Macrophages regulate PD-1 and CTLA-4 expression on ILC2s and their responsiveness in the tumor microenvironment

To validate our previous findings of high-level EGFR expression in GCCC using an expanded cohort of specimens and to further examine the molecular and cellular features of this aggressive malignancy to identify potentially actionable therapeutic targets. The SEER database was queried to obtain the epidemiological data regarding the current national survival trends for GCCC. Immunohistochemistry (IHC) was used to examine the expression of EGFR, PD-1, and PD-L1. CiberSort analysis was used to analyze a previously published RNA-sequencing dataset obtained from a single patient diagnosed with GCCC. In comparison to squamous cell carcinomas and adenocarcinoma/adenosquamous carcinomas, GCCC was observed in younger patients (p < 0.001) and demonstrated inferior survival (p < 0.001). All (100%) of the specimens (8/8) exhibited immunoreactivity when stained for CD3ε (T-cell marker), EGFR, PD-1, and PD-L1 whereas CTLA4 expression was not detected. Analysis of RNA-sequencing data revealed that cetuximab and erlotinib altered the chemokine profile, lymphocyte abundance, and expression of inhibitory immune checkpoints in a single patient when combined with cytotoxic chemotherapy in a single patient. The data from this descriptive study suggests that immune checkpoint blockade, whether single agent or in combination, may be a suitable therapeutic option for a disease for which targeted approaches do not currently exist.

The present study sought to evaluate the expression of PD-1 and CTLA-4 in blood T lymphocytes during the periparturient period and their relationship with uterine health in dairy cows, as determined by endometrial cytology and serum concentrations of β-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFAs), which are indicators of a negative energy balance. The second objective of this study was to investigate whether the expression of PD-1 and CTLA-4 in T lymphocytes is associated with the serum acute phase-protein haptoglobin concentration during the periparturient period. To address these objectives, 26 clinically healthy dairy cows were used. Peripheral blood was collected 14 days prepartum (T-14), at calving (T0), and 30 days postpartum (T30) to measure the expression of PD-1 and CTLA-4 in blood T lymphocytes by flow cytometry. In addition, we collected blood at T0, 10 days after parturition (T10), and T30 to obtain serum and determine the serum concentrations of NEFA, BHB, and Hp. Endometrial cytology was performed at T10, 20 days after parturition (T20), and T30. In the present study, we observed higher expression of CTLA-4 and PD-1 in T lymphocytes at parturition and in the prepartum period, which could indicate a relationship between these immune checkpoints and immunological tolerance during gestation in dairy cattle. In addition, a negative association between the expression of these immune checkpoints prepartum or at parturition and endometrial cytology at T20 and T30 was observed, indicating the negative implications of these immune response regulators in susceptibility to infections. This finding was further corroborated by the relationship between the serum concentration of haptoglobin and the expression of CTLA-4 and PD-1 by T lymphocytes. However, we did not observe a relationship between the indicators of negative energy balance, evaluated by the serum concentrations of BHB and NEFA, and the expression of the immune checkpoint markers studied. Thus, our findings represent an initial step that paves the way for the development of new therapeutic alternatives directed by the host with the objective of increasing the resistance of dairy cattle to infections in this critical period of life.

Primary central nervous system germ cell tumors (CNS GCTs) are rare intracranial malignancies, and their tumor microenvironment plays a crucial role in tumor initiation and progression. However, the specific characteristics of the immune microenvironment and their clinical significance remain poorly understood. This study included 93 paraffin-embedded tissue samples from 90 patients diagnosed with CNS GCTs. Immunohistochemistry and immunofluorescence staining were used to assess the infiltration patterns of T cell subsets (CD3+, CD4+, CD8+, Foxp3+) and the expression levels of immune checkpoints (CTLA-4, PD-1, PD-L1). Additionally, the study explored the relationship between these immune features and the patient's clinical characteristics and prognosis. The study revealed that germinomas exhibited significantly higher infiltration of CD4+ and Foxp3+ T cells compared to non-germinomatous GCTs (NGGCTs). Additionally, CTLA-4 expression was detected in 58.06% of cases, while PD-1 and PD-L1 were expressed in over 90%, with higher CTLA-4 levels in germinomas and elevated PD-L1 levels in NGGCTs. T cell infiltration was positively correlated with immune checkpoint expression, particularly in germinomas. The results also highlighted the strong immunosuppressive nature of the CNS GCTs' tumor microenvironment. Furthermore, T cell infiltration and immune checkpoint expression were closely associated with clinical characteristics and prognosis. Notably, PD-1 expression was identified as an independent prognostic factor for progression-free survival (PFS) and recurrence-free survival (RFS). Our study highlighted the distinct characteristics of T cell infiltration and the significant expression of immune checkpoints in CNS GCTs, revealing the highly heterogeneous and immunosuppressive nature of the tumor microenvironment. PD-1 expression was identified as an independent prognostic predictor, offering a foundation for enhancing risk stratification in CNS GCT patients. These findings also support the potential for future clinical applications of immune checkpoint inhibitors, such as PD-1 monoclonal antibodies.

Immune checkpoint(s) blockade is becoming the therapeutic mainstay in melanoma and lung cancer. Based on these important clinical achievements, immunotherapy with immunomodulating monoclonal antibodies (mAb) is being explored as a new therapeutic modality in most human malignancies, either alone or in combination with other immunomodulating agents. We have provided extensive evidence that the second generation DNA hypomethylating agent (DHA), guadecitabine (SGI-110), plays a promising role in potentiating the immunogenicity and the immune recognition of human malignancies through the up-regulation of the expression of different immune molecules on cancer cells. These findings led us to hypothesize that DHA could represent ideal partners for immune checkpoint(s) blocking agents to improve their therapeutic efficacy. Here, we studied the expression of programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1), and 2 (PD-L2), and cytotoxic T lymphocyte-associated antigen 4 (CTLA4) in peripheral blood mononuclear cells of acute myeloid leukemia (AML) patients (N = 23), enrolled in a phase 1-2 study with guadecitabine. Patients included in this preliminary analysis were selected based on their responsiveness to therapy. Quantitative RT-PCR analyses showed a constitutive expression (gene/β-actin molecules &amp;gt;7.5E-05) of PD1, PD-L1, PD-L2 and CTLA-4 in 82.6%, 100%, 47.8% and 8.7% patients, respectively. Guadecitabine therapy up-regulated (≥2 fold) the expression of PD1, PD-L1, PD-L2 and CTLA-4 in 57.9%, 56.5%, 18.2% and 0% patients, respectively, at any of the time-points investigated during treatment. De novo expression of PD1, PD-L2 and CTLA-4 was observed in 100%, 41.6% and 38.1% of the immune checkpoint(s)-negative patients, respectively. Though preliminary, these results further support the strong link between epigenetics/DNA methylation and immune response, in cancer patients, and strengthen the therapeutic potential of epigenetic immunomodulation, with “consolidated” and emerging immune checkpoint(s) blocking mAb. Citation Format: Carolina Fazio, Alessia Covre, Maria Lofiego, Pietro Taverna, Mohammad Azab, James N. Lowder, Sandra Coral, Michele Maio. Immune checkpoint(s) expression in AML patients enrolled in a phase 1-2 study with guadecitabine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2325.

An increasing number of therapy regimens using a combination of different immune checkpoint inhibitors (ICIs) have shown remarkable results in several different tumor entities. However, the likelihood of a positive response rate to combined ICIs is poor in most tumor entities and depends on several parameters including the tumor microenvironment. Particularly little is known about the spatial orchestration and spatial interplay between different immune checkpoint expressing cells. Given that the T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is expressed on both immune cells as well as tumor cells and that several phase I/II studies are currently evaluating anti-TIM3 drugs, the interplay between these immune checkpoints in human cancers is of topical interest. To study the spatial orchestration and interplay between TIM3, CTLA-4, PD-1, and PD-L1 expression on T-cell subsets, macrophage subsets, CD11c+ dendritic cells, CD20+B-cells in relation to panCK+ malignant cells, CD31+ vessels and other structural tumor compartments, a multiplex fluorescence immunohistochemistry approach was used to stain 18 different antibodies on a set of tissue microarrays containing samples from more than 3000 carcinoma samples. In addition, a deep learning-based framework for cell type identification was developed and validated in this study. TIM3, PD-1, PD-L1, and CTLA-4 expression was measured on tumor cells (panCK+), cytotoxic T-cells (CD3+CD8+), T-helper cells (CD3+CD4+), regulatory T-cells (CD3+CD4+FOXP3+), subsets of macrophages (CD68+CD163+/CD68+iNOS+) and dendritic cells (CD11c+). Interestingly, TIM3 as well as CTLA-4 expression on CD3+CD8+ cytotoxic T-cells and CD3+CD4+FOXP3+ regulatory T-cells showed a spatially more diverse expression pattern - particularly inverse expression profile - compared to PD-1 expression on all analyzed T-cells subsets that was consistently accompanied by PD-L1 expression on immune and tumor cells (p&amp;lt;0.001). Combined analysis of cell densities, expression patterns, intensity measurements, interaction and distance analysis between immune cells and tumor cells revealed distinct changes in the immune cell infiltration pattern that was linked to several major immune checkpoint receptor expression profiles. Previously uncharacterized immune cell-composition dynamics in clustered tumor phenotypes, according to the immune checkpoint expression, were detected. This included for instance, a significant inverse association between CTLA-4 expression on T-cells and high expression levels of the PD-1/PD-L1 axis. In conclusion, deep profiling of 18 biomarkers in more than 40 different carcinoma entities revealed complex changes in the spatial orchestration of a wide range of immune cell subsets that was driven by the expression profile and composition of TIM3, PD-1, PD-L1, and CTLA-4. Citation Format: Nicolaus F. Debatin, Elena Bady, Tim Mandelkow, Magalie C. Lurati, Ronald Simon, Claudia Hube-Magg, Maximilian Lennartz, Guido Sauter, Niclas C. Blessin. Interplay between TIM3+ immune cells and other immune checkpoints in more than 40 different human carcinoma entities using 18+1 BLEACH&amp;STAIN mfIHC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1935.

Introduction. The first clinical trials of the treatment of patients with prostate cancer with checkpoint inhibitors show that only a few patients benefit from this type of treatment. This implies the need to find predictive biomarkers that would help identify patients for whom treatment with checkpoint inhibitors could be effective. Our study aimed to assess the level of PD-1, PD-L, and CTLA-4 expression on peripheral blood mononuclear cells of patients with primary prostate cancer and to demonstrate their applicability in clinical practice. Material and methods. Fifty men with primary prostate cancer were enrolled in the study. The control group consisted of 20 healthy men. The material for the study was peripheral blood from which mononuclear cells were isolated by flow cytometry, and the expression of PD-1, CTLA-4, and PD-L1 on them was assessed. Results. High PD-L1 expression on lymphoid dendritic cells has been demonstrated in patients with prostate cancer in comparison to the control group (p = 0.015) and high PD-L1 expression has been demonstrated in the following groups: high risk (p = 0.026), T2/T3 (p = 0.011), and Gleason 7 (p = 0.027). There was no high PD-1 expression on T lymphocytes among patients with prostate cancer in comparison to the control group, but positive correlations were found between PD-1 expression on CD3+ T cells and PSA (p = 0.049), risk group (p = 0.002), and TNM (p = 0.050). Low CTLA-4 expression was found on CD3+ lymphocytes among patients with prostate cancer in comparison to the control group (p = 0.006). Conclusions. Several groups of patients with prostate cancer have been identified, showing high PD-L1 and PD-1 expression on peripheral blood mononuclear cells, and a relationship between PD-1 and PD-L1 expression and the tumor aggressiveness potential has been demonstrated. This means that the assessment of PD-1 and PD-L1 expression can be used as a prognostic and predictive biomarker in prostate cancer.

To study the expression and significance of PD-1, PD-L1 and CTLA-4 tumor-associated antigens in multiple myeloma. Bone marrow specimens from 122 patients with multiple myeloma were collected and divided into new-onset group (NDMM), complete remission group (CRMM) and relapsed and refractory group (RRMM) according to the disease progression stage. The proportion of CD4+ T lymphocytes, CD8+ T lymphocytes, Treg cells and plasma cells in the specimens and the expressions of PD-1, PD-L1 and CTLA-4 were detected by multi-parameter flow cytometry. There was no significant difference in the proportion of CD8+T and Treg cells among the three groups (P>0.05), while the proportions of CD4+T cells and PC in NDMM group were significantly higher than those in the CRMM group (P<0.05), the ratios of CD4+ to CD8+T in the NDMM and RRMM groups were significantly higher than those in the CRMM group (P<0.05). The expressions of PD-1, PD-L1 and CTLA-4 in CD8+ T cells was no significant difference among NDMM, CRMM and RRMM groups (P>0.05). While the expressions of PD-1, PD-L1 and CTLA-4 in CD4+ T cells and PC in the NDMM group were significantly lower than that in the CRMM group (P<0.05). There was significantly difference among the three groups in the expression of PD-1 in Treg cells, of which the NDMM group was significantly lower than that of the CRMM group (all P<0.05). The expressions of PD-1 and CTLA-4 in PC were significantly higher than those in CD8+ T, CD4+ T and Treg cells (P<0.05), the expression of PD-L1 in CD8+ T cells was significantly higher than that in CD4+ T and Treg cells (P<0.05). There is a correlation between the immune status of multiple myeloma and the expressions of PD-1, PD-L1 and CTLA-4 in plasma cells and lymphocyte subsets in vivo.

Effect of boswellic acids on the expression of PD-1 and TIGIT immune checkpoints on activated human T cells.

5536 Background: Immune checkpoint blockade (ICB) is being explored as a treatment option in ovarian cancer, but objective response rates for single agent ICB are modest at around 10-15%. Validated biomarkers are needed to predict which patients will respond to ICB. BRCA mutations and homologous recombination deficiency (HRD) status are the only validated integral biomarkers in ovarian cancer. HRD tumors exhibit defective DNA repair mechanisms that promote increased mutational burden, which we postulate may correlate with higher neoantigen load and increased expression of targetable immune checkpoints. Methods: The Cancer Genome Atlas (TCGA) ovarian cancer dataset was evaluated and previously published, well annotated samples were obtained for HRD status. HLA type was determined with OptiType. Nonsynonymous mutations were annotated with Ensembl VEP. pVAC-Seq using NetMHCpan algorithm predicted neoepitopes 9 amino acids in length for MHC class I, reporting only those with a predicted IC50 less than 500 nM. Immune checkpoint gene expression counts were normalized with TCGAbiolinks. Correlation between HRD status and neoantigen load was assessed by Wilcoxon test. After log2 transformation, Wilcoxon tests evaluated for association between HRD status and expression of immune checkpoints. The relationship between HRD status and PD-L1 protein abundance with reverse phase protein array was measured. Results: Data from 154 HRD positive and 198 HRD negative tumors were analyzed. HRD positive status correlated with higher neoantigen load (p = 0.038) and increased expression of the immune checkpoints CTLA4 (p = 0.024), TIGIT (p = 0.027), and PVR (p = 0.002), but not PD-L1 (p = 0.238), LAG3 (p = 0.583), HVEM (p = 0.805), GAL9 (p = 0.750), NECTIN2 (p = 0.874), VSIG3 (p = 0.438), PSGL1 (p = 0.205) or VISTA (p = 0.531). TIM3 (p = 0.064) and B7H3 (p = 0.052) both demonstrated a trend towards increased expression in HRD tumors. Interestingly, HRD status showed a negative association with PVRIG (p = 0.028). There was no association between PD-L1 protein abundance and HRD status. Conclusions: HRD positive ovarian tumors demonstrate higher neoantigen load than HRD negative tumors, as well as increased expression of certain immune checkpoints. This supports the hypothesis that increased neoantigen load leads to compensatory induction of immune checkpoints, and suggests that HRD status may predict response to ICB, particularly to drugs that target CTLA4, TIGIT, PVR, TIM3 and B7H4.

Deep Phenotyping Reveals Expansion of T Follicular Regulatory Cells and Triple Positive (CTLA4, TIGIT, PD1) Exhausted Effector Memory T Cells Characterizing the Immunosuppressive Microenvironment in Follicular Lymphoma

BackgroundThe tumor microenvironment has an important role in the growth and progression of diffuse large B-cell lymphoma (DLBCL). Immune checkpoint molecules, including PD1, LAG3, and CTLA4, are crucial to regulate the T cells function in the tumor microenvironment. Exploring the expression of these molecules in DLBCL microenvironment is crucial for developing targeted therapies enhancing anti-tumor immune responses.AimThis study aims to evaluate the immunohistochemical (IHC) expression of PD1, LAG3, and CTLA4 in DLBCL, assess the relation of their expression to different clinicopathological parameters and evaluate their prognostic significance.MethodsThis retrospective study encompassed 103 cases diagnosed as de novo DLBCL. Clinicopathologic and survival data were gathered. IHC for PD1, LAG3, and CTLA4 was performed.ResultsPD1, LAG3, and CTLA4 positive reaction was observed in tumor-infiltrating lymphocytes (TILs) in 68.9% (71/103), 82.5% (85/103), and 92.2% (95/103) of DLBCL cases, respectively. PD1 expression in TILs was significantly associated with hepatitis C virus (HCV) positivity and prolonged overall survival (OS) in univariate analysis. LAG3 expression in TILs was significantly associated with IPI score and tended towards shorter OS (not statistically significant). LAG3 expression in tumor cells was significantly associated with shorter disease-free survival (DFS). CTLA4 expression in TILs was significantly associated with advanced disease stage (III/IV).ConclusionPD1 and LAG3 are expressed mainly in TILs. PD1 expression (in TILs and tumor cells) is associated with prolonged OS, while LAG3 expression (in tumor cells) is associated with shorter DFS and its expression in TILs tended towards shorter OS. CTLA4 expression is associated with advanced disease stage but not associated with OS. These findings may suggest that immune checkpoint inhibitors targeting LAG3 may offer therapeutic potential in DLBCL by enhancing the antitumor immune response. Additional research is needed to assess the effectiveness of inhibition of these checkpoint molecules in combination with existing treatment modalities.

Immune Checkpoint (PD-1, PD-L1, PD-L2, and CTLA-4) Expression in Plasma Cell Myeloma

The expression of checkpoint blockade molecules PD-1, PD-L1, CTLA-4, and foxp3+CD25+CD4+ T cells (Tregs) regulate donor T cell activation and graft-vs-host disease (GvHD) in allogeneic hematopoietic stem cell transplant (allo-HSCT). Detailed kinetics of PD-1-, CTLA-4-, and PD-L1 expression on donor and host cells in GvHD target organs have not been well studied. Using an established GvHD model of allo-HSCT (B6 → CB6F1), we noted transient increases of PD-1- and CTLA-4-expressing donor CD4+ and CD8+ T cells on day 10 post transplant in spleens of allo-HSCT recipients compared with syngeneic HSCT (syn-HSCT) recipients. In contrast, expression of PD-1- and CTLA-4 on donor T cells was persistently increased in bone marrow (BM) of allo-HSCT recipients compared with syn-HSCT recipients. Similar differential patterns of donor T cell immune response were observed in a minor histocompatibility (miHA) mismatched transplant model of GvHD. Despite higher PD-1 and CTLA-4 expression in BM, numbers of foxp3+ T cells and Tregs were much lower in allo-HSCT recipients compared with syn-HSCT recipients. PD-L1-expressing host cells were markedly decreased concomitant with elimination of residual host hematopoietic elements in spleens of allo-HSCT recipients. Allo-HSCT recipients lacking PD-L1 rapidly developed increased serum inflammatory cytokines and lethal acute GvHD compared with wild-type (WT) B6 allo-HSCT recipients. These data suggest that increased expression of checkpoint blockade molecules PD-1 and CTLA-4 on donor T cells is not sufficient to prevent GvHD, and that cooperation between checkpoint blockade signaling by host cells and donor Tregs is necessary to limit GvHD in allo-HSCT recipients.

BackgroundImmune checkpoints are a set of costimulatory and inhibitory molecules that maintain self-tolerance and regulate immune homeostasis. The expression of immune checkpoints on T cells in malignancy, chronic inflammation, and neurodegenerative diseases has gained increasing attention.ResultsTo characterize immune checkpoints in neurodegenerative diseases, we aimed to examine the expression of the immune checkpoint PD-1/PD-L1 in peripheral T cells in different Alzheimer’s disease (AD) patients. To achieve this aim, sixteen AD patients and sixteen age-matched healthy volunteers were enrolled to analyze their CD3+ T cells, CD3+CD56+ (neural cell adhesion molecule, NCAM) T cells, CD4+/CD8+ T cells, and CD4+/CD8+CD25+ (interleukin-2 receptor alpha, IL-2RA) T cells in this study. The expression of PD-1 on T cells was similar between the AD patients and healthy volunteers, but increased expression of PD-L1 on CD3+CD56+ T cells (natural killer T cells, NKT-like), CD4+ T cells (helper T cells, Th), CD4+CD25+ T cells, and CD8+ T cells (cytotoxic T lymphocytes, CTL) was detected in the AD patients. In addition, we found negative correlations between the AD patients’ cognitive performance and both CD8+ T cells and CD8+CD25+ T cells. To identify CD8+ T-cell phenotypic and functional characteristic differences between the healthy volunteers and AD patients in different stages, a machine learning algorithm, t-distributed stochastic neighbor embedding (t-SNE), was implemented. Using t-SNE enabled the above high-dimensional data to be visualized and better analyzed. The t-SNE analysis demonstrated that the cellular sizes and densities of PD-1/PD-L1 on CD8+ T cells differed among the healthy, mild AD, and moderate AD subjects.ConclusionsOur results suggest that changes in PD-1/PD-L1-expressing T cells in AD patients’ peripheral blood could be a potential biomarker for monitoring disease and shed light on the AD disease mechanism. Moreover, these findings indicate that PD-1/PD-L1 blockade treatment could be a novel choice to slow AD disease deterioration.

Comparison of Anti-PD1, Anti-CTLA4 and Anti-TIM3 in Treatment of AML Patients with Single-Cell Transcriptomics Displays Different Effects on Immune Subtypes