Siglecs as Immune Cell Checkpoints in Disease.

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are expressed on the majority of white blood cells of the immune system and play critical roles in immune cell signaling. Through recognition of sialic acid-containing glycans as ligands, they help the immune system distinguish between self and nonself. Because of their restricted cell type expression and roles as checkpoints in immune cell responses in human diseases such as cancer, asthma, allergy, neurodegeneration, and autoimmune diseases they have gained attention as targets for therapeutic interventions. In this review we describe the Siglec family, its roles in regulation of immune cell signaling, current efforts to define its roles in disease processes, and approaches to target Siglecs for treatment of human disease.

Neuropilin-1 is a T cell memory checkpoint limiting long-term antitumor immunity.

Robust CD8+ T cell memory is essential for long-term protective immunity but is often compromised in cancer, where T cell exhaustion leads to loss of memory precursors. Immunotherapy via checkpoint blockade may not effectively reverse this defect, potentially underlying disease relapse. Here we report that mice with a CD8+ T cell-restricted neuropilin-1 (NRP1) deletion exhibited substantially enhanced protection from tumor rechallenge and sensitivity to anti-PD1 immunotherapy, despite unchanged primary tumor growth. Mechanistically, NRP1 cell-intrinsically limited the self-renewal of the CD44+PD1+TCF1+TIM3- progenitor exhausted T cells, which was associated with their reduced ability to induce c-Jun/AP-1 expression on T cell receptor restimulation, a mechanism that may contribute to terminal T cell exhaustion at the cost of memory differentiation in wild-type tumor-bearing hosts. These data indicate that blockade of NRP1, a unique 'immune memory checkpoint', may promote the development of long-lived tumor-specific Tmem that are essential for durable antitumor immunity.

Antigen and checkpoint receptor engagement recalibrates T cell receptor signal strength.

How T cell receptor (TCR) signal strength modulates T cell function and to what extent this is modified by immune checkpoint blockade (ICB) are key questions in immunology. Using Nr4a3-Tocky mice, we characterized early quantitative and qualitative changes that occur in CD4+ T cells in relation to TCR signaling strength. We captured how dose- and time-dependent programming of distinct co-inhibitory receptors rapidly recalibrates T cell activation thresholds and visualized the immediate effects of ICB on T cell re-activation. Our findings reveal that anti-PD1 immunotherapy leads to an increased TCR signal strength. We defined a strong TCR signal metric of five genes upregulated by anti-PD1 in T cells (TCR.strong), which was superior to a canonical T cell activation gene signature in stratifying melanoma patient outcomes to anti-PD1 therapy. Our study therefore reveals how analysis of TCR signal strength-and its manipulation-can provide powerful metrics for monitoring outcomes to immunotherapy.

Next generation of immune checkpoint molecules in maternal-fetal immunity.

Successful pregnancy is a unique situation requires the maternal immune system to recognize and tolerate a semi-identical fetus and allow normal invasion of trophoblast cells. Although efforts have been made, the deep mechanisms of the maternal-fetal crosstalk have not yet been fully deciphered. Immune checkpoint molecules (ICMs) are a group of negative modulators of the immune response that avoid immune damage. They have been extensively studied in the fields of oncology and transplantation, while the latest evidence suggests that they are closely associated with pregnancy outcomes via multiple inhibitory mechanisms. Although studies have mostly demonstrated the regulatory role of the well-known PD-1, CTLA-4 at the maternal-fetal interface, what is unique about the newly discovered multiple ICMs remains a mystery. Here, we review the latest knowledge on ICMs, focusing on the first generation of checkpoints (PD-1, CTLA-4) and the next generation (Tim-3, Tigit, Lag-3, VISTA) highlighting their immunoregulatory roles in maternal-fetal tolerance and decidual vascular remodeling, and their involvement in pathological pregnancies. The content covers three aspects: the characteristics they possess, the dynamic expression profile of their expression at the maternal-fetal interface, and their involvement in pathological pregnancy. In immunotherapy strategies for pregnancy complications, upregulation of immune checkpoints may play a role. Meanwhile, the impact on pregnancy outcomes when using ICMs in clinical cancer treatment during pregnancy is a topic worth exploring. These may serve as a guide for future basic research and clinical applications of maternal-fetal immunity.

ADAR-mediated RNA editing regulates PVR immune checkpoint in colorectal cancer.

Recent studies have revealed that tumor immunotherapy resistance is influenced by ADAR-mediated RNA editing, but its targets remain unelucidated. Our current study identified the poliovirus receptor (PVR) oncogene, which encodes an immune checkpoint in colorectal cancer (CRC), as a potential target for RNA editing. We performed transcriptome sequencing analysis and experimental validation in two Chinese CRC cohorts. PVR and ADAR expressions significantly increased in CRC tumors and showed positive correlations in both cohorts, coupled with upregulated PVR RNA editing in CRC tumors. Manipulation of ADAR expression by over-expression or knockdown substantially changed PVR expression and RNA editing in HTC116 CRC cells. Luciferase reporter and actinomycin D assays further revealed that RNA editing in PVR 3'-UTR could upregulate PVR RNA expression, probably by increasing the RNA stability. By increasing PVR expression, ADAR-mediate RNA editing might contribute to tumor- and immune-related gene functions and pathways in CRC. Moreover, a signature combining PVR RNA editing and expression showed promising predictive performance in CRC diagnosis in both Chinese CRC cohorts. Our findings thus highlight the importance of ADAR-mediated RNA editing in PVR up-regulation in CRC tumors and provide new insight into the application of PVR RNA editing as a novel diagnostic biomarker for CRC.

The role of extracellular vesicle immune checkpoints in cancer.

Immune checkpoints (ICPs) play a crucial role in regulating the immune response. In the tumor, malignant cells can hijack the immunosuppressive effects of inhibitory ICPs to promote tumor progression. Extracellular vesicles (EVs) are produced by a variety of cells and contain bioactive molecules on their surface or within their lumen. The expression of ICPs has also been detected in EVs. In vitro and in vivo studies have shown that extracellular vesicle immune checkpoints (EV ICPs) have immunomodulatory effects and are involved in tumor immunity. EV ICPs isolated from the peripheral blood of cancer patients are closely associated with the tumor progression and the prognosis of cancer patients. Blocking inhibitory ICPs has been recognized as an effective strategy in cancer treatment. However, the efficacy of immune checkpoint inhibitors (ICIs) in cancer treatment is hindered by the emergence of therapeutic resistance, which limits their widespread use. Researchers have demonstrated that EV ICPs are correlated with clinical response to ICIs therapy and were involved in therapeutic resistance. Therefore, it is essential to investigate the immunomodulatory effects, underlying mechanisms, and clinical significance of EV ICPs in cancer. This review aims to comprehensively explore these aspects. We have provided a comprehensive description of the cellular origins, immunomodulatory effects, and clinical significance of EV ICPs in cancer, based on relevant studies.

Mutual exclusivity and co-occurrence patterns of immune checkpoints indicate NKG2A relates to anti-PD-1 resistance in gastric cancer.

BACKGROUND: An increasing number of clinical studies have begun to explore combination strategies with immune checkpoint inhibitors, aiming to present new opportunities for overcoming anti-PD-1 treatment resistance in gastric cancer. Unfortunately, the exploration of certain immune checkpoint inhibitor combination strategies has yielded suboptimal results. Therefore, it is necessary to comprehensively analyze the expression patterns of immune checkpoints and identify optimal combination regimens of anti-PD-1 inhibitors with other immune checkpoint inhibitors. METHODS: Leveraging single-cell RNA sequencing (scRNA-seq) and multivariate linear regression interaction models, we dissected the immune checkpoint expression characteristics of CD8+ T cells in gastric cancer and the immune checkpoint expression pattern (ICEP) mediating anti-PD-1 treatment resistance. Furthermore, we employed transcription factor analysis and CellOracle to explore the transcriptional regulatory mechanisms governing CD8+ T cell differentiation fates. Finally, we utilized Nichenet and spatial transcriptomic analysis to investigate the spatial expression patterns of immune checkpoints. RESULTS: Interaction analysis indicated that, among the known immune checkpoints, co-expression of NKG2A and PD-1 might exert a more profound inhibitory effect on the proliferative capacity of CD8+ T cells. The co-expression analysis revealed differential co-expression pattern of PD-1 and NKG2A, defined as ICEP1 (CD8+ T cells co-expressing PD-1, CTLA-4, TIGIT, LAG-3 or CD38) and ICEP2 (CD8+ T cells solely expressing NKG2A or co-expressing with other immune checkpoints), reflecting the co-occurrence pattern of PD-1 and the mutual exclusivity of NKG2A. Further, these two ICEP CD8+ T cell subsets represented distinct CD8+ T cell differentiation fates governed by MSC and RUNX3. Notably, ICEP2 CD8+ T cells were associated with anti-PD-1 therapy resistance in gastric cancer. This phenomenon may be attributed to the recruitment of LGMN+ macrophages mediated by the CXCL16-CXCR6 signaling pathway. CONCLUSION: This study unveiled two distinct ICEPs and the mutually exclusivity and co-occurrence characteristics of CD8+ T cells in gastric cancer. The ICEP2 CD8+ T cell subset, highly expressed in gastric cancer patients resistant to anti-PD-1 therapy, may be recruited by LGMN+ macrophages through CXCL16-CXCR6 axis. These findings provide evidence for NKG2A as a novel immunotherapeutic target in gastric cancer and offer new insights into combination strategies for immune checkpoint inhibitors in gastric cancer.

Soluble form of immune checkpoints in autoimmune diseases.

Immune checkpoints are essential regulators of immune responses, either by activating or suppressing them. Consequently, they are regarded as pivotal elements in the management of infections, cancer, and autoimmune disorders. In recent years, researchers have identified numerous soluble immune checkpoints that are produced through various mechanisms and demonstrated biological activity. These soluble immune checkpoints can be produced and distributed in the bloodstream and various tissues, with their roles in immune response dysregulation and autoimmunity extensively documented. This review aims to provide a thorough overview of the generation of various soluble immune checkpoints, such as sPD-1, sCTLA-4, sTim-3, s4-1BB, sBTLA, sLAG-3, sCD200, and the B7 family, and their importance as indicators for the diagnosis and prediction of autoimmune conditions. Furthermore, the review will investigate the potential pathological mechanisms of soluble immune checkpoints in autoimmune diseases, emphasizing their association with autoimmune diseases development, prognosis, and treatment.

Redundancy and absurd names in immunology.

In this short review, examples of unnecessary multiple names of cell membrane molecules, for example, immune checkpoints and cytokines, are presented. Moreover, ridiculous or inaccurate names, such as 'Regulated on activation, normal T-cell expressed and secreted' and 'tissue factor', are discussed.

Bioinformatic analysis of the role of immune checkpoint genes and immune infiltration in the pathogenesis and development of premature ovarian insufficiency.

PURPOSE: With advances in immunology, increasing evidence suggests that immunity is involved in premature ovarian insufficiency (POI) pathogenesis. This study investigated the roles of immune checkpoint genes and immune cell infiltration in POI pathogenesis and development. METHODS: The GSE39501 dataset and immune checkpoint genes were obtained from the Gene Expression Omnibus database and related literature. The two datasets were intersected to obtain immune checkpoint-related differentially expressed genes (ICRDEGs), which were analyzed using Gene Ontology and Kyoto Encyclopedia of Gene and Genomes enrichment analysis, weighted correlation network analysis, protein-protein interaction and related microRNAs, transcription factors, and RNA binding proteins. The immune cell infiltration of ICRDEGs was explored, and receiver operating characteristic curves were used to validate the diagnostic value of ICRDEGs in POI. RESULTS: We performed ICRDEG functional enrichment analysis and found that these genes were closely related to immune processes, such as T cell activation. Specifically, they are enriched in various biological processes and pathways, such as cell adhesion molecule and T cell receptor signaling pathways. Weighted correlation network analysis identified seven hub genes: Cd200, Cd274, Cd28, neurociliary protein-1, Cd276, Cd40lg, and Cd47. Furthermore, we identified 112 microRNAs, 17 RNA-binding proteins, and 101 transcription factors. Finally, immune infiltration analysis showed a clear positive correlation between hub genes and multiple immune cell types. CONCLUSION: Bioinformatic analysis identified seven potential ICRDEGs associated with POI, among which the immune checkpoint molecules CD200 and neurociliary protein-1 may be involved in the pathogenesis of POI.

Immune Checkpoints in Endometriosis-A New Insight in the Pathogenesis.

Endometriosis (EMS) is an oestrogen-dependent, chronic disease affecting women of a reproductive age. One of the important factors involved in the development of this disease is the complex disorders associated with the functioning of the immune system. Recent evidence has shown that EMS development is associated with changes in systemic and local immunity, including functional disturbances of effector and antigen-presenting cells. One of the reasons for immune imbalance can be the improper expression of immune checkpoints (ICPs). ICPs and their ligands are responsible for maintaining self-tolerance and the modulation of the initiation, duration, and magnitude of the immune response of effector cells in normal tissues to avoid tissue damage. Considering the complex nature of co-stimulatory or co-inhibitory ICPs and the signalling between effector cells and APCs, we hypothesise that changes in cells' activity caused by ICPs may lead to serious immune system disturbances in patients with endometriosis. Moreover, both upregulation and downregulation in the expression of ICPs may be implicated in this process, including the reduced activity of effector cells against endometrial implants and disturbances in the antigen-presenting process. In this narrative review, we discuss, for the first time, key findings from the emerging literature, describing the associations between ICPs and their possible implication in the pathogenesis of endometriosis.

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.

Differential Immune Checkpoint Protein Expression in HNSCC: The Role of HGF/MET Signaling.

Although inhibitors targeting the PD1/PD-L1 immune checkpoint are showing comparably good outcomes, a significant percentage of head and neck squamous cell carcinoma (HNSCC) patients do not respond to treatment. Apart from using different treatment strategies, another possibility would be to target other immune checkpoints operating in these non-responding tumors. To obtain an overview of which checkpoint ligands are expressed on HNSCC tumor cells and if these ligands are affected by HGF/MET signaling, we used mRNA sequencing and antibody-based techniques for identifying checkpoint ligands in six HNSCC tumor cell lines. Furthermore, we compared our results to mRNA sequencing data. From the checkpoint ligands we investigated, VISTA was expressed the highest at the RNA level and was also the most ubiquitously expressed. PD-L2 and B7-H3 were expressed comparably lower and were not present in all cell lines to the same extent. B7-H4, however, was only detectable in the Detroit 562 cell line. Concerning the effect of HGF on the ligand levels, PD-L2 expression was enhanced with HGF stimulation, whereas other checkpoint ligand levels decreased with stimulation. B7-H4 levels in the Detroit 562 cell line drastically decreased with HGF stimulation. This is of interest because both the checkpoint ligand and the growth factor are reported to be connected to epithelial-mesenchymal transition in the literature.

The role of metabolic checkpoint regulators in B cell survival and transformation.

In response to mitogenic stimulation, B cells activate different pro-anabolic signaling pathways such as c-Myc- and mTORC1-dependent networks to satisfy the energetic demands of biomass synthesis and proliferation. In order to preserve viability and function, cell growth cannot progress unchecked and must be adjusted according to the availability of nutrients. Nutrient-sensing proteins such as AMPK antagonize mTORC1 activity in response to starvation. If pro-anabolic signaling pathways are aberrantly activated, B cells may lack the metabolic capacity to accommodate their energetic needs, which can lead to cell death. On the other hand, metabolic hyperactivation is a salient feature of cancer cells, suggesting that mechanisms exist, which allow B cells to cope with metabolic stress. The aim of this review is to discuss how B cells respond to a mismatch between energy supply and demand and what the consequences are of metabolic dysregulation in normal and malignant B cells.

Genetic Screening for Novel Regulators of Immune Checkpoint Molecules.

Inhibitory and stimulatory immune checkpoint molecules play important roles in regulating immune responses. An increasing number of these immune regulators are currently being evaluated as targets in putative anti-cancer therapies. Recently, sophisticated genetic screens have been performed to increase our understanding of immune checkpoint pathways and their immunomodulatory regulators. Here, we summarize novel insights obtained by these screens and discuss new directions to advance possible strategies to treat malignancies.

Cbl-b Is Upregulated and Plays a Negative Role in Activated Human NK Cells.

The E3 ubiquitin ligase Cbl-b has been characterized as an intracellular checkpoint in T cells; however, the function of Cbl-b in primary human NK cells, an innate immune anti-tumor effector cell, is not well defined. In this study, we show that the expression of Cbl-b is significantly upregulated in primary human NK cells activated by IL-15, IL-2, and the human NK cell-sensitive tumor cell line K562 that lacks MHC class I expression. Pretreatment with JAK or AKT inhibitors prior to IL-15 stimulation reversed Cbl-b upregulation. Downregulation of Cbl-b resulted in significant increases in granzyme B and perforin expression, IFN-γ production, and cytotoxic activity against tumor cells. Collectively, we demonstrate upregulation of Cbl-b and its inhibitory effects in IL-15/IL-2/K562-activated human NK cells, suggesting that Cbl-b plays a negative feedback role in human NK cells.

Impact of human papillomavirus on the tumor microenvironment in oropharyngeal squamous cell carcinoma.

Increasing evidence has elucidated the clinicopathological significance of tumor microenvironment (TME) cells. However, TME differences associated with human papillomavirus (HPV) infection in oropharyngeal squamous cell carcinoma (OPSCC) have not been well characterized. In our study, we comprehensively determined the TME infiltration patterns in 315 OPSCC patients, and systematically correlated the TME phenotypes with genomic characteristics and clinical features of OPSCCs. In this way, we observed the enrichment of high endothelial cells and adaptive immune cells in HPV-positive (HPV+) OPSCCs, in contrast to the enrichment of fibroblasts and capillary endothelial cells in HPV- negative (HPV-) OPSCCs. By focusing on immune checkpoint genes, we constructed a coexpression network using genes that were differentially expressed between HPV+ and HPV- OPSCCs. Functional analysis of the network indicated that HPV+ OPSCCs had elevated immune activities by promoting adaptive immune response and suppressing activities related to extracellular matrix organization. Subsequently, clinical analysis showed that identified TME-relevant genes were closely associated with the prognosis and therapy response in OPSCC. Importantly, results from the TME analysis were further validated using an independent OPSCC cohort.

An aging-related signature predicts favorable outcome and immunogenicity in lung adenocarcinoma.

Aging has been demonstrated to play vital roles in the prognosis and treatment efficacy of cancers, including lung adenocarcinoma (LUAD). This novel study aimed to construct an aging-related risk signature to evaluate the prognosis and immunogenicity of LUAD. Transcriptomic profiles and clinical information were collected from a total of 2518 LUAD patients from 12 independent cohorts. The risk signature was developed by combining specific gene expression with the corresponding regression coefficients. One cohort treated with the immune checkpoint inhibitor (ICI) was also used. Subsequently, a risk signature was developed based on 21 aging-related genes. LUAD patients with low-risk scores exhibited improved survival outcomes in both the discovery and validation cohorts. Further immunology analysis revealed elevated lymphocyte infiltration, decreased infiltration of immune-suppressive cells, immune response-related pathways, and favorable ICI predictor enrichment in the low-risk subgroup. Genomic mutation exploration indicated the enhanced mutation burden and higher mutation rates in significantly driver genes of TP53, KEAP1, SMARCA4, and RBM10 were enriched in patients with a low-risk signature. In the immunotherapeutic cohort, it was observed that low-risk aging scores were markedly associated with prolonged ICI prognosis. Overall, the estimated aging signature proved capable of evaluating the prognosis, tumor microenvironment, and immunogenicity, which further provided clues for tailoring prognosis prediction and immunotherapy strategies, apart from promoting individualized treatment plans for LUAD patients.

Increased TIGIT expressing NK cells with dysfunctional phenotype in AML patients correlated with poor prognosis.

AML is the most common blood cancer in adults with a high relapse and an overall poor survival rate. NK cells have been demonstrated to have the capacity to eradicate AML blast, and an impaired NK cell function is involved in AML development and progression. Immune checkpoints are involved in immune escape in various cancers. Immune checkpoints blockade therapy mainly aimed to unleash CD8+T cells function, but NK cells have emerged as new target. However, immune checkpoints profile on NK cells has not been observed in AML patients. Here, we studied the immune checkpoints expression of NK cells from AML patients at initial diagnosis and found increased PD-1, TIGIT and TIM-3 expression compared to NK cells from healthy donors. Further analysis showed that TIGIT expressing NK cells from AML patients had a dysfunctional phenotype, as TIGIT+NK cells exhibit lower antileukemia effect, cytokine production and degranulation compared to TIGIT-NK cells. TIGIT blockade could significantly enhance the function of NK cells. Moreover, AML patients with high frequency of TIGIT+NK cells had higher frequency of poor prognosis risk. Further analysis found that IL-10 upregulated TIGIT expression on NK cells. Thus, TIGIT blockade alone or in combination with other therapy might be potential strategy to treat AML.

CD155 expression impairs anti-PD1 therapy response in non-small cell lung cancer.

CD155 is an immune checkpoint protein expressed in tumor cells that interacts with its ligand TIGIT, and inhibition of this point presents a new and novel way for cancer therapy. At present, whether the expression of CD155 affects the response to anti(α)-PD1 treatment in non-small cell lung cancer (NSCLC) patients is unclear. This observational study characterizes the expression of CD155 in NSCLC patients and its responses to PD1 inhibitors. We retrospectively detected the expression of CD155 and tumor-infiltrated lymphocyte (TIL) TIGIT by immunohistochemistry in advanced NSCLC patients who had received αPD1 therapy. The patients with CD155 positive had a significantly worse response to αPD1 therapy compared with CD155-negative patients (ORR: 25.6% vs 54.8%, P < 0.01; median PFS: 5.1 vs 7.1 months, HR = 2.322; 95% CI 1.396-3.861, P = 0.001). This effect is more prominent in PD-L1 positive patients. In PD-L1-positive patients, CD155 expression is associated with a poor response to αPD1 therapy in both LUAC (lung adenocarcinoma) and LUSC (lung squamous cell carcinoma); meanwhile, the expression of CD155 was associated with a poor response to the first-line αPD1 therapy, posterior-line αPD1 therapy, and αPD1 combination therapy. Furthermore, the expression of TIGIT was not correlated with the therapeutic effect of αPD1. Our pilot study suggests that CD155 expression attenuates the therapeutic effect of αPD1 therapy and is associated with a higher risk of progression. The CD155 pathway may be a promising immunotherapeutic target and simultaneously targeting CD155/TIGIT and PD1/PD-L1 can improve the effect of immunotherapy.