Newly Emerging Immune Checkpoints: Promises for Future Cancer Therapy.
ABSTRACT: Cancer immunotherapy has been a great breakthrough, with immune checkpoint inhibitors leading the way. Despite the clinical effectiveness of certain immune checkpoint inhibitors, the overall response rate remains low, and the effectiveness of immunotherapies for many tumors has been disappointing. There is substantial interest in looking for additional immune checkpoint molecules that may act as therapeutic targets for cancer. Recent advances during the last decade have identified several novel immune checkpoint targets, including lymphocyte activation gene-3 (LAG-3), B and T lymphocyte attenuator (BTLA), programmed death-1 homolog (PD-1H), T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIM-3)/carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), and the poliovirus receptor (PVR)-like receptors. The investigations into these molecules have generated promising results in preclinical studies. Herein, we will summarize our current progress and understanding of these newly-characterized immune checkpoints and their potential application in cancer immunotherapy.
Project description:Antibody based immune-checkpoint blockade therapy is a major breakthrough in oncology, leading to clinical benefit for cancer patients. Among the growing family of inhibitory receptors, the B and T lymphocyte attenuator (BTLA), which interacts with herpes virus entry mediator (HVEM), is a promising target for immunotherapy. Indeed, BTLA inhibits T-cell proliferation and cytokine production. The crystal structure of the BTLA/HVEM complex has shown that the HVEM(26-38) fragment is directly involved in protein binding. We designed and analyzed the capacity of several analogs of this fragment to block the ligation between BTLA and HVEM, using competitive ELISA and cellular assay. We found that the HVEM(23-39) peptide can block BTLA/HVEM ligation. However, the blocking ability was due to the Cys encompassed in this peptide and that even free cysteine targeted the BTLA protein and blocked its interaction with HVEM. These data highlight a Cys-related artefact in vitro, which should be taken in consideration for future development of BTLA/HVEM blocking compounds.
Project description:Endogenous control mechanisms, including immune checkpoints and immunosuppressive cells, are exploited in the process of tumorigenesis to weaken the anti-tumor immune response. Cancer treatment by chemotherapy or immune checkpoint inhibition can lead to changes of checkpoint expression, which influences therapy success. Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) were isolated from head and neck squamous cell carcinoma (HNSCC) patients (n = 23) and compared to healthy donors (n = 23). Immune checkpoint expression (programmed cell death ligand 1 (PD-1), tumor necrosis factor receptor (TNFR)-related (GITR), CD137, tumor necrosis factor receptor superfamily member 4 (TNFRSF4) (OX40), t-cell immunoglobulin and mucin-domain containing-3 (TIM3), B- and T-lymphocyte attenuator (BTLA), lymphocyte-activation gene 3 (LAG3)) was determined on immune cells by flow cytometry. PD-L1 expression was detected on tumor tissue by immunohistochemistry. Immune cells were treated with immuno- and chemotherapeutics to investigate treatment-specific change in immune checkpoint expression, in vitro. Specific changes of immune checkpoint expression were identified on PBL and TIL of HNSCC patients compared to healthy donors. Various chemotherapeutics acted differently on the expression of immune checkpoints. Changes of checkpoint expression were significantly less pronounced on regulatory T cells compared to other lymphocyte populations. Nivolumab treatment significantly reduced the receptor PD-1 on all analyzed T cell populations, in vitro. The specific immune checkpoint expression patterns in HNSCC patients and the investigated effects of immunomodulatory agents may improve the development and efficacy of targeted immunotherapy.
Project description:B- and T-lymphocyte attenuator (BTLA) is an immune-regulatory receptor, similar to CTLA-4 and PD-1, and is mainly expressed on B-, T-, and all mature lymphocyte cells. Herpes virus entry mediator (HVEM)-BTLA plays a critical role in immune tolerance and immune responses which are areas of intense research. However, the mechanisms of the BTLA and the BTLA/HVEM signaling pathway in human diseases remain unclear. This review describes the research milestones of BTLA and HVEM in chronological order and their role in chronic HBV infection.
Project description:Background: Cancer patients often display dysfunctional antitumor T-cell responses. Because noteworthy benefits of immune checkpoint pathway blockade, such as programmed cell death protein 1 (PD-1) inhibitors, have been achieved in multiple advanced cancers, the next critical question is which mono-blockade or combinatorial blockade regimens may reinvigorate antitumor T-cell immunity in those cancer patients while limiting immune-related adverse effects. Method: This study recruited, in total, 172 primary cancer patients (131 were blood-tumor-matched patients) who were treatment-naïve prior to the surgeries or biopsies covering the eight most prevalent types of cancer. With access to fresh surgical samples, this study simultaneously investigated the ex vivo expression level of eight known immune checkpoint receptors [PD-1, cytotoxic T-lymphocyte antigen-4 [CTLA-4], T-cell immunoglobulin and mucin-domain containing-3 [Tim-3], 2B4, killer cell lectin like receptor G1 [KLRG-1], TIGIT, B- and T-lymphocyte attenuator [BTLA], and CD160] on tumor-infiltrating T cells (TILs) and paired circulating T cells in blood from a 131-patient cohort. Results: We found increased an expression of PD-1 and Tim-3 but a decreased expression of BTLA on TILs when compared with peripheral blood from multiple types of cancer. Moreover, our co-expression analysis of key immune checkpoint receptors delineates "shared" subsets as PD-1+Tim-3+TIGIT+2B4+KLRG-1-CTLA-4- and PD-1+TIGIT+2B4+Tim-3-KLRG-1-CTLA-4- from bulk CD8 TILs. Furthermore, we found that a higher frequency of advanced differentiation stage T cells (CD27-CCR7-CD45RA-) among the "shared" subset (PD-1+Tim-3+TIGIT+2B4+KLRG-1-CTLA-4-) in bulk CD8 TILs was associated with poorly differentiated cancer type in cervical cancer patients. Conclusions: To our knowledge, our study is the first comprehensive analysis of key immune checkpoint receptors on T cells in treatment-naïve, primary cancer patients from the eight most prevalent types of cancer. These findings might provide useful information for future design of mono-blockade/combinatorial blockades and/or genetically modified T-cell immunotherapy.
Project description:Chronic lymphocytic leukemia (CLL) is characterized by the peripheral accumulation of neoplastic B cells and is frequently complicated by the systemic immunosuppression associated with an impairment in B and T lymphocyte activation. We hypothesized that the expression of immune checkpoint suppressors B and T lymphocyte attenuator (BTLA) and cytotoxic T lymphocyte antigen (CTLA-4) is disturbed in both lymphocyte subpopulations in CLL. The expression of CTLA-4 and BTLA mRNA was determined by real-time PCR, while CTLA-4 protein expression (surface or intracellular) was estimated in BTLA+ lymphocytes by flow cytometry. In CLL patients, we observed a higher gene transcript level of BTLA and CTLA-4 than in healthy individuals in both freshly isolated and PMA stimulated B and T cells. Remarkably, lower amounts of both inhibitory proteins were found in peripheral blood (PB) CLL B cells, whereas normal BTLA and elevated CTLA-4 were found in T cells. Consistently, there was a prevalence of CTLA-4+ cells within circulating BTLA+ T cells cells of patients confronting PB healthy cells. After in vitro stimulation, the only change found in CLL patients was a decrease in BTLA expression in B and T lymphocytes. In contrast, healthy lymphocytes responded more vigorously as regards the BTLA and CTLA expression with substantially higher frequency of CD69+ cells under the stimulating condition compared to corresponding cells from the CLL group. Our results indicate that CLL development is associated with the affected expression of BTLA and CTLA-4 checkpoint receptors in PB and its impaired expression might be associated with lowering of the threshold for B cell activation and proliferation, while upregulated CTLA-4 expression in CLL peripheral BTLA+ T cells may contribute to suppressed T cell effector functions. This hypothesis needs to be validated in future studies, which would allow us to explain how the increased or decreased expression of these molecules affects the cell function.
Project description:Antibodies that block T cell inhibition via the immune checkpoints CTLA-4 and PD-1 have revolutionized cancer therapy during the last 15 years. T cells express additional inhibitory surface receptors that are considered to have potential as targets in cancer immunotherapy. Antibodies against LAG-3 and TIM-3 are currently clinically tested to evaluate their effectiveness in patients suffering from advanced solid tumors or hematologic malignancies. In addition, blockade of the inhibitory BTLA receptors on human T cells may have potential to unleash T cells to effectively combat cancer cells. Much research on these immune checkpoints has focused on mouse models. The analysis of animals that lack individual inhibitory receptors has shed some light on the role of these molecules in regulating T cells, but also immune responses in general. There are current intensive efforts to gauge the efficacy of antibodies targeting these molecules called immune checkpoint inhibitors alone or in different combinations in preclinical models of cancer. Differences between mouse and human immunology warrant studies on human immune cells to appreciate the potential of individual pathways in enhancing T cell responses. Results from clinical studies are not only highlighting the great benefit of immune checkpoint inhibitors for treating cancer but also yield precious information on their role in regulating T cells and other cells of the immune system. However, despite the clinical relevance of CTLA-4 and PD-1 and the high potential of the emerging immune checkpoints, there are still substantial gaps in our understanding of the biology of these molecules, which might prevent the full realization of their therapeutic potential. This review addresses PD-1, CTLA-4, BTLA, LAG-3, and TIM-3, which are considered major inhibitory immune checkpoints expressed on T cells. It provides summaries of our current conception of the role of these molecules in regulating T cell responses, and discussions about major ambiguities and gaps in our knowledge. We emphasize that each of these molecules harbors unique properties that set it apart from the others. Their distinct functional profiles should be taken into account in therapeutic strategies that aim to exploit these pathways to enhance immune responses to combat cancer.
Project description:Expression of immune checkpoint molecules, including programmed cell death protein-1 (PD-1), has been reported on T cells in various types of cancer. However, the expression status of these molecules in the tumor microenvironment of epithelial ovarian cancer (EOC) has not yet been studied. A total of 54 cases of malignant ascites from patients with EOC were analyzed in the present study. The expression of PD-1, lymphocyte-activation gene-3 (LAG-3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and B and T lymphocyte attenuator (BTLA) on cluster of differentiation (CD)4+ and CD8+ T cells in malignant EOC ascites were investigated using multicolor flow cytometric analysis. The expression of PD-L1 in tumor cells, PD-L2 in HLA-DR-positive cells and galectin-9 in ascitic fluid was also analyzed. In addition, cytokine profiling of ascitic fluid was performed to understand the immune microenvironment of EOC. PD-1, LAG-3 TIM-3, and BTLA were expressed on 65.8, 10.6, 4.3 and 37.6% of CD4+ T cells, and on 57.7, 5.0, 4.9 and 15.7% of CD8+ T cells, respectively. Programmed cell death protein-1 (PD-1), LAG-3 and BTLA were more frequently expressed on CD4+ compared with CD8+ T cells. The co-expression of immune checkpoints was further investigated and results indicated that 39 (72.2%) and 37 patients (68.5%) expressed multiple immune checkpoints on CD4+ T cells and CD8+ T cells, respectively. In addition, lower levels of TNF-? and interleukin-6 in ascitic fluid were significantly associated with multiple immune checkpoint expression on CD8+ T cells. The present findings indicated that multiple immune checkpoint molecules were expressed on T cells in the EOC tumor microenvironment and the results may suggest the significance of simultaneous blockade of immune checkpoints to control EOC.
Project description:The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.
Project description:Background:B and T lymphocyte attenuator (BTLA) is a co-signaling protein belonging to the CD28 immunoglobulin superfamily. However, the role of BTLA in prognosis and immunotherapy of colorectal cancer (CRC) remains unclear. Methods:We evaluated the expression of BTLA via the Oncomine and the cancer genome atlas (TCGA) database. We research the outcome among different BTLA expression patients by Kaplan-Meier curve. We used the Chi-Squared test and Cox regression analysis to identify potential risk factors. Besides, the correlations between BTLA and cancer immune infiltration were investigated via CIBERSORT. Results:Various cohorts showed that BTLA expression was lower in CRC compared to corresponding normal tissue. Moreover, low BTLA expression was correlated with poor overall survival in TCGA cohorts and Gene Expression Omnibus cohorts (GSE29623 and GSE17536). Low BTLA expression was associated with less lymph node metastasis (p = 0.0123). In the Cox proportional hazards model, BTLA was identified as a favorable prognostic factor. Naive B cells, memory B cells, CD8 T cells, CD4 memory resting T cells, follicular helper T (Tfh) cells, monocytes, resting natural killing (NK) cells, M0 macrophages, M1 macrophages, resting mast cells, and activated mast cells were affected by BTLA expression (all p < 0.01). Correlated immune markers and functional enrichment analysis revealed BTLA functioned in the T cell receptor signaling pathway, B cell receptor signaling pathway, and NK cell-mediated cytotoxicity pathway. Conclusion:These analyses suggest BTLA is a potential factor for extended survival and closely related to CD8 T cells, Tfh cells, B cells, and NK cells in CRC. We summarize these results that BTLA can be used as a prognostic biomarker and might contribute to developing novel CRC immunological treatment strategies.
Project description:We analyzed gene expression profiles of young and aged mouse CD8+ T cells specific for the nucleoprotein (NP) of influenza A/PR8/34 virus. CD8+ T cells were stimulated either by the NP antigen expressed in its native form or fused into the herpes virus (HSV)-1 glycoprotein D (gD) protein, which blocks signaling through the immunoinhibitory B and T lymphocyte attenuator (BTLA) and CD160 pathways. We show that NP-specific CD8+ T cells from aged mice exhibit numerous differences in gene expression compared to NP-specific CD8+ T cells from young mice, including a significant reduction of expression in genes involved in T cell receptor (TcR) and CD28 signaling. We also show that these changes can be reversed in a sub-population (~50%) of the aged mice by a BTLA/CD160 checkpoint blockade. These results suggest that BTLA/CD160 checkpoint blockade has potential value as a vaccine additive to induce better CD8+ T cell responses in the aged.