Expression Of Checkpoint Markers Research Articles

Identification of Cellular Senescence-Related Critical Genes and Molecular Classification and Revealing the Drug-Resistant Therapeutic Effect of IGFBP2 in Chronic Myeloid Leukemia.

The occurrence and development of hematologic tumors are closely linked to cellular senescence. However, the molecular characteristics associated with this phenomenon in chronic myeloid leukemia (CML) have not been thoroughly investigated. The cellular senescence score was calculated using gene set variation analysis. Consensus clustering algorithm was used to identify the molecular subtypes associated with cellular senescence. Clinical samples were collected for sequencing analysis to verify the expression of critical cellular senescence-related genes (CSRG). The effect of targeted inhibition of IGFBP2 on the malignant phenotype of CML-resistant cells was studied by cell experiments. The cellular senescence score in CML samples was significantly lower compared to normal samples. Higher expression of immune checkpoint markers correlated with increased cellular senescence scores. We identified two distinct molecular subtypes (C1 and C2) related to cellular senescence. The C1 subtype exhibited enhanced metabolic function and DNA damage repair capacity, while the C2 subtype showed higher infiltration of immune effector cells and activity in immune-related signaling pathways. We also discovered a group of drugs that displayed significant sensitivity differences between these two molecular subtypes, with the C2 subtype showing greater responsiveness to immunotherapy. Four critical cellular senescence-related genes (CSRGs), namely IGFBP2, IL7R, PLAU, and SUN1 demonstrated high diagnostic value for CML. We validated the expression levels of these four genes using clinical samples and confirmed through cell experiments that targeted inhibition of IGFBP2 effectively suppressed proliferation of resistant CML cells, promoted apoptosis, and enhanced therapeutic sensitivity to imatinib. Our study conducted a comprehensive analysis on CSRG expression characteristics in CML and explored potential correlations between cellular senescence and immune function. The identification of molecular subtypes provides valuable insights into assessing individual patients' biological characteristics for guiding clinical treatment decisions. Additionally, IGFBP2has emerged as a promising therapeutic target for therapy-resistant cases of CML.

The carcinogenesis of esophageal squamous cell cancer is positively regulated by USP13 through WISP1 deubiquitination.

The objective was to determine whether USP13 stabilizes WISP1 protein and contributes to tumorigenicity and metastasis in ESCC through the Wnt/CTNNB1 signaling pathway. ESCC cell lines (KYSE150 and TE10) were treated with the proteasome inhibitor MG-132, followed by siRNA screening of deubiquitinases (DUBs) to identify regulators of WISP1. Mass spectrometry, immunoprecipitation, and in vitro functional assays were conducted to explore the interaction between USP13 and WISP1 and to assess the effects of USP13 downregulation on cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and apoptosis. Additionally, in vivo experiments using mouse models were performed to evaluate the impact of USP13 knockdown on tumor growth and metastasis. USP13 was identified as a key regulator of WISP1, stabilizing its protein levels through deubiquitination. Downregulation of USP13 resulted in reduced WISP1 protein stability, decreased cell proliferation, migration, and EMT, and increased apoptosis in vitro. In vivo, USP13 knockdown significantly inhibited tumor growth and lung metastasis. WISP1 overexpression in USP13-knockdown cells partially rescued these phenotypes, confirming the functional role of the USP13/WISP1 axis. Furthermore, knockdown of USP13 or WISP1 impaired the activation of the Wnt/CTNNB1 signaling pathway and reduced immune checkpoint marker expression, indicating a mechanism by which USP13 promotes immune evasion in ESCC. USP13 stabilizes WISP1 through deubiquitination, enhancing ESCC progression by activating the Wnt/CTNNB1 pathway and promoting immune evasion, making USP13 a potential therapeutic target in ESCC.

Inhibition of ATM or ATR in combination with hypo-fractionated radiotherapy leads to a different immunophenotype on transcript and protein level in HNSCC.

The treatment of head and neck tumors remains a challenge due to their reduced radiosensitivity. Small molecule kinase inhibitors (smKI) that inhibit the DNA damage response, may increase the radiosensitivity of tumor cells. However, little is known about how the immunophenotype of the tumor cells is modulated thereby. Therefore, we investigated whether the combination of ATM or ATR inhibitors with hypo-fractionated radiotherapy (RT) has a different impact on the expression of immune checkpoint markers (extrinsic), the release of cytokines or the transcriptome (intrinsic) of head and neck squamous cell carcinoma (HNSCC) cells. The toxic and immunogenic effects of the smKI AZD0156 (ATMi) and VE-822 (ATRi) in combination with a hypo-fractionated scheme of 2x5Gy RT on HPV-negative (HSC4, Cal-33) and HPV-positive (UM-SCC-47, UD-SCC-2) HNSCC cell lines were analyzed as follows: cell death (necrosis, apoptosis; detected by AnxV/PI), expression of immunostimulatory (ICOS-L, OX40-L, TNFSFR9, CD70) and immunosuppressive (PD-L1, PD-L2, HVEM) checkpoint marker using flow cytometry; the release of cytokines using multiplex ELISA and the gene expression of Cal-33 on mRNA level 48h post-RT. Cell death was mainly induced by the combination of RT with both inhibitors, but stronger with ATRi. Further, the immune phenotype of cancer cells, not dying from combination therapy itself, is altered predominantly by RT+ATRi in an immune-stimulatory manner by the up-regulation of ICOS-L. However, the analysis of secreted cytokines after treatment of HNSCC cell lines revealed an ambivalent influence of both inhibitors, as we observed the intensified secretion of IL-6 and IL-8 after RT+ATRi. These findings were confirmed by RNAseq analysis and further the stronger immune-suppressive character of RT+ATMi was enlightened. We detected the down-regulation of a central protein of cytoplasmatic sensing pathways of nucleic acids, RIG-1, and found one immune-suppressive target, EDIL3, strongly up-regulated by RT+ATMi. Independent of a restrictive toxicity, the combination of RT + either ATMi or ATRi leads to comprehensive and immune-modulating alterations in HNSCC. This includes pro-inflammatory signaling induced by RT + ATRi but also anti-inflammatory signals. These findings were confirmed by RNAseq analysis, which further highlighted the immune-suppressive nature of RT + ATMi.

Sirolimus reduces T cell cycling, immune checkpoint marker expression, and HIV-1 DNA in people with HIV

Key HIV cure strategies involve reversing immune dysfunction and limiting the proliferation of infected Tcells. We evaluate the safety of sirolimus, a mammalian target of rapamycin (mTOR) inhibitor, in people with HIV (PWH) and study the impact of sirolimus on HIV-1 reservoir size and HIV-1-specific immunity in a single-arm study of 20weeks of treatment in PWH on antiretroviral therapy (ART). Sirolimus treatment does not impact HIV-1-specific CD8 Tcell responses but leads to a significant decrease in CD4+ Tcell-associated HIV-1 DNA levels at 20weeks of therapy in the primary efficacy population (n= 16; 31% decline, p=0.008). This decline persists for at least 12weeks following cessation of the study drug. Sirolimus treatment also leads to a significant reduction in CD4+ Tcell cycling and PD-1 expression on CD8+ lymphocytes. These data suggest that homeostatic proliferation of infected cells, an important mechanism for HIV persistence, is an intriguing therapeutic target.

Expression of immune checkpoint markers in peripheral blood lymphocytes of women with fibroadenoma of the breast

Changes of the immune state in patients with fibroadenoma (FA) or other benign lesions of the breast, as well as the involvement of immune checkpoints in pathogenesis of these lesions, remain underexplored. The aim of this study was to compare the expression level of the key immune checkpoint markers CD279/PD-1, CD274/PD-L1, CD366/TIM3, and CD223/LAG3 in total circulating lymphocytes, T cell population as well as in its CD4 and CD8 subsets in peripheral blood from women with breast FA and healthy controls. Blood samples were taken from 12 women diagnosed with FA of the breast (aged 23-54 years, FA group) and 15 healthy women (aged 22-52 years, control group). Sample uptake was performed immediately before surgery, and samples were further analyzed by multicolor flow cytometry using monoclonal antibodies CD3-VioBlue, CD4/CD8-FITC, PD1-PE, PD-L1-PerCP-Cy.5.5, and TIM3/LAG3-APC. Each sample was incubated with 4 antibody combinations: CD3/CD4/PD1/PD-L1/TIM3, CD3/CD4/PD1/PD-L1/LAG3, CD3/CD8/PD1/PD-L1/TIM3, and CD3/CD8/PD1/PD-L1/LAG3. First, mono-expression of each of the 4 immune checkpoint markers was evaluated in the lymphocyte gate from both investigated groups. In FA samples, a significant increase in PD-L1 expression (assessed as percent of positive cells and fluorescence intensity change) was observed. Regarding expression of immune checkpoints in CD3+ T cells, along with significantly increased %PD-L1+, elevated numbers of PD1+T cells were detected. As for the differences in immune checkpoint expression changes between CD4+ and CD8+ T cell subsets, FA patient group demonstrated a more prominent increase in the amount of CD8+PD1+T cells relative to CD4 subset. The profiles of PD-L1 changes in CD4 and CD8 subpopulations were comparable showing, in both cases, a significant increase in FA sample group. We also analyzed changes in co-expression of any two immune checkpoint markers in CD4+ and CD8+ T cell subsets. The most noticeable was as increase in the prevalence of PD1+PD-L1+ phenotype in both T cell subpopulations from FA patients compared to the controls. With respect to co-expression of 3 checkpoint markers in CD4+ and CD8+ T cells, a significant increase in the percentage of PD1+PD-L1+TIM3+ cells among CD4 T helpers was found in FA. Thus, specific changes of T cell phenotype related to (co-)expression of immune checkpoint regulators may occur in systemic circulation of women with breast FA.

Immune landscape of oncohistone-mutant gliomas reveals diverse myeloid populations and tumor-promoting function

Histone H3-mutant gliomas are deadly brain tumors characterized by a dysregulated epigenome and stalled differentiation. In contrast to the extensive datasets available on tumor cells, limited information exists on their tumor microenvironment (TME), particularly the immune infiltrate. Here, we characterize the immune TME of H3.3K27M and G34R/V-mutant gliomas, and multiple H3.3K27M mouse models, using transcriptomic, proteomic and spatial single-cell approaches. Resolution of immune lineages indicates high infiltration of H3-mutant gliomas with diverse myeloid populations, high-level expression of immune checkpoint markers, and scarce lymphoid cells, findings uniformly reproduced in all H3.3K27M mouse models tested. We show these myeloid populations communicate with H3-mutant cells, mediating immunosuppression and sustaining tumor formation and maintenance. Dual inhibition of myeloid cells and immune checkpoint pathways show significant therapeutic benefits in pre-clinical syngeneic mouse models. Our findings provide a valuable characterization of the TME of oncohistone-mutant gliomas, and insight into the means for modulating the myeloid infiltrate for the benefit of patients.

Assessment of Immune Status in Patients with Mismatch Repair Deficiency Endometrial Cancer.

This study introduced a novel subtype classification method for endometrial cancer (EC) with mismatch repair deficiency (MMRd) by employing immune status and prognosis as the foundational criteria. The goal was to enhance treatment guidance through precise subtype delineation. Study Cohort: This study encompassed a cohort of 119 patients diagnosed with MMRd-EC between 2015 and 2022. Analyses using t-tests and Mann-Whitney U-tests were performed to assess prognostic markers and peripheral blood immune cell profiles in patients with MutS deficiency (MutS-d) versus those with MutL deficiency (MutL-d). Logistic regression analysis was used to identify independent risk factors. Bioinformatics Analysis: An online database was used to assess the prognostic implications, immune cell infiltration, and immune checkpoint involvement associated with the deficiency of MutS versus MutL in EC. Patients with MutL-d exhibited heightened risk factors, including elevated cancer grade and increased myometrial invasion, leading to poorer prognosis and shorter overall survival and progression-free survival. Regarding systemic immune status, patients with MutL-d demonstrated decreased peripheral blood lymphocyte percentage, lymphocyte count, and CD8+ T cell percentage. For local immunity, the infiltration of natural killer cells, CD8+ T cells, and cytotoxic T lymphocytes in the tumor tissue was reduced in patients with MutL-d. Additionally, patients with MutL-d exhibited lower expression of immune checkpoint markers. The composition of immune subtypes and survival outcomes also indicate that patients with MutL-d have a poorer immune status and prognosis than the patients with MutS-d. Patients with MMRd-EC can be subclassified according to MutS or MutL deficiency. Patients with MutS-d exhibited better immune status, prognosis, and immunotherapy benefits than those with MutL-d. These results can help guide patients to a more precise treatment.

KEAP1-Mutant Lung Cancers Weaken Anti-Tumor Immunity and Promote an M2-like Macrophage Phenotype.

Considerable advances have been made in lung cancer therapies, but there is still an unmet clinical need to improve survival for lung cancer patients. Immunotherapies have improved survival, although only 20-30% of patients respond to these treatments. Interestingly, cancers with mutations in Kelch-like ECH-associated protein 1 (KEAP1), the negative regulator of the nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor, are resistant to immune checkpoint inhibition and correlate with decreased lymphoid cell infiltration. NRF2 is known for promoting an anti-inflammatory phenotype when activated in immune cells, but the study of NRF2 activation in cancer cells has not been adequately assessed. The objective of this study was to determine how lung cancer cells with constitutive NRF2 activity interact with the immune microenvironment to promote cancer progression. To assess, we generated CRISPR-edited mouse lung cancer cell lines by knocking out the KEAP1 or NFE2L2 genes and utilized a publicly available single-cell dataset through the Gene Expression Omnibus to investigate tumor/immune cell interactions. We show here that KEAP1-mutant cancers promote immunosuppression of the tumor microenvironment. Our data suggest KEAP1 deletion is sufficient to alter the secretion of cytokines, increase expression of immune checkpoint markers on cancer cells, and alter recruitment and differential polarization of immunosuppressive macrophages that ultimately lead to T-cell suppression.

Deciphering m6A signatures in hepatocellular carcinoma: Single-cell insights, immune landscape, and the protective role of IGFBP3.

RNA m6 methyladenosine (m6A) modifications impact tumor biology and immune processes, particularly in hepatocellular malignant tumors. Using a consensus clustering algorithm on 371 hepatocellular carcinoma (HCC) samples, we identified three m6A-modified subtypes and correlated them with positive tumor microenvironment (TME) markers for distinct immune phenotypes. Stratifying patients based on m6A scores revealed a low presentation group with better immune penetration, lower tumor mutation load, and increased expression of immune checkpoint markers like CTLA-4 and PD-1, suggesting enhanced responsiveness to immunization therapy. A machine-learning model of 23 m6A genes was constructed. Single-cell analysis revealed a surprising enrichment of IGFBP3 in astrocytes, prompting the exploration of associated signaling pathways. Experimental verification shows that IGFBP3 is significantly enhanced in normal tissues, while immunohistochemical analysis shows that its expression is lower in tumor tissues, indicating its protective effect in HCC and a good prognosis. Importantly, high IGFBP3 expression is associated with better outcomes in patients receiving immunotherapy. Moreover, cytotoxic T lymphocyte (CTL) experiments have confirmed that high expression of IGFBP3 is associated with stronger T cell-killing ability. In summary, the comprehensive evaluation of m6A modification, immune characteristics, and single-cell analysis in this study not only revealed the TME of HCC but also made significant contributions to the progress of personalized HCC immunotherapy targeting IGFBP3. This study provides a solid theoretical foundation for clinical translation and emphasizes its potential impact on developing effective treatment strategies.

Itaconate impairs immune control of Plasmodium by enhancing mtDNA-mediated PD-L1 expression in monocyte-derived dendritic cells

Severe forms of malaria are associated with systemic inflammation and host metabolism disorders; however, the interplay between these outcomes is poorly understood. Using a Plasmodium chabaudi model of malaria, we demonstrate that interferon (IFN) γ boosts glycolysis in splenic monocyte-derived dendritic cells (MODCs), leading to itaconate accumulation and disruption in the TCA cycle. Increased itaconate levels reduce mitochondrial functionality, which associates with organellar nucleic acid release and MODC restraint. We hypothesize that dysfunctional mitochondria release degraded DNA into the cytosol. Once mitochondrial DNA is sensitized, the activation of IRF3 and IRF7 promotes the expression of IFN-stimulated genes and checkpoint markers. Indeed, depletion of the STING-IRF3/IRF7 axis reduces PD-L1 expression, enabling activation of CD8+ Tcells that control parasite proliferation. In summary, mitochondrial disruption caused by itaconate in MODCs leads to a suppressive effect in CD8+ Tcells, which enhances parasitemia. We provide evidence that ACOD1 and itaconate are potential targets for adjunct antimalarial therapy.

CDKL3 is a promising biomarker for diagnosis and prognosis prediction in patients with hepatocellular carcinoma.

Cyclin-dependent kinase-like 3 (CDKL3) has been identified as an oncogene in certain types of tumors. Nonetheless, its function in hepatocellular carcinoma (HCC) is poorly understood. In this study, we conducted a comprehensive analysis of CDKL3 based on data from the HCC cohort of The Cancer Genome Atlas (TCGA). Our analysis included gene expression, diagnosis, prognosis, functional enrichment, tumor microenvironment and metabolic characteristics, tumor burden, mRNA expression-based stemness, alternative splicing, and prediction of therapy response. Additionally, we performed a cell counting kit-8 assay, TdT-mediated dUTP nick-end Labeling staining, migration assay, wound healing assay, colony formation assay, and nude mouse experiments to confirm the functional relevance of CDKL3 in HCC. Our findings showed that CDKL3 was significantly upregulated in HCC patients compared to controls. Various bioinformatic analyses suggested that CDKL3 could serve as a potential marker for HCC diagnosis and prognosis. Furthermore, CDKL3 was found to be involved in various mechanisms linked to the development of HCC, including copy number variation, tumor burden, genomic heterogeneity, cancer stemness, and alternative splicing of CDKL3. Notably, CDKL3 was also closely correlated with tumor immune cell infiltration and the expression of immune checkpoint markers. Additionally, CDKL3 was shown to independently function as a risk predictor for overall survival in HCC patients by multivariate Cox regression analysis. Furthermore, the knockdown of CDKL3 significantly inhibited cell proliferation in vitro and in vivo, indicating its role as an oncogene in HCC. Taken together, our findings suggest that CDKL3 shows promise as a biomarker for the detection and treatment outcome prediction of HCC patients.

γδ T cells mediate robust anti-HIV functions during antiretroviral therapy regardless of immune checkpoint expression.

Although antiretroviral therapy (ART) efficiently suppresses HIV viral load, immune dysregulation and dysfunction persist in people living with HIV (PLWH). γδ T cells are functionally impaired during untreated HIV infection, but the extent to which they are reconstituted upon ART is currently unclear. Utilising a cohort of ART-treated PLWH, we assessed the frequency and phenotype, characterised in vitro functional responses and defined the impact of immune checkpoint marker expression on effector functions of both Vδ1 and Vδ2 T cells. We additionally explore the in vitro expansion of Vδ2 T cells from PLWH on ART and the mechanisms by which such expanded cells may sense and kill HIV-infected targets. A matured NK cell-like phenotype was observed for Vδ1 T cells among 25 ART-treated individuals (PLWH/ART) studied compared to 17 HIV-uninfected controls, with heightened expression of 2B4, CD160, TIGIT and Tim-3. Despite persistent phenotypic perturbations, Vδ1 T cells from PLWH/ART exhibited strong CD16-mediated activation and degranulation, which were suppressed upon Tim-3 and TIGIT crosslinking. Vδ2 T cell degranulation responses to the phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate at concentrations up to 2 ng mL-1 were significantly impaired in an immune checkpoint-independent manner among ART-treated participants. Nonetheless, expanded Vδ2 T cells from PLWH/ART retained potent anti-HIV effector functions, with the NKG2D receptor contributing substantially to the elimination of infected cells. Our findings highlight that although significant perturbations remain within the γδ T cell compartment throughout ART-treated HIV, both subsets retain the capacity for robust anti-HIV effector functions.

PD-1 Expression By Dendritic Cells Is a Key Regulator of T-Cell Immunity in Cancer

Programmed cell death 1 (PD-1) is an inhibitory receptor expressed on a variety of immune cells and a therapeutic target of checkpoint immunotherapy for cancer patients. PD-1 engagement by PD-L1 or PD-L2 can inhibit activation and expansion of tumor antigen-specific T cells. PD-1 expression in tumor-associated macrophages (TAM) correlates with impaired phagocytosis and antigen presenting function and enhanced immunosuppression. PD-1 is also expressed in dendritic cells (DC) and has been shown to correlate negatively with the outcome of DC-based cancer vaccines. On DC, PD-1 can interact with PD-L1 in cis when both of these receptors are expressed on the same cell surface. DCs are the major antigen presenting cells for cross-presenting tumor antigens to T cells, and PD-L1 upregulation protects them from killing by cytotoxic T lymphocytes, yet dampens the antitumor responses. Very little is known about the specific role of PD-1 on DCs function. In the present study, we sought to understand the role of PD-1 on DCs in the context of cancer. We generated mice with conditional targeting of the Pdcd1 gene (encoding for PD-1) and crossed them with CD11cCre ( Pdcd1 f/fCD11cCre) or Clec9Cre ( Pdcd1 f/fClec9Cre) resulting in specific deletion of PD-1 in DC cells. We used the MC17 fibrosarcoma syngeneic mouse model to investigate the tentative implications of DC-specific PD-1 ablation on anti-tumor responses. We found larger tumor sizes and weights in tumor-bearing Pdcd1 f/fCD11cCre mice compared to their Pdcd1 wt/wtCD11cCre control counterparts. Pdcd1 f/fCD11cCre tumor-bearing mice had significantly higher fractions of DCs in spleens and tumor-draining lymph nodes (tdLNs) but only minimal differences in their activation state and checkpoint marker expression. Similarly, CD4 + and CD8 + T cells from spleens and tdLNs were comparable in percentages and activation states. However, characterization of the immune cells at the tumor site, indicated that Pdcd1 f/fCD11cCre tumor-bearing mice had a more immunosuppressive tumor microenvironment with increased fractions of CD11b +Ly6C hiLy6G -monocytic myeloid derived suppressor cells (M-MDSCs) compared to control tumor-bearing mice. Moreover, flow cytometric analysis revealed that intratumoral M-MDSC in Pdcd1 f/fCD11cCre mice displayed enhanced expression of PD-L1 and CD38, consistent with a more activated and more immunosuppressive phenotype. Characterization of tumor infiltrating CD4 + and CD8 + T cells also revealed vast differences in composition and activation states. Specifically, Pdcd1 f/fCD11cCre mice had larger populations of intratumoral CD3 + T cells, with a greater CD4/CD8 T cell ratio, and higher percentages of CD4 + T regulatory cells. Conversely, intratumoral CD8 + T cells were reduced, and were characterized by diminished expansion of CD8 + T effector cells, and a less activated phenotype as determined by lower expression of CD25, CD69, and GITR. In syngeneic tumor experiments with B16 melanoma expressing ovalbumin (B16-Ova), Pdcd1 f/fCD11cCre mice and Pdcd1 f/fClec9Cre mice similarly had greater tumor sizes and weights compared to their control counterparts. These results indicate that selective ablation of PD-1 in DCs cells confers a more immunosuppressive tumor microenvironment by promoting T regulatory cell expansion and diminishing CD8 + T effector cell activation compromising anti-tumor responses.

IMMU-25. ANALYSIS OF IMMUNOSUPPRESSIVE MYELOID CELLS IN THE GLIOBLASTOMA TUMOR MICROENVIRONMENT REVEALS THERAPEUTICALLY TARGETABLE IMMUNOMODULATORY SIGNALS ACROSS POPULATIONS

Abstract Immunosuppression in glioblastoma (GBM) remains a critical and poorly understood barrier to the development of effective novel therapies. Increasing evidence points to the critical role of immunosuppressive myeloid cells in the immunosuppressive tumor microenvironment and an intriguing target for ameliorating tumor-mediated immune suppression. Efforts to characterize the myeloid milieu in the tumor microenvironment have revealed a highly heterogenous population of cells with the capacity to exert immunosuppressive effects. In order to maximize the efficacy of potential therapeutic targets, we sought to identify immune checkpoint markers expressed across a range of immunomodulatory myeloid cell populations in the tumor microenvironment of patients undergoing resection for GBM. Freshly collected ultrasonic aspirate obtained during the primary surgery for GBM resection (based on preoperative imaging and intraoperative frozen section, later confirmed on final pathology) was prepared for high dimensional flow cytometry to quantify the immune checkpoint marker expression profile of a number of described immunosuppressive myeloid cell populations, including both monocytic and granulocytic myeloid-derived suppressor cells (mMDSC and gMDSC), and CD16+ non-classical monocytes (NCM). mMDSCs expressed particularly high levels of TIGIT (53.9±19.27%), which was also highly expressed in the NCM population (25.03±5.44%). NCMs also demonstrated high levels of B7H3 expression (39.85±18.05%), which has recently been implicated as an important immune checkpoint protein in GBM. gMDSCs demonstrated a rather different expression profile, with significantly higher levels of VISTA expression than other immunomodulatory markers (p< 0.05). These findings underscore the diversity of immunomodulatory signaling in the tumor-associated myeloid cell compartment, emphasizing the challenges in developing single-target immune checkpoint inhibitor therapy in GBM. Nevertheless, this data points to some common patterns of expression across major immunomodulatory cell populations that remain understudied in GBM and could herald more promising avenues of immunotherapy development.

Specific PD-1 Deletion on Regulatory T Cells Leads to Enhanced Anti-Tumor Responses

Programmed cell death 1 (PD-1) is a T cell inhibitory receptor and the most extensively exploited therapeutic target of checkpoint immunotherapy in cancer. Ligation of PD-1 by its ligands PD-L1 or PD-L2 leads to inhibition of CD4 + and CD8 + T cell responses. However, very little is known about the specific role of PD-1 in regulatory T (Treg) cells. Previous studies have shown that PD-1 engagement during induction of Treg polarization by TCR/CD28 signaling in the presence of IL-2 and TGF-β promoted the generation and expansion of Treg cells with potent suppressive function. It has also been shown that patients with high numbers of PD-1 + Treg cells have unfavorable responses to PD-1 checkpoint immunotherapy. Moreover, patients who developed hyperprogressive disease after PD-1 blocking immunotherapy exhibited enhanced Treg cell proliferative ability compared to patients with non-hyperprogressive disease. In the present study, we sought to understand the role of PD-1 on Treg cells in the context of cancer. We generated mice with conditional targeting of the Pdcd1 gene (encoding for PD-1) and crossed them with FoxP3Cre-YFP ( Pdcd1 f/fFoxP3Cre) resulting in specific deletion of PD-1 in Treg cells. Under steady-state conditions, the percentage of Treg cells in the spleen and lymph nodes remained comparable between Pdcd1 f/fFoxP3Cre and Pdcd1wt/wtFoxP3Cre control mice. However, PD-1-deficient Treg cells had a more activated phenotype compared to their counterparts in control mice, with higher expression of CD69, ICOS, and PD-L1. Furthermore, Treg cells in Pdcd1 f/fFoxp3Cre mice expressed increased levels of GITR, CTLA-4, ICOS, and PD-L1 compared to endogenous CD4 + T conventional cells (CD4 +Foxp3 -, Tconv) and CD8 + T cells. To investigate the implications of these immunological changes in anti-tumor responses, we used the MC38 colon adenocarcinoma syngeneic mouse model. We found a smaller tumor size in Pdcd1 f/fFoxP3Cre mice compared to their control counterparts. Treg cells from spleens and tumor-draining lymph nodes (tdLNs) showed minimal differences in their activation state, expression of checkpoint markers, and proliferation levels, as determined by direct ex vivo assessment. Similarly, CD4 + Tconv and CD8 + T cells from spleens and tdLNs showed a very low level of activation that was comparable between the two experimental groups. In the tumor site, however, Pdcd1 f/fFoxp3Cre tumor-bearing mice had a significant reduction in the fractions of CD4 + Tconv and Treg cells and a significant increase in the fraction of CD8 + T cells compared to control tumor-bearing mice. Detailed analysis revealed that in tumor-bearing Pdcd1 f/fFoxp3Cre mice intratumoral naive and central memory CD4 + T cells were reduced whereas CD4 + T effector cells were increased. Similarly, in Pdcd1 f/fFoxp3Cre tumor-bearing mice we noticed a reduction in naïve and central memory CD8 + T cells but a significant expansion in the effector CD8 + T cells . No significant differences were detected in the expression of activation markers in tumor infiltrating Treg cells between the two groups. In contrast, in Pdcd1 f/fFoxp3Cre tumor-bearing mice, tumor infiltrating CD4 + Tconv cells displayed an activated phenotype with upregulation of PD-L1, ICOS, TIM3, CD69, and CTLA-4, downregulated PD-1, and increased IFN-γ production. Tumor infiltrating CD8 + T cells also displayed an activated phenotype with upregulation of activation markers such as CD69, ICOS, PD-L1, TIM3, and LAG3 but reduced PD-1 expression. Notably, both CD4 + Tconv and CD8 + T cells proliferated more robustly in the Pdcd1 f/fFoxp3Cre tumor-bearing mice as determined by the high expression of the proliferation marker Ki67. These results indicate that selective ablation of PD-1 in Treg cells confers a less suppressive immunological environment and allows for expansion and activation of CD4 + and CD8 + T cells, leading to anti-tumor responses from CD4 + and CD8 + T effector cells.

CD33-TIM3 Dual CAR T Cells: Enhancing Specificity While Maintaining Efficacy Against AML

Background: The myeloid associated antigen CD33 is overexpressed in over 88% of Acute myeloid leukemia (AML) patients, making it a suitable target antigen for CAR T cell therapy across different genetic subtypes. Despite the development of clinical trial data, there remains a concern about on-target off-leukemia toxicity. To enhance specificity, we generated dual targeting CAR T cells that target CD33 and/or T-cell immunoglobulin and mucin-domain containing-3 (TIM3). The latter is known to be expressed on LSCs, but not on healthy hematopoietic precursor or stem cells. Notably, TIM3 is recognized for its inhibitory immunomodulatory role further enhancing the suitability of this target antigen. The objective of this research was to explore various CD33 and TIM3 dual-targeting CAR T cells approaches that enhance AML specificity and maintain high anti-AML activity. Methods: The hybridoma technology was used to generate antibodies against TIM3 immunized mice. Antibodies were screened for TIM3 specificity via ELISA and FACS. The anti-TIM3 single-chain variable fragment (scFv) DNA sequence was sequenced from hybridoma cells. Furthermore, the binding ability to TIM3 antigen (PDB ID: 5F71) was evaluated using AlphaFold. The CD33 scFv was derived from Gemtuzumab ozogamicin (clone: hP67.6). ScFv sequences and co-stimulation domains (CD28 or 4-1BB) were cloned into a pMP71 vector. Retrovirus for transduction was produced using the 293Vec-GALV and RD114 retroviral production system. In vitro co-culture assays of CAR T cells and target cells were performed to study the efficacy of CAR T cells. The cytotoxicity against wild-type and TIM3 transduced AML cell lines (THP-1 and OCI-AML3) was assessed by multiparameter flow cytometry (MPFC). The secretion of effector cytokines (IFN-γ, TNF, IL-2) was analyzed via CBA assays. In addition, avidity between CAR T cells and target cells was determined by Z-Movie analyzer. The different CAR constructs were screened for on-target off-tumor toxicity in colony forming unit assays (CFU) using isolated CD34 + hematopoietic stem and progenitor cells (HSPCs) from healthy doners after 14 days. In order to compare CAR constructs regarding long-term efficacy in antigen restimulation assays, CAR T cells were co-cultured with irradiated TIM3 transduced OCI-AML3 every 4 days at an E: T ratio of 1:1 for 24 days. Moreover, CAR T-cell proliferation, checkpoint marker expression and T-cell subset differentiation were analyzed via MPFC. Results: All dual CAR T cells (compound, split, tandem, pooled) were generated with high and robust transduction efficacy (Figure 1A). TIM3-dependent fratricide was not observed during CAR T cell manufacturing. Compared to single antigen targeting CAR T cells, dual CAR T cells showed enhanced cytotoxicity against AML cell lines and primary AML cells (Figure 1B). Moreover, we also observed a strong increase in the secretion of proinflammatory cytokines (IFN-γ and IL-2) and higher avidity of dual CAR T cells. Notably, split CAR T cells demonstrated greater specificity in cocultures directed against mono vs. dual-target antigen expression cell lines. In accordance with these observations, split CAR T cells did not exert on-target off-leukemia toxicity against healthy HSC in CFU assays. In the antigen restimulation assay, we observed that compound CAR T cells exhibited diminished expanding capacity and heightened expression of exhaustion markers in comparison to the other CAR T constructs. Conclusion: We developed multiple CD33 and TIM3 dual CAR T cells using both “AND” and “OR” gating strategies in this study. Our findings revealed that dual CAR T cells provided higher avidity and cytotoxicity compared to single targeting CAR T cells against dual antigen expressing target cells in vitro. Importantly, only the split CAR T constructs demonstrated specific killing of CD33 +TIM3 + cell lines and primary AML cells while sparing HSPCs. Prospectively, compound, tandem, pooled CD33 TIM3 specific CAR T cells might be helpful in a bridge to transplant setting, whereas split CAR T cells might provide a higher safety profile, thereby allowing a transplant independent approach. To advance our concepts, we are currently conducting in vivo experiments in an NSG mouse model.

Differentiation of BCMA-specific induced pluripotent stem cells into rejuvenated CD8αβ+ T cells targeting multiple myeloma

AbstractA major hurdle in adoptive T-cell therapy is cell exhaustion and failure to maintain antitumor responses. Here, we introduce an induced pluripotent stem cell (iPSC) strategy for reprogramming and revitalizing precursor exhausted B-cell maturation antigen (BCMA)-specific T cells to effectively target multiple myeloma (MM). Heteroclitic BCMA72-80 (YLMFLLRKI)–specific CD8+ memory cytotoxic T lymphocytes (CTL) were epigenetically reprogrammed to a pluripotent state, developed into hematopoietic progenitor cells (CD34+ CD43+/CD14− CD235a−), differentiated into the T-cell lineage and evaluated for their polyfunctional activities against MM. The final T-cell products demonstrated (1) mature CD8αβ+ memory phenotype, (2) high expression of activation or costimulatory molecules (CD38, CD28, and 41BB), (3) no expression of immune checkpoint and senescence markers (CTLA4, PD1, LAG3, and TIM3; CD57), and (4) robust proliferation and polyfunctional immune responses to MM. The BCMA-specific iPSC–T cells possessed a single T-cell receptor clonotype with cognate BCMA peptide recognition and specificity for targeting MM. RNA sequencing analyses revealed distinct genome-wide shifts and a distinctive transcriptional profile in selected iPSC clones, which can develop CD8αβ+ memory T cells. This includes a repertoire of gene regulators promoting T-cell lineage development, memory CTL activation, and immune response regulation (LCK, IL7R, 4-1BB, TRAIL, GZMB, FOXF1, and ITGA1). This study highlights the potential application of iPSC technology to an adaptive T-cell therapy protocol and identifies specific transcriptional patterns that could serve as a biomarker for selection of suitable iPSC clones for the successful development of antigen-specific CD8αβ+ memory T cells to improve the outcome in patients with MM.

Differential Gene Expression of Checkpoint Markers and Cancer Markers in Mouse Models of Spontaneous Chronic Colitis.

The presence of checkpoint markers in cancer cells aids in immune escape. The identification of checkpoint markers and early cancer markers is of utmost importance to gain clarity regarding the relationship between colitis and progressive inflammation leading to cancer. Herein, the gene expression levels of checkpoint makers, cancer-related pathways, and cancer genes in colon tissues of mouse models of chronic colitis (Winnie and Winnie-Prolapse mice) using next-generation sequencing are determined. Winnie mice are a result of a Muc2 missense mutation. The identification of such genes and their subsequent expression and role at the protein level would enable novel markers for the early diagnosis of cancer in IBD patients. The differentially expressed genes in the colonic transcriptome were analysed based on the Kyoto Encyclopedia of Genes and Genomes pathway. The expression of several oncogenes is associated with the severity of IBD, with Winnie-Prolapse mice expressing a large number of key genes associated with development of cancer. This research presents a number of new targets to evaluate for the development of biomarkers and therapeutics.

Deciphering Treg cell roles in esophageal squamous cell carcinoma: a comprehensive prognostic and immunotherapeutic analysis.

Background: Esophageal squamous cell carcinoma (ESCC) is a prevalent and aggressive form of cancer that poses significant challenges in terms of prognosis and treatment. Regulatory T cells (Treg cells) have gained attention due to their influential role in immune modulation within the tumor microenvironment (TME). Understanding the intricate interactions between Treg cells and the tumor microenvironment is essential for unraveling the mechanisms underlying ESCC progression and for developing effective prognostic models and immunotherapeutic strategies. Methods: A combination of single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq analysis was utilized to explore the role of Treg cells within the TME of ESCC. The accuracy and applicability of the prognostic model were assessed through multi-dimensional evaluations, encompassing an examination of the model's performance across various dimensions, such as the mutation landscape, clinical relevance, enrichment analysis, and potential implications for immunotherapy strategies. Results: The pivotal role of the macrophage migration inhibitory factor (MIF) signaling pathway within the ESCC TME was investigated, with a focus on its impact on Treg cells and other subpopulations. Through comprehensive integration of bulk sequencing data, a Treg-associated signature (TAS) was constructed, revealing that ESCC patients with elevated TAS (referred to as high-TAS individuals) experienced significantly improved prognoses. Heightened immune infiltration and increased expression of immune checkpoint markers were observed in high-TAS specimens. The model's validity was established through the IMvigor210 dataset, demonstrating its robustness in predicting prognosis and responsiveness to immunotherapy. Heightened therapeutic benefits were observed in immune-based interventions for high-TAS ESCC patients. Noteworthy differences in pathway enrichment patterns emerged between high and low-TAS cohorts, highlighting potential avenues for therapeutic exploration. Furthermore, the clinical relevance of key model genes was substantiated by analyzing clinical samples from ten paired tumor and adjacent tissues, revealing differential expression levels. Conclusion: The study established a TAS that enables accurate prediction of patient prognosis and responsiveness to immunotherapy. This achievement holds significant implications for the clinical management of ESCC, offering valuable insights for informed therapeutic interventions.

SAT-374 - Interplay between hepatitis B virus replication and intrahepatic expression of VISTA and TIM-3 immune checkpoint markers in chronic hepatitis B patients

SAT-374 - Interplay between hepatitis B virus replication and intrahepatic expression of VISTA and TIM-3 immune checkpoint markers in chronic hepatitis B patients