B And T Lymphocyte Attenuator Research Articles

THEMIS promotes T cell development and maintenance by rising the signaling threshold of the inhibitory receptor BTLA

The current paradigm about the function of T cell immune checkpoints is that these receptors switch on inhibitory signals upon cognate ligand interaction. We here revisit this simple switch model and provide evidence that the T cell lineage protein THEMIS enhances the signaling threshold at which the immune checkpoint BTLA (B- and T-lymphocyte attenuator) represses T cell responses. THEMIS is recruited to the cytoplasmic domain of BTLA and blocks its signaling capacity by promoting/stabilizing the oxidation of the catalytic cysteine of the tyrosine phosphatase SHP-1. In contrast, THEMIS has no detectable effect on signaling pathways regulated by PD-1 (Programmed cell death protein 1), which depend mainly on the tyrosine phosphatase SHP-2. BTLA inhibitory signaling is tuned according to the THEMIS expression level, making CD8+ T cells more resistant to BTLA-mediated inhibition than CD4+ T cells. In the absence of THEMIS, the signaling capacity of BTLA is exacerbated, which results in the attenuation of signals driven by the T cell antigen receptor and by receptors for IL-2 and IL-15, consequently hampering thymocyte positive selection and peripheral CD8+ T cell maintenance. By characterizing the pivotal role of THEMIS in restricting the transmission of BTLA signals, our study suggests that immune checkpoint operability is conditioned by intracellular signal attenuators.

153 Spatial Transcriptomic Profiling of T Cell Exhaustion Markers in Atypical Teratoid Rhabdoid Tumor Subtypes

INTRODUCTION: Atypical teratoid/rhabdoid tumor (ATRT) is one of the most common brain tumors in pediatric populations. Despite advances in treatment, overall survival remains poor and novel effective therapies are needed. METHODS: Tumor purities of 49 atypical ATRT tumors, encompassing 3 epigenetic subtypes (MYC, SHH, TYR) were estimated using known mRNA expression signatures of stromal and immune cells. Using the purity estimates and bulk tumor expression values, non-negative linear regression was implemented to estimate the average expression of each gene in the stromal and tumor compartments for each subtype. RESULTS: Compared to other subtypes, SHH-ATRTs expressed relatively higher levels of T-cell immunoglobulin domain and mucin domain protein 3 (TIM-3) and its ligand GAL9. MYC-ATRTs expressed higher levels of numerous T cell inhibitory receptors including lymphocyte activation gene 3 protein (LAG-3), band T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte antigen-4 (CTLA-4), and programmed cell death protein 1 (PD-1). CONCLUSIONS: The predicted stromal and tumor compartment deconvolution yielded previously unrecognized expression patterns of T cell exhaustion markers which should be evaluated further to develop effective targeted therapies for ATRT.

Abstract 120: A closer Look at colorectal cancer risk: Insights from the complex relationship of HIV, helminths, and immune responses

Abstract The advent of antiretroviral therapy has markedly increased the survival rates of individuals with human immunodeficiency virus (HIV). However, this demographic, particularly in low-middle-income countries (LMICs) where HIV-helminth co-infections are prevalent, remains susceptible to infections. The intricate interplay between helminth infections and HIV susceptibility, often inducing immunological phenotypes that heighten vulnerability, remains poorly understood. Studies have shown the association between HIV-helminth co-infections and elevated susceptibility to colorectal cancer (CRC), introducing an additional layer of health concerns for this already vulnerable population. This retrospective observational study delves into the profound effects of HIV-helminth co-infections on immune responses, with a specific focus on type 2 T helper (Th2) cell immunomodulation and susceptibility to CRC. Utilizing samples from a master project (n=414), 80 systematically randomized individuals were categorized into uninfected control, HIV-infected only, helminth-infected only, and HIV-helminth co-infected groups. Parasite detection employed the Kato-Katz technique, and serum testing was conducted for helminth-specific antibodies. Serological HIV testing, viral load categorization, white blood cell counts, and differential analyses were performed. Human ProcartaPlex Panel 1 was used for cytokine profiling of immune stimulatory and inhibitory soluble proteins. A significant reduction in B and T lymphocyte attenuator (BTLA) (p=0.049) in helminth infections compared to the control. The association of BTLA with its ligand, herpesvirus entry mediator (HVEM), establishes a direct link between the CD28 and tumor necrosis factor receptor (TNFR) families, triggering wide-ranging and robust immune responses. Subsequently, a notable reduction in CD28 was observed in HIV-infected subjects (p=0.003), followed by helminth-infected and HIV-helminth co-infected individuals (p=0.03) compared to the control. Furthermore, programmed death ligand-2 (PD-L2) expression, a major contributor to alternatively activated macrophage polarization during helminth infections, was significantly increased in the HIV-helminth co-infected group (p = 0.05), favoring a shift towards Th2 immune responses. These observed alterations underscore the complexity of the immunological interplay in co-infected individuals, emphasizing an additional risk factor for colorectal cancer development. This necessitates a comprehensive understanding, providing valuable insights for the development of therapeutic strategies in LMICs and similar settings, addressing the heightened risk of CRC within this population. Citation Format: Botle P. Damane, Thanyani V. Mulaudzi, Sayed S. Kader, Ropo E. Ogunsakin, Pragalathan Naidoo, Zodwa Dlamini, Zilungile L. Mkhize-Kwitshana. A closer Look at colorectal cancer risk: Insights from the complex relationship of HIV, helminths, and immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 120.

Viewing the immune checkpoint VISTA: landscape and outcomes across cancers

Optimizing immune checkpoint inhibitor (ICI) therapy may require identification of co-targetable checkpoint pathways via immune profiling. Herein, we analyzed the transcriptomic expression and clinical correlates of V-domain immunoglobulin suppressor of T-cell activation (VISTA), a promising targetable checkpoint. RNA sequencing was carried out on 514 tissues reflecting diverse advanced/metastatic cancers. Expression of eight immune checkpoint markers [lymphocyte-activation gene 3 (LAG-3), tumor necrosis factor receptor superfamily 14 (TNFRSF14), programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), B- and T-lymphocyte attenuator (BTLA), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), cytotoxic T-lymphocyte antigen 4 (CTLA-4)], in addition to VISTA, was analyzed, along with clinical outcomes. High VISTA RNA expression was observed in 32% of tumors (66/514) and was the most common highly expressed checkpoint among the nine assessed. High VISTA expression was independently correlated with high BTLA, TIM-3, and TNFRSF14, and with a diagnosis of pancreatic, small intestine, and stomach cancer. VISTA transcript levels did not correlate with overall survival (OS) from metastatic/advanced disease in the pan-cancer cohort or with immunotherapy outcome (progression-free survival and OS from the start of ICI) in 217 ICI-treated patients. However, in ICI-treated pancreatic cancer patients (n = 16), median OS was significantly shorter (from immunotherapy initiation) for the high- versus not-high-VISTA groups (0.28 versus 1.21 years) (P= 0.047); in contrast, VISTA levels were not correlated with OS in 36 pancreatic cancer patients who did not receive ICI. High VISTA expression correlates with high BTLA, TIM-3, and TNFRSF14 checkpoint-related molecules and with poorer post-immunotherapy survival in pancreatic cancer, consistent with prior literature indicating that VISTA is prominently expressed on CD68+ macrophages in pancreatic cancers and requiring validation in larger prospective studies. Immunomic analysis may be important for individualized precision immunotherapy.

The role of the BTLA-HVEM complex in the pathogenesis of breast cancer.

Breast cancer (BC) is widely recognized as a prevalent contributor to cancer mortality and ranks as the second most prevalent form of cancer among women across the globe. Hence, the development of innovative therapeutic strategies is imperative to effectively manage BC. The B- and T-lymphocyte attenuator (BTLA)-Herpesvirus entry mediator (HVEM) complex has garnered significant scientific interest as a crucial regulator in various immune contexts. The interaction between BTLA-HVEM ligand on the surface of T cells results in reduced cellular activation, cytokine synthesis, and proliferation. The BTLA-HVEM complex has been investigated in various cancers, yet its specific mechanisms in BC remain indeterminate. In this study, we aim to examine the function of BTLA-HVEM and provide a comprehensive overview of the existing evidence in relation to BC. The obstruction or augmentation of these pathways may potentially enhance the efficacy of BC treatment.

BTLA contributes to acute-on-chronic liver failure infection and mortality through CD4+ T-cell exhaustion

B- and T-lymphocyte attenuator (BTLA) levels are increased in patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF). This condition is characterized by susceptibility to infection and T-cell immune exhaustion. However, whether BTLA can induce T-cell immune exhaustion and increase the risk of infection remains unclear. Here, we report that BTLA levels are significantly increased in the circulating and intrahepatic CD4+ T cells from patients with HBV-ACLF, and are positively correlated with disease severity, prognosis, and infection complications. BTLA levels were upregulated by the IL-6 and TNF signaling pathways. Antibody crosslinking of BTLA activated the PI3K-Akt pathway to inhibit the activation, proliferation, and cytokine production of CD4+ T cells while promoting their apoptosis. In contrast, BTLA knockdown promoted their activation and proliferation. BTLA-/- ACLF mice exhibited increased cytokine secretion, and reduced mortality and bacterial burden. The administration of a neutralizing anti-BTLA antibody reduced Klebsiella pneumoniae load and mortality in mice with ACLF. These data may help elucidate HBV-ACLF pathogenesis and aid in identifying novel drug targets.

The Role of the CD28 Family Receptors in T-Cell Immunomodulation.

The CD28 family receptors include the CD28, ICOS (inducible co-stimulator), CTLA-4 (cytotoxic T-lymphocyte antigen-4), PD-1 (programmed cell death protein 1), and BTLA (B- and T-lymphocyte attenuator) molecules. They characterize a group of molecules similar to immunoglobulins that control the immune response through modulating T-cell activity. Among the family members, CD28 and ICOS act as enhancers of T-cell activity, while three others-BTLA, CTLA-4, and PD-1-function as suppressors. The receptors of the CD28 family interact with the B7 family of ligands. The cooperation between these molecules is essential for controlling the course of the adaptive response, but it also significantly impacts the development of immune-related diseases. This review introduces the reader to the molecular basis of the functioning of CD28 family receptors and their impact on T-cell activity.

Clinical significance of BTLA gene expression and rs1982809 polymorphism in pan-cancer

The association between the B and T lymphocyte attenuator (BTLA) gene rs1982809 polymorphism and cancer susceptibility has been reported, but these findings are inconsistent. In addition to clarifying the relationship between the rs1982809 polymorphism and cancer susceptibility, the current study also explored the clinical significance of BTLA gene expression. The GSCA tool and Stata software were used to explore the association between BTLA gene expression and tumor stage, immune infiltration, survival prognosis, and drug sensitivity for pan-cancer, and the association of BTLA gene rs1982809 polymorphism with cancer susceptibility, respectively. BTLA gene expression was associated not only with the pathologic stages of thyroid carcinoma, skin cutaneous melanoma, and kidney renal clear cell carcinoma, but also with immune infiltration in 33 types of cancers. In addition, BTLA gene expression was linked to survival prognosis in 8 types of cancers and the sensitivity of 255 drugs such as 5-Fluorouracil, docetaxel, and methotrexate. A meta-analysis of 7 relevant studies with 4002 cancer patients and 5278 healthy controls showed that the BTLA gene rs1982809 polymorphism was unrelated to cancer susceptibility under all genetic models. However, a country-based stratification analysis suggested that the rs1982809 polymorphism could reduce cancer susceptibility in Polish and Tunisian populations. In conclusion, BTLA is expected to serve as a prognostic marker and therapeutic target for certain cancers, and the rs1982809 polymorphism is expected to serve as a cancer susceptibility marker in Polish and Tunisian populations.

Modulation of the Btla-HVEM Axis to Enhance CAR T Cell Immunotherapy Against Cancer

Introduction: The efficacy of adoptive T cell immunotherapies against cancer, such as chimeric antigen receptor (CAR) T cells, is severely blunted by the immunosuppressive tumor microenvironment (TME). We sought to investigate the role of the TME in cancer resistance to effector T cells in order to define actionable targets to enhance CAR T cell immunotherapies. We initially used Hodgkin lymphoma (HL) as an ideal tumor model since it is characterized by a TME that is profoundly infiltrated by immunosuppressive cells, and then we expanded our findings to multiple cancer models. We first sought to identify the dominant interactions of immunosuppressive cellular compartments and effector T cells by analyzing single-cell RNA sequencing data on a total of 26 (4 exploratory + 22 validation) HL patient tumor biopsies. Using the CellPhoneDB algorithm, we inferred that the ligand B- and T-lymphocyte attenuator (BTLA) on effector T cells and the receptor Herpesvirus entry mediator (HVEM, TNFRSF14) on immunosuppressive cells (e.g., regulatory T cells, monocytes) strongly interact in the TME and promote T cell dysfunction ( Fig 1a). Akin to the canonical checkpoint PD1, BTLA recruits two potent tyrosine phosphatases, SHP-1 and SHP-2, to disable early T cell activation. Thus, we rationalized that BTLA expression on T cells might reduce their anti-tumor function. We hypothesized that deleting BTLA in CAR T cells would abolish BTLA-HVEM trans interactions at the immunological synapse and unleash the cytotoxic potential of CAR T cells. Methods and Results: We first generated BTLA KO anti-CD30 CAR T (CART30) cells against HL. To test their function in vivo, 15x10 6 HDLM-2 (CD30+HVEM+ HL) cells were subcutaneously implanted into NSG mice on day 0, and on day 62, 3x10 5 CAR30+ T cells were infused intravenously. Our results demonstrated that BTLA KO significantly enhances the function of anti-CD30 CAR T cells in HVEM+ HL ( Fig 1b), as assessed via tumor size (caliper) and CART30 expansion in the peripheral blood (flow cytometry). Additionally, we generated BTLA KO 4-1BBζ CART19 cells, which showed greater in vivo anti-tumor function in a subcutaneous tumor model of DLBCL (CD19+ HVEM+ OCI-Ly18). Serum collected from OCI-Ly18-bearing NSG mice infused with BTLA KO CART19 was enriched in effector cytokines (e.g., TNF, IFNγ, IL-2) as measured by Luminex. We then extended these findings into HVEM+ solid tumor models. BTLA KO improved tumor control in vitro in short-term killing experiments (Incucyte SX5) for both CAR and TCR T cells, respectively directed against prostate cancer (HER2+ PC-3) and melanoma (GP-100+ DM-6). Importantly, we demonstrated that BTLA KO in primary BALB/c-derived murine CAR T cells enhances tumor control in A20 murine lymphoma. In this model, BTLA KO tumor-infiltrating CAR T cells showed substantially reduced exhaustion in the TME relative to wild-type CAR T cells. Mechanistically, we showed that targeted mutations in BTLA intracellular tyrosine motifs effectively reduce the recruitment of SHP-1/2 and preserve the docking domain of the pro-stimulatory Grb2, thus maintaining high NFAT signaling and increased persistence in vivo. These effects were dependent on the expression of HVEM on tumor cells. Finally, we found that high BTLA RNA expression in tisagenlecleucel infusion products correlates with poor response to treatment in patients with DLBCL and FL (NCT02030834). Conclusion: Our results reveal a critical role of the BTLA-HVEM axis in inhibiting CAR T cell function, and demonstrate that CRISPR-Cas9 deletion of BTLA leads to enhanced anti-tumor efficacy in multiple models of cancer. The key mechanism is the reduction of SHP-1/2 recruitment and the consequent increase in CAR T cell activation. The results of this study will be translated into a first in human clinical trial of BTLA-deficient CAR T cells for relapsed or refractory cancer.

Vactosertib, a Novel TGFb Type I Receptor Kinase Inhibitor, Improves T-Cell Fitness:a Single-Arm, Phase 1b Trial in Relapsed/Refractory Multiple Myeloma

The prognosis for Multiple Myeloma (MM) patients (pts) has improved substantially over the past two decades with the success of immunomodulatory drugs (IMiDs), monoclonal antibodies, chimeric antigen receptor (CAR) T-cells and bispecific T-cell engagers (BiTEs) that broke the circulus vitiosus of tumor-induced immunosuppression to reengage the immune system. Functional blockade of the transforming growth factor-beta (TGF) signaling pathway improves the efficacy of cytotoxic and immune therapies, therefore we investigator the impact of TGF-inhibition in an steroid free regimn. Methods: We conducted a phase 1b study to determine the safety, efficacy, and maximal tolerated dose (200 mg po bid) of the potent, orally-available TGF type I receptor kinase inhibitor vactosertib in relapsed and/or refractory multiple myeloma (RRMM) pts who had received -2 lines of chemoimmunotherapy in combination with standard dosage of Pomalidomide without any corticosteroids. Results: Twenty one pts were enrolled in the phase 1b dose escalation cohort and received vactosertib plus pomalidomide as indicated (Fig. 1A). Pts' characteristics are described in Table 1The summary of grade 1-4 adverse events is shown (Table 2). A single grade 4 elevated bilirubin (5%) and a single grade 4 elevated lipase (5%) were observed as well as four grade 4 events of decreased neutrophils (19%). Four pts demonstrated a PR, two had a MR, nine had SD and four had disease progression (Fig. 1B). The median time to response for all pts and the responders eight weeks and median duration of response was ~12 weeks. The best overall response for individual pts to treatment is shown (Fig. 1C). Clinical activity was observed across all four dose levels (Table 3). To probe the tumor intrinsic anti-myeloma activity of vactosertib, we first determined the relative effect of vactosertib compared to the IMiDs pomalidomide and lenalidomide (Fig. 2A, B). The effect of vactosertib was greater than that observed with either lenalidomide or pomalidomide. MMCLs and patient CD138 + cells were then treated with vactosertib in combination with pomalidomide, and importantly, vactosertib with pomalidomide synergistically reduced myeloma viability (Fig. 3A). We next probed the systemic, tumor extrinsic effects of vactosertib by analyzing cytokines immune and tumor cells present within patient BM samples. (Fig. 4A). (Fig. 4B). Therefore, we explored the immunophenotype of CD8 + T-cells isolated from patient BM prior to and at the EOT. The relative percentage of T-cells that expressed the immunosuppressive markers programmed cell death-1 (PD-1), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), B and T lymphocyte attenuator (BTLA), and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) was reduced following treatment relative to their expression prior to treatment (Fig. 4C). Results indicated that vactosertib and pomalidomide treatment in vivo reduced PD-1, TIM3, BTLA and CTLA-4 expression on patient CD8 + T-cells. Next, CD8 + T-cells were isolated from patient peripheral blood and cultured in the presence of TGF-1, vactosertib, pomalidomide, or vactosertib plus pomalidomide (Fig. 5B). Treatment of patient CD8 + T-cells with vactosertib decreased PD-1 expression up to 40% and decreased TIM3 expression up to 65%. Pomalidomide alone did not significantly reduce PD-1, TIM3, BTLA, or LAG-3 on patient CD8 + T-cells. Vactosertib and pomalidomide also reduced TIM3 levels on CD8 + T-cells from most pts. (Fig. 5C, D). (Fig. 5 G, H). Results indicated that vactosertib or pomalidomide did not increase CD138 + cell death (Fig. 5I). Conclusion: Vactosertib combined with pomalidomide was well-tolerated at all doses, had a manageable adverse event profile and induced durable responses with 80% progression-free survival (PFS-6) at 6 months, Vactosertib reduced TGF- in patient bone marrow and suppressed PD-1 expression on CD8 + T-cells and lead to reduction of PD-L1/PD-L2 expression on CD138 + cells and enhanced autologous T-cell cytotoxicity. Taken together, our results support the safety and efficacy of vactosertib to treat RRMM and revealed that vactosertib modulates the T-cell immunophenotype and reinvigorates T-cell fitness.

Anti-Btla Antibody Tifcemalimab As a Single Agent or in Combination with Toripalimab in Relapsed/Refractory Lymphomas

Background: The B- and T-lymphocyte attenuator (BTLA) is an inhibitory receptor expressed on B, T and NK cells. Using Peripheral Blood Mononuclear Cell (PBMC) derived from cancer patients, co-blockade of the BTLA and PD-1 pathways improved antigen specific T cell response compared to either blockade alone. Tifcemalimab (JS004 or TAB004) is a humanized IgG4 monoclonal antibody with a hinge mutation (S228P) that binds BTLA and blocks its interaction with its ligand Herpesvirus Entry Mediator (HVEM). Tifcemalimab is the first BTLA antibody to enter the clinical development stage. Here, we report the safety and efficacy of tifcemalimab as a single agent or in combination with toripalimab (anti-PD-1) in patients with relapsed/refractory (R/R) lymphoma after long term follow-up. Methods: Eligible patients with R/R lymphoma were enrolled in this open-label, multicenter phase 1 study (NCT04477772). Tifcemalimab was administered as a monotherapy at escalating doses of 1, 3 and 10 mg/kg intravenously Q3W, followed by 3 mg/kg and 200 mg monotherapy dose expansion until disease progression or intolerable toxicity. During combination dose escalation, patients received ascending doses of tifcemalimab (100mg and 200mg) plus toripalimab (240mg). Dose-limiting toxicity (DLT) was evaluated by a safety monitoring committee. Study objectives included safety, pharmacokinetics, and efficacy. Results: By the cutoff date of July 19, 2023, a total of 71 patients were enrolled, including 25 in the monotherapy and 46 in the combination cohorts. The median follow-up time was 57 weeks. The median age was 38 (range 19-70) years with 50 (70%) male patients. The median prior line of therapy was 4 with 54 (76%) progressed upon prior anti-PD-1/L1 therapy. No DLT was observed in either monotherapy or combination dose escalation. Recommended phase 2 dose (RP2D) was determined to be 200 mg tifcemalimab as monotherapy or in combination with toripalimab. Sixty-seven (94%) patients experienced treatment emergent adverse events (TEAEs), 22 (31%) of whom experienced grade 3 or above TEAEs. The most common TEAEs were COVID-19 infection (30%), anemia (30%) and Infusion related reaction (27%). Four treatment-related adverse events led to the discontinuation of the study drug. Among 25 patients receiving monotherapy, 1 PR (follicular lymphoma) and 7 SD were observed with median progression-free survival (PFS) 2.1 months. Among 46 patients receiving combination therapy, 1 CR,16 PR (ORR 37.0%) and 20 SD (DCR 80.4%) were observed and estimated median PFS was 14.7 months. Thirty-four patients with Hodgkin's Lymphoma received RP2D of combination therapy and all of them had received previous chemotherapy and anti-PD-1 or anti-PD-L1 antibody, and 12 PR (ORR 35.3%) and 17 SD (DCR 85.3%) were observed with estimated median progression-free survival was 16.2 months. Conclusions: Tifcemalimab alone or in combination with toripalimab were well tolerated in all doses evaluated. The combination regimen showed favorable safety and durable efficacy in lymphoma patients with heavily pretreatment, providing evidence for future investigation.

Herpesvirus Entry Mediator As a Novel Therapeutic Target in Multiple Myeloma

The use of immune checkpoint inhibitors has markedly improved the survival outcomes of many patients with solid tumors. However, in multiple myeloma (MM), the use of such inhibitors has failed to achieve survival benefit. We previously showed that oncolytic herpes simplex virus 1 (oHSV-1) can effectively infect MM cells and induce apoptosis through the receptors for oHSV-1, NECTIN-1 and HVEM (herpes virus entry mediator). HVEM, a member of the TNF receptor superfamily (TNFRSF14), is an immune checkpoint present on T cells, B cells, NK cells and macrophages. Depending on HVEM's interaction with its major inhibitory ligand, B- and T-lymphocyte attenuator (BTLA) or its major activating ligand, tumor necrosis factor superfamily member 14 (TNFSF14, LIGHT), it is pivotal in downmodulating or stimulating the immune cells. Accordingly, targeting HVEM to block its interaction with BTLA is an area of therapeutic interest. Flow cytometry analysis showed that HVEM was consistently expressed on the surface of all tested MM cell lines (n=9). Mass cytometry analysis of bone marrow (BM) from multi-relapsing MM patients revealed that HVEM was also expressed in different immune populations and that its expression was increased in T lymphocytes upon relapse from the anti-CD38 antibody daratumumab. Loss of function experiments by silencing RNA (siRNA) showed that, after 48hrs, HVEM knockdown significantly reduced the proliferation of the MM cell lines MM.1S (48% decrease vs control si-RNA; P˂0.0001) and H929 (35% decrease vs control si-RNA; P˂0.0001), as indicated by the MTS assay. To this end, we developed several anti-HVEM monoclonal antibodies directed against the extracellular domain of human HVEM and used surface plasmon resonance experiments and CyTOF to validate their binding. Among all antibodies tested, HVEM5 had one of the highest binding affinities, with minimal cross-reactivity with other non-cancer tissues as assessed on tissue array by imaging mass cytometry. An ELISA-based BTLA:HVEM inhibitor screening assay showed that HVEM5, but not a non-binding control (HVEM4), resulted in a 100% blockade of the interaction of HVEM with recombinant BTLA protein. Moreover, HVEM5 elicited potent antibody-dependent cellular cytotoxicity by primary NK cells against MM cell lines (MM.1S and H929), in contrast to a control human IgG (p=0.0022 and 0.0061, respectively), and was comparable to that of daratumumab (Fig.1A). Flow cytometry data showed that ex vivo HVEM5 treatment of tumor-associated macrophages (TAMs) polarized them from a M2/repair-type to a M1/kill-type, as indicated by their decreased CD163 (69% increase vs. control IgG; P=0.0011; n=4) and increased CD80 (30% increase vs control IgG; P=0.0436; n=4) expression. Furthermore, HVEM5 (10 µg/mL) treatment of peripheral blood mononuclear cells almost doubled CD8+ T lymphocytes proliferation ex vivo. To assess whether HVEM5 exerts an agonistic effect and activates HVEM-NF-κB downstream activation signaling, we used a luciferase reporter assay in a Jurkat cell line overexpressing HVEM. HVEM5 binding induced a 1.5-fold increase in NF-κB promoter activity (1.50±0.07) compared to a control human IgG (1.02±0.02) or the non-binding antibody HVEM4 (1.09±0.07) (p=0.0005). This increase was equivalent to that observed using the HVEM immune activator, LIGHT (Fig.1B). In line with these results, immunofluorescence staining of primary monocytes showed that HVEM5 treatment increased the nuclear translocation of the NF-kB p65 transcriptional subunit, which was greater than that of LIGHT-treated cells (Fig.1B). Furthermore, HVEM5 generated with a mouse-reactive Fc fragment induced a significantly longer overall survival (median 22 days) and tumor reduction in an NCr nude mouse model when injected intravenously twice a week (3 mg/kg, Mon-Fri), whereas the median survival was 15 days in the IgG-treated control animals (p=0.01) (Fig.1A; right trace). Further in vivo studies in HVEM/BTLA double humanized immune competent animal models and in HVEM knock-out mouse models are ongoing, and the results will be presented at the meeting. To our knowledge, the present work is the first to report that MM cells ubiquitously express HVEM, which could be exploited as a novel therapeutic opportunity to not only target it as an immune checkpoint, but also as a direct MM-targeting strategy.

Impact of Genetic Mutations on Survival Following Receipt of Commercial CART19 for Patients Diagnosed with Aggressive B Cell Lymphoma

Introduction Anti-CD19 chimeric antigen receptor-modified T cell therapies (CART19) have revolutionized treatment of patients (pts) with relapsed/refractory (R/R) diffuse large B cell lymphoma (DLBCL) and high grade B cell lymphoma (HGBL). However, progression of disease following receipt of CART19 is common with only approximately one-third of pts achieving long-term remission in the standard-of-care setting (Transplant Cell Ther. 2022 Sep;28(9):581.e1-581.e8, Blood (2022) 140 (Supplement 1): 1584-1587). While loss of CD19 antigen expression antigen is one mechanism of CART19 failure, other tumor-intrinsic factors which may result in CART19 failure remain poorly defined. Clinical laboratory mutation analysis (CLMA) performed on tumor specimens can identify recurring genetic mutations which may predict for clinical outcomes; however, the predictive value of CMLA has not been well-established for DLBCL/HGBL pts treated with CART19. Here, we analyze the impact of genetic mutations as detected by CLMA, along with other clinicopathologic characteristics, on survival outcomes of DLBCL/HGBL pts following receipt of CART19. Methods We collected data from 46 pts with DLBCL or HGBL treated at the University of Pennsylvania between 2018 and 2021. Patient tumors were sequenced with one of three sequencing panels comprised of 41-116 genes prior to receiving commercial CART19. Cox proportional hazards regression was used to assess the impact of clinicopathologic characteristics on disease progression and death, with characteristics demonstrating p <0.10 on univariate analysis included in multivariate analysis. Estimated progression free survival (PFS) and overall survival (OS) from the time of CART19 infusion were calculated using Kaplan-Meier estimates. Results Baseline clinicopathologic characteristics are listed in Table 1. A total of 30 of 46 pts had at least one tumor mutation detected prior to receiving CART19, with 22 genes mutated in at least two pts (Figure 1). The most frequent mutations were TP53 (15 of 46, 32.7%), BCL2 (3 of 12, 25%), MYC (4 of 20, 20%), SOCS1 (5 of 38, 13.2%), CD79B (5 of 38, 13.2%), CREBBP (5 of 38, 13.2%), EZH2 (5 of 46, 10.9%), STAT3 (4 of 38, 10.5%), TNFRSF14 (4 of 38, 10.5%) and DNMT3A (2 of 20, 10%). The median length of follow-up from the time of CART19 infusion was 29.6 months. For all pts, the rates of 2 year (y) PFS and OS were 21% (95% confidence interval [CI] 10-34%) and 56% (95% CI 40-69%), respectively. Univariate analysis of mutations present in ≥10% of cases revealed that TNFRSF14 mutations predicted for lower risk of disease progression at 2y (HR 0.16, 95% CI 0.02-1.186, p = 0.07). No other mutation was associated with either disease progression or death at 2y. Univariate analysis of baseline clinicopathologic characteristics also identified International Prognostic Index (IPI) score ≥3 at the time of relapse preceding CART19, HGBL classification, no history of indolent lymphoma (IL), MYC rearrangement (-R) and double hit lymphoma (DHL) as predictive of disease progression at 2y; however, none of these characteristics remained predictive on multivariate analysis. Additionally, IPI score ≥3 at the time of relapse preceding CART19, HGBL classification, no history of IL, MYC-R and DHL were also predictive of death at 2y on univariate analysis; however, none of these characteristics remained predictive on multivariate analysis. Conclusions Tumor characteristics, including recurring genetic mutations as detected by CLMA, may not independently predict for survival outcomes for DLBCL/HGBL pts following receipt of CART19. However, our finding that TNFRSF14 mutations predict for a lower rate of disease progression at 2y on univariate analysis may be explained by loss of the inhibitory effect of HVEM on T helper cells through binding of B and T lymphocyte attenuator (BTLA) (Cell. 2016 Oct 6;167(2):405-418.e13). Additionally, our finding that TP53 mutations did not predict for inferior PFS following receipt of CART19 is in agreement with that of a prior analysis (J Clin Oncol. 2022 Feb 1;40(4):369-381). Performance of CLMA on a larger number of tumor samples from DLBCL/HGBL pts subsequently treated with CART19, which is planned, may be informative.

The Methylation in B7-H4 and BTLA Genes are Associated with the Risk of Pulmonary Tuberculosis.

The important roles of B7 homologous body 4 (B7-H4), B and T lymphocyte attenuator (BTLA) in patients with pulmonary tuberculosis (PTB) have been reported. This study aims to evaluate the association among B7-H4 and BTLA genes polymorphism, methylation and PTB susceptibility. Here, we assessed the possible relationship of 10 single nucleotide polymorphisms (SNPs) in B7-H4, BTLA genes with PTB susceptibility in a Chinese population (496 PTB patients and 502 controls) by SNPscan technique. Then, the B7-H4, BTLA genes methylation levels among 98 PTB patients and 97 controls were detected using MethylTarget technique. This study found no significant differences in allele and genotype frequencies of B7-H4 gene rs10754339, rs10801935, rs10923223, rs1937956, rs3738414, BTLA gene rs1982809, rs2971205, rs75368388, rs9288953 variants between PTB patients and controls. Haplotype analysis suggested that the lower frequencies of B7-H4 AATTG haplotype, BTLA GATT haplotype and the higher frequency of BTLA AGTC haplotype were found in PTB patients when compared with controls. We also found that the frequency of BTLA gene rs9288953 C allele was significantly increased in PTB patients with drug resistance. Moreover, the methylation levels of B7-H4 and BTLA genes in PTB patients were greater than that in controls, and rs10754339 variant in B7-H4 gene could affect its methylation level in PTB patients. B7-H4, BTLA genes polymorphism might not affect PTB susceptibility, while the abnormal methylation levels of B7-H4, BTLA genes were associated with the genetic background of PTB.

In vitro vascularized immunocompetent patient-derived model to test cancer therapies

This work describes a patient-derived tumoroid model (PDTs) to support precision medicine in lung oncology. The use of human adipose tissue-derived microvasculature and patient-derived peripheral blood mononuclear cells (PBMCs) permits to achieve a physiologically relevant tumor microenvironment. This study involved ten patients at various stages of tumor progression. The vascularized, immune-infiltrated PDT model could be obtained within two weeks, matching the requirements of the therapeutic decision. Histological and transcriptomic analyses confirmed that the main features from the original tumor were reproduced. The 3D tumor model could be used to determine the dynamics of response to antiangiogenic therapy and platinum-based chemotherapy. Antiangiogenic therapy showed a significant decrease in vascular endothelial growth factor (VEGF)-A expression, reflecting its therapeutic effect in the model. In an immune-infiltrated PDT model, chemotherapy showed the ability to decrease the levels of lymphocyte activation gene-3 protein (LAG-3), B and T lymphocyte attenuator (BTLA), and inhibitory receptors of Tcells functions.

Novel PD-L1/BTLA Checkpoint Axis Exploited for Bacterial Immune Escape by Restraining CD8+ T Cell-Initiated Adaptive Immunity in Zebrafish.

Programmed death-ligand 1/programmed cell death 1 (PD-L1/PD-1) is one of the most important immune checkpoints in humans and other mammalian species. However, the occurrence of the PD-L1/PD-1 checkpoint in evolutionarily ancient vertebrates remains elusive because of the absence of a PD-1 homolog before its appearance in tetrapods. In this article, we identified, to our knowledge, a novel PD-L1/B and T lymphocyte attenuator (BTLA) checkpoint in zebrafish by using an Edwardsiella tarda-induced bacterial infection model. Results showed that zebrafish (Danio rerio) PD-L1 (DrPD-L1) and BTLA (DrBTLA) were differentially upregulated on MHC class II+ macrophages (Mϕs) and CD8+ T cells in response to E. tarda infection. DrPD-L1 has a strong ability to interact with DrBTLA, as shown by the high affinity (KD = 5.68 nM) between DrPD-L1/DrBTLA proteins. Functionally, the breakdown of DrPD-L1/DrBTLA interaction significantly increased the cytotoxicity of CD8+BTLA+ T cells to E. tarda-infected PD-L1+ Mϕ cells and reduced the immune escape of E. tarda from the target Mϕ cells, thereby enhancing the antibacterial immunity of zebrafish against E. tarda infection. Similarly, the engagement of DrPD-L1 by soluble DrBTLA protein diminished the tolerization of CD8+ T cells to E. tarda infection. By contrast, DrBTLA engagement by a soluble DrPD-L1 protein drives aberrant CD8+ T cell responses. These results were finally corroborated in a DrPD-L1-deficient (PD-L1-/-) zebrafish model. This study highlighted a primordial PD-L1/BTLA coinhibitory axis that regulates CD8+ T cell activation in teleost fish and may act as an alternative to the PD-L1/PD-1 axis in mammals. It also revealed a previously unrecognized strategy for E. tarda immune evasion by inducing CD8+ T cell tolerance to target Mϕ cells through eliciting the PD-L1/BTLA checkpoint pathway.

Dual inhibition of BTLA and PD-1 can enhance therapeutic efficacy of paclitaxel on intraperitoneally disseminated tumors

BackgroundExpression of immune checkpoints in the tumor microenvironment is one mechanism underlying paclitaxel (PTX) chemoresistance. This study aimed to investigate whether the addition of checkpoint blockade to PTX can improve...

Stimulation of Immune Checkpoint Molecule B and T-Lymphocyte Attenuator Alleviates Experimental Crescentic Glomerulonephritis.

Modulating T-lymphocytes represents a promising targeted therapeutic option for glomerulonephritis (GN) because these cells mediate damage in various experimental and human GN types. The immune checkpoint molecule B and T-lymphocyte attenuator (BTLA) has shown its potential to restrain inflammation in other T-cell-mediated disease models. Its role in GN, however, has not been investigated. We induced nephrotoxic nephritis (NTN), a mouse model of crescentic GN, in Btla-deficient (BtlaKO) mice and wild-type littermate controls and assessed disease severity using functional and histologic parameters at different time points after disease induction. Immunologic changes were comprehensively evaluated by flow cytometry, RNA sequencing, and in vitro assays for dendritic cell and T-cell function. Transfer experiments into Rag1KO mice confirmed the observed in vitro findings. In addition, we evaluated the potential of an agonistic anti-BTLA antibody to treat NTN in vivo. The BtlaKO mice developed aggravated NTN, driven by an increase of infiltrating renal Th1 cells. Single-cell RNA sequencing showed increased renal T-cell activation and positive regulation of the immune response. Although BTLA-deficient regulatory T cells (Tregs) exhibited preserved suppressive function in vitro and in vivo, BtlaKO T effector cells evaded Treg suppression. Administration of an agonistic anti-BTLA antibody robustly attenuated NTN by suppressing nephritogenic T effector cells and promoting Treg expansion. In a model of crescentic GN, BTLA signaling effectively restrained nephritogenic Th1 cells and promoted regulatory T cells. Suppression of T-cell-mediated inflammation by BTLA stimulation may prove relevant for a broad range of conditions involving acute GN.

Epitope topography of agonist antibodies to the checkpoint inhibitory receptor BTLA

B and T lymphocyte attenuator (BTLA) is an attractive target for a new class of therapeutics that attempt to rebalance the immune system by agonizing checkpoint inhibitory receptors (CIRs). Herpesvirus entry mediator (HVEM) binds BTLA in both trans- and cis-orientations. We report here the development and structural characterization of three humanized BTLA agonist antibodies, 22B3, 25F7, and 23C8. We determined the crystal structures of the antibody-BTLA complexes, showing that these antibodies bind distinct and non-overlapping epitopes of BTLA. While all three antibodies activate BTLA, 22B3 mimics HVEM binding to BTLA and shows the strongest agonistic activity in functional cell assays and in an imiquimod-induced mouse model of psoriasis. 22B3 is also capable of modulating HVEM signaling through the BTLA-HVEM cis-interaction. The data obtained from crystal structures, biochemical assays, and functional studies provide a mechanistic model of HVEM and BTLA organization on the cell surface and informed the discovery of a highly active BTLA agonist.

BTLA transcriptomic expression correlates with high levels of PD-1, PD-L2, and CTLA-4 checkpoints and with its ligand, HVEM, across diverse solid cancers.

e14641 Background: B and T lymphocyte attenuator (BTLA) is an immune checkpoint that is highly expressed on tumor-specific T cells and has been shown to be upregulated in several cancer types. The binding of BTLA to its ligand herpesvirus entry mediator (HVEM, also known as TNFRSF14) inhibits activation and proliferation of T lymphocytes, leading to decreased tumor killing. Early clinical trials of BTLA inhibitors are ongoing with preliminary results showing modest effects in advanced solid tumors. More recent clinical data suggests that combination checkpoint therapy with anti-BTLA and anti-PD-1 treatment provides greater benefit compared to monotherapy in patients with lymphoma. Methods: We analyzed RNA expression levels of 397 genes in various solid tumor types from 514 patients with various types of solid tumors seen at University of San Diego (UCSD), Moores Cancer Center. RNA expression was quantified at a CLIA-approved laboratory Omniseq (https://www.omniseq.com). Transcript abundance was normalized to internal housekeeping gene profiles and ranked (0-100 percentile) in a standardized manner to a reference population of 735 tumors spanning 35 histologies. We analyzed tumors with “High” (75-100 percentile rank) BTLA expression profiles. Odds ratio for high BTLA expression was calculated and multivariate analysis was utilized to determine significant correlations for multiple genes. Results: A total of 96 (18.7%) tumors had high BTLA (>75) expression across 32 different histologies. Cancers with the highest proportion of BTLA expression were small intestine (42%), breast (31%), pancreatic (29%), stomach (24%), neuroendocrine (20%), lung (20%), head and neck (17%) and colorectal (13%). There was significant association for BTLA and high PD-1 (p < 0.001), high CTLA-4 (p < 0.001), high PD-L2 (p = 0.05) and its ligand HVEM (p < 0.01). There was no significant association between BTLA expression and PD-L1, gender or stomach, neuroendocrine, head and neck, and lung histologies. Conclusions: High BTLA expression was found in a large number of tumor samples and significantly associated with other immune checkpoints with FDA-approved drugs. Furthermore, HVEM expression (ligand for BTLA) significantly correlates with high BTLA transcriptome levels. The BTLA/HVEM pathway has been associated with suppression of T cell-mediated anti-tumor activity and resistance to anti-PD-1 immunotherapy leading to poor clinical outcomes. The utilization of combination checkpoint therapy involving BTLA inhibitors and specific checkpoint blockade is currently being explored. Our results suggest that patient selection based on the RNA expression level of immune markers matched to corresponding immunotherapy agent, rather than a population-based approach, may be ideal for optimizing treatment response.