PD-1 Expression Research Articles

Study on correlation between CXCL13 and prognosis and immune characteristics of ovarian cancer.

Ovarian cancer (OC) has a limited immunotherapeutic response; hence, this study aimed to investigate the relationship between CXC-chemokine ligand 13 (CXCL13) expression and overall survival (OS) rate, key immune pathways, degree of immune cell infiltration, and progressive disease (PD)-1 checkpoint blockade. A total of 703 differentially expressed genes were obtained from "The Cancer Genome Atlas" (TCGA) database based on the immune and stromal scores of 379 OC patients for getting the targeted gene CXCL13. The association between CXCL13 and OS in OC patients, biological function annotation of CXCL13, and its correlation with immune components were assessed. The results indicated that upregulated CXCL13 expression was positively correlated with better OC patient prognosis. CXCL13 expression was associated with 6 immune-related pathways, 10 immune cells, and PD-1 expression of OC micro-environment. Moreover, high expression of CXCL13 was related to a better tumor response and more extended tumor-stable stage after PD-1 blocking therapy in IMvigor210. The study concluded that CXCL13 could be a prognostic marker and a potential immunotherapy target for OC patients, especially PD-1 checkpoint blockade.

How Co-Stimulatory/Inhibitory Molecules Vary Across Immune Cell Subtypes in the Severity of Systemic Lupus Erythematosus Compared to Controls

Background: Co-stimulatory and co-inhibitory molecules are critical to T cell responses and involved in the pathogenesis of systemic lupus erythematosus (SLE). This study aimed to comprehensively analyze the surface expression of these molecules in various phenotypic immune cells, comparing the differences between various levels of the severity in SLE and control groups. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque from blood samples of severe SLE patients (treatment with immunosuppressants), mild SLE patients (excluding those with persistent proteinuria or thrombocytopenia), and healthy controls (n = 10 each). PBMCs were stimulated for 48 h. The cells were stained with anti-CD3, CD4, CD28, PD-1, and CTLA-4 antibodies and analyzed by flow cytometry. Differences between groups were assessed using the Kruskal–Wallis test and Mann–Whitney U-test, with median values and statistical significance (p < 0.05) reported. Results: The results showed that CD28 expression was significantly higher in SLE patients compared to controls, with the highest levels in mild SLE. However, CD3+ CD28+ and CD4+ CD28+ cells were more prevalent in controls (p = 0.032 and 0.017, respectively). Mild SLE patients exhibited the highest CTLA-4 expression, with significant differences from severe SLE and controls (p = 0.030 and 0.037, respectively). PD-1 expression was lowest in severe SLE but highest in mild SLE within CD3+ CD4+ cells (p = 0.001). After 48 h of activation, CD4+ CTLA4+ and CD3+ CTLA4+ expression levels were significantly higher in controls compared to SLE groups. Conclusions: Our study highlighted that the expression of CD28, CTLA-4, and PD-1 in lymphocytes and specific T cell subsets was various according the severity of SLE in patients, underscoring their roles in disease pathogenesis.

A splicing isoform of PD-1 promotes tumor progression as a potential immune checkpoint

The immune checkpoint receptor, programmed cell death 1 (PD-1, encoded by PDCD1), mediates the immune escape of cancer, but whether PD-1 splicing isoforms contribute to this process is still unclear. Here, we identify an alternative splicing isoform of human PD-1, which carries a 28-base pairs extension retained from 5′ region of intron 2 (PD-1^28), is expressed in peripheral T cells and tumor infiltrating lymphocytes. PD-1^28 expression is induced on T cells upon activation and is regulated by an RNA binding protein, TAF15. Functionally, PD-1^28 inhibits T cell proliferation, cytokine production, and tumor cell killing in vitro. In vivo, T cell-specific exogenous expression of PD-1^28 promotes tumor growth in both a syngeneic mouse tumor model and humanized NOG mice inoculated with human lung cancer cells. Our study thus demonstrates that PD-1^28 functions as an immune checkpoint, and may contribute to resistance to immune checkpoint blockade therapy.

The prognostic and therapeutic value of the tumor microenvironment and immune checkpoints in pancreatic neuroendocrine neoplasms

Pancreatic neuroendocrine neoplasms (Pan-NEN) represent a group of highly heterogeneous cancers, characterized by complex and diverse biological behavior. The objective of this study was to systematically investigate the immunological features of the tumor microenvironment (TME) in Pan-NEN, aiming to identify prognostic biomarkers and explore the therapeutic potential of immunotherapy for Pan-NEN. Tumor and adjacent normal tissues were collected from 56 patients with Pan-NEN. Immunohistochemical analysis was conducted on tumor tissues using a panel of monoclonal antibodies targeting key immune markers. The expression levels of these markers were quantitatively assessed and correlated with clinicopathological features and overall survival. Low expression of CD3, CD8, CD4, CD68, CD163, Foxp3, CD56, CD69, GZMB, HLA-1, HLA-II, PD-1, and PD-L1 were observed in the majority of Pan-NEN patient samples. PD-1 expression was positively correlated with CD4 and Foxp3 expression, while PD-L1 expression was positively correlated with CD68, CD163, and Foxp3 expression; HLA-II expression was positively correlated with GZMB expression. Infiltration of lymphocytes (CD3 + or CD8+) and macrophages (CD68 + or CD163+) and expression of PD-1/PD-L1 were more pronounced in poorly differentiated neuroendocrine carcinoma (Pan-NEC) compared to well-differentiated neuroendocrine tumors (Pan-NET), while CD68 and PD-L1 correlated with advanced disease stage. Conversely, HLA-I antigen expression was commonly downregulated in Pan-NEC. Univariate Cox proportional hazard analysis demonstrated that tumor grade, stage; CD4+, CD68+, and CD163 + cell count; and expression of PD-1 and PD-L1 were significantly associated with poor survival outcomes, while the positive expression of HLA-I was correlated with a more favorable survival prognosis. Furthermore, multivariate Cox proportional hazard analyses revealed that tumor grade, stage, and PD-1 expression are independent prognostic factors. The immunological landscape of Pan-NEN offers potential prognostic value and therapeutic targets. The findings suggest that immunotherapy, particularly targeting the PD-1/PD-L1 pathway, may serve as a promising strategy for the treatment of Pan-NEN, especially for Pan-NEC patients.

High Expression of Immune Checkpoint Molecules in Different Types of Thyroid Cancer

This study aimed to investigate the expression of programmed cell death protein-1 (PD-1) and its ligand (PD-L1) immune checkpoint molecules in thyroid carcinomas and determine their association with the clinicopathological characteristics of patients. Thyroid tissue specimens from 100 patients diagnosed with primary thyroid carcinomas including papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), medullary thyroid carcinoma (MTC), and anaplastic thyroid carcinoma (ATC) were collected. Sections were prepared from formalin-fixed paraffin-embedded samples, and PD-1 and PD-L1 expressions were examined using immunohistochemistry. PD-1 was detected in tumor-infiltrating lymphocytes (TILs) in 88% of the patients and tumor cells in 28% of the patients with 10% in PTC, 5% in FTC, 5% in MTC, and 8% in ATC). PD-L1 was found in tumor cells and TILs in 30% and 79% of the patients, respectively. Moreover, a significant difference was observed in PD-1 and PD-L1 expression between tumor cells and TILs across different tumor types. However, their expression in tumor cells and TILs was significantly higher in ATC compared to other tumor types. Additionally, the expression of PD-1 and PD-L1 was significantly associated with an advanced stage, higher tumor size, tumor necrosis, and mitosis. A significant positive correlation was also observed between the expression of PD-1 and PD-L1 in tumor cells and TILs. The higher expression of PD-1 and PD-L1 may contribute to tumor progression. Therefore, combinational immunotherapy by these immune checkpoint inhibitors might be a promising strategy for clinical improvement in patients with thyroid cancer, especially those with ATC.

Modification with IL-21 and CCL19 enhances killing efficiency and tumor infiltration of NKP30 CAR-T cells in lung cancer

To investigate whether modification with IL-21 and CCL19 enhances killing and tumor-infiltrating efficiency of NKP30 CAR-T cells in lung cancer. The modified IL-21-CCL19 NKP30 CAR-T cells expressing IL-21 and CCL19 fusion gene was constructed based on NKP30 CAR-T cells and stimulated with CD3CD28 antibodies and IL-2. The immunophenotype and migration of the cells in the presence of IL-21 were investigated using flow cytometry and migration experiments. Lactate dehydrogenase (LDH) release and sphere formation assays were used to assess the killing and infiltration capabilities of CAR-T cells, and the secretion levels of IFN-γ, IL-21 and CCL19 were determined with enzyme-linked immunospot assay (ELISPOT) and ELISA. A zebrafish model bearing HCG-27 cell xenograft was established by microinjection of the tumor cells into the yolk sac followed 24 h later by injection of the immune cells at the same site, and the fluorescence signals were captured using a fluorescent microscopy. The NKP30 ligand B7H6, which was almost undetectable in normal tissues and blood cells, was highly expressed (over 90%) in lung cancer cells. Compared with NKP30 CAR-T cells and conventional T cells, IL-21-CCL19 NKP30 CAR-T cells exhibited stronger proliferative and migration capabilities with the formation of central memory T cells. The reduced expressions of CTLA4 and PD1 in the constructed cells resulted in enhanced killing efficiency against lung cancer cells accompanied by significantly increased production of IFN-γ, IL-21 and CCL19. In the zebrafish models, CAR-T cells exhibited stronger cytotoxicity and proliferative abilities than typical T cells, but these differences were not statistically significant between the two CAR-T cells. Modification of NKP30 CAR-T cells with IL-21 and CCL19 facilitates their access into solid tumors for more effective tumor cell killing while producing a large number of memory T cells.

Myeloid gasdermin D drives early-stage T cell immunity and peripheral inflammation in a mouse model of Alzheimer's disease.

It is now realized that peripheral inflammation and abnormal immune responses, especially T cells, contribute to the development of Alzheimer's disease (AD). Gasdermin D (GSDMD) -mediated pyroptosis has been associated with several neuroinflammatory diseases, but whether GSDMD is involved in the peripheral inflammation and T cell immunity during AD remains unclear. We dynamically investigated GSDMD activation in the peripheral and central nervous system of 5×FAD mouse model and dissected the role of myeloid GSDMD using genetic knockout mice, especially its influence on peripheral T cell responses and AD inflammation. RNA sequencing and in vitro coculture were used to elucidate the underlying immune mechanisms involved. Targeted inhibitor experiments and clinical correlation analysis were used to further verify the function of GSDMD in AD. In the present study, caspase activated GSDMD in the spleen of 5×FAD mice earlier than in the brain during disease progression. Loss of myeloid cell GSDMD was shown to impair early-stage effector T cell activation in the periphery and prevent T cell infiltration into the brain, with an overall reduction in neuroinflammation. Furthermore, myeloid cell GSDMD induced T cell PD-1 expression through the IL-1β/NF-κB pathway, restricting regulatory T cells. The administration of a GSDMD inhibitor combined with an anti-PD-1 antibody was found to mitigate the development of AD-associated inflammation. In some AD patients, plasma sPD-1 is positively correlated with IL-Iβ and clinical features. Our study systematically identified a role for GSDMD in the AD-related peripheral inflammation and early-stage T cell immunity. These findings also suggest the therapeutic potential of targeting GSDMD for the early intervention in AD.

Abstract A047: Activation of conventional type 2 dendritic cells (cDC2) by anti-CD47 may enhance antitumor T cell function in mouse liver cancer models

Abstract Anti-CD47 has been extensively studied for its effects on activating phagocytosis by blocking the ‘don’t eat me’ signal presented by CD47 expressed on cancer cells. This study explored the effects of anti-CD47 on enhancing antigen presentation by dendritic cells and improving antigen-specific antitumor immunity. Mouse Hepa1-6 liver cancer cells with or without over-expression of LCMV-GP protein and splenocytes from P14 transgenic mice were used to test antigen-specific antitumor immunity. Bone marrow-derived conventional type 1 and type 2 dendritic cells (cDC1 and cDC2) or MutuDC1/ DC2 cell lines were co-cultured with CD8 T cells with or without exhaustion. Effects of anti-CD47 on the cross-priming ability of DC were analyzed by T cell proliferation (CSFE labeling), activation (expression of IFNγ, granzyme B, TNFa, and IL-2), cell killing (in vitro cytotoxicity assay) and T cell exhaustion (LAG3, TOX, PD1 expression). In vivo studies indicated that anti-CD47 treatment may suppress tumor growth, activate intra-tumoral cDC2 but not type 1 dendritic cells (cDC1), and delay CD8 T cells from entering terminal exhaustion in the tumor microenvironments. In vitro studies indicated that anti-CD47 may enhance the priming ability of cDC2, but not cDC1, and increase ISG15 expression in cDC2. When co-cultured with exhausted T cells, anti-CD47-treated cDC2 may delay the exhaustion process and restore antigen-specific effector function and cytotoxic capacity. Conclusion: Anti-CD47 may enhance antitumor T cell function in mouse liver cancer models by activation of cDC2, which may serve as a new strategy of developing immunotherapeutic agents for liver cancer (supported by grants NSTC 111- 2314-B-002-039 and 113-2314-B-002-113-MY3). Citation Format: Cheng-Zhe Jian, Da-Liang Ou, Chia-Lang Hsu, Chien-Kuo Lee, Chiun Hsu. Activation of conventional type 2 dendritic cells (cDC2) by anti-CD47 may enhance antitumor T cell function in mouse liver cancer models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A047.

Prognostic Impact of Acute and Chronic Inflammatory Interleukin Signatures in the Tumor Microenvironment of Early Breast Cancer

Interleukins play dual roles in breast cancer, acting as both promoters and inhibitors of tumorigenesis within the tumor microenvironment, shaped by their inflammatory functions. This study analyzed the subtype-specific prognostic significance of an acute inflammatory versus a chronic inflammatory interleukin signature using microarray-based gene expression analysis. Correlations between these interleukin signatures and immune cell markers (CD8, IgKC, and CD20) and immune checkpoints (PD-1) were also evaluated. This study investigated the prognostic significance of an acute inflammatory IL signature (IL-12, IL-21, and IFN-γ) and a chronic inflammatory IL signature (IL-4, IL-5, IL-10, IL-13, IL-17, and CXCL1) for metastasis-free survival (MFS) using Kaplan–Meier curves and Cox regression analyses in a cohort of 461 patients with early breast cancer. Correlations were analyzed using the Spearman–Rho correlation coefficient. Kaplan–Meier curves revealed that the prognostic significance of the acute inflammatory IL signature was specifically pronounced in the basal-like subtype (p = 0.004, Log Rank). This signature retained independent prognostic significance in multivariate Cox regression analysis (HR 0.463, 95% CI 0.290–0.741; p = 0.001). A higher expression of the acute inflammatory IL signature was associated with longer MFS. The chronic inflammatory IL signature showed a significant prognostic effect in the whole cohort, with higher expression associated with shorter MFS (p = 0.034). Strong correlations were found between the acute inflammatory IL signature and CD8 expression (ρ = 0.391; p < 0.001) and between the chronic inflammatory IL signature and PD-1 expression (ρ = 0.627; p < 0.001). This study highlights the complex interaction between acute and chronic inflammatory interleukins in breast cancer progression and prognosis. These findings provide insight into the prognostic relevance of interleukin expression patterns in breast cancer and may inform future therapeutic strategies targeting the immune–inflammatory axis.

Assessment of anti-CD69 antibody therapy alone or in combination with anti-PD-1 in murine GBM

ABSTRACT Background Glioblastoma (GBM) is an aggressive cancer with limited treatment options. Immunotherapy targeting CD69, an early activation marker on T cells, has shown promise in preclinical models of non-CNS malignancies. This study investigates anti-CD69 therapy alone or in combination with anti-PD-1 in a preclinical GBM model. Research design and methods CD69 expression in GBM patient tissues was analyzed using the TCGA database. Therapeutic efficacy of anti-CD69 was tested in a murine GBM model with different regimens. Immune cell populations in the tumor microenvironment (TME) were assessed by flow cytometry. Results Increased CD69 expression was observed in GBM patients compared to normal brain tissue and was associated with worse prognosis. Anti-CD69 treatment reduced percentages of CD69+ immune cells but did not improve survival in GBM-bearing mice. Increased PD-1 expression on NK cells was observed following anti-CD69 treatment. Anti-CD69 treatment was not improved by the addition of anti-PD-1 in vivo. Conclusions This is the first study evaluating anti-CD69 therapy in a preclinical GBM model. Despite promising preclinical data in other cancers, anti-CD69 monotherapy or combination therapy with anti-PD-1 did not improve survival in this GBM model.

P53 ABNORMAL ENDOMETRIAL CANCERS – ANALYSIS OF PD-1 CO-EXPRESSION BY CD4 HELPER AND CD8 CYTOTOXIC T-CELL SUBTYPES IN THE TUMOR MICROENVIRONMENT

Abstract Introduction/Objective Endometrial cancer is classified into four molecular subtypes of which the p53 abnormal variant is biologically aggressive. Programmed cell death protein 1 (PD-1) is a cell surface receptor and a vital inhibitor of the immune response. Programmed death ligand 1 (PD-L1) is a trans-membrane co-inhibitory factor of the immune response. PD-1 / PDL-1 interaction inhibits the activation and survival of T cells and decreases cytokine secretion. Some cancer cells express PD-L1, which then bind to PD-1 expressed on tumor specific T cells, and escape destruction by the T cells. The CD4/CD8 T cell immune response in the p53 abnormal variant has not been adequately evaluated with respect to the PD-1/PD-L1 pathway. Understanding this immune microenvironment is important to assess the potential use of anti-PD-1 targeted tumor therapy. Methods/Case Report Twenty cases of p53 abnormal endometrial cancers were obtained. All cases were PDL-1 +ve by immunohistochemistry. Two dual stains were performed - PD-1/CD4 and PD-1/CD8 antibodies. Six high-power fields (40x objective) with the greatest concentration of lymphocytes were counted. The total number of CD4 or CD8-only cells was recorded for each of the two stained slides for each case. The total number of PD- 1/CD4 and PD-1/CD8 positive cells were counted and recorded. The results were tabulated, and a paired t-test statistical analysis was performed to compare the total number of CD4 and CD8 cells and the percentage of PD-1/CD4 cells with the percentage of PD-1/CD8 positive cells. Results (if a Case Study enter NA) A statistically significant increase in the number of CD8 (Mean = 383) compared to CD4 cells (Mean = 317) was seen in the tumor microenvironment (p-value=0.0023). Evaluating PD-1 expression, the converse was true, and the percentage of PD-1/CD4 cells (Mean = 13.035%) was increased compared to the percentage of PD-1/CD8 cells (Mean = 9.205%), achieving statistical significance (p-value=0.0116). Conclusion The statistically significant increase in CD8 cells in the tumor and its stroma suggests that a robust cytotoxic response is being mounted to this high-risk molecular variant. The concurrent increase in CD4/PD-1 positive lymphocytes provides an appropriate microenvironment for the use of anti-PD-1 immunotherapy, since blockade of PD-1 on CD4 helper T cells may augment the tumor ablative cytotoxic properties of the CD8 positive lymphocytes. This includes reinvigoration of exhausted CD8+ T cells. Anti-PD-1 may also prevent the inhibition of PD-1 positive CD8 cells.

The tumor immune microenvironment of SCLC is not associated with its molecular subtypes

IntroductionSmall-cell lung carcinoma (SCLC) is a high-grade neuroendocrine carcinoma of poor prognosis. Although immune checkpoint blockers have shown promising results in advanced SCLC, the tumor immune microenvironment (TME) remains poorly understood, with no validated prognostic or predictive biomarkers of efficacy. MethodsThis retrospective study included surgically samples from 48 SCLC patients between 2009 and 2018. We assessed the TME using two quantitative 7-plex immunofluorescence panels focusing on T and B cells, and compared it to NSCLC (N = 10). Molecular subtypes were determined by assessing the expression of ASCL1, NEUROD1 and YAP1 using immunohistochemistry. ResultsImmune-hot SCLC were defined as those exhibiting the highest immune cell and immune-related marker densities. They were associated with longer overall survival, significantly more frequently detected at early stages, and exhibited high PD-L1 expression in immune cells, but were not associated with molecular subtypes. Compared to NSCLC, SCLC had significantly lower densities of CD20 + cells and higher density of PD1 + cells, with no significant differences in CD4 + , CD8 + and plasma cell densities. In univariate analysis, the highest OS was significantly associated with early stage (p < 0.001), low expression of NEUROD1 (p = 0.047), high PD1 + cell density (p < 0.001) and high PD-L1 immune cell expression (p = 0.04). Only stage and PD1 + cell density emerged as independent prognostic markers. ConclusionSCLC TME is highly heterogeneous. Immune-hot tumors were associated with OS but not with molecular classification. PD1 expression and PD-L1 expression by immune cells may thus serve as a prognostic marker.

Hemodiafiltration May Be Associated with Senescence-Related Phenotypic Alterations of Lymphocytes, Which May Predict Mortality in Patients Undergoing Dialysis.

Senescence-resembling alterations on the lymphocytes of patients undergoing dialysis have been widely described. However, the pathophysiology behind these phenomena has not been clarified. In this study, we examined the impact of dialysis prescription on T and B lymphocytes, in patients undergoing dialysis.: T and B cell subsets were determined with flow cytometry in 36 patients undergoing hemodialysis and 26 patients undergoing hemodiafiltration, according to the expression of CD45RA, CCR7, CD31, CD28, CD57, and PD1 for T cells, and IgD and CD27 for B cells. The immune phenotype was associated with dialysis modality, hemofiltration volume, and mortality. Compared with hemodialysis, patients undergoing hemodiafiltration had a significantly decreased percentage of CD4+CD28-CD57- T cells [3.8 (2.4-5.3) vs. 2.1 (1.3-3.3)%, respectively, p = 0.002] and exhausted CD4+ T cells [14.1 (8.9-19.4) vs. 8.5 (6.8-11.7)%, respectively, p = 0.005]. Additionally, the hemofiltration volume was negatively correlated with CD8+ EMRA T cells (r = -0.46, p = 0.03). Finally, the increased exhausted CD4+ T cell percentage was associated with increased all-cause mortality in patients undergoing dialysis, independent of age. Hemodiafiltration, especially with high hemofiltration volume, may have beneficial effects on senescence-related immune phenotypes. Immune phenotypes may also be a predicting factor for mortality in patients undergoing dialysis.

LncRNA MEG3 suppresses hepatocellular carcinoma by stimulating macrophage M1 polarization and modulating immune system via inhibiting CSF-1 in vivo/vitro studies

Hepatocellular carcinoma (HCC) is characterized by a complex tumor microenvironment (TME), and long non-coding RNAs (lncRNAs) MEG3 emerged as regulators of macrophage polarization with a negative relationship with colony-stimulating factor 1 (CSF-1). Few studies are on the interplay among MEG3, CSF-1, T helper cells (Th), and the programmed cell death protein 1 and its ligands (PD-1/PD-Ls) in TME of HCC.MEG3 expression in THP-1 macrophages, monitored polarization, and PD-1/PD-Ls expression were through flow cytometry, WB, and RT-qPCR. In co-cultures, the interaction of MEG3, macrophage, and HCC was assayed by ELISA. The invasive and migratory of HCC were assessed through experiments such as CCK-8, clonogenic assay, wound healing, and Transwell. A xenograft mouse model of HCC was established, administered with MEG3 overexpression (OE) or knockdown (KD) constructs, and monitored tumor growth. In vitro, MEG3 OE induced a robust M1 macrophage phenotype, evidenced by elevated expression of M1 markers and a significant increase in Th1 cytokines, with a concomitant decrease in Th2 cytokines. This was paralleled by reduced CSF-1 and PD-1/PD-Ls expression. In contrast, MEG3 KD promoted an M2 phenotype with increased CSF-1 and PD-1/PD-Ls expression, and an upregulation of Th2 cytokines. MEG3 OE inhibited the growth, invasion, and migration of HCC, while the opposite was observed when MEG3 was downregulated. In vivo, MEG3 OE resulted in significantly reduced tumor growth, with decreased PD-1/PD-Ls expression on macrophages and enhanced Th1 response. Conversely, MEG3 KD promoted tumor growth with increased PD-1/PD-Ls and a Th2-skewed immune response. MEG3 modulates the TME by affecting TAMs through CSF-1, thereby influencing the balance of Th1/Th2 cells and altering the expression of PD-1/PD-L1s. This study demonstrates that targeting MEG3 is an effective therapeutic strategy for HCC.

Novel Compound Heterozygous Variants in the FAS Gene Lead to Fetal Onset of Autoimmune Lymphoproliferative Syndrome (ALPS).

FAS gene defects lead to autoimmune lymphoproliferative syndrome (ALPS), which is often inherited in an autosomal dominant and rarely in an autosomal recessive manner. We report a case of a newborn girl with novel compound heterozygous variants in FAS and reveal the underlying mechanism. Whole-exome sequencing (WES) was used to identify pathogenic variants. Multiparametric flow cytometry analysis, phosflow analysis, and FAS-induced apoptosis assays were used to explore the effects of the variants on FAS expression, apoptosis, and immunophenotype. The HEK293T cells were used to assess the impact of the variants on protein expression and FAS-induced apoptosis. The patient was born with hepatosplenomegaly, anemia, and thrombocytopenia. She also experienced COVID-19, rotavirus infection, herpes simplex virus infection, and severe pneumonia. The proportion of double-negative T cells (DNTs) was significantly elevated. Novel FAS compound heterozygous variants c.310T > A (p.C104S) and c.702_704del (p.T235del) were identified. The apoptotic ability of T cells was defective, and FAS expression on the surface of T cells was deficient. The T235del variant decreased FAS expression, and the C104S protein remained in the endoplasmic reticulum (ER) and could not translocate to the cell surface. Both mutations resulted in loss-of-function in terms of FAS-induced apoptosis in HEK293T cells. The DNTs were mainly terminally differentiated T (TEMRA) and CD45RA+HLA-DR+, with high expression of CD85j, PD-1, and CD57. The percentage of Th1, Tfh, and autoreactive B cells were significantly increased in the patient. The abnormal immunophenotyping was partially attenuated by sirolimus treatment. We identified two variants that significantly affect FAS expression or localization, leading to early disease onset of in the fetus. Abnormalities in the mTOR pathway are associated with a favorable response to sirolimus.

Circulating mucosal-associated invariant T cell alterations in adults with recent-onset and long-term oral lichen planus

BackgroundMucosal-associated invariant T (MAIT) cells play key roles in many inflammatory diseases. However, their effects on the long-term course of oral lichen planus (OLP) and recent-onset OLP remain unclear. In this study, we aimed to investigate the function of MAIT cells in the different processes of OLP and to explore the immunological background of this disease.MethodsThe frequency, phenotype, cytokine secretion, and clinical relevance of MAIT cells were investigated. MAIT cells were collected from the peripheral blood of 14 adults with recent-onset OLP (7–120 days after disease onset) and 16 adults with long-term course OLP (>2 years after diagnosis) using flow cytometry and compared with 15 healthy blood donors. Statistical analyses were performed using the GraphPad Prism software.ResultsMAIT cells from adults with recent-onset OLP exhibited an activated phenotype, as indicated by an increased frequency of CD69+ (p < 0.05) and CD38+MAIT cells (p < 0.01) and elevated production of the proinflammatory cytokine IL-17 A (p < 0.01), compared with healthy adult donors. In adults with long-term OLP, MAIT cells exhibited an activated and exhausted phenotype, characterized by high expression of CD69 (p < 0.01) and PD-1 (p < 0.001) and increased production of granzyme B released (p < 0.01). Compared with recent-onset OLP patients, long-term OLP patients showed a decreased production of CD8+, and CD4−CD8− cells, but an increase in PD-1+ production (p < 0.05).ConclusionsCirculating MAIT cells exhibited activation in OLP patients across varying disease durations. Given that PD-1 expression is elevated in adults with long-term OLP, it is reasonable to infer that circulating MAIT cells in long-term OLP may exhibit a more exhausted state than those in recent-onset OLP.

6413 Anti-PD1 induces T1D in an HLA-DQ8, HLA-A2.1 mouse model

Abstract Disclosure: P. Houeiss: None. S. luce: None. C. Boitard: None. Anti-PD1 is an immune checkpoint inhibitor that has revolutionized cancer treatment. It blocks the interaction between PD1, a receptor on the surface of activated or exhausted T cells, and its ligands PDL1 and PDL2, to inhibit the immune response. However, anti-PD1 induces T1D in 0.4-2% of patients raising questions about the risk factors and the mechanisms behind T1D development. Since the pancreas of these patients is inaccessible, we focus on autoimmune diabetes induction in a transgenic mouse model modified to express the T1D susceptibility genes HLA-DQ8, HLA-A2.1 and the human preproinsulin and on identifying new targets to prevent or treat this disease. Thus, 8 to 12 weeks old mice were treated initially with 500 micrograms of anti-PD1 followed by 250 micrograms every other day for 10 days. Control mice received 6 injections of PBS. Mice were followed up for evidence of hyperglycemia for 60 days. Cells and tissues from the pancreas, and the spleens were isolated 15-, 30- or 60-days post-treatment initiation and analyzed by flow cytometry and immunohistochemistry. Although diabetes incidence in treated mice was 14% (n=14), insulitis incidence progressively increased over time reaching 86% on D60 (p=0.005). On Day 15, in the spleen, a significant 10% increase of CD4+Foxp3+CD25- T cells at the expense of the CD4+Foxp3- T cells were seen in the treatment group as compared to the control (37.1±4.25 vs 26±3.5, p&amp;lt;0.01, n=5 per group). In the treatment group, PD1 expression significantly increased on CD4+Foxp3+ T cells whereas PDL1 expression increased on CD4+Foxp3- and CD8+ T cells (p&amp;lt;0.01). On D30, in the pancreas, infiltrates show positive staining for F4/80+, CD4+ and CD8+ T cells with scarce foxp3+ T cells. While the absolute percentage of CD4+Foxp3+ T cells increased in the insulitis and diabetes group, their percentage among CD3+ T cells decreased in the insulitis (10.2%) and diabetes group (3.9%) as compared to the control group (13.5%). Infiltrates were concentrated around the islets and the efferent and afferent vessels, and extended to the exocrine pancreas, leading to secondary acinar destruction. In the pancreatic infiltrates from anti-PD1 treated mice, we noted a significant increase in CD4+ T cells recognizing Ins13-21 monomers as compared to the negative control. PD1 blockade induced an insulitis characterized by a Treg to effector T cells imbalance. Thus, it acted on the early phase of T1D. In conclusion, this model describes a new T1D endotype that shares similarities with ICI induced diabetes in patients. Presentation: 6/2/2024

9300 Anti-PD1 induces T1D in an HLA-DQ8, HLA-A2.1 mouse model

Abstract Disclosure: P. Houeiss: None. S. luce: None. C. Boitard: None. Anti-PD1 is an immune checkpoint inhibitor that has revolutionized cancer treatment. It blocks the interaction between PD1, a receptor on the surface of activated or exhausted T cells, and its ligands PDL1 and PDL2, to inhibit the immune response. However, anti-PD1 induces T1D in 0.4-2% of patients raising questions about the risk factors and the mechanisms behind T1D development. Since the pancreas of these patients is inaccessible, we focus on autoimmune diabetes induction in a transgenic mouse model modified to express the T1D susceptibility genes HLA-DQ8, HLA-A2.1 and the human preproinsulin and on identifying new targets to prevent or treat this disease. Thus, 8 to 12 weeks old mice were treated initially with 500 micrograms of anti-PD1 followed by 250 micrograms every other day for 10 days. Control mice received 6 injections of PBS. Mice were followed up for evidence of hyperglycemia for 60 days. Cells and tissues from the pancreas, and the spleens were isolated 15-, 30- or 60-days post-treatment initiation and analyzed by flow cytometry and immunohistochemistry. Although diabetes incidence in treated mice was 14% (n=14), insulitis incidence progressively increased over time reaching 86% on D60 (p=0.005). On Day 15, in the spleen, a significant 10% increase of CD4+Foxp3+CD25- T cells at the expense of the CD4+Foxp3- T cells were seen in the treatment group as compared to the control (37.1±4.25 vs 26±3.5, p&amp;lt;0.01, n=5 per group). In the treatment group, PD1 expression significantly increased on CD4+Foxp3+ T cells whereas PDL1 expression increased on CD4+Foxp3- and CD8+ T cells (p&amp;lt;0.01). On D30, in the pancreas, infiltrates show positive staining for F4/80+, CD4+ and CD8+ T cells with scarce foxp3+ T cells. While the absolute percentage of CD4+Foxp3+ T cells increased in the insulitis and diabetes group, their percentage among CD3+ T cells decreased in the insulitis (10.2%) and diabetes group (3.9%) as compared to the control group (13.5%). Infiltrates were concentrated around the islets and the efferent and afferent vessels, and extended to the exocrine pancreas, leading to secondary acinar destruction. In the pancreatic infiltrates from anti-PD1 treated mice, we noted a significant increase in CD4+ T cells recognizing Ins13-21 monomers as compared to the negative control. PD1 blockade induced an insulitis characterized by a Treg to effector T cells imbalance. Thus, it acted on the early phase of T1D. In conclusion, this model describes a new T1D endotype that shares similarities with ICI induced diabetes in patients. Presentation: 6/2/2024

Vaccination generates functional progenitor tumor-specific CD8 T cells and long-term tumor control

BackgroundImmune checkpoint blockade (ICB) therapies are an important treatment for patients with advanced cancers; however, only a subset of patients with certain types of cancer achieve durable remission. Cancer vaccines...

Effects of PACAP Deficiency on Immune Dysfunction and Peyer's Patch Integrity in Adult Mice.

PACAP (pituitary adenylate cyclase activating polypeptide) is a widespread neuropeptide with cytoprotective and anti-inflammatory effects. It plays a role in innate and adaptive immunity, but data are limited about gut-associated lymphoid tissue. We aimed to reveal differences in Peyer's patches between wild-type (WT) and PACAP-deficient (KO) mice. Peyer's patch morphology from young (3-months-old) and aging (12-15-months-old) mice was examined, along with flow cytometry to assess immune cell populations, expression of checkpoint molecules (PD-1, PD-L1, TIM-3, Gal-9) and functional markers (CD69, granzyme B, perforin) in CD3+, CD4+, and CD8+ T cells. We found slight differences between aging, but not in young, WT, and KO mice. In WT mice, aging reduced CD8+ T cell numbers frequency and altered checkpoint molecule expression (higher TIM-3, granzyme B; lower Gal-9, CD69). CD4+ T cell frequency was higher with similar checkpoint alterations, indicating a regulatory shift. In PACAP KO mice, aging did not change cell population frequencies but led to higher TIM-3, granzyme B and lower PD-1, PD-L1, Gal-9, and CD69 expression in CD4+ and CD8+ T cells, with reduced overall T cell activity. Thus, PACAP deficiency impacts immune dysfunction by altering checkpoint molecules and T cell functionality, particularly in CD8+ T cells, suggesting complex immune responses by PACAP, highlighting its role in intestinal homeostasis and potential implications for inflammatory bowel diseases.