, p = 0.0003; nAb: 5246.11 vs. 2592.07 IU/mL, p = 0.0002). The use of inactivated vaccines followed by an Ad5-nCoV booster in PLTRs is generally safe and elicits a robust humoral response, albeit with limited T-cell responses.

Phase I study targeting newly diagnosed grade 4 astrocytoma with bispecific antibody armed T cells (EGFR BATs) in combination with radiation and temozolomide

PurposeThe purpose of this study was to determine the safety, feasibility, and immunologic responses of treating grade 4 astrocytomas with multiple infusions of anti-CD3 x anti-EGFR bispecific antibody (EGFRBi) armed T cells (EGFR BATs) in combination with radiation and chemotherapy.MethodsThis phase I study used a 3 + 3 dose escalation design to test the safety and feasibility of intravenously infused EGFR BATs in combination with radiation and temozolomide (TMZ) in patients with newly diagnosed grade 4 astrocytomas (AG4). After finding the feasible dose, an expansion cohort with unmethylated O6-methylguanine-DNA methyltransferase (MGMT) tumors received weekly EGFR BATs without TMZ.ResultsThe highest feasible dose was 80 × 109 EGFR BATs without dose-limiting toxicities (DLTs) in seven patients. We could not escalate the dose because of the limited T-cell expansion. There were no DLTs in the additional cohort of three patients with unmethylated MGMT tumors who received eight weekly infusions of EGFR BATs without TMZ. EGFR BATs infusions induced increases in glioma specific anti-tumor cytotoxicity by peripheral blood mononuclear cells (p < 0.03) and NK cell activity (p < 0.002) ex vivo, and increased serum concentrations of IFN-γ (p < 0.03), IL-2 (p < 0.007), and GM-CSF (p < 0.009).ConclusionTargeting AG4 with EGFR BATs at the maximum feasible dose of 80 × 109, with or without TMZ was safe and induced significant anti-tumor-specific immune responses. These results support further clinical trials to examine the efficacy of this adoptive cell therapy in patients with MGMT-unmethylated GBM.ClinicalTrials.gov Identifier: NCT03344250

Synchronous targeted delivery of TGF-β siRNA to stromal and tumor cells elicits robust antitumor immunity against triple-negative breast cancer by comprehensively remodeling the tumor microenvironment

The poor permeability of therapeutic drugs, limited T-cell infiltration, and strong immunosuppressive tumor microenvironment of triple-negative breast cancer (TNBC) acts as a prominent barrier to the delivery of drugs and immunotherapy including programmed cell death ligand-1 antibody (anti-PD-L1). Transforming growth factor (TGF)-β, an important cytokine produced by cancer-associated fibroblasts (CAFs) and tumor cells contributes to the pathological vasculature, dense tumor stroma and strong immunosuppressive tumor microenvironment (TME). Herein, a nanomedicine platform (HA-LSL/siTGF-β) employing dual-targeting, alongside hyaluronidase (HAase) and glutathione (GSH) triggered release was elaborately constructed to efficiently deliver TGF-β small interference RNA (siTGF-β). It was determined that this system was able to improve the efficacy of anti-PD-L1. The siTGF-β nanosystem efficiently silenced TGF-β-related signaling pathways in both activated NIH 3T3 cells and 4T1 cells in vitro and in vivo. This occurred firstly, through CD44-mediated uptake, followed by rapid escape mediated by HAase in endo/lysosomes and release of siRNA mediated by high GSH concentrations in the cytoplasm. By simultaneous silencing of TGF-β in stromal and tumor cells, HA-LSL/siTGF-β dramatically reduced stroma deposition, promoted the penetration of nanomedicines for deep remodeling of the TME, improved oxygenation, T cells infiltration and subsequent anti-PD-L1 deep penetration. The double suppression of TGF-β has been demonstrated to promote blood vessel normalization, inhibit an epithelial-to-mesenchymal transition (EMT), and further modify the immunosuppressive TME, which was supported by an overall increase in the proportion of dendritic cells and cytotoxic T cells. Further, a reduction in the proportion of immunosuppression cells such as regulatory T cells and myeloid-derived suppressor cells was also observed in the TME. Based on the comprehensive remodeling of the tumor microenvironment by this nanosystem, subsequent anti-PD-L1 therapy elicited robust antitumor immunity. Specifically, this system was able to suppress the growth of both primary and distant tumor while preventing tumor metastasis to the lung. Therefore, the combination of the dual-targeted siTGF-β nanosystem, alongside anti-PD-L1 may serve as a novel method to enhance antitumor immunotherapy against stroma-rich TNBC.

Search of a genomic sequence database for potential novel blood group antigens: Investigation into why some amino acid substitutions are not immunogenic.

Polypeptide blood group antigens are typically identified through investigation of the antibodies they induce. Human genome sequence databases are a new tool to identify AA substitutions that potentially create blood group antigens. The Erythrogene genomic sequence database was searched for missense mutations not known to be blood group antigens in the extracellular domains of selected RBC proteins in European populations. Any mutations found with prevalence of 1%-90% and not known to have induced antibodies in transfusion practice were analyzed using protein structural analysis and epitope prediction programs to determine why they apparently lack immunogenicity. Thirteen missense mutations not known to create blood group antigens were identified in the extracellular domains of Kell, BCAM, and RhD proteins, but not in RhCE, Urea Transporter 1 (Kidd), Atypical Chemokine Receptor 1 (Duffy), glycophorin A or glycophorin B. While 11 of the 13 mutations had low prevalence (<1%), a Kell Ser726Pro substitution and a BCAM Val196Ile substitution had predicted phenotype prevalences of 43.2% and 5.7%, respectively. Ser726Pro had multiple properties of a linear B-cell epitope, but possible suboptimal protein location for B-cell receptor binding and limited T-cell epitope possibilities. Val196Ile was not predicted to be in a linear B-cell epitope. Multiple potential new blood group antigens of low prevalence were identified. Whether they are antigenic remains to be determined. Two higher prevalence variants in Kell and BCAM are unlikely antigens, otherwise their antibodies presumably would already have been identified. Possible reasons for their poor immunogenicity were identified.

2-D-gal Targets Terminal Fucosylation to Inhibit T-cell Response in a Mouse Skin Transplant Model.

Organ allograft rejection is mainly driven by T-cell response. Studies have shown that fucosylation plays essential roles in the immune cell development and function. Terminal fucosylation inhibitor, 2-deoxy-D-galactose (2-D-gal), has been reported to suppress immunoresponse of macrophages, but its effects on T-cell-mediated immune response and transplant rejection have not been fully explored. The terminal fucosylation level in T cells was detected through ulex europaeus agglutinin-I staining. The consequences of 2-D-gal on murine T-cell proliferation, activation, cytokine secretion, and cell cycle were investigated in vitro. T-cell receptor signaling cascades were examined. Last, mouse skin transplant model was utilized to evaluate the regulatory effects of 2-D-gal on T-cell response in vivo. The expression of fucosyltransferase1 was upregulated in CD3/CD28-activated T cells along with an elevation of α(1,2)-fucosylation level as seen by ulex europaeus agglutinin-I staining. Furthermore, 2-D-gal suppressed T-cell activation and proliferation, decrease cytokines production, arrest cell cycle, and prevent the activation of T-cell receptor signaling cascades. In vivo experiments showed that 2-D-gal limited T-cell proliferation to prolong skin allograft in mice. This was accompanied by lower level of inflammatory cytokines, and were comparable to those treated with Cyclosporin A. Terminal fucosylation appears to play a role in T-cell activation and proliferation, and its inhibitor, 2-D-gal, can suppress T-cell activation and proliferation both in vitro and in vivo. In a therapeutic context, inhibiting terminal fucosylation may be a potential strategy to prevent allogeneic transplant rejection.

TCRvβ-CART therapy mediates high-precision targeting of malignant T-cell clones.

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of lymphoid malignancies associated with poor prognosis due to ineffective treatment options and high rates of relapse. The success of chimeric antigen receptor T-cell (CART) therapy for certain hematologic malignancies makes it an attractive treatment option for PTCLs. However, shared expression of potential target antigens by both malignant and healthy T cells poses a challenge. Current prospective CART approaches cause a high degree of on-target, off-tumor activity, resulting in fratricide during CART expansion, depletion of healthy Tcells invivo, and immune compromise in the patient. To limit off-tumor targeting, we sought to develop a CART platform specific for a given T-cell receptor vβ (TCRvβ) family thatwould endow CAR-modified T cells with the ability to mediate lysis of the clonal malignant population while preserving the majority of healthy T cells. Here, CAR constructs specific for multiple TCRvβ family members were designed and validated. Our results demonstrate that TCRvβ-family-specific CARTs (TCRvβ-CARTs) recognize and kill TCRvβ-expressing target cells. This includes specific self-depletion of the targeted cell subpopulation in the CART product and lysis of cell lines engineered to express a target TCRvβ family. Furthermore, TCRvβ-CARTs eliminated the dominant malignant TCRvβ clonein 2 patient samples. Finally, in immunodeficient mice, TCRvβ-CARTs eradicated malignant cells in a TCRvβ-dependent manner. Importantly, the nontargeted TCRvβ families were spared in all cases. Thus, TCRvβ-CART therapy provides a potential option for high-precision treatment of PTCL with limited healthy T-cell depletion.

Abstract 1778: B7-H3-CAR T-cell therapy in immune-competent osteosarcoma models: Regnase-1 KO overcomes limited CAR T-cell expansion

Abstract Chimeric antigen receptor (CAR) T-cell therapy offers promise to improve outcomes for pediatric patients with recurrent/refractory osteosarcoma (OS), a ‘poor-prognosis’ cancer. However, early-phase clinical studies have shown limited activity despite potent antitumor activity in preclinical xenograft models, highlighting the need to develop better preclinical models. B7-H3 has emerged as a promising immunotherapeutic target for pediatric solid tumors including OS, and we and other investigators are actively exploring B7-H3-CAR T cell therapies in early-phase clinical studies. Our goal was to develop a murine immunocompetent OS model (F331) for the realistic pre-clinical evaluation of B7-H3-CAR T cells and to explore additional genetic modifications to improve their effector function. We generated murine B7-H3-CAR T cells expressing a 2nd generation B7-H3-CD28.ζ CAR by retroviral transduction. In vitro, B7-H3-CAR T cells recognized B7-H3-positive F331 cells as judged by IFNγ production and cytolytic activity in contrast to B7-H3-negative tumor cells; Control (ctrl) CAR T-cells recognizing an irrelevant antigen (SP6) did not recognize or kill B7-H3-positive tumor cells confirming specificity. Since lung is the primary OS metastasis site, we focused on the intravenous (i.v.) disseminated lung F331 OS metastasis model. In this model, a single i.v. infusion of 5x106 CD8+ B7-H3-CAR T cells demonstrated significant antitumor activity compared to CD8+ ctrl CAR T cells, resulting in a significant survival advantage (median: 70 vs 38 days respectively). Unfortunately, tumors invariably recurred. We found limited B7-H3-CAR T cell expansion and persistence, even though recurring tumors still expressed B7-H3, excluding tumor antigen escape mechanism. We therefore embarked on a comprehensive screen to knock out (KO) known negative regulators of T-cell function, starting with Regnase-1 (Reg-1). Reg-1 is known to have RNase activity and to regulate activation of immune cells. KO of Reg-1 in CD8+ B7-H3-CAR T cells improved their expansion post-infusion in spleen and lungs as judged by flow cytometric analysis without systemic toxicities based on mice body weight assessment. This resulted in a significant improvement in overall survival in comparison to control KO B7-H3-CAR T cells at a cell dose (1x106) at which ctrl-KO CD8+ B7-H3-CAR T cells were ineffective. Reg1-KO ctrl CAR T cells had no therapeutic benefit, excluding nonspecific effects. In summary, we have established an immune-competent OS model to evaluate the effector function of B7-H3-CAR T cells and have demonstrated the advantage given by 2nd genetic modifications to enhance their antitumor activity. We are currently using this model to define mechanisms of immune resistance with the goal of further enhancing OS-redirected CAR T-cell therapy. Citation Format: Adeleye O. Adeshakin, Peipei Zhou, Jean-Yves Métais, Phuong Nguyen, Scott Perry, Heather Sheppard, Xiang Sun, Trevor Cunningham, Hao Shi, Jessica Wagner, Jason T. Yustein, Christopher DeRenzo, Giedre Krenciute, Hongbo Chi, Stephen Gottschalk. B7-H3-CAR T-cell therapy in immune-competent osteosarcoma models: Regnase-1 KO overcomes limited CAR T-cell expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1778.

PEPITEM modulates leukocyte trafficking to reduce obesity-induced inflammation.

Dysregulation of leukocyte trafficking, lipid metabolism, and other metabolic processes are the hallmarks that underpin and drive pathology in obesity. Current clinical management targets alternations in lifestyle choices (e.g. exercise, weight loss) to limit the impact of the disease. Crucially, re-gaining control over the pathogenic cellular and molecular processes may offer an alternative, complementary strategy for obese patients. Here we investigate the impact of the immunopeptide, PEPITEM, on pancreas homeostasis and leukocyte trafficking in mice on high-fed obesogenic diet (HFD). Both prophylactic and therapeutic treatment with PEPITEM alleviated the effects of HFD on the pancreas, reducing pancreatic beta cell size. Moreover, PEPITEM treatment also limited T-cell trafficking (CD4+ T-cells and KLRG1+ CD3+ T-cells) to obese visceral, but not subcutaneous, adipose tissue. Similarly, PEPITEM treatment reduced macrophage numbers within the peritoneal cavity of mice on HFD diet at both 6 and 12 weeks. By contrast, PEPITEM therapy elevated numbers of T and B cells were observed in the secondary lymphoid tissues (e.g. spleen and inguinal lymph node) when compared to the untreated HFD controls. Collectively our data highlights the potential for PEPITEM as a novel therapy to combat the systemic low-grade inflammation experienced in obesity and minimize the impact of obesity on pancreatic homeostasis. Thus, offering an alternative strategy to reduce the risk of developing obesity-related co-morbidities, such as type 2 diabetes mellitus, in individuals at high risk and struggling to control their weight through lifestyle modifications.

Runx3-overexpression cooperates with ex vivo AKT inhibition to generate receptor-engineered T cells with better persistence, tumor-residency, and antitumor ability

BackgroundSolid tumors pose unique roadblocks to treatment with chimeric antigen receptor (CAR) T cells, including limited T-cell persistence, inefficient tumor infiltration, and an immunosuppressive tumor microenvironment. To date, attempts to...

The Immunosuppressive Potential of Cholesterol Sulfate Through T Cell Microvilli Disruption.

Cholesterol (CL) is required for various biomolecular production processes, including those of cell membrane components. Therefore, to meet these needs, CL is converted into various derivatives. Among these derivatives is cholesterol sulfate (CS), a naturally produced CL derivative by the sulfotransferase family 2B1 (SULT2B1), which is widely present in human plasma. CS is involved in cell membrane stabilization, blood clotting, keratinocyte differentiation, and TCR nanocluster deformation. This study shows that treatment of T cells with CS resulted in the decreased surface expression of some surface T-cell proteins and reduced IL-2 release. Furthermore, T cells treated with CS significantly reduced lipid raft contents and membrane CLs. Surprisingly, using the electron microscope, we also observed that CS led to the disruption of T-cell microvilli, releasing small microvilli particles containing TCRs and other microvillar proteins. However, in vivo, T cells with CS showed aberrant migration to high endothelial venules and limited infiltrating splenic T-cell zones compared with the untreated T cells. Additionally, we observed significant alleviation of atopic dermatitis in mice injected with CS in the animal model. Based on these results, we conclude that CS is an immunosuppressive natural lipid that impairs TCR signaling by disrupting microvillar function in T cells, suggesting its usefulness as a therapeutic agent for alleviating T-cell-mediated hypersensitivity and a potential target for treating autoimmune diseases.

A Sampling of Highlights from the Literature

CRISPR-based screening can enhance understanding of T-cell biology (from marius walter via Wikimedia Commons)Using CRISPR-based screening, two groups have identified canonical BRG1/BRM-associated factor (cBAF) complex components as regulators of T-cell differentiation and function that are important for antitumor immunity. Guo et al. find cBAF levels influence the fate of activated CD8+ T cells: high levels promote effector T-cell differentiation and low levels promote memory T-cell differentiation. Belk et al. show cBAF complex components, including Arid1a, promote T-cell acquisition of epigenetic features of exhaustion and limit T-cell persistence. Reducing cBAF activity, either pharmacologically (Guo et al.) or genetically (Belk et al.), improves tumor control in mouse models. The two studies suggest modulating the cBAF complex may improve T cell–based cancer immunotherapy.Guo A, …, Green DR. Nature 2022 June 22;607:135–141.Belk JA, …, Satpathy AT. Cancer Cell 2022 July 11;40:768–86.E7.S. epidermis specific T-cell responses can drive skin irAEs (from NIAID via NIH Flickr)Increasing our understanding of the mechanisms underlying immune-related adverse events (irAEs) is important for optimizing the use of immune checkpoint inhibitors (ICI). Using a mouse model of skin irAEs, Hu et al. find that exposure to Staphylococcus epidermis at the time of anti-CTLA4 treatment induces an inflammatory response resembling skin irAEs in patients treated with ICIs. The response is driven by S. epidermis–specific T cells that produce IL17 and leads to the emergence of S. epidermis–specific memory T cells. Further analysis will determine whether modulating cross-talk between the host immune system and microbiota can alleviate skin irAEs.Hu ZI, …, Belkaid Y. PNAS 2022 June 21;119:e2200348119.Phase II clinical trial results testing chemoimmuno-therapy in PDAC (via Rawpixel)Immunotherapy has so far been ineffective in the treatment of pancreatic ductal adenocarcinoma (PDAC). Padrón et al. report results from a phase II clinical trial evaluating chemotherapy combined with the anti–PD-1 nivolumab or the CD40-agonist sotigalimab, or with both together. The only group of patients among whom the 1-year overall survival rate was significantly greater than a historical control rate of 35% was the group assigned nivolumab/chemotherapy. Analysis of circulating and tumor biomarkers identified distinct immune signatures of improved outcome for patients receiving nivolumab/chemotherapy or sotigalimab/chemotherapy that could be used for patient selection in future clinical trials.Padrón LJ, …, Vonderheide RH. Nat Med 2022 June 3;28:1167–77.B cells can promote or suppress tumors depending on the TME (Carsten Tolkmit via Flickr)The role of B cells in the suppression or promotion of tumors remains unclear. In colorectal cancer (CRC), Wang et al. discover a specific regulatory B-cell subset expressing leucine-tRNA-synthase-2 (LARS2) and TGFβ1 that contributes to CRC progression by altering metabolic processes in the tumor microenvironment (TME). In a second study, Sagiv-Barfi et al. demonstrate that B-cell activation and antigen presentation are key to inducing antitumor T-cell responses, and both B and T cells are needed for efficacy of combination therapy with IL12, anti-OX40, and CpG in breast cancer. The two studies highlight how B-cell responses in the TME not only depend on cancer type, but also on B-cell phenotype and regulatory function.Wang Z, …, Chu Y. Immunity 2022 June 14;55:1067–81.E8.Sagiv-Barfi I, …, Levy R. Sci Immunol 2022 May 27;7:eabn5859.Profiling the diversity of tumor antigens (Andy Mitchell via Wikimedia Commons)A greater understanding of in vivo cancer-specific patterns of peptide presentation by MHC class I complexes is needed to improve the development of antigen-specific immunotherapies. Jaeger et al. develop and use a new genetically engineered mouse model to profile the cancer immunopeptidome from autochthonous pancreatic ductal adenocarcinoma and lung adenocarcinomas. The authors find little correlation between mRNA abundance and peptide detection, suggesting significant posttranslational regulation of tumor-specific peptide presentation. Dendritic cell vaccines loaded with lung adenocarcinoma peptides uniquely identified by the assay induce antigen-specific T-cell responses ex vivo, highlighting the model’s ability to capture the cancer immunopeptidome.Jaeger AM, …, Jacks T. Nature 2022 June 15;607:149–55.A new map of human immune cell development (tzunghaor via openclipart)Understanding how and where the immune system develops can provide a better understanding of cancer origin and facilitate the development of novel immunotherapies. Through single-cell profiling and spatial transcriptomics of nine prenatal tissues, Suo et al. have generated a comprehensive atlas of the developing human immune system. Highlights of the findings include the discovery that human immune development occurs across multiple tissue sites, including sites not considered primary hematopoietic organs; the identification of a population of prenatal B1 cells; and the demonstration that unconventional T cells are selected on thymocytes. This road map provides a resource for advancing immune-based science and medicine.Suo C, …, Teichmann SA. Science 2022 May 12;376:eabo0510.

Engineering naturally occurring CD7− T cells for the immunotherapy of hematological malignancies

AbstractChimeric antigen receptor (CAR) T-cell therapy targeting T-cell acute lymphoblastic leukemia (T-ALL) faces limitations such as antigen selection and limited T-cell persistence. CD7 is an attractive antigen for targeting T-ALL, but overlapping expression on healthy T cells leads to fratricide of CD7-CAR T cells, requiring additional genetic modification. We took advantage of naturally occurring CD7− T cells to generate CD7-CAR (CD7-CARCD7−) T cells. CD7-CARCD7− T cells exhibited a predominantly CD4+ memory phenotype and had significant antitumor activity upon chronic antigen exposure in vitro and in xenograft mouse models. Based on these encouraging results, we next explored the utility of CD7− T cells for the immunotherapy of CD19+ hematological malignancies. Direct comparison of nonselected (bulk) CD19-CAR and CD19-CARCD7− T cells revealed that CD19-CARCD7− T cells had enhanced antitumor activity compared with their bulk counterparts in vitro and in vivo. Lastly, to gain insight into the behavior of CD19-CAR T cells with low levels of CD7 gene expression (CD7lo) in humans, we mined single-cell gene and T-cell receptor (TCR) expression data sets from our institutional CD19-CAR T-cell clinical study. CD19-CARCD7lo T cells were present in the initial CD19-CAR T-cell product and could be detected postinfusion. Intriguingly, the only functional CD4+ CD19-CAR T-cell cluster observed postinfusion exhibited CD7lo expression. Additionally, samples from patients responsive to therapy had a higher proportion of CD7lo T cells than nonresponders (NCT03573700). Thus, CARCD7− T cells have favorable biological characteristics and may present a promising T-cell subset for adoptive cell therapy of T-ALL and other hematological malignancies.

Advances in Our Understanding of the Interaction of Drugs with T-cells: Implications for the Discovery of Biomarkers in Severe Cutaneous Drug Reactions.

Drugs can activate different cells of the immune system and initiate an immune response that can lead to life-threatening diseases collectively known as severe cutaneous adverse reactions (SCARs). Antibiotics, anticonvulsants, and antiretrovirals are involved in the development of SCARs by the activation of αβ naïve T-cells. However, other subsets of lymphocytes known as nonconventional T-cells with a limited T-cell receptor repertoire and innate and adaptative functions also recognize drugs and drug-like molecules, but their role in the pathogenesis of SCARs has only just begun to be explored. Despite 30 years of advances in our understanding of the mechanisms in which drugs interact with T-cells and the pathways for tissue injury seen during T-cell activation, at present, the development of useful clinical biomarkers for SCARs or predictive preclinical in vitro assays that could identify immunogenic moieties during drug discovery is an unmet goal. Therefore, the present review focuses on (i) advances in the understanding of the pathogenesis of SCARs reactions, (ii) a description of the interaction of drugs with conventional and nonconventional T-cells, and (iii) the current state of soluble blood circulating biomarker candidates for SCARs.