Immune Lymphocytes Research Articles

Pla2g2a promotes innate Th2-type immunity lymphocytes to increase B1a cells

Newborns require early generation of effective innate immunity as a primary physiological mechanism for survival. The neonatal Lin28+Let7– developmental pathway allows increased generation of Th2-type cells and B1a (B-1 B) cells compared to adult cells and long-term maintenance of these initially generated innate cells. For initial B1a cell growth from the neonatal to adult stage, Th2-type IL-5 production from ILC2s and NKT2 cells is important to increase B1a cells. The Th17 increase is dependent on extracellular bacteria, and increased bacteria leads to lower Th2-type generation. Secreted group IIA-phospholipase A2 (sPLA2-IIA) from the Pla2g2a gene can bind to gram-positive bacteria and degrade bacterial membranes, controlling microbiota in the intestine. BALB/c mice are Pla2g2a+, and express high numbers of Th2-type cells and B1a cells. C57BL/6 mice are Pla2g2a-deficient and distinct from the SLAM family, and exhibit fewer NKT2 cells and fewer B1a cells from the neonatal to adult stage. We found that loss of Pla2g2a in the BALB/c background decreased IL-5 from Th2-type ILC2s and NKT2s but increased bacterial-reactive NKT17 cells and MAIT cells, and decreased the number of early-generated B1a cells and MZ B cells and the CD4/CD8 T cell ratio. Low IL-5 by decreased Th2-type cells in Pla2g2a loss led to low early-generated B1a cell growth from the neonatal to adult stage. In anti-thymocyte/Thy-1 autoreactive μκ transgenic (ATAμκ Tg) Pla2g2a+ BALB/c background C.B17 mice generated NKT2 cells that continuously control CD1d+ B1 B cells through old aging and lost CD1d in B1 B cells generating strong B1 ATA B cell leukemia/lymphoma. Pla2g2a-deficient ATAμκTg C57BL/6 mice suppressed the initial B1a cell increase, with low/negative spontaneous leukemia/lymphoma generation. These data confirmed that the presence of Pla2g2a to control bacteria is important to allow the neonatal to adult stage. Pla2g2a promotes innate Th2-type immunity lymphocytes to increase early generated B1a cells.

Identification of Adipocytokine Pathway-Related Genes in Epilepsy and Its Effect on the Peripheral Immune Landscape

Epilepsy is a multifactorial neurological disorder with recurrent epileptic seizures. Current research stresses both inflammatory and autoimmune conditions as enablers in the pathophysiological process of epilepsy. In view of the growing concern about the role of adipocytokines in antiepileptic and modulating immune responses, we aimed to investigate the relevance of the adipocytokine signaling pathway in the pathological process of epilepsy and its impacts on peripheral immune characteristics. In this study, expression profiles of 142 peripheral blood samples were downloaded from the Gene Expression Omnibus (GEO) database. Adipocytokine pathway-related genes were screened out by feature selection using machine-learning algorithms. A nomogram was then constructed and estimated for the efficacy of diagnosis. Cluster analysis was employed for the recognization of two distinct epilepsy subtypes, followed by an estimation of the immune cell infiltration levels using single-sample gene-set enrichment analysis (ssGSEA). The biological characteristics were analyzed by functional enrichment analysis. The aberrant regulation of adipocytokine signaling pathway was found in the peripheral blood of patients with epilepsy. Twenty-one differently expressed adipocytokine pathway-related genes were identified and five (RELA, PRKAB1, TNFRSF1A, CAMKK2, and CPT1B) were selected to construct a nomogram. Subsequent validations of its forecasting ability revealed that this model has satisfactory predictive value. The immune cell infiltration degrees, such as those of innate immune cells and lymphocytes, were found to significantly correlate to the levels of adipocytokine pathway-related genes. Additionally, 239 differentially expressed genes (DEGs) were identified and their biological functions were mainly enriched in the regulation of the immune response. In conclusion, our results confirmed the predictive value of adipocytokine pathway-related genes for epilepsy and explored their effects on immune infiltration, thereby improving our understanding of the pathogenesis of epilepsy and providing assistance in the diagnosis and treatment of epilepsy.

Identify metabolism-related genes IDO1, ALDH2, NCOA2, SLC7A5, SLC3A2, LDHB, and HPRT1 as potential prognostic markers and correlate with immune infiltrates in head and neck squamous cell carcinoma

Hypopharyngeal squamous cell carcinoma (HSCC) is a kind of head and neck squamous cell carcinoma (HNSCC) with poor prognosis. Metabolic reprogramming may regulate the tumor microenvironment (TME) by adapting quickly to cellular stress and regulating immune response, but its role in HSCC has not been reported. We used the nCounter® Metabolic Pathways Panel to investigate metabolic reprogramming, cellular stress, and their relationship in HSCC tissues and adjacent normal tissues. Metabolism-related pathways nucleotide synthesis and glycolysis pathways were significantly upregulated, while amino acid synthesis and fatty acid oxidation pathways were significantly downregulated in HSCC tissues compared to adjacent normal tissues. There is a significant correlation between metabolism-related pathways and cellular stress pathways. Enrichment of immune cell and tumor infiltrating lymphocyte (TIL) analysis showed changes in immune responses between HSCC tissues and adjacent normal tissues. Overall survival analysis showed that upregulated genes CD276, LDHB, SLC3A2, EGFR, SLC7A5, and HPRT1 are potential unfavorable prognostic markers in HNSCC, while downregulated genes EEA1, IDO1, NCOA2, REST, CCL19, and ALDH2 are potential favorable prognostic markers in HNSCC. Moreover, metabolism-related genes IDO1, ALDH2, NCOA2, SLC7A5, SLC3A2, LDHB, and HPRT1 are correlated with immune infiltrates in HNSCC. These results suggest that metabolic reprogramming occurs and correlates with cellular stress and immune response in HSCC, which may help researchers understand mechanisms of metabolic reprogramming and develop effective immunotherapeutic strategies in HNSCC.

WD repeat domain 6 as a novelty prognostic biomarker correlates with immune infiltration in lung cancer: A preliminary study.

BackgroundWD repeat domain 6 (WDR6), a novel human WD‐repeat gene, encodes a member of the WD repeat protein family, and its tumorigenic effect has rarely been reported so far.MethodsOur study used Oncomine, TIMER2.0, GEPIA2, Kaplan–Meier plotter, PrognoScan, and TISIDB tools to analyze the differential expression between pan‐cancer, especially lung cancer, and corresponding normal tissue, and further explore the prognostic and immunological role of WDR6 expression.ResultsOur results showed WDR6 was lower expressed in lung squamous cell carcinoma than in normal tissue, but WDR6 expression was correlated obviously with clinical stage in Lung adenocarcinoma. The overall survival, first progression, postprogression survival, and Relapse‐free survival of lung cancer patients were longer in the WDR6 high‐expression group than in the low‐expression group. We found the expression of WDR6 significantly correlated with immune molecules, including immunomodulators, lymphocytes, and chemokines in lung cancer.ConclusionWDR6 can be used as a prognostic marker for lung cancer and is significantly associated with immune cell infiltration.

Heat-killed Limosilactobacillus reuteri PSC102 Ameliorates Impaired Immunity in Cyclophosphamide-induced Immunosuppressed Mice.

The immune functions of heat-killed Limosilactobacillus reuteri PSC102 (hLR) were investigated in cyclophosphamide (CP)-treated immunosuppressed mice. BALB/c mice were randomly divided into five groups: normal control group, CP group, CP treated with levamisole (positive control group), and CP treated with low- and high-dose hLR. After receiving the samples for 21 days, mice were sacrificed, and different parameters, such as immune organ index, immune blood cells, splenocyte proliferation, lymphocyte subpopulations, cytokines, and immunoglobulins, were analyzed. Results showed that the immune organ (thymus and spleen) indices of hLR treatment groups were significantly increased compared to the CP group (p < 0.05). hLR administration prevented CP-induced reduction in the numbers of white blood cells, lymphocytes, midrange absolute, and granulocytes, providing supporting evidence for hematopoietic activities. Splenocyte proliferation and T-lymphocyte (CD4+ and CD8+) subpopulations were also significantly augmented in mice treated with hLR compared to the CP group (p < 0.05). Moreover, Th1-type [interferon-γ, interleukin (IL)-2, and tumor necrosis factor-α] and Th2-type (IL-4 and IL-10) immune factors and immunoglobulin (IgG) showed significant increasing trends (p < 0.05). Additionally, the other proinflammatory cytokines (IL-1β and IL-6) were also significantly elevated (p < 0.05). Taken together, this investigation suggested that orally administered hLR could recover immunosuppression caused by CP and be considered a potential immunostimulatory agent for the treatment of immunosuppressive disorders.

Comprehensive Analysis Identifies and Validates the Tumor Microenvironment Subtypes to Predict Anti-Tumor Therapy Efficacy in Hepatocellular Carcinoma

ObjectiveThe objective of this study was to explore and verify the subtypes in hepatocellular carcinoma based on the immune (lymphocyte and myeloid cells), stem, and stromal cells in the tumor microenvironment and analyze the biological characteristics and potential relevance of each cluster.MethodsWe used the xCell algorithm to calculate cell scores and got subtypes by k-means clustering. In the external validation sets, we verified the conclusion stability by a neural network model. Simultaneously, we speculated the inner connection between clusters by pseudotime trajectory analysis and confirmed it by pathway enrichment, TMB, CNV, etc., analysis.ResultAccording to the results of the consensus cluster, we chose k = 4 as the optimal value and got four different subtypes (C1, C2, C3, and C4) with different biological characteristics based on infiltrating levels of 48 cells in TME. In univariable Cox regression, the hazard ratio (HR) value of C3 versus C1 was 2.881 (95% CI: 1.572–5.279); in multivariable Cox regression, we corrected the age and TNM stage, and the HR value of C3 versus C1 was 2.510 (95% CI: 1.339–4.706). C1 and C2 belonged to the immune-active type, C3 and C4 related to the immune-insensitive type and the potential conversion relationships between clusters. We established a neural network model, and the area under the curves of the neural network model was 0.949 in the testing cohort; the same survival results were also observed in the external validation set. We compared the differences in cell infiltration, immune function, pathway enrichment, TMB, and CNV of four clusters and speculated that C1 and C2 were more likely to benefit from immunotherapy and C3 may benefit from FGF inhibitors.DiscussionOur analysis provides a new approach for the identification of four tumor microenvironment clusters in patients with liver cancer and identifies the biological differences and predicts the immunotherapy efficacy between the four subtypes.

Immune Lymphocyte Infiltrate and its Prognostic Value in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) occurs more frequently in young (<50 years) non-Hispanic black and Hispanic/Latina women. It is considered the most aggressive subtype of breast cancer, although, recently, immune infiltrate has been associated with long-term survival, lower risk of death and recurrence, and response to neoadjuvant chemotherapy. The aim of this review was to evaluate the clinical impact of the immune infiltrate in TNBC by discussing whether its prognostic value varies across different populations. A comprehensive systematic search in databases such as PubMed and Web of Science was conducted to include papers focused on tumor-infiltrating lymphocytes (TILs) in TNBC in different population groups and that were published before January 2021. TNBC patients with higher levels of TILs had longer overall survival and disease-free survival times compared with TNBC patients with low TIL levels. Similar results were observed for CD4+, CD8+ TIL populations. On the other hand, patients with high TIL levels showed a higher rate of pathological complete response regardless of the population group (Asian, European, and American). These results altogether suggest that TIL subpopulations might have a prognostic role in TNBC, but the underlying mechanism needs to be elucidated. Although the prognosis value of TILs was not found different between the population groups analyzed in the revised literature, further studies including underrepresented populations with different genetic ancestries are still necessary to conclude in this regard.

Cross-Activation of Hemichannels/Gap Junctions and Immunoglobulin-Like Domains in Innate-Adaptive Immune Responses.

Hemichannels (HCs)/gap junctions (GJs) and immunoglobulin (Ig)-like domain-containing proteins (IGLDCPs) are involved in the innate–adaptive immune response independently. Despite of available evidence demonstrating the importance of HCs/GJs and IGLDCPs in initiating, implementing, and terminating the entire immune response, our understanding of their mutual interactions in immunological function remains rudimentary. IGLDCPs include immune checkpoint molecules of the immunoglobulin family expressed in T and B lymphocytes, most of which are cluster of differentiation (CD) antigens. They also constitute the principal components of the immunological synapse (IS), which is formed on the cell surface, including the phagocytic synapse, T cell synapse, B cell synapse, and astrocytes–neuronal synapse. During the three stages of the immune response, namely innate immunity, innate–adaptive immunity, and adaptive immunity, HCs/GJs and IGLDCPs are cross-activated during the entire process. The present review summarizes the current understanding of HC-released immune signaling factors that influence IGLDCPs in regulating innate–adaptive immunity. ATP-induced “eat me” signals released by HCs, as well as CD31, CD47, and CD46 “don’t eat me” signaling molecules, trigger initiation of innate immunity, which serves to regulate phagocytosis. Additionally, HC-mediated trogocytosis promotes antigen presentation and amplification. Importantly, HC-mediated CD4+ T lymphocyte activation is critical in the transition of the innate immune response to adaptive immunity. HCs also mediate non-specific transcytosis of antibodies produced by mature B lymphocytes, for instance, IgA transcytosis in ovarian cancer cells, which triggers innate immunity. Further understanding of the interplay between HCs/GJs and IGLDCPs would aid in identifying therapeutic targets that regulate the HC–Ig-like domain immune response, thereby providing a viable treatment strategy for immunological diseases. The present review delineates the clinical immunology-related applications of HC–Ig-like domain cross-activation, which would greatly benefit medical professionals and immunological researchers alike. HCs/GJs and IGLDCPs mediate phagocytosis via ATP; “eat me and don’t eat me” signals trigger innate immunity; HC-mediated trogocytosis promotes antigen presentation and amplification in innate–adaptive immunity; HCs also mediate non-specific transcytosis of antibodies produced by mature B lymphocytes in adaptive immunity.

Dihydromyricetin Improves High-Fat Diet-Induced Hyperglycemia through ILC3 Activation via a SIRT3-Dependent Mechanism.

Previous studies indicate that dihydromyricetin (DHM) effectively improved glucose homeostasis and alleviated insulin resistance in population-intervened trials, yet the underlying mechanism remains obscure. Wild-type male mice and recombinase activating gene 1(Rag1)-/- mice (lacking adaptive immunity lymphocytes) are fed with control, high-fat diet (HFD), or HFD+DHM diets for 8 weeks. DHM effectively protects HFD feeding mice against hyperglycemia by promoting group 3 innate lymphoid cells (ILC3s) cells proliferation and interleukin 22 (IL-22) production. Furthermore, IL-22 secretion induced by DHM increases the expression levels of the tight junction (TJs) molecules to protect the intestinal barrier integrity, thereby decreasing the level of lipopolysaccharides (LPS), an endotoxin that is involved in the regulation of chronic tissue inflammation and insulin resistance. In addition, silent mating-type information regulation 2 homolog 3 (SIRT3) deficiency results in more serious obesity and intestinal barrier damage following HFD feeding and abolished DHM-mediated increase in IL-22 expression levels of ILC3 cells in SIRT3 knockout (SIRT3KO) mice. DHM reduces metabolic stress and enhances mitochondrial respiratory capacity to promote cell proliferation and IL-22 secretion by activating SIRT3 in ILC3 cells CONCLUSIONS: DHM improves IL-22 production of ILC3 cells and subsequently inhibits intestinal barrier dysfunction to alleviate hyperglycemia partially mediated by SIRT3.

LAG3 ectodomain structure reveals functional interfaces for ligand and antibody recognition.

The immune checkpoint receptor lymphocyte activation gene 3 protein (LAG3) inhibits T cell function upon binding to major histocompatibility complex class II (MHC class II) or fibrinogen-like protein 1 (FGL1). Despite the emergence of LAG3 as a target for next-generation immunotherapies, we have little information describing the molecular structure of the LAG3 protein or how it engages cellular ligands. Here we determined the structures of human and murine LAG3 ectodomains, revealing a dimeric assembly mediated by Ig domain 2. Epitope mapping indicates that a potent LAG3 antagonist antibody blocks interactions with MHC class II and FGL1 by binding to a flexible 'loop 2' region in LAG3 domain 1. We also defined the LAG3-FGL1 interface by mapping mutations onto structures of LAG3 and FGL1 and established that FGL1 cross-linking induces the formation of higher-order LAG3 oligomers. These insights can guide LAG3-based drug development and implicate ligand-mediated LAG3 clustering as a mechanism for disrupting T cell activation.

Analysis of the immune checkpoint lymphocyte activation gene-3 (LAG-3) in endometrial cancer: An emerging target for immunotherapy

BackgroundLymphocyte activation gene-3 (LAG-3) is a novel molecule that participates in the immune escape of tumor cells and is a target for immunotherapy. However, the expression of LAG-3 in patients with endometrial cancer (EC) has not been comprehensively characterized. ObjectivesWe elucidated the expression of LAG-3 and investigated its correlation with clinicopathological parameters, ProMisE subtypes, CD8+ T-cell infiltration and relapse-free survival (RFS) in a retrospective cohort of 421 patients with endometrial cancer. MethodsNext-generation sequencing of the polymerase epsilon (POLE) and immunohistochemistry of mismatch repair (MMR)-related protein (MLH1, PMS2, MSH2, and MSH6), p53, CD8 and LAG-3 protein in whole sections were performed. ResultsPositive LAG-3 was detected in tumor cells (TCs) and immune cells (ICs) in 31.6% (133/421) and 24.0% (101/421) of the patients, respectively. LAG-3 positivity in ICs was more common in high-grade, high-intermediate risk, high-risk, and advanced/metastatic subgroups and was relevant to lymphovascular space invasion, while that in TCs was more common in older individuals (≥54 years). LAG-3 expression was more prevalent in POLE ultramutated (POLEmut) and MMR-deficient (MMRd) EC than in p53-abnormal (p53abn) and p53-wild (p53wt) EC in TCs (34.4 % and 66.3% in POLEmut and MMRd versus 28.6% and 19.5% in p53abn and p53wt, P < 0.001) and ICs (78.1 % and 65.1% in POLEmut and MMRd versus 2.9% and 5.2% in p53abn and p53wt, P < 0.001). Positive expression of LAG-3 in TCs and ICs was associated with high levels of tumor-associated CD8+ T-cell immune infiltration. Additionally, LAG-3 positivity in TCs was related to improved RFS. ConclusionsThis study suggests that immunotherapy targeting LAG-3 may play a role in EC patients with POLEmut or MMRd molecular markers. Positive LAG-3 expression in TCs may be a predictor of improved RFS.

Intratumoral fibrosis and patterns of immune infiltration in clear cell renal cell carcinoma

BackgroundIntratumoral fibrosis was positively correlated with histological grade of renal clear cell carcinoma (ccRCC) and intratumoral inflammation. However, the association of intratumoral fibrosis with the immune infiltration of ccRCC was few evaluated.MethodsWe used the second harmonic generation (SHG)-based imaging technology and evaluated the intratumoral fibrosis in ccRCC, and then divided the patients into the high fibrosis group (HF) and the low fibrosis group (LF). Meanwhile, the Kaplan–Meier survival curve analysis was performed to analyze the relationship between intratumoral fibrosis and the disease-free survival rate. Antibody arrays were used for seeking difference in cytokines and immune infiltration between the HF group (N = 11) and LF group (N = 11). The selected immune infiltration marker was then verified by immunohistochemistry (IHC) staining in 45 ccRCC samples.ResultsOut of 640 cytokines and immune infiltration markers, we identified 115 proteins that were significantly different in quantity between ccRCC and adjacent normal tissues. In addition, the Venn diagram indicated that six proteins, including Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4), were significantly associated with intratumoral fibrosis (p < 0.05). The GO/KEGG enrichment analysis indicated that the proteins associated with intratumoral fibrosis were involved in the immunity and tumor-infiltrating lymphocytes. The expression of the CTLA4 was negatively correlated with collagen level, confirmed by IHC staining of CTLA4 (p < 0.05).ConclusionsThe study indicated that the intratumoral fibrosis level was negatively correlated with the expression of CTLA4 in the tumor immune microenvironment of the ccRCC, which posed the potential value of targeting the stroma of the tumor, a supplement to immunotherapy. However, the specific mechanism of this association is still unclear and needs further investigation.

Abstract 3104: Inverse correlation of circulating CD8+ T cells with tumor burden from immune checkpoint treatment in humanized orthotopic model of renal cell carcinoma

Abstract Background: Renal cell carcinoma (RCC) is a highly heterogeneous and metastatic disease with a widely varying prognosis. RCC is the first solid tumor treated with immune checkpoint blockade and approved by the FDA for showing improvement of clinical outcomes. Immune checkpoints physiologically regulate immune response via appropriate signals to T cells and maintain homeostasis, while pathologically they can be co-opted by cancer cells to evade the immune system. Here we developed a humanized (Hu)-mice model with adaptive transferred human immune cells to test the role of ICB [anti-PD-1/PD-L1, or anti-CTLA-4 antibody (Ab)] in an orthotopic human RCC tumor-bearing xenograft mouse model and analyzed the correlation of innate immune cells with tumor burden. Methods: Luciferase-tagged RCC cell line 786-O were injected intra-kidney into immune-deficient Rag2 mice. After tumor formation, mice were humanized by intraperitoneal injection of donor PBMCs (20×106/per mouse). Treatment groups included control, anti-PD-1/PD-L1 Abs or anti-CTLA-4 Ab (all Abs 200 µg/mouse, iv) weekly for 4 weeks. Tumor growth was measured by weekly bioluminescent imaging (BLI) and tumor weight at necropsy. The presence of human immune cells and tumor infiltrating lymphocytes was confirmed by flow cytometry and immunohistochemistry staining. The significance of the differences between groups was determined by student t test. The Pearson correlation coefficient was obtained by analysis of correlation between different study parameters. Results: BLI level showed a steady xenograft tumor progression for nine weeks. Anti-PD-1/PD-L1 Abs treatment trended toward reducing tumor BLI level while anti-CTLA-4 Ab significantly decreased tumor BLI level (p&amp;lt;0.01). Furthermore, anti-PD-1/PD-L1 Abs reduced lung metastasis with a significant decrease of BLI level compared to control group (p&amp;lt;0.05). Meanwhile, humanization was evidenced by robust, elevated levels of human CD45+ lymphocytes in the peripheral blood compared to non-reconstituted Rag2 mice. Infiltrated human CD3+CD8+ cells were found in mouse blood and spleen. Granzyme B+ cells, CD31+ cells, and pan-cytokeratin+ cells were found in tumors. Interestingly, we observed inverse correlation between circulating human CD8+ T cells and mouse RCC tumor burden. Conclusion: We successfully generated a humanized RCC mouse model which allows the RCC xenograft to grow steadily within the human immune system reconstituted Rag2 mice. Circulating CD8+ T cells are inversely correlated to tumor growth in this model. The PD-1/PD-L1 or CTLA-4-targeted immunotherapies showed distinct therapeutic effects in our humanized orthotopic xenograft model, which may lead to personalized treatment targeting different immune checkpoints to reinvigorate anti-tumor responses in RCC patients. Citation Format: Xin Zhang, Amita Bhattarai, Ravan Moret, Grace Maresh, Alicia Nicole Ray, David Woods, Rachel Graham, Maria Latis, Stephen Bardot, Li Li. Inverse correlation of circulating CD8+ T cells with tumor burden from immune checkpoint treatment in humanized orthotopic model of renal cell carcinoma [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 3104.

Abstract 774: Microdissected gene expression profiling of recurrent nasopharyngeal carcinoma

Abstract Nasopharyngeal cancer (NPC) is an EBV-driven epithelial carcinoma with a recurrence rate of 30%. Biological pathways and predictive markers for recurrence remain understudied. Further, NPC tumors are associated with an intense inflammatory infiltrate, masking gene expression signals from the tumor epithelial compartment. We perform laser-capture microdissection on a preliminary cohort of 12 tumors, including primary tumors from recurrent and non-recurrent cases matched for stage of disease. Smart-3SEQ, a novel 3’ end RNA-Seq technique which allows for the accurate quantification of transcript abundance in FFPE samples, was performed to obtain gene expression libraries. Tumor epithelial and microenvironment regions from were separately microdissected, giving a total of 42 histologically-pure gene expression libraries. Raw reads were mapped to the human hg19 and EBV genomes and gene expression counts were obtained with Subread. Differential gene expression and gene set enrichment was performed in R with DESeq2 and fgsea respectively. We observed that primary NPC tumors that did not recur were significantly enriched for immune processes, including leukocyte mediated immunity, innate immune response and lymphocyte activation (padj &amp;lt; 1E-9 for all). The top differentially expressed genes in this group included mediators of inflammation such as STAT1 (padj = 0.0204). Critically, these gene expression changes were observed within the tumor epithelial compartment. In contrast, primary NPC tumors that eventually recurred were enriched for known oncogenes including BCL2 (padj = 0.000420). These findings suggest that heterogeneity in gene expression between NPC tumors play an important role in treatment response. Further work to define gene expression subtypes of NPC tumors will be helpful for patient stratification and personalized care. Citation Format: Joshua Tay, Wei Keat Teo, Chuan Keng Goh, Bing Cheng Wu, Kwok Seng Loh. Microdissected gene expression profiling of recurrent nasopharyngeal carcinoma [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 774.

Abstract 269: PD-L1 dominated tumor-CAF-immune cell crosstalk of stromal microenvironment: Endometrial tumor vs. tumor-adjacent normal

Abstract Background: In endometrial cancers (EC), the mechanistic contribution of PD-L1/L2 and PD-1 signaling of host’s stromal microenvironment (SME) [cancer-associated fibroblast (CAF) and immune cells] in relation to the tumor (T) cells is elusive. Aim: To understand the tumor-stroma-immune crosstalk in EC, we studied the compartmental pattern of PD-L1, PD-L2, and PD-1 expression in EC tissues and their matched CAFs from both T and tumor-adjacent normal (N) tissues. Method: Over 116 surgically resected T and N tissues were obtained from consented patients with EC at the Avera Cancer Institute (March ‘16 and September ’21). IHC expression was performed in T, N-epithelium, SME (mesenchyme) in the T and N tissues. The staining intensity and distribution pattern were evaluated by a pathologist using a standard scoring system (1% cut-off value). Since fibroblasts are the most abundant cells of SME, the PD-L1 was parallelly evaluated in the multiple passages of cultured T and N derived CAFs by the qRT-PCR, flow-cytometry, and ICC. Result: The expression of 3 immune checkpoint proteins in the epithelial and mesenchyme of N was limited to only rare and focal positivity for PD-L1. The epithelial compartments of the T tissue, in contrast, had 70% positivity for PD-L1, which was independent of differentiation status, presence of TILs, or lymphovascular invasion. PD-L1 positivity was 90% of the SME of tested samples, identified predominantly in the (1) immune component, including macrophages and lymphocytes, and (2) EpCAM-/SMA+/FAP+/S100A4+ CAFs. Both PD-L1 and PD-L2 were identified by flow, ICC, and qRT-PCR in NCAF and TCAF. The overall expression of PD-L2 was found to be qualitatively and quantitatively lesser than that of PD-L1, primarily contributed by SME/stromal macrophages and lymphocytes. PD-1 expression was restricted to the SME/stromal immune components, predominantly lymphocytes. The expression for PD-L1 and PD-L2 mRNA was higher in TCAF than NCAF in successive passages, which ICC and flow cytometry confirmed. Summary: The expression of immune checkpoint proteins, PD-L1, and PD-L2, are primarily tumor-driven and associated with the endometrioid (invasive) and serous adenocarcinomas of grades II and above with myometrial invasion. The expression of PD-L1 and PD-L2 are distributed in both tumor and SME/stromal components, while PD-1 expression is exclusively SME/stromal. Citation Format: Xiaoqian Lin, Jennifer Aske, Adam Dale, Nischal Koirala, Ethan Thompson, Mary Fagerness, Natasha Flier, Paige Davies, Stacy Kehoe, Cheryl Ageton, Kris Gaster, Luis Rojas Espaillat, David Starks, Raed Sulaiman, Pradip De, Nandini Dey. PD-L1 dominated tumor-CAF-immune cell crosstalk of stromal microenvironment: Endometrial tumor vs. tumor-adjacent normal [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 269.

Neoadjuvant chemo-immunotherapy increases tumor immune lymphocytes infiltration in resectable NSCLC.

e20573 Background: NCIT (neoadjuvant Chemo-immunotherapy treatment) has revolutionized the treatment of resectable NSCLC due to its better MPR (major pathologic response) rate and pCR (pathologic complete response) rate over neoadjuvant chemotherapy or immunotherapy. However, the intrinsic mechanism underlying this treatment remains unclear. In recent years, the immune microenvironment has attracted considerable attention in the field of immunotherapy. In this study, we explored the relationship between tumor immune lymphocytes (TILs), tertiary lymphoid structures (TLS), and response to NCIT in resectable NSCLC and the change in TILs before and after NCIT. Methods: Twenty-five patients with stage IIA-IIIC NSCLC who underwent surgery after NCIT were enrolled in this retrospective study. Multiplex immunofluorescence (mIF) staining and image analysis assays were performed on the samples collected before and after NCIT for each patient. Results: Among the enrolled patients, 10 achieved MPR or pCR which were defined as response group, whereas 15 did not respond well to NCIT which were defined as non-response group. The results of the mIF assays revealed that NCIT was associated with an increase in immune lymphocytes, including CD3+ (tumor, p = 0.23; stroma, p &lt; 0.01), CD4+ (tumor, p = 0.011; stroma, p &lt; 0.001), CD4+Foxp3+ (tumor, p = 0.63; stroma, p &lt; 0.001), CD8+ (tumor, p = 0.00031; stroma, p &lt; 0.001), CD8+PD-1+ (tumor, p = 0.13; stroma, p = 0.022), PD-L1+CD68+ (tumor, p = 0.85; stroma, p = 0.041), and TLS (tertiary lymphoid structures, p = 0.00027). The comparison of TIL between response group and non-response group revealed that CD3, FOXP3+, and CD8+PD-1+ may serve as predictors of the response to neoadjuvant immunotherapy. Conclusions: To our knowledge, this is the first study to explore the relationship between TILs, TLS, and response to NCIT and TIL changes during NCIT in resectable Chinese NSCLC. In resectable NSCLC, the infiltration of immune cells before NCIT was correlated with the pathologic complete response, which enhanced the TILs as a promising predictor for selecting patients who were more likely to benefit from NCIT.

Modulation of lymphocyte subpopulations in the small intestine of mice treated with probiotic bacterial strains and infected with Trichinella spiralis.

To study the local intestinal lymphocyte immunity in mice with trichinellosis affected by probiotic bacteria. Enterococcus faecium CCM8558, Enterococcus durans ED26E/7, Limosilactobacillus fermentum CCM7421 and Lactiplantibacillus plantarum 17L/1 were administered daily (109 CFUml-1 ) and mice were infected with Trichinella spiralis (400 larvae) on the 7th day of treatment. T. spiralis infection significantly inhibited lymphocyte subpopulations from 5 to 25days postinfection (dpi). L. fermentum CCM7421 and L. plantarum 17L/1 restored the CD4+ T cell numbers in the epithelium and lamina propria at the control level from 11 dpi. All strains stimulated the CD8+ T cells numbers in infected mice, which were restored in the lamina propria on 11 dpi and in the epithelium only on 32 dpi. B cells (CD19+ ) inhibition after T. spiralis infection was not affected by treatment till 25 dpi. The strain-specific immunomodulatory effect of tested bacteria was confirmed. L. fermentum CCM7421 and L. plantarum 17L/1 showed the greatest immunomodulatory potential on CD4+ and CD8+ T lymphocytes in trichinellosis. E. faecium CCM8558 and E. durans ED26E/7 activated only CD8+ T cells in the lamina propria. Positive modulation of the gut lymphocyte immunity in T. spiralis infection with bacterial strains showed their beneficial effect with the host's antiparasitic defence.

Efficiency of the original flavonoids composition based on Curcumin in chronic alcohol intoxication

IntroductionMultiple tissue and organ damage induced by ethanol toxic effects requires prescription of a wide range of drugs that have a positive symptomatic effect, while simultaneously increasing the toxic load on the body, which necessitates the search for new approaches to the therapy of alcoholism, possibly with the use of parapharmaceuticals.ObjectivesThe anti-inflammatory, antioxidant and neuroprotective properties of curcuminoids, as well as their ability to influence the epigenetic mechanisms of gene expression regulation, can provide a positive therapeutic effect in alcoholism by influencing the pathogenetic mechanisms of this pathology, which determines the relevance and prospects of studying their effects during chronic ethanol intoxication.Methods(CBAxC57Bl/6) F1 male mice with 6-month 10% ethanol exposure were undergoing administration of original composition of flavonoids based on Curcumin for 40 days. Animal’s alcohol consumption, behavior and immune parameters were estimated.Results As a result of flavonoids original composition administration, correction of the changes in the nervous system functional activity, caused by the chronic toxic effect of ethanol has been achieved (decrease in alcoholic motivation, exploratory behavior stimulation, modulation of the cytokines level in brain, indicating a neuroinflammation decrease). We also registered the immune system functional activity modulation (stimulation of immune response and lymphocytes proliferative activity) in mice with alcohol dependence.ConclusionsThe original composition of flavonoids against the background of long-term alcohol consumption has a positive psychoneuroimmunomodulatory effect, which serves as an experimental substantiation of the prospects of using the composition as an adjuvant in the treatment of chronic alcoholism.DisclosureNo significant relationships.

A lncRNA signature associated with tumor immune heterogeneity predicts distant metastasis in locoregionally advanced nasopharyngeal carcinoma

Increasing evidence has revealed the roles of long noncoding RNAs (lncRNAs) as tumor biomarkers. Here, we introduce an immune-associated nine-lncRNA signature for predicting distant metastasis in locoregionally advanced nasopharyngeal carcinoma (LA-NPC). The nine lncRNAs are identified through microarray profiling, followed by RT–qPCR validation and selection using a machine learning method in the training cohort (n = 177). This nine-lncRNA signature classifies patients into high and low risk groups, which have significantly different distant metastasis-free survival. Validations in the Guangzhou internal (n = 177) and Guilin external (n = 150) cohorts yield similar results, confirming that the signature is an independent risk factor for distant metastasis and outperforms anatomy-based metrics in identifying patients with high metastatic risk. Integrative analyses show that this nine-lncRNA signature correlates with immune activity and lymphocyte infiltration, which is validated by digital pathology. Our results suggest that the immune-associated nine-lncRNA signature can serve as a promising biomarker for metastasis prediction in LA-NPC.

Immunometabolic Markers in a Small Patient Cohort Undergoing Immunotherapy.

Although the discovery of immune checkpoints was hailed as a major breakthrough in cancer therapy, generating a sufficient response to immunotherapy is still limited. Thus, the objective of this exploratory, hypothesis-generating study was to identify potentially novel peripheral biomarkers and discuss the possible predictive relevance of combining scarcely investigated metabolic and hormonal markers with immune subsets. Sixteen markers that differed significantly between responders and non-responders were identified. In a further step, the correlation with progression-free survival (PFS) and false discovery correction (Benjamini and Hochberg) revealed potential predictive roles for the immune subset absolute lymphocyte count (rs = 0.51; p = 0.0224 *), absolute basophil count (rs = 0.43; p = 0.04 *), PD-1+ monocytes (rs = −0.49; p = 0.04 *), hemoglobin (rs = 0.44; p = 0.04 *), metabolic markers LDL (rs = 0.53; p = 0.0224 *), free androgen index (rs = 0.57; p = 0.0224 *) and CRP (rs = −0.46; p = 0.0352 *). The absolute lymphocyte count, LDL and free androgen index were the most significant individual markers, and combining the immune subsets with the metabolic markers into a biomarker ratio enhanced correlation with PFS (rs = −0.74; p ≤ 0.0001 ****). In summary, in addition to well-established markers, we identified PD-1+ monocytes and the free androgen index as potentially novel peripheral markers in the context of immunotherapy. Furthermore, the combination of immune subsets with metabolic and hormonal markers may have the potential to enhance the power of future predictive scores and should, therefore, be investigated further in larger trials.