The Immune Checkpoint BTLA in Oral Cancer: Expression Analysis and Its Correlation to Other Immune Modulators.

In oral squamous cell carcinoma (OSCC) tissues, an immunotolerant situation triggered by immune checkpoints (ICPs) can be observed. Immune checkpoint inhibitors (ICIs) against the PD1/PD-L axis are used with impressive success. However, the response rate is low and the development of acquired resistance to ICI treatment can be observed. Therefore, new treatment strategies especially involving immunological combination therapies need to be developed. The novel negative immune checkpoint BTLA has been suggested as a potential biomarker and target for antibody-based immunotherapy. Moreover, improved response rates could be displayed for tumor patients when antibodies directed against BTLA were used in combination with anti-PD1/PD-L1 therapies. The aim of the study was to check whether the immune checkpoint BTLA is overexpressed in OSCC tissues compared to healthy oral mucosa (NOM) and could be a potential diagnostic biomarker and immunological target in OSCC. In addition, correlation analyses with the expression of other checkpoints should clarify more precisely whether combination therapies are potentially useful for the treatment of OSCC. A total of 207 tissue samples divided into 2 groups were included in the study. The test group comprised 102 tissue samples of OSCC. Oral mucosal tissue from 105 healthy volunteers (NOM) served as the control group. The expression of two isoforms of BTLA (BTLA-1/2), as well as PD1, PD-L1/2 and CD96 was analyzed by RT-qPCR. Additionally, BTLA and CD96 proteins were detected by IHC. Expression levels were compared between the two groups, the relative differences were calculated, and statistical relevance was determined. Furthermore, the expression rates of the immune checkpoints were correlated to each other. BTLA expression was significantly increased in OSCC compared to NOM (pBTLA_1 = 0.003; pBTLA_2 = 0.0001, pIHC = 0.003). The expression of PD1, its ligands PD-L1 and PD-L2, as well as CD96, were also significantly increased in OSCC (p ≤ 0.001). There was a strong positive correlation between BTLA expression and that of the other checkpoints (p < 0.001; ρ ≥ 0.5). BTLA is overexpressed in OSCC and appears to be a relevant local immune checkpoint in OSCC. Thus, antibodies directed against BTLA could be potential candidates for immunotherapies, especially in combination with ICI against the PD1/PD-L axis and CD96.

The effect of gD-derived peptides on T cell immune response mediated by BTLA-HVEM protein complex in melanoma patients.

Introduction: The effector function of T cells is regulated via immune checkpoints, activating or inhibiting the immune response. The BTLA-HVEM complex, the inhibitory immune checkpoint, may act as one of the tumor immune escape mechanisms. Therefore, interfering with the binding of these proteins can prove beneficial in cancer treatment. Our study focused on peptides interacting with HVEM at the same place as BTLA, thus disrupting the BTLA-HVEM interaction. These peptides' structure and amino acid sequences are based on the gD protein, the ligand of HVEM. Here, we investigated their immunomodulatory potential in melanoma patients. Methods: Flow cytometry analyses of activation, proliferation, and apoptosis of T cells from patients were performed. Additionally, we evaluated changes within the T cell memory compartment. Results: The most promising compound - Pep(2), increased the percentages of activated T cells and promoted their proliferation. Additionally, this peptide affected the proliferation rate and apoptosis of melanoma cell line in co-culture with T cells. Discussion: We conclude that the examined peptide may act as a booster for the immune system. Moreover, the adjuvant and activating properties of the gD-derived peptide could be used in a combinatory therapy with currently used ICI-based treatment. Our studies also demonstrate that even slight differences in the amino acid sequence of peptides and any changes in the position of the disulfide bond can strongly affect the immunomodulatory properties of compounds.

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.

Targeting BTLA with the peptide inhibitor HVEM(14-39) - A new way to restore the activity of T cells in melanoma.

The complex of B- and T-lymphocyte attenuator (BTLA) and herpes virus entry mediator (HVEM) plays a critical role in immune regulation and has emerged as a promising therapeutic target for cancer treatment. In this study, we investigated the potential of the peptide inhibitor HVEM(14-39) to restore peripheral T cell activity in patients with advanced melanoma. In these patients, CD8+ T cells downregulated BTLA expression and increased HVEM expression upon activation. The addition of HVEM(14-39) reduced the percentage of BTLA+ CD8+ T cells and increased the subpopulation of HVEM+ CD8+ T cells. Additionally, HVEM(14-39) enhanced T cell activation, proliferation, and the shift toward effector memory T cell subpopulations. Finally, this peptide affected the proliferation rate and late apoptosis of melanoma cell line in co-culture with T cells. These findings suggest that HVEM(14-39) can overcome T cell exhaustion and improve antitumor responses. Peptide-based immunotherapy targeting the BTLA-HVEM complex offers a promising alternative to monoclonal antibody-based therapies, with the potential for fewer side effects and higher treatment efficacy.

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.

The BTLA-HVEM complex - The future of cancer immunotherapy.

The BTLA-HVEM complex plays a pivotal role in cancer and cancer immunotherapy by regulating immune responses. Dysregulation of BTLA and HVEM expression contributes to immunosuppression and tumor progression across various cancer types. Targeting the interaction between BTLA and HVEM holds promise for enhancing anti-tumor immune responses. Disruption of this complex presents a valuable avenue for advancing cancer immunotherapy strategies. Aberrant expression of BTLA and HVEM adversely affects immune cell function, particularly T cells, exacerbating tumor evasion mechanisms. Understanding and modulating the BTLA-HVEM axis represents a crucial aspect of designing effective immunotherapeutic interventions against cancer. Here, we summarize the current knowledge regarding the structure and function of BTLA and HVEM, along with their interaction with each other and various immune partners. Moreover, the expression of soluble and transmembrane forms of BTLA and HVEM in different types of cancer and their impact on the prognosis of patients is also discussed. Additionally, inhibitors of the proteins binding that might be used to block BTLA-HVEM interaction are reviewed. All the presented data highlight the plausible clinical application of BTLA-HVEM targeted therapies in cancer and autoimmune disease management. However, further studies are required to confirm the practical use of this concept. Despite the increasing number of reports on the BTLA-HVEM complex, many aspects of its biology and function still need to be elucidated. This review can be regarded as an encouragement and a guide to follow the path of BTLA-HVEM research.

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.

Costimulatory receptors in the channel catfish: CD28 family members and their ligands.

The CD28-B7 interaction is required to deliver a second signal necessary for T-cell activation. Additional membrane receptors of the CD28 and B7 families are also involved in immune checkpoints that positively or negatively regulate leukocyte activation, in particular T lymphocytes. BTLA is an inhibitory receptor that belongs to a third receptor family. Fish orthologs exist only for some of these genes, and the potential interactions between the corresponding ligands remain mostly unclear. In this work, we focused on the channel catfish (Ictalurus punctatus), a long-standing model for fish immunology, to analyze these co-stimulatory and co-inhibitory receptors. We identified one copy of cd28, ctla4, cd80/86, b7h1/dc, b7h3, b7h4, b7h5, two btla, and four b7h7 genes. Catfish CD28 contains the highly conserved mammalian cytoplasmic motif for PI3K and GRB2 recruitment, however this motif is absent in cyprinids. Fish CTLA4 share a C-terminal putative GRB2-binding site but lacks the mammalian PI3K/GRB2-binding motif. While critical V-domain residues for human CD80 or CD86 binding to CD28/CTLA4 show low conservation in fish CD80/86, C-domain residues are highly conserved, underscoring their significance. Catfish B7H1/DC had a long intracytoplasmic domain with a P-loop-NTPase domain that is absent in mammalian sequences, while the lack of NLS motif in fish B7H4 suggests this protein may not regulate cell growth when expressed intracellularly. Finally, there is a notable expansion of fish B7H7s, which likely play diverse roles in leukocyte regulation. Overall, our work contributes to a better understanding of fish leukocyte co-stimulatory and co-inhibitory receptors.

BTLA biology in cancer: from bench discoveries to clinical potentials.

Immune checkpoints play a critical role in maintaining the delicate balance of immune activation in order to prevent potential harm caused by excessive activation, autoimmunity, or tissue damage. B and T lymphocyte attenuator (BTLA) is one of crucial checkpoint, regulating stimulatory and inhibitory signals in immune responses. Its interaction with the herpes virus entry mediator (HVEM) plays an essential role in negatively regulating immune responses, thereby preserving immune homeostasis. In cancer, abnormal cells evade immune surveillance by exploiting checkpoints like BTLA. Upregulated BTLA expression is linked to impaired anti-tumor immunity and unfavorable disease outcomes. In preclinical studies, BTLA-targeted therapies have shown improved treatment outcomes and enhanced antitumor immunity. This review aims to provide an in-depth understanding of BTLA's biology, its role in various cancers, and its potential as a prognostic factor. Additionally, it explores the latest research on BTLA blockade in cancer immunotherapy, offering hope for more effective cancer treatments.

Analysis of the immunological markers BTLA, TIM-3, and PD-L1 at the invasion front and tumor center in clear cell renal cell carcinoma.

PURPOSE: Immune checkpoint inhibitors (ICI) are then backbone in the therapy of metastatic renal cell carcinoma (RCC). The aim of this analysis was to explore the different expression of the ICI PD-L1, BTLA, and TIM-3 at the different tumor locations of the invasion front and the tumor center. METHODS: Large-area sections of the tumor center and invasion front of 44 stage pT1-4 clear cell RCCs were examined immunohistochemically using antibodies against BTLA, TIM-3, and PD-L1 and subsequently correlated with clinicopathologic data. RESULTS: TIM-3 was most strongly expressed at the invasion front (mean ± SD: 84.1 ± 46.6, p = 0.094). BTLA expression was highest in normal tissue, with weak staining in the tumor center and at the invasion front [110.2 vs. 18.6 (p < 0.001) vs. 32.2 (p = 0.248)]. PD-L1 was weakly expressed at the tumor center (n = 5/44) and at the invasion front (n = 5/44). Correlation with clinicopathological parameters revealed significantly higher BTLA expression in ≥ T3 tumors compared to T1/2 tumors (tumor center p = 0.009; invasion front p = 0.005). BTLA-positive tumors at the tumor center correlated with worse CSS (median 48.46 vs. 68.91 months, HR 4.43, p = 0.061). PD-L1 expression was associated with worse CSS (median 1.66 vs. 4.5 years, HR 1.63, p = 0.652). For TIM-3, there were no significant associations with clinicopathological parameters and survival. CONCLUSION: The present results show heterogeneous intratumoral and intertumoral expression of the investigated checkpoint receptors PD-L1, BTLA, and TIM-3. In the clinical practice tumor sampling should include different tumor locations, and multiple inhibition of different checkpoint receptors seems reasonable to increase the therapeutic success.

Difficult-to-express antigen generation through a co-expression and disassociation methodology.

Extracellular domain (ECD) antigens are crucial components for antibody discovery, in vitro assays, and epitope mapping during therapeutical antibody development. Oftentimes, those antigens are difficult to produce while retaining the biologic function/activity upon extracellular secretion in commonly used expression systems. We have developed an effective method to cope with the challenge of generating quality antigen ECDs. In this method, a monoclonal antibody (Mab) or antibody fragment antigen-binding (Fab) region acts as a "chaperone" to stabilize the antigen ECD through forming an antibody:antigen complex. This methodology includes transient co-expression of the complex in Chinese hamster ovary cells and then dissociation of the purified complex into individual components by low pH treatment in the presence of arginine. The antigen is then separated from the chaperone on a preparative size exclusion chromatography (pSEC) followed by an optional affinity chromatography process to remove residual Mab or Fab. We demonstrate this co-expression/disassociation methodology on two difficult-to-express antigen ECDs from cluster-of-differentiation/cytokine family and were successful in producing stable, biologically active antigens when the common methods using Histidine-tagged and/or Fc-fused protein failed. This can be applied as a general approach for antigen production if a Mab or binding partner is available.

Peptides targeting the BTLA-HVEM complex can modulate T cell immune response.

Immune checkpoints secure the proper function of the immune system and the maintenance of the BTLA-HVEM complex, an inhibitory immune checkpoint, is one of the pathways vital for T cell responsiveness to various stimuli. The present study reports the immunomodulatory potential of five peptides targeting the BTLA-HVEM complex on the activity of human T cells. Isolated T cells were exposed to the peptides alone or combined with CD3/CD28 mAb for 72 h or 120 h. The flow cytometry was used to evaluate the activation markers (CD69, CD62L, CD25), changes within the T cell memory compartment, proliferation rate, and apoptosis of T cells. The immunomodulatory effect of the peptides was visible as an increase in the percentage of CD4+ and CD8+ T cells expressing CD69 or CD25, a boost in T cell proliferation, and shifts in the T cell memory compartment. Pep(2) and Pep(5) were the most promising compounds, displaying a putative immune-restoring function.

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.

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

Background: 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. Methodology: 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. Results: 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. Conclusion: 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.

Variation in the gene encoding the co-inhibitory molecule BTLA is associated with survival in patients treated for clear cell renal carcinoma - results of a prospective cohort study.

Introduction: The successful introduction of immune checkpoint blockade approaches to renal-cell carcinoma (RCC) treatment indicates the importance of molecules regulating the T cell response to RCC risk and progression. Material and methods: In this study, we evaluate the association of variations in the CTLA-4, BTLA and CD28 genes with overall survival (OS) of RCC patients and specifically clear cell RCC (ccRCC) patients. The following single nucleotide polymorphisms (SNPs) previously genotyped using the RFLP method or TaqMan SNP Genotyping Assays were analyzed: CTLA-4 gene: c.49A>G (rs231775), g.319C>T (rs5742909), g.*6230G>A (CT60; rs3087243), g.*10223G>T (Jo31; rs11571302); CD28 gene: c.17+3T>C (rs3116496), c.-1042G>A (rs3181098); BTLA gene: rs2705511, rs1982809, rs9288952, rs9288953, rs2705535 and rs1844089. Results: During long term observation (6.5 years) we discovered that possessing the A allele at BTLA rs1844089 SNP, together with advanced disease (stage ≥ 3, tumor grade > 3, tumor diameter ≥ 70 mm), is an independent risk factor of death which increases the hazard ratio (HR) of death by more than two-fold (HR = 2.21, 95% CI: 1.28-3.83). Furthermore, the OS of patients bearing this allele is 6 months shorter than for homozygous (GG) patients (42.5 vs. 48.2 months). Conclusions: Our results indicate for the first time that genetic variation within the gene encoding BTLA is significantly associated with overall survival in clear cell renal cell carcinoma patients.

Beyond the anti-PD-1/PD-L1 era: promising role of the BTLA/HVEM axis as a future target for cancer immunotherapy.

Recent introduction of monoclonal antibodies targeting immune checkpoints to harness antitumor immunity has revolutionized the cancer treatment landscape. The therapeutic success of immune checkpoint blockade (ICB)-based therapies mainly relies on PD-1/PD-L1 and CTLA-4 blockade. However, the limited overall responses and lack of reliable predictive biomarkers of patient´s response are major pitfalls limiting immunotherapy success. Hence, this reflects the compelling need of unveiling novel targets for immunotherapy that allow to expand the spectrum of ICB-based strategies to achieve optimal therapeutic efficacy and benefit for cancer patients. This review thoroughly dissects current molecular and functional knowledge of BTLA/HVEM axis and the future perspectives to become a target for cancer immunotherapy. BTLA/HVEM dysregulation is commonly found and linked to poor prognosis in solid and hematological malignancies. Moreover, circulating BTLA has been revealed as a blood-based predictive biomarker of immunotherapy response in various cancers. On this basis, BTLA/HVEM axis emerges as a novel promising target for cancer immunotherapy. This prompted rapid development and clinical testing of the anti-BTLA blocking antibody Tifcemalimab/icatolimab as the first BTLA-targeted therapy in various ongoing phase I clinical trials with encouraging results on preliminary efficacy and safety profile as monotherapy and combined with other anti-PD-1/PD-L1 therapies. Nevertheless, it is anticipated that the intricate signaling network constituted by BTLA/HVEM/CD160/LIGHT involved in immune response regulation, tumor development and tumor microenvironment could limit therapeutic success. Therefore, in-depth functional characterization in different cancer settings is highly recommended for adequate design and implementation of BTLA-targeted therapies to guarantee the best clinical outcomes to benefit cancer patients.

Retroviral b-Zip protein (HBZ) contributes to the release of soluble and exosomal immune checkpoint molecules in the context of neuroinflammation.

HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive, neuroinflammatory demyelinating condition of the spinal cord. We have previously shown that aberrant expression and activity of immune checkpoint (ICP) molecules such as PD-1 and PD-L1/PD-L2, negatively associates with the cytolytic potential of T cells in individuals with HAM/TSP. Interestingly, ICPs can exist in a soluble cell-free form and can be carried on extracellular vesicles (EVs) and exosomes (small EVs, <300nm) while maintaining their immunomodulatory activity. Therefore, we investigated the role of soluble and exosomal ICPs in HTLV-1 associated neuroinflammation. For the very first time, we demonstrate a unique elevated presence of several stimulatory (CD27, CD28, 4-1BB) and inhibitory (BTLA, CTLA-4, LAG-3, PD-1, PD-L2) ICP receptors in HAM/TSP sera, and in purified exosomes from a HAM/TSP-derived HTLV-1-producing (OSP2) cells. These ICPs were found to be co-localized with the endosomal sorting complex required for transport (ESCRT) pathway proteins and exhibited functional binding with their respective ligands. Viral proteins and cytokines (primarily IFNγ) were found to be present in purified exosomes. IFNγ exposure enhanced the release of ICP molecules while antiretroviral drugs (Azidothymidine and Lopinavir) significantly inhibited this process. HTLV-1 b-Zip protein (HBZ) has been linked to factors that enhance EV release and concurrent knockdown here led to the reduced expression of ESCRT associated genes (eg. Hrs, Vsp4, Alix, Tsg101) as well as abrogated the release of ICP molecules, suggesting HBZ involvement in this process. Moreso, exosomes from OSP2 cells adversely affected CD8 T-cell functions by dimishing levels of cytokines and cytotoxic factors. Collectively, these findings highlight exosome-mediated immunmodulation of T-cell functions with HBZ and ESCRT pathways as an underlying mechanism in the context of HTLV-1-induced neuroinflammation.

BTLA-derived peptides as inhibitors of BTLA/HVEM complex formation - design, synthesis and biological evaluation.

Immune checkpoints can be divided into co-stimulatory and co-inhibitory molecules that regulate the activation and effector functions of T cells. The co-inhibitory pathways mediated by ICPs are used by cancer cells to escape from immune surveillance, and therefore the blockade of these receptor/ligand interactions is one of the strategies used in the treatment of cancer. The two main pathways currently under investigation are CTLA-4/CD80/CD86 and PD-1/PD-L1, and the monoclonal Abs targeting them have shown potent immunomodulatory effects and activity in clinical environments. Another interesting target in cancer treatment is the BTLA/HVEM complex. Binding of BTLA protein on T cells to HVEM on cancer cells leads to inhibition of T cell proliferation and cytokine production. In the presented work, we focused on blocking the HVEM protein using BTLA-derived peptides. Based on the crystal structure of the BTLA/HVEM complex and MM/GBSA analysis performed here, we designed and synthesized peptides, specifically fragments of BTLA protein. We subsequently checked the inhibitory capacities of these compounds using ELISA and a cellular reporter platform. Two of these peptides, namely BTLA(35-43) and BTLA(33-64)C58Abu displayed the most promising properties, and we therefore performed further studies to evaluate their affinity to HVEM protein, their stability in plasma and their effect on viability of human PBMCs. In addition, the 3D structure for the peptide BTLA(33-64)C58Abu was determined using NMR. Obtained data confirmed that the BTLA-derived peptides could be the basis for future drugs and their immunomodulatory potential merits further examination.

T Cells Expressing CAR Equipped With Extracellular Domain of BTLA Are Effective Against HVEM-over-expressing Melanoma Cell Lines.

BACKGROUND/AIM: Several chimeric antigen receptor (CAR) T cells have been used to treat melanoma but have not shown favorable results. This study investigated whether Herpes virus entry mediator (HVEM), which is overexpressed in melanoma, is a potential novel antigen for CAR T cell therapy. MATERIALS AND METHODS: A CAR construct, composed of the BTLA extracellular domain for HVEM recognition (BTLA-28z), was developed and tested. RESULTS: Jurkat cells transduced with BTLA-28z exhibited enhanced IL-2 secretion when incubated with HVEM-over-expressing melanoma cells. KHYG-1 cells transduced with BTLA-28z also lysed melanoma cell lines. Using primary T cells, we generated CAR T cells targeting HVEM. BTLA-28z CAR T cells exhibited excellent lytic activities against melanoma cell lines. CONCLUSION: HVEM-targeting CAR T cells may be useful for the treatment of melanoma.

Lymphocyte HVEM/BTLA co-expression after critical illness demonstrates severity indiscriminate upregulation, impacting critical illness-induced immunosuppression.

Introduction: The co-regulatory molecule, HVEM, can stimulate or inhibit immune function, but when co-expressed with BTLA, forms an inert complex preventing signaling. Altered HVEM or BTLA expression, separately have been associated with increased nosocomial infections in critical illness. Given that severe injury induces immunosuppression, we hypothesized that varying severity of shock and sepsis in murine models and critically ill patients would induce variable increases in HVEM/BTLA leukocyte co-expression. Methods: In this study, varying severities of murine models of critical illness were utilized to explore HVEM+BTLA+ co-expression in the thymic and splenic immune compartments, while circulating blood lymphocytes from critically ill patients were also assessed for HVEM+BTLA+ co-expression. Results: Higher severity murine models resulted in minimal change in HVEM+BTLA+ co-expression, while the lower severity model demonstrated increased HVEM+BTLA+ co-expression on thymic and splenic CD4+ lymphocytes and splenic B220+ lymphocytes at the 48-hour time point. Patients demonstrated increased co-expression of HVEM+BTLA+ on CD3+ lymphocytes compared to controls, as well as CD3+Ki67- lymphocytes. Both L-CLP 48hr mice and critically ill patients demonstrated significant increases in TNF-α. Discussion: While HVEM increased on leukocytes after critical illness in mice and patients, changes in co-expression did not relate to degree of injury severity of murine model. Rather, co-expression increases were seen at later time points in lower severity models, suggesting this mechanism evolves temporally. Increased co-expression on CD3+ lymphocytes in patients on non-proliferating cells, and associated TNF-α level increases, suggest post-critical illness co-expression does associate with developing immune suppression.