Expression Of BTLA Research Articles

Circulating B Cells Display Differential Immune Regulatory Molecule Expression in Granulomatosis with Polyangiitis.

Granulomatosis with polyangiitis (GPA) is a B cell-mediated, relapsing, autoimmune disease. There is a need for novel therapeutic approaches and relapse markers to achieve durable remission. B cells express immune regulatory molecules that modulate their activation and maintain tolerance. While recent studies show dysregulation of these molecules in other autoimmune diseases, data on their expression in GPA are limited. This study aimed to map the expression of surface immune regulatory molecules on circulating B cell subsets in GPA and correlate their expression with clinical parameters. Immune regulatory molecule expression on circulating B cell subsets was comprehensively examined in active GPA (n=16), GPA in remission (n=16), and healthy controls (HCs, n=16) cross-sectionally using a 35-color B cell-specific spectral flow cytometry panel. Our supervised and unsupervised in-depth analysis revealed differential expression of inhibitory and stimulatory immune molecules on distinct B cell populations in GPA, with the most notable differences observed in active GPA. These differences include the upregulation of FcγRIIB on non-mature B cells, downregulation of CD21 and upregulation of CD86 on antigen-experienced B cells, and elevated CD22 expression on various populations. Additionally, we found a strong association between FcγRIIB, BTLA, and CD21 expression on specific B cell populations and disease activity in GPA. Together, these findings provide novel insights into the immune regulatory molecule expression profile of B cells in GPA, and could potentially form the foundation for new therapeutic approaches and disease monitoring markers.

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.

Pan-cancer B- and T-cell transcriptome analysis of CXCL13 as a predictive marker for immune checkpoint inhibitor response.

2626 Background: Maturation of lymphoid structures and tumor-antigen presentation via B cells in the tumor microenvironment (TME) have been recognized as factors in tumor immunity. The presence of B cells and tertiary lymphoid structures appears to result in better prognosis in patients with cancer but their role in response to immunotherapy remains subject to research. This study aimed to leverage the transcriptome expression of B and T cell markers and elucidate their impact on response to immune checkpoint inhibitors (ICIs). Methods: Among 514 patients with cancer included in the Profile-Related Evidence determining Individualized Cancer therapy study (NCT02478931), 208 with advanced cancer and available date treated with ICIs were analyzed. Patients were divided into responders (progression-free survival [PFS] after initiation of ICI therapy: 6 or more months) and non-responders (PFS: less than 6 months). Transcriptome expression of 34 immunoregulatory markers associated with B and T cells was compared between these groups using odds ratios (ORs) of “High” transcriptome expression [“High” (75-100 percentile), “Intermediate” (25-74), and “Low” (0-24), rank compared to 735 controls] of each immune marker for immunotherapy responders. A logistic regression model was used to perform a multivariable analysis of ORs. Clinical characteristics between responders and non-responders were summarized, and rate of “High” transcriptome expression of identified markers contributing to immunotherapy response was summarized by cancer type. Results: In total, 82 and 126 patients were classified into responder and non-responder groups. Median age was 65.0 and 61.0, and women were 58.5% (48/82) and 54.8% (69/126) in each group, respectively. Cancer type in both groups was well balanced. In univariate analysis, “High” transcriptome expression of CXCL13, CD3, BTLA, CTLA-4, and PD-1 was significantly more frequent in the responder than non-responder group. Cluster heatmaps in both groups revealed more B and T cell enriched population in the responder group. In multivariable analysis, patients with “High” CXCL13 expression were more likely to be immunotherapy responders (OR: 3.91, 95% confidence interval [CI]=1.02-15.04, p=0.044). High CXCL13 transcriptome expression was most common in head and neck (25%, 3/12), breast (22.4%, 11/49), neuroendocrine (20%, 3/15), and lung (20%, 4/20) tumors. Conclusions: This pan-cancer analysis found an association between high CXCL13 mRNA expression and better response to ICI therapy. CXCL13 role in promoting a lymphoid structure in the TME by facilitating B-cell recruitment could enable anti-tumor immunity, resulting in better response to immunotherapy. This indicates that dissection of not only T cell but also B cell regulatory factors is necessary to cancer immunotherapy response and resistance mechanisms. Clinical trial information: NCT02478931 .

The BTLA-HVEM axis restricts CAR T cell efficacy in cancer.

The efficacy of T cell-based immunotherapies is limited by immunosuppressive pressures in the tumor microenvironment. Here we show a predominant role for the interaction between BTLA on effector T cells and HVEM (TNFRSF14) on immunosuppressive tumor microenvironment cells, namely regulatory T cells. High BTLA expression in chimeric antigen receptor (CAR) T cells correlated with poor clinical response to treatment. Therefore, we deleted BTLA in CAR T cells and show improved tumor control and persistence in models of lymphoma and solid malignancies. Mechanistically, BTLA inhibits CAR T cells via recruitment of tyrosine phosphatases SHP-1 and SHP-2, upon trans engagement with HVEM. BTLA knockout thus promotes CAR signaling and subsequently enhances effector function. Overall, these data indicate that the BTLA-HVEM axis is a crucial immune checkpoint in CAR T cell immunotherapy and warrants the use of strategies to overcome this barrier.

B- and T-Lymphocyte Attenuator in Systemic Lupus Erythematosus Disease Pathogenesis.

B- and T-lymphocyte attenuator (BTLA; CD272) is an immunoglobulin superfamily member and part of a family of checkpoint inhibitory receptors that negatively regulate immune cell activation. The natural ligand for BTLA is herpes virus entry mediator (HVEM; TNFRSF14), and binding of HVEM to BTLA leads to attenuation of lymphocyte activation. In this study, we evaluated the role of BTLA and HVEM expression in the pathogenesis of systemic lupus erythematosus (SLE), a multisystem autoimmune disease. Peripheral blood mononuclear cells from healthy volunteers (N = 7) were evaluated by mass cytometry by time-of-flight to establish baseline expression of BTLA and HVEM on human lymphocytes compared with patients with SLE during a self-reported flare (N = 5). High levels of BTLA protein were observed on B cells, CD4+, and CD8+ T cells, and plasmacytoid dendritic cells in healthy participants. HVEM protein levels were lower in patients with SLE compared with healthy participants, while BTLA levels were similar between SLE and healthy groups. Correlations of BTLA-HVEM hub genes' expression with patient and disease characteristics were also analyzed using whole blood gene expression data from patients with SLE (N = 1,760) and compared with healthy participants (N = 60). HVEM, being one of the SLE-associated genes, showed an exceptionally strong negative association with disease activity. Several other genes in the BTLA-HVEM signaling network were strongly (negative or positive) correlated, while BTLA had a low association with disease activity. Collectively, these data provide a clinical rationale for targeting BTLA with an agonist in SLE patients with low HVEM expression.

Everolimus treatment enhances inhibitory immune checkpoint molecules’ expression in monocyte-derived dendritic cells

BackgroundAntigen-specific T-cell immunity is provided by dendritic cells (DCs), which are specialized antigen-presenting cells. Furthermore, they establish a link between innate and adaptive immune responses. Currently, DC modification is a new approach for the therapy of several disorders. During solid organ transplantation, Everolimus, which is a mammalian target of rapamycin (mTOR) inhibitor, was initially utilized to suppress the immune system's functionality. Due to the intervention of Everolimus in various signaling pathways in cells and its modulatory properties on the immune system, this study aims to investigate the effect of treatment with Everolimus on the maturation and expression of immune checkpoint genes in monocyte-derived DCs. MethodsTo isolate monocytes from PBMCs, the CD14 marker was used via the MACS method. Monocytes were cultured and induced to differentiate into monocyte-derived DCs by utilizing GM-CSF and IL-4 cytokines. On the fifth day, immature DCs were treated with Everolimus and incubated for 24 h. On the sixth day, the flow cytometry technique was used to investigate the effect of Everolimus on the phenotypic characteristics of DCs. In the end, the expression of immune checkpoint genes in both the Everolimus-treated and untreated DCs groups was assessed using the real-time PCR method. ResultsThe findings of this research demonstrated that the administration of Everolimus to DCs led to a notable rise in human leukocyte antigen (HLA)-DR expression and a decrease in CD11c expression. Furthermore, there was a significant increase in the expression of immune checkpoint molecules, namely CTLA-4, VISTA, PD-L1, and BTLA, in DCs treated with Everolimus. ConclusionThe findings of this study show that Everolimus can target DCs and affect their phenotype and function in order to shift them toward a partially tolerogenic state. However, additional research is required to gain a comprehensive understanding of the precise impact of Everolimus on the activation status of DCs.

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

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. 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. 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. 68.91months, HR 4.43, p = 0.061). PD-L1 expression was associated with worse CSS (median 1.66 vs. 4.5years, HR 1.63, p = 0.652). For TIM-3, there were no significant associations with clinicopathological parameters and survival. 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.

88 ANB032, a novel BTLA agonist monoclonal antibody, inhibits T cell proliferation, reduces inflammatory cytokines, and down modulates BTLA expression on circulating T and B cells

88 ANB032, a novel BTLA agonist monoclonal antibody, inhibits T cell proliferation, reduces inflammatory cytokines, and down modulates BTLA expression on circulating T and B cells

Patient-derived lymphoma spheroids integrating immune tumor microenvironment as preclinical follicular lymphoma models for personalized medicine

BackgroundFollicular lymphoma (FL), the most common indolent non-Hodgkin’s Lymphoma, is a heterogeneous disease and a paradigm of the contribution of immune tumor microenvironment to disease onset, progression, and therapy resistance....

B- and T-lymphocyte attenuator could be a new player in accelerated atherosclerosis associated with chronic kidney disease.

In chronic kidney disease (CKD), cardiovascular morbi-mortality is higher than in general population. Atherosclerotic cardiovascular disease is accelerated in CKD, but specific CKD-related risk factors for atherosclerosis are unknown. CKD patients from the NEFRONA study were used. We performed mRNA array from blood of patients free from atheroma plaque at baseline, with (n=10) and without (n=10) de novo atherosclerotic plaque development 2 years later. Selected mRNA candidates were validated in a bigger sample (n=148). Validated candidates were investigated in vivo in an experimental model of CKD-accelerated atherosclerosis, and in vitro in murine macrophages. mRNA array analysis showed 92 up-regulated and 67 down-regulated mRNAs in samples from CKD patients with de novo plaque development. The functional analysis pointed to a paramount role of the immune response. The validation in a bigger sample confirmed that B- and T-lymphocyte co-inhibitory molecule (BTLA) down-regulation was associated with de novo plaque presence after 2 years. However, BTLA down-regulation was not found to be associated with atherosclerotic progression in patients with plaque already present at baseline. In a model of CKD-accelerated atherosclerosis, mRNA and protein expression levels of BTLA were significantly decreased in blood samples and atheroma plaques. Plaques from animals with CKD were bigger, had more infiltration of inflammatory cells, higher expression of IL6 and IL17 and less presence of collagen than plaques from control animals. Incubation of macrophages with rat uremic serum decreased BTLA expression. BTLA could be a potential biomarker or therapeutic target for atherosclerosis incidence in CKD patients.

TIGIT may Serve as a Potential Target for the Immunotherapy of Renal Cell Carcinoma.

This study aims to explore whether TIGIT is an effective target for the immunotherapy of renal cell cancer (RCC) with PD-1 as a positive control. The expression of TIGIT and PD-1 in RCC and peripheral blood mononuclear cells (PBMC) and the correlation between TIGIT and PD-1 are evaluated. The expression of TIGIT and PD-1 is inhibited, and then the proliferation, apoptosis, and migration are assessed. TIGIT expression is positively related to the expression of PDCD1, BTLA, ICOS, and FOXP3 (p<0.05). TIGIT expression in the PBMC, TIL, RCC, and adjacent normal tissues is higher than PD-1 expression. Blocking the TIGIT and PD-1 signaling pathways significantly inhibits the proliferation, migration, and invasion of RCC cells and promotes their apoptosis. These effects are more evident in TIGIT inhibitors than in PD-1 inhibitors. TIGIT inhibitor mainly regulates the expression of differential genes to achieve the reconstruction of immune killing and restore the killing effect on the RCC, and its mechanism by which TIGIT functions overlap that of PD-1 inhibitor. TIGIT may become a target for the immunotherapy of RCC, and there is a theoretical basis for the combination of TIGIT inhibitors and PD-1 inhibitors for the treatment of RCC.

Differentiation and traffic of IgM+ B cells between focal dark spots in skeletal muscle of Atlantic salmon, lymphoid and adipose tissues.

Focal dark spots (DS) in farmed Atlantic salmon fillets contain a significant number of B cells as revealed by the high abundance of immunoglobulin (Ig) transcripts in transcriptome data. The immune response in DS remains unknown while they represent a major problem in commercial aquaculture. Here, we characterized the diversity and clonal composition of B cells in DS. Sixteen gene markers of immune cells and antigen presentation were analyzed with RT-qPCR. All genes expression showed a positive correlation with DS area and intensity. The flatter the DS, the higher the expression of cd28, csfr, ctla, igt, and sigm, the lower expression of cd83 and btla, and the larger the cumulative frequency within DS. The expression of most of the analyzed immune genes, including three Ig types and markers of B cells was lower in DS than in the lymphatic organs, head kidney and spleen, but significantly higher compared to skeletal muscle. High levels of ctla4 and cd28 in DS might indicate the recruitment of T cells. Sequencing of IgM repertoire (Ig-seq) assessed migration of B cells by co-occurrence of identical CDR3 sequences in different tissues. The combination of gene expression and Ig-seq revealed the presence of several stages of B cell differentiation in DS. B cells at the earliest stage, with high ratio of membrane to secretory IgM (migm and sigm), showed minor Ig repertoire overlap with other tissues. Further differentiation stage (increased sigm to migm ratio and high expression of pax5 and cd79) was associated with active movement of B cells from DS towards lymphatic organs and visceral fat. Traffic and expression of immune genes decreased at later stages. These B cells could be involved in a response directed against viruses, pathogenic or opportunistic bacteria in DS. Seven of eight fish were positive for salmon alphavirus, and levels were higher in DS than in unstained muscle. PCR with universal primers to the 16S rRNA gene did not detect bacteria in DS. Although the evolution of DS most likely implies local exposure to antigens, neither this nor previous studies have found a necessary association between DS and pathogens or self-antigens.

Prognostic Immune Effector Signature in Adult Acute Lymphoblastic Leukemia Patients Is Dominated by γδ T Cells.

The success of immunotherapy has highlighted the critical role of the immune microenvironment in acute lymphoblastic leukemia (ALL); however, the immune landscape in ALL remains incompletely understood and most studies have focused on conventional T cells or NK cells. This study investigated the prognostic impact of circulating γδ T-cell alterations using high-dimensional analysis in a cohort of newly diagnosed adult ALL patients (10 B-ALL; 9 Philadelphia+ ALL; 9 T-ALL). Our analysis revealed common alterations in CD8+ T cells and γδ T cells of relapsed patients, including accumulation of early stage differentiation and increased expression of BTLA and CD73. We demonstrated that the circulating γδ T-cell signature was the most discriminating between relapsed and disease-free groups. In addition, Vδ2 T-cell alterations strongly discriminated patients by relapse status. Taken together, these data highlight the role of ɣδ T cells in adult ALL patients, among whom Vδ2 T cells may be a pivotal contributor to T-cell immunity in ALL. Our findings provide a strong rationale for further monitoring and potentiating Vδ2 T cells in ALL, including in the autologous setting.

Exploring the potential of combination immune checkpoint strategies in non-small cell lung cancer (NSCLC).

9037 Background: The clinical outcomes for patients with NSCLC remain suboptimal despite recent advancements in anti-PD-1/PD-L1 immunotherapies. Thus, identification of rational combinations of novel immune checkpoint (IC) strategies is imperative. This study aimed to comprehensively characterize the expression of IC targets to inform drug development and design effective combinatorial strategies, to advance the search for optimized therapies for NSCLC. Methods: We analyzed the differential gene expression of IC targets in 537 primary lung adenocarcinoma (LUAD) samples and 59 normal lung tissue samples pre-immunotherapy receipt using RNA-sequencing data from The Cancer Genome Atlas. Gene expression of IC-targets including PDCD1 (PD-1), CD274 (PD-L1), CTLA4, TIGIT, LAG3, HAVCR2 (TIM-3), VSIR (VISTA), ICOS, CD276 (B7-H3), BTLA, IDO1, KLRD1 (CD94/NKG2A), NT5E (CD73), TNFRSF4 (OX-40), TNFRSF18 (GITR), and TNFRSF9 (4-1BB) were obtained. Differential gene expression was determined intra-tumorally by comparison of transcripts per million and inter-tumorally using the DESeq2 algorithm. Log2 fold change &gt;0.5 and false discovery rate adjusted P&lt;0.05 were used as criteria for significant differential expression. Hierarchical clustering was used to identify subsets of tumors with similar profiles of IC-targetable gene expression. Results: Tumors showed significantly increased expression of TNFRSF18 (GITR), NT5E (CD73), TNFRSF9 (4-1BB), LAG3, TIGIT, CTLA4, PD-1, BTLA, B7-H3, and OX-40 (Table 1). On the other hand, tumors exhibited significant downregulation of CD274 (PD-L1), HAVCR2 (TIM-3), ICOS, KLRD1 (CD94/NKG2A), and VSIR (VISTA). Hierarchical clustering revealed recurrent patterns of IC gene overexpression which were characterized by: 1) IDO1-hi, 2) CD276-mid + TNFRSF18-mid, 3) VSIR-hi, 4) CD276-hi, 5) NT5E-hi, and 6) CD276-mid + NT5E-mid. Interestingly, patterns of IC gene expression distinguish tumors from controls. Of note, high VSIR expression was specific to normal tissue rather than tumors. Conclusions: Our study identified many IC targetable genes that were significantly overexpressed in tumors. However, subsets of tumors showed distinct and segregable combinations of overexpressed IC genes. These results suggest heterogeneous mechanisms of immune evasion in NSCLC, which may explain suboptimal outcomes with anti-PD-1/PD-L1 based strategies in this patient population. Our findings emphasize the importance of biomarker-guided approaches for the optimal selection of ICs and the development of rational combinatorial immunotherapies in NSCLC.

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

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. 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. 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. 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.

617 ANB032, a novel BTLA agonist monoclonal antibody, inhibited T cell proliferation, reduced inflammatory cytokines, and down modulated BTLA expression on circulating T and B cells in phase 1, progresses into a phase 2 study in atopic dermatitis

617 ANB032, a novel BTLA agonist monoclonal antibody, inhibited T cell proliferation, reduced inflammatory cytokines, and down modulated BTLA expression on circulating T and B cells in phase 1, progresses into a phase 2 study in atopic dermatitis

BTLA dysregulation correlates with poor outcome and diminished T cell-mediated antitumor responses in chronic lymphocytic leukemia

Patients with chronic lymphocytic leukemia (CLL) progressively develop marked immunosuppression, dampening innate and adaptive-driven antitumor responses. However, the underlying mechanisms promoting immune exhaustion are largely unknown. Herein, we provide new insights into the role of BTLA/HVEM axis promoting defects in T cell-mediated responses against leukemic cells. Increased expression of BTLA, an inhibitory immune checkpoint, was detected on the surface of CD4 + and CD8 + T lymphocytes in patients with CLL. Moreover, high levels of BTLA on CD4 + T cells correlated with diminished time to treatment. Signaling through BTLA activation led to decreased IL-2 and IFN-γ production ex vivo, whereas BTLA/HVEM binding disruption enhanced IFN-γ + CD8 + T lymphocytes. Accordingly, BTLA blockade in combination with bispecific anti-CD3/anti-CD19 antibody promoted CD8 + T cell-mediated anti-leukemic responses. Finally, treatment with an anti-BLTA blocking monoclonal antibody alone or in combination with ibrutinib-induced leukemic cell depletion in vitro. Altogether, our data reveal that BTLA dysregulation has a prognostic role and is limiting T cell-driven antitumor responses, thus providing new insights about immune exhaustion in patients with CLL.

A lipid metabolism–based prognostic risk model for HBV–related hepatocellular carcinoma

BackgroundUp to 85% of hepatocellular carcinoma (HCC) cases in China can be attributed to infection of hepatitis B virus (HBV). Lipid metabolism performs important function in hepatocarcinogenesis of HBV–related liver carcinoma. However, limited studies have explored the prognostic role of lipid metabolism in HBV–related HCC. This study established a prognostic model to stratify HBV–related HCC based on lipid metabolisms.MethodsBased on The Cancer Genome Atlas HBV–related HCC samples, this study selected prognosis-related lipid metabolism genes and established a prognosis risk model by performing uni- and multi-variate Cox regression methods. The final markers used to establish the model were selected through the least absolute shrinkage and selection operator method. Analysis of functional enrichment, immune landscape, and genomic alteration was utilized to investigate the inner molecular mechanism involved in prognosis.ResultsThe risk model independently stratified HBV-infected patients with liver cancer into two risk groups. The low–risk groups harbored longer survival times (with P < 0.05, log–rank test). TP53, LRP1B, TTN, and DNAH8 mutations and high genomic instability occurred in high–risk groups. Low–risk groups harbored higher CD8 T cell infiltration and BTLA expression. Lipid–metabolism (including “Fatty acid metabolism”) and immune pathways were significantly enriched (P < 0.05) in the low–risk groups.ConclusionsThis study established a robust model to stratify HBV–related HCC effectively. Analysis results decode in part the heterogeneity of HBV–related liver cancer and highlight perturbation of lipid metabolism in HBV–related HCC. This study’s findings could facilitate patients’ clinical classification and give hints for treatment selection.

Abstract PD2-10: MiR-150 expression in breast cancer cells attract infiltration of lymphocytes to tumor microenvironment and is associated with better survival of the patients

Abstract MicroRNA (miRNA) are known as a key player in tumor biology and is shown to epigenetically regulate a large number of protein-coding genes, including tumor-related genes. MiR-150, a hematopoietic cell-specific miRNA, has been suggested to have various effects on cell proliferation, differentiation, apoptosis, migration, and invasion. However, there has been no study that investigated the role of miR-150 in the tumor microenvironment (TME) of breast cancer patients. We hypothesized that miR-150 expressed in breast cancer cells attract infiltration of lymphocytes to tumor microenvironment and is associated with better survival of the patients. In silico analyses of 1961 breast cancer patients were performed using multiple independent large cohorts, and in vitro studies by overexpressing miR-150 using its mimic in triple negative breast cancer (TNBC) cell lines were conducted. We found that miR-150 expression in patient breast tumors were strongly correlated with immune-related gene set scores, Allograft rejection, IL6/JAK/STAT3 signaling, Interferon-γ response, Inflammatory response, IL2/STAT5 signaling, and complement, in Hallmark collection of gene set variation analyses consistently in both METABRIC and TCGA cohorts (all spearman’s rank correlation coefficient (r) &amp;gt; 0.50, all p &amp;lt; 0.01). MiR-150 expression was also strongly correlated with cytolytic activity (CYT) score in both cohorts (r = 0.824 and 0.786, respectively, both p &amp;lt; 0.01). Furthermore, miR-150 expression was significantly correlated with infiltrating fraction of CD8+ T cells (r = 0.799 and 0.525, respectively), CD4+ memory T cells (r = 0.759 and 0.656, respectively), dendritic cells (r = 0.735 and 0.696, respectively), and B cells (r = 0.759 and 0.576, respectively), as well as mRNA expression of major immune checkpoint molecules, including PD-1, CTLA4, IDO1, TIGIT, BTLA, and LAG3, in both cohorts (all r &amp;gt; 0.50, and all p &amp;lt; 0.01). MiR-150 expression in triple negative breast cancer (TNBC) was the highest among the subtypes (both p &amp;lt; 0.001). MiR-150 expression level was significantly correlated with Nottingham histologic grade (both p &amp;lt; 0.001). MiR-150 high tumor enriched not only immune-related gene sets but also apoptosis, KRAS signaling up, MTORC1, and p53 pathway by gene set enrichment analysis in both cohorts. High miR-150 expression patients were significantly associated with better overall survival (OS) in both cohorts (p &amp;lt; 0.001 and p = 0.030, respectively). Subgroup analysis revealed that a high miR-150 was associated with better OS in ER-positive/HER2-negative breast cancer in both cohorts (p = 0.002 and 0.044, respectively), and in TNBC in the METABRIC cohort (p = 0.006). Unexpectedly, overexpression of miR-150 by its mimic shown no significant effect on cell proliferation, migration, nor invasion consistently in two TNBC cell lines. On the other hand, overexpression of miR-150 by its mimic significantly attracted Jurkart immortalized lymphocytes consistently in two cell lines, which effect was abolished by addition of miR-150 inhibitor. Finally, we found that mimic overexpression of miR-150 significantly expressed multiple cytokines and multiple inflammatory signaling pathways which at least partially explained the increased attraction of T cells in patient samples and in vitro condition. In conclusion, miR-150 expression in breast cancer cells may be associated with better patient survival outcomes by attracting and activating immune cells in breast cancer TME. Citation Format: Masanori Oshi, Aparna Maiti, Raj.G Vaghjiani, Rongrong Wu, Li Yan, Akimitsu Yamada, Nitai Hait, Takashi Ishikawa, Itaru Endo, Kazuaki Takabe, Yoshihisa Tokumaru. MiR-150 expression in breast cancer cells attract infiltration of lymphocytes to tumor microenvironment and is associated with better survival of the patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-10.

Improved MAIT cell functions following fecal microbiota transplantation for metastatic renal cell carcinoma.

Strategies to modify the gut microbiome in cancer patients using fecal microbiota transplantation (FMT) have gained momentum as a therapeutic intervention. However, how FMT impacts innate-like, antimicrobial T lymphocytes is unclear. In this study, we assessed peripheral blood (PB) mucosa-associated invariant T (MAIT) cell frequencies and functions in patients with metastatic renal cell carcinoma (mRCC) before and seven days after they received FMT as part of a clinical trial. We found comparable MAIT cell frequencies in healthy controls and mRCC patients. In contrast, γδ T cells exhibited a numerical decline in mRCC, which was partially reversed by FMT. We also found a significant increase in the PB CD4+ MAIT cell compartment of mRCC patients with or without FMT. Paired sample analyses revealed CD69 upregulation on MAIT cells accompanied by decreased PD-1 levels post-FMT. These changes were unique to MAIT cells as non-MAIT T lymphocytes showed either no trend or a trend in the opposite direction. Importantly, FMT did not render MAIT cells exhausted as also judged by their stable expression of TIM-3, LAG-3, BTLA, CTLA-4, TIGIT and VISTA. These findings were corroborated in functional assays in which MAIT cells were stimulated with MR1 ligands or with a combination of IL-12 and IL-18 to produce inflammatory cytokines and granzyme B. Indeed, when stimulated ex vivo with IL-12 and IL-18, MAIT cells mounted a more rigorous TNF-α response post-FMT. In conclusion, FMT improves MAIT cell functions, which should serve patients well in subsequent microbial challenges in the face of cancer-elicited immunosuppression. Trial Registration: https://clinicaltrials.gov/ Identifier: NCT04163289 (registration date: November 14, 2019).