Proportion Of Immune Cell Subsets Research Articles

Predicting immunotherapy toxicity through single-cell sequencing of peripheral immune cells: A pilot study.

e14538 Background: Immune checkpoint inhibitors are widely used in oncology. However, immune-related adverse events (iRAEs) are idiosyncratic and can be severe. The overall incidence is 27%, with severe iRAEs occurring in 6% of patients. We hypothesised that distinct immune cell phenotypes (gene expression) correlate with immune-related toxicity from checkpoint inhibitor immunotherapy. Single-cell RNA sequencing (scRNA-seq) is a powerful tool that can assay the gene expression of thousands of individual cells. RPS26 expression mediates CD4/CD8 T cell activation through a known trans-expressive quantitative trait locus at rs1131017 and is linked with autoimmunity. FOS and JUN encode for c-Fos and c-Jun, which dimerise to create the activator protein-1 complex, a well-established transcriptional factor of the immune system. c-Fos is an immunoregulator known to suppress pro-inflammatory cytokines, suggesting that c-FOS deficiency plays a crucial role in developing autoimmunity. We aimed to analyse the transcriptomic differences of peripheral immune cells between patients who experienced severe checkpoint inhibitor therapy iRAE and those who did not. Methods: We identified a pilot cohort of eight patients who developed grade three or above toxicity due to immune checkpoint inhibitors and eight controls who were matched for age, cancer and treatment type. Peripheral blood mononuclear cells were collected prospectively at baseline and two subsequent treatment cycles. Cells were captured on the 10x Chromium platform, and libraries were sequenced to 30,000 reads per cell. Samples were demultiplexed, and the data was analysed with the Seurat package on R statistical software (version 4.3.0). Results: Five patients had baseline samples only, two patients had two samples from two cycles, and one patient had samples from three cycles. After quality control filtering, we identified 36,288 cells from 16 individuals and 24 samples. Immune cells were annotated with the Azimuth package into 29 subtypes. We identified differences in cell proportions of immune cell subsets in the toxicity vs control group, including B naive cells (5.57% vs 3.71% p=0.01) and reduced cytotoxic CD4 T lymphocyte cells (0.66% vs 2.26%, p=0.005). Differential gene expression analysis identified a significantly high expression of ribosomal genes, particularly RPS26, in multiple cell types at baseline (p<0.01) and reduced expression of FOS and JUN-related genes in CD14 and CD16 monocytes, CD4 and CD8 T cells in the toxicity cohort (p<0.01). Conclusions: Overexpression of ribosomal genes and reduced expression of FOS/ JUN genes amongst PBMCs at baseline are potentially predictive biomarkers of severe checkpoint inhibitor immunotherapy toxicity. Validation of these findings in a larger cohort is in progress.

Implication of m6A Methylation Regulators in the Immune Microenvironment of Bronchopulmonary Dysplasia.

N6-methyladenosine (m6A) regulates gene expression and governs many important biological processes. However, the function of m6A in the development of bronchopulmonary dysplasia (BPD) remains poorly characterized. Thus, the purpose of this investigation was to evaluate the effects of m6A RNA methylation regulators on the development of BPD. BPD-related transcriptome data were downloaded from the GEO database. Differentially expressed m6A methylation regulators between BPD and control group were identified. Consensus clustering was conducted for the classification of BPD and association between clusters and BPD phenotypes were explored. Analysis of differentially expressed genes (DEGs) and immune-related DEGs was performed. The GSEA, GO and KEGG analyses were used to interpret the functional enrichments. The composition of immune cell subtypes in BPD subsets was predicted by CIBERSORT analysis. Compared with the control group, expression of most m6A regulators showed significant alteration, especially for IGF2BP1/2/3. BPD was classified into 2 subsets, and cluster 1 was correlated with severe BPD. Furthermore, the results of functional enrichment analyses showed a disturbed immune-related signaling pathway. Based on CIBERSORT analysis, we found that the proportion of immune cell subsets changed between cluster 1 and cluster 2. Our study revealed the implication of m6A methylation regulators in the development of BPD, which might provide a novel insight for the diagnosis and treatment of BPD.

Association of Claudin-1 overexpression with immune exclusion in nasopharyngeal carcinoma and its potential as a novel therapeutic target.

151 Background: Claudin-1 (CLDN1) is a tight junction protein overexpressed in several cancers, where it is involved in the tumor microenvironment remodeling and stiffening of the tumor-stroma interface. This potentially prevents penetration of tumor infiltrating immune cells, in turn hindering the efficacy of immune checkpoint inhibitors (ICI). Given recent evidence supporting ICI efficacy in nasopharyngeal carcinoma (NPC), we investigated whether CLDN1 is overexpressed in NPC and its association with the tumor immune microenvironment (TME). Methods: 131 NPC patients treated at a single institution were prospectively recruited. TME profiling was performed using deconvolutional methods (CIBERSORT, quanTIseq) on tumor transcriptomes to estimate proportions of immune cell subsets, and expression of immune-related genes and pathways. Transcriptome profiling was performed on tumors with >70% cellularity and normal controls using Illumina Truseq (CA). We stratified tumors by their median CLDN1 expression and compared the immune profile of CLDN1high versus CLDN1low tumors. Cox regression model was used to analyze disease-free survival (DFS) as the primary clinical endpoint. Results: We observed CLDN1 overexpression in NPC versus normal tissues ( P=0.0046) that was independent of known prognostic clinical parameters e.g., TNM-stage and EBV DNA levels. Consistent with our hypothesis, CLDN1 expression was negatively correlated with total immune cell fraction in the tumor ( P<0.001). We observed reduced abundance of most immune subsets in CLDN1high tumors, notably CD8 T cells ( P<0.001), activated CD4 memory T cells ( P<0.001), and M1 and M2 macrophages ( P=0.048 and P<0.001). There was an overall downregulation of both stimulatory and suppressive GO immune-related gene-sets and genes in CLDN1high versus CLDN1low tumors, thereby corroborating our preceding observation of reduced immune infiltration in the TME in CLDN1 overexpressing NPC. Our findings were validated in a published dataset of 113 NPC tumors (Zhang et al., 2017). Of clinical relevance, reduced immune infiltration in the TME was prognostic for DFS; HR of CLDN1high, CD8 T celllow (stratified by median) NPC was 2.1 (95% CI:0.9-5.0) when compared with CLDN1low, CD8 T cellhigh NPC. Conclusions: Here, we report the overexpression of CLDN1 in NPC that is ubiquitous across disease stages and associated with an immune-low TME. This influence on the TME is associated with an inferior outcome of NPC patients. Given the role of the tumor TME in ICI response, our findings provide a rationale for future trials investigating combination of CLDN1 targeting with ICI to reverse this immune exclusion effect, potentially leading to improved outcomes.

Gene Signatures Associated with Temporal Rhythm as Diagnostic Markers of Major Depressive Disorder and Their Role in Immune Infiltration.

Temporal rhythm (TR) is involved in the pathophysiology and treatment response of major depressive disorder (MDD). However, there have been few systematic studies on the relationship between TR-related genes (TRRGs) and MDD. This study aimed to develop a novel prognostic gene signature based on the TRRGs in MDD. We extracted expression information from the Gene Expression Omnibus (GEO) database and retrieved TRRGs from GeneCards. Expressed genes (TRRDEGs) were identified differentially, and their potential biological functions were analyzed. Subsequently, association analysis and receiver operating characteristic (ROC) curves were adopted for the TRRDEGs. Further, upstream transcription factor (TF)/miRNA and potential drugs targeting MDD were predicted. Finally, the CIBERSORT algorithm was used to estimate the proportions of immune cell subsets. We identified six TRRDEGs that were primarily involved in malaria, cardiac muscle contraction, and the calcium-signaling pathway. Four genes (CHGA, CCDC47, ACKR1, and FKBP11) with an AUC of >0.70 were considered TRRDEGs hub genes for ROC curve analysis. Outcomes showed that there were a higher ratio of T cells, gamma-delta T cells, monocytes, and neutrophils, and lower degrees of CD8+ T cells, and memory resting CD4+ T cells in TRRDEGs. Four new TRRDEG signatures with excellent diagnostic performance and a relationship with the immune microenvironment were identified.

Distribution and clinical significance of circulating CD8+CD28− regulatory T cells in the peripheral blood of patients with pulmonary tuberculosis

BackgroundRegulatory T cells (Treg cells) in the peripheral blood of patients with pulmonary tuberculosis (PTB) may be closely related to the progression of PTB. In this study, the distribution characteristics and clinical importance of CD8+CD28− Treg cells in patients with tuberculosis were systematically analyzed, and the role and importance of CD8+CD28− Treg cells in influencing the immune response and progression of tuberculosis were discussed, which will provide immunological indices and reference values for the clinical diagnosis of tuberculosis.MethodsFlow cytometry, sputum smears and computed tomography imaging were used to analyze the distribution characteristics of CD8+CD28− Treg cells in the peripheral blood of patients with PTB and the correlation between CD8+CD28−Treg cells and clinical and immune indices.ResultsThe percentages of CD4+CD25high and CD8+CD28− Treg cells in the peripheral blood of patients with PTB were significantly higher than those in the healthy control (HC) group. Further analysis showed that the percentage of CD4+CD25highTreg cells in the Stage II group was significantly higher than that in the HC group. The percentages of CD4+CD25high and CD8+CD28− Treg cells increased significantly in patients in the Stage II group. The proportion of CD8+CD28− Treg cells was directly proportional to the degree of positivity in sputum smears, while CD4+CD25highTreg cells did not exhibit this trend.The correlations between the percentage of CD4+CD25high and CD8+CD28− Treg cells and the percentage of lymphocyte subsets were examined. The percentage of CD8+CD28− Treg cells was negatively correlated with the percentage of CD4+T cells and positively correlated with the CD8+T cell percentage in the HC and PTB groups. The percentage of CD4 + CD25highTreg cells was positively correlated with the percentage of CD4+T cells only in the PTB group.ConclusionsThis study was the first to show that the proportion of CD8+CD28− Treg cells in the peripheral blood of patients with PTB was significantly increased, and the increase in CD8+CD28− Treg cells was related to the progression of PTB, which may affect the proportion of immune cell subsets by inhibiting the immune response, resulting in the progression of PTB.

Transplantation of insulin-producing cells derived from human mesenchymal stromal/stem cells into diabetic humanized mice

BackgroundThe purpose of this study was to investigate allogenic immune responses following the transplantation of insulin-producing cells (IPCs) differentiated from human adipose tissue-derived stem cells (hAT-MSCs) into humanized mice.MethodshAT-MSCs were isolated from liposuction aspirates obtained from HLA-A2-negative healthy donors. These cells were expanded and differentiated into IPCs. HLA-A2-positive humanized mice (NOG-EXL) were divided into 4 groups: diabetic mice transplanted with IPCs, diabetic but nontransplanted mice, nondiabetic mice transplanted with IPCs and normal untreated mice. Three million differentiated cells were transplanted under the renal capsule. Animals were followed-up to determine their weight, glucose levels (2-h postprandial), and human and mouse insulin levels. The mice were euthanized 6–8 weeks posttransplant. The kidneys were explanted for immunohistochemical studies. Blood, spleen and bone marrow samples were obtained to determine the proportion of immune cell subsets (CD4+, CD8+, CD16+, CD19+ and CD69+), and the expression levels of HLA-ABC and HLA-DR.ResultsFollowing STZ induction, blood glucose levels increased sharply and were then normalized within 2 weeks after cell transplantation. In these animals, human insulin levels were measurable while mouse insulin levels were negligible throughout the observation period. Immunostaining of cell-bearing kidneys revealed sparse CD45+ cells. Immunolabeling and flow cytometry of blood, bone marrow and splenic samples obtained from the 3 groups of animals did not reveal a significant difference in the proportions of immune cell subsets or in the expression levels of HLA-ABC and HLA-DR.ConclusionTransplantation of IPCs derived from allogenic hAT-MSCs into humanized mice was followed by a muted allogenic immune response that did not interfere with the functionality of the engrafted cells. Our findings suggest that such allogenic cells could offer an opportunity for cell therapy for insulin-dependent diabetes without immunosuppression, encapsulation or gene manipulations.Graphical

Feiyanning downregulating CXCLs/CXCR2 axis to suppress TANs infiltration in the prevention of lung cancer metastasis

Ethnopharmacological relevanceTumor-associated neutrophils (TANs) play an important role in tumor metastasis. The Traditional Chinese medicine (TCM) Feiyanning (FYN) has been clinically proven to effectively prevent the recurrence and metastasis of lung cancer, improve immunity, and prolong the survival period of lung cancer patients. However, its anti-metastatic immune mechanism has not been fully elucidated. To this end, we studied the mechanism of FYN's regulation of neutrophils infiltration in the tumor microenvironment (TME). Aim of the studyTo explore the anti-metastatic mechanism of FYN from the perspective of anti-immunosuppressive phenotype neutrophils infiltration in the TME. Materials and methodsTCM network pharmacological analysis was used to predict Feiyanning effective target. Flow cytometry was used to detect the proportion of immune cell subsets in the TME. Lung metastases were investigated in C57 mice by tail vein injection. Protein expression was evaluated by immunohistochemistry and Western blot. Gene expression was evaluated by qRT-PCR. ResultsFYN could reshape the tumor immune microenvironment. It prevents Tregs, M2 macrophages, and neutrophils infiltration, as well as recruits T cells, NK cells, and DCs, and improves DCs activation. In addition, FYN could regulate the polarization of TANs, inhibit the infiltration of neutrophils with an immunosuppressive phenotype, downregulate CXCLs/CXCR2 axis and inhibitory factors like Arg-1 and TGF-β, and up-regulate the immune effector molecule ICAM-1. Furthermore, FYN increases anti-tumor immune effects in the TME to prevent tumor cells from spreading to the lungs. ConclusionThis study clarifies the potential mechanism of FYN in regulating neutrophils infiltration and anti-metastasis. FYN may regulate neutrophils infiltration in the TME by regulating CXCLs/CXCR2 axis.

Altered ocular surface immune cell profile in patients with dry eye disease

PurposeAberrant inflammation and immune dysregulation are known pathogenic contributors in dry eye disease (DED). Aim of the study was to determine the proportions of immune cell subsets on the ocular surface (OS) of DED patients. Methods15 healthy controls (22 eyes) and 48 DED subjects (36 eyes with evaporative DED – EDED; 60 eyes with aqueous deficient DED – ADED) were included in the study. Tear break up time (TBUT), Schirmer's test 1 (ST1), corneal staining (CS) and ocular surface disease index (OSDI) scoring were recorded. OS wash was used to collect immune cells on the OS of study subjects. The cells immunophenotyped using flow cytometry include leukocytes, neutrophils, macrophages, natural killer–NK cells and T cell subsets (CD4; CD8; double positive–DP; gamma delta–γδ and NK T cells). ResultsSignificantly higher proportions of leukocytes, neutrophils, CD4 T cells, CD8 T cells, DP T cells and CD4/CD8 T cells ratio were observed in EDED and/or ADED patients. Significantly higher proportions of neutrophils and lower proportions of NK cells were observed in ADED subjects with corneal staining compared to those without and controls. Neutrophils/NK cells ratio was significantly higher in EDED and ADED subjects compared to controls. Correlation analysis revealed pathological relationships between proportions of leukocytes, neutrophils, CD4 T cells and Neutrophil/NK cells ratio with DED clinical parameters. ConclusionOS immune cell subset proportion changes in DED patients were associated with DED types and severity. The data suggests the potential for a new generation of therapies targeting immune cells on the ocular surface.

Yu-Ping-Feng Formula Exerts Antilung Cancer Effects by Remodeling the Tumor Microenvironment through Regulating Myeloid-Derived Suppressor Cells.

Yu-Ping-Feng (YPF) formula is a classical prescription used for enhancing the body's immunity function in traditional Chinese medicine (TCM). In clinical practice, the YPF formula has been reported to exhibit antilung cancer and immunomodulatory effect. However, the relationship between them remains unclear. The present study aimed to investigate the antilung cancer effect of the YPF formula and its immune-related mechanisms. The C57BL/6 tumor-bearing mice model was established and randomly divided into the YPF group and the control group. Tumor volume, spleen weight, and survival in both groups were measured and evaluated during 28 days of consecutive intervention. Flow cytometry was used to detect the proportion of immune cell subsets. Myeloid-derived suppressor cells (MDSCs) were induced in vitro from bone marrow cells. After intervention by the YPF formula, CCK-8 and flow cytometry analyses were performed to detect proliferation and apoptosis of MDSCs. A coculture system containing T cells and MDSCs was established to further study the role of MDSCs in the regulation of T-cell subsets proportion by the YPF formula. The expressions of MDSCs-related genes and proteins were detected by RT-PCR and Western blotting. The results showed the YPF formula inhibited tumor growth, reduced spleen weight, and prolonged the survival of mice. Besides, the proportions of MDSCs subsets and Regulatory T (Treg) in the YPF group decreased, whereas those of CD4+T and CD8+T increased both in vitro and in vivo. CCK-8 and flow cytometry demonstrated that the YPF formula could inhibit proliferation and promote apoptosis of MDSCs. The coculture experiments further confirmed that MDSCs served a critical role in regulating the tumor microenvironment by the YPF formula. RT-PCR and Western blotting indicated that the levels of MDSCs' activation and proliferation-related proteins and genes were downregulated in the YPF group. Therefore, our results demonstrated that the YPF formula could promote apoptosis and inhibit the proliferation of MDSCs. As a result, the negative regulatory effect on the positive immune cells induced by MDSCs was weakened, thus achieving the antilung cancer effect by remodeling the tumor microenvironment.

Synergistic effects of AAGL and anti-PD-1 on hepatocellular carcinoma through lymphocyte recruitment to the liver.

Objective:Therapy for hepatocellular carcinoma (HCC) is a major challenge, and targeted therapies provide only a modest benefit in terms of overall survival. Treatment with antibodies to programmed cell death protein 1 (PD-1)/PD-L1 can restore the functions of tumor-infiltrating T cells in HCC and has shown clinical efficacy in 20% of patients with advanced HCC. Novel approaches are urgently needed to treat HCC and to augment the efficacy of immunotherapy.Methods:Tumor-bearing mice were treated with Agrocybe aegerita galectin (AAGL) alone or in combination with anti-PD-1, and the tumor sizes and lifespans of mice were determined. Transcriptome analysis, cytokine analysis, flow cytometry analysis of the number and proportion of immune cell subsets in the liver and spleen, and molecular and cellular analyses of tumors were used to define the underlying mechanisms.Results:AAGL significantly inhibited the growth of liver tumors in a dose-dependent manner. Furthermore, AAGL increased the expression of multiple cytokines and chemokines in tumor-bearing mouse livers; this effect was associated with the activation and migration of T cells and macrophages, in agreement with the in vitro results. Importantly, the aggregation of T cells and macrophages induced by AAGL in tumor-bearing mouse livers clearly enhanced the response to PD-1 blockade immunotherapy.Conclusions:The results showed that AAGL induced the activation and migration of lymphocytes to the liver, and that the combination of AAGL and anti-PD-1 may be a promising strategy for HCC treatment.

Changes in subsets of immunocytes in endometrial hyperplasia.

Endometrial hyperplasia (EH) is characterized by an endometrial gland-to-stroma ratio >1 and is one of the most common gynecological diseases in the world. The role of immunocyte subsets in the development of EH remains unknown. Patients who underwent dilatation and curettage due to abnormal uterine bleeding were recruited in the present study. Alterations in the numbers of different types of immune cell subsets in the endometrium of patients were analyzed by flow cytometry. The present study included 48 patients who were divided into three groups, based on the pathological results: (a) proliferative period (PP, n=12); (b) simple EH (SEH, n=30); and (c) complex EH (CEH, n=6). The results showed that immune cell subpopulations were significantly different between these three groups. Compared with the PP group, the proportion of CD45+ cells and neutrophils and the subtypes of T cells and macrophages were significantly increased in the SEH patients. Compared with the PP and SEH groups, subsets of immunocytes in the CEH group were significantly decreased, including the population of CD45+ cells and the subtypes of T cells and natural killer cells; in contrast, the proportion of macrophages was significantly increased. There were no significant differences between the other cell subsets in each group. The changes in immune cell subsets may be closely associated with the progression of EH. Although the specific role of different immune cell subsets in the development of the diseases requires further study, the changes in the proportions of immune cell subsets should not be ignored.

OX40 and LAG3 are associated with better prognosis in advanced gastric cancer patients treated with anti-programmed death-1 antibody

BackgroundAnti-PD-1 monoclonal antibody, nivolumab, has shown efficacy for advanced gastric cancer (AGC). However, the specific immune cell subsets predominantly activated during the period of anti-PD-1 therapy for AGC have not been clarified.MethodsPeripheral blood of 30 AGC patients treated with nivolumab was prospectively obtained before the initial and second administrations and at the time of progressive disease (PD). The proportions of immune cell subsets and the serum concentrations of cytokines were systematically analysed by flow cytometry. Associations of subsets and serum cytokines with therapeutic effects were evaluated.ResultsAfter the initial administration, significant increases in activated central/effector memory, activated effector T cells, and activated T-helper 1 subsets were observed. At the time of PD, activated regulatory T cells, LAG3-positive CD4+/CD8+ T cells, and TIM3-positive CD4+/CD8+ T cells increased significantly. Significant positive correlations were shown between progression-free survival and proportions of LAG3-positive CD4+/CD8+ T cells and of OX40-positive CD4+/CD8+ T cells (log-rank p = 0.0008, 0.0003, 0.0035 and 0.0040).ConclusionsNivolumab therapy enhances activation of central/effector memory and effector subsets of CD4+/CD8+ T cells. The expression levels of LAG-3 and OX40 on T cells correlated with the efficacy of nivolumab therapy and could be reasonable biomarkers for anti-PD-1 therapy.

Activation of memory/effector T cells and association between prognosis and OX40-positive T cells in advanced head and neck cancer patients treated with anti-programmed death-1 antibody.

35 Background: Anti-programmed death-1 (PD-1) monoclonal antibody, nivolumab, enhances anti-tumor activity by inhibiting the interaction of PD-1 and programmed death-1 ligand 1 and has shown efficacy for platinum-refractory recurrent or advanced head and neck cancer (HNC). However, subsets of immune cells predominantly activated during the period of anti-PD-1 therapy for HNC and specifically associated with the prognosis have not been clarified. Methods: Peripheral blood mononuclear cells of 15 HNC patients treated with nivolumab were prospectively obtained before the initial and second administrations of nivolumab, and at the time of progressive disease (PD). We performed comprehensive analysis of the proportion of immune cell subsets by flow cytometry, including the expression of coinhibitory and costimulatory molecules such as T-cell immunoglobulin and mucin domain 3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), B and T lymphocyte attenuator (BTLA), CD28, OX40, inducible T cell costimulator (ICOS). Association between changes in the proportion of the subsets and therapeutic effect were also analyzed. Results: Median progression free survival (PFS) of the whole patients was 96 days (95% CI 70–308). After a single course of nivolumab, patients showed a significant increase in activated central memory and effector subsets of CD4+/CD8+ T cells and activated helper T1 cells (p = 0.0039, 0.0078, 0.0273, 0.0391, 0.0391). A trend of increase of activated effector memory CD4+/CD8+ T cell was observed (p = 0.4961, 0.3594). At the time of PD, effector regulatory T cells, LAG3 positive CD4+/CD8+ T cells, TIM-3 positive CD4+/CD8 T cells and BTLA positive CD4+/CD8+ T cells significantly increased. Significant positive correlations were found between PFS and the proportion of OX40 positive CD4+/CD8+ T cells before nivolumab therapy (p = 0.0239, 0.0134). Conclusions: Nivolumab therapy enhances activation of central memory and effector subsets of CD4+/CD8+ T cells. The expression level of OX40 on T cells was correlated with efficacy of nivolumab therapy in HNC patients.

Role of Immune Response, Inflammation, and Tumor Immune Response–Related Cytokines/Chemokines in Melanoma Progression

To investigate the role of tumor cytokines/chemokines in melanoma immune response, we estimated the proportions of immune cell subsets in melanoma tumors from The Cancer Genome Atlas, followed by evaluation of the association between cytokine/chemokine expression and these subsets. We then investigated the association of immune cell subsets, chemokines, and cytokines with patient survival. Finally, we evaluated the immune cell tumor-infiltrating lymphocyte (TIL) score for correlation with melanoma patient outcome in a separate cohort. There was good agreement between RNA sequencing estimation of T-cell subset and pathologist-determined TIL score. Expression levels of cytokines IL-12A, IFNG, and IL-10, and chemokines CXCL9 and CXCL10 were positively correlated with PDCD1, CTLA-4, and CD8+ T-cell subset, but negatively correlated with tumor purity (Bonferroni-corrected P < 0.05). In multivariable analysis, higher expression levels of cytokines IFN-γ and TGFB1, but not chemokines, were associated with improved overall survival. A higher expression level of CD8+ T-cell subset was also associated with improved overall survival (hazard ratio [HR]= 0.06, 95% confidence interval [CI]= 0.01-0.35, P= 0.002). Finally, multivariable analysis showed that patients with a brisk TIL score had improved melanoma-specific survival than those with a nonbrisk score (HR= 0.51, 95% CI= 0.27-0.98, P= 0.0423). These results suggest that the expression of specific tumor cytokines represents important biomarkers of melanoma immune response.

Activation of central/effector memory T cells in advanced gastric cancer patients treated with antiprogrammed death-1 antibody.

54 Background: Anti-programmed death-1 (PD-1) monoclonal antibody, nivolumab, enhances antitumor activity by inhibiting the interaction of PD-1 and programmed death-1 ligand 1 (PD-L1) and has shown efficacy for advanced gastric cancer (AGC) in the salvage line. However, specific subsets of immune cells predominantly activated during the period of anti-PD-1 therapy for AGC have not been clarified. Methods: Peripheral blood mononuclear cells of 20 AGC patients treated with nivolumab were prospectively obtained before the initial and second administrations of nivolumab, and at the time of progressive disease (PD). The proportion of immune cell subsets were systematically analyzed by flow cytometry, including the expression of costimulatory and coinhibitory molecules such as T-cell immunoglobulin and mucin domain 3 (TIM-3), Lymphocyte-activation gene 3 (LAG-3), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), cytotoxic T cell antigen-4 (CTLA-4), CD28, OX40, and inducible T cell costimulator (ICOS). Association between changes in the proportion of the subsets and therapeutic effect were analyzed. Results: Median progression free survival (PFS) of the whole patients was 51 days (95% CI 35–83). After a single course of nivolumab, patients showed a significant increase in activated effector memory and activated effector subsets of CD4+/CD8+ T cells (p = 0.018, 0.018, 0.032, 0.024). At the time of PD, proportions of myeloid dendritic cell, IgM memory B cell and Tfh-Th1/17 cell subsets decreased (p = 0.024, 0.013, 0.0039). On the other hand, LAG3 positive CD4+/CD8+ T cells, TIM-3 positive CD4+/CD8 T cells increased at the time of PD (p = 0.013, 0.032, 0.042, 0.042). Significant positive correlations were found between PFS and the proportion of LAG3 positive CD4+/CD8+ T cells (p = 0.0056, 0.0054), OX 40 positive CD4+/CD8+ T cells (p = 0.0034, 0.0006) prior to the initial nivolumab therapy. Conclusions: Nivolumab therapy enhances activation of effector memory and effector subsets of CD4+/CD8+ T cells. The expression level of LAG3 and OX40 on T cells might be correlated with efficacy of nivolumab therapy.

Immune Phenotype by Flow Cytometry of Paediatric Kidney Transplant Recipients and Healthy Adult Controls

Introduction The proportion of immune cell subsets in paediatric kidney transplant recipients could help monitor the best approach to allow tolerance to renal allografts. Aim To determine/establish the differences in the immunophenotype of pediatric kidney transplant recipients and healthy adult controls. Methods Absolute cell count (TruCount) and seven leukocyte-profiling panels containing 8-10 marker-antigens (46 of which were unique within the panels) consisting of Tregs, NKT, B, NK, DCs, and monocyte subsets were used to monitor the immune profiles of 9 paediatric kidney transplant and 8 adult control samples. Whole-blood samples, 100-300μl/panel, were stained and acquired on a BD-LSRFortessa and Flowjo was used for data analysis. Results Differences in subpopulations between pediatric and healthy adult control can be seen in Table 1. This showed a significant increase in absolute numbers of Granulocytes, CD14+Monocytes, double negative NKT cells and CD56+HICD16+ Intermediate NK cells. B cell panel showed a significant increase in naïve B cells, IgD+IgM+ B cells, and IgM+CD27- B cells. The naïve CD4+ (CD45RA+CCR7+/CD62+) and naïve CD8+ (CD45RA+CCR7+/CD62+) T cells, and naïve Tregs (CD4+CD25+hiCD127lowCD45RA+CCR7+/CD62+ and CD4+FOXP3+CD45RO-) which were higher than in adult controls. Moreover, naïve Foxp3 Tregs in pediatric transplanted patients had higher CD25 expression than naive Tregs in adult controls. Memory CD4+ T cells (CD45RA-CD4+) in pediatric patients has a similar HLA-DR expression as adult controls (Fig. 1).Further we found a significant decrease in the following cell populations in our pediatric kidney transplant patients compare to healthy adult controls: Non-classical monocytes, CD8+NKT, memory B cells (IgD-CD27+), IgD-IgM- B cells, CD27+CD38low Class-switched memory. A decreased proportion in CD27-CD28+, CD25+ of CD4+, CD57+CD27-CD28+ and CD25+ of CD8, CCR7-/CD62L-CD45RA- of CD4 and CD8, and CXCR3+CD45RO+ of CD4 and of CD8 (Fig.2), and CD127+CD45RO+ and CD25+CD45RO+ on CD4 T cells (effector Foxp3 Tregs) suggested less memory and effector memory T cells in our pediatric kidney transplant patients than controls (Fig.3). Conclusion Immune profiling of pediatric kidney transplant recipients demonstrated more naïve T cells, B cells and Tregs and less memory and effector memory T cells compared to healthy adult controls. Immunophenotyping by flow cytometry is a non-invasive technique requiring less than 1.5ml of whole blood and maybe a useful tool to monitor determine the differences in the immunophenotype in transplant recipients. Birgit Sawitzki. Mathias Streitz.

Elevated frequencies of CD8 T cells expressing PD-1, CTLA-4 and Tim-3 within tumour from perineural squamous cell carcinoma patients.

Perineural spread of tumour cells along cranial nerves is a severe complication of primary cutaneous squamous cell carcinomas of the head and neck region. While surgical excision of the tumour is the treatment of choice, removal of all the tumour is often complicated by the neural location and recurrence is frequent. Non-invasive immune treatments such as checkpoint inhibitor blockade may be useful in this set of tumours although little is understood about the immune response to perineural spread of squamous cell carcinomas. Immunohistochemistry studies suggest that perineural tumour contains a lymphocyte infiltrate but it is difficult to quantitate the different proportions of immune cell subsets and expression of checkpoint molecules such as PD-1, Tim-3 and CTLA-4. Using flow cytometry of excised perineural tumour tissue, we show that a T cell infiltrate is prominent in addition to less frequent B cell, NK cell and NKT cell infiltrates. CD8 T cells are more frequent than other T cells in the tumour tissue. Amongst CD8 T cells, the frequency of Tim-3, CTLA-4 and PD-1 expressing cells was significantly greater in the tumour relative to the blood, a pattern that was repeated for Tim-3, CTLA-4 and PD-1 amongst non-CD8 T cells. Using immunohistochemistry, PD-1 and PD-L1-expression could be detected in close proximity amongst perineural tumour tissue. The data suggest that perineural SCC contains a mixture of immune cells with a predominant T cell infiltrate containing CD8 T cells. Elevated frequencies of tumour-associated Tim-3+, CTLA-4+ and PD-1+ CD8 T cells suggests that a subset of patients may benefit from local antibody blockade of these checkpoint inhibitors.

Effect of PPARγ agonist (rosiglitazone) on the secretion of Th2 cytokine in asthma mice.

To explore the effect of PPARγ agonist (rosiglitazone) on the secretion of Th2 cytokines and the proportion of immune cell subsets in asthma mice. Ovalbumin (OVA)-sensitized mice were used to build asthma models. Those mice were divided into the normal control group, model group and rosiglitazone group. Differences of the changes in lung histopathology of mice in the three groups were observed through hematoxylin and eosin (HE) strain, and the numbers of the total cells, eosinophils and neutrophils in BALF of mice in the three groups were compared. ELISA and real-time PCR were employed to detect the protein levels of interleukin (IL)-5, IL-13, IL-4 and IL-10 and mRNA level, respectively. Flow cytometry number was implied to analyze the proportion of immune cell subsets in peripheral blood of mice. Compared with the mice in the control group, and mice of the model group, the infiltration of inflammatory cells in BALF increased, bronchial smooth muscle became thickened, a large amount of collagen deposited, the secretion of Th2 cytokine increased significantly, the ratio of regulatory T cells (Treg) decreased, the ratio of T17 cells rose distinctly; while in mice of the rosiglitazone group, the changes of their lung histopathology were improved obviously, the number of infiltration of inflammatory cells declined, the thickened smooth muscle relieved, the deposition of collagen decreased, the secretion of Th2 cytokine was inhibited, the ratio of Treg went up, and the increased of the ratio of T17 cells was inhibited but still not return to normal level. Rosiglitazone can regulate the proportion of Treg and Th17 cells and inhibit the secretion of Th2 cytokines, which inhibit the airway inflammatory response for asthma mice effectively.

Impact of NK cells on control of HIV infection in humanized BLT mice (P6207)

Abstract Evidence suggests natural killer (NK) cells can mediate antiviral activity in HIV-infected humans. Here, we sought to investigate whether the level of NK cell activation in primary HIV infection not only influences the NK cell mediated elimination of HIV infected cells, but also affects the longevity and effectiveness of HIV-specific T cell responses. To address this hypothesis, we monitored HIV viral loads and HIV-specific adaptive immune responses in a humanized BLT mouse model of HIV infection, comparing NK cell-depleted and control mice. Depletion was achieved through the use of an anti-NKp46 antibody prior to HIV infection. Proportions of immune cell subsets and viremia were assessed at baseline and then once a week for at least 5 weeks post-infection using flow cytometry and qRT-PCR. We observed that the impact of NK cells on plasma viral loads varied between batches of mice reconstituted with different tissues. One set of NK cell depleted mice displayed a 4-fold increase in viremia as well as decreased levels of T cell immune activation at 3 weeks post-infection. In a second group of mice reconstituted with independent human tissue, NK cell depletion had no significant effect on viral loads. These results suggest that further investigation is warranted to identify factors accounting for an increased control of HIV replication associated with NK cells in some individuals and not others, such as expression of a protective combination of KIR and HLA alleles.

Multiple sclerosis: reduced proportion of circulating plasmacytoid dendritic cells expressing BDCA-2 and BDCA-4 and reduced production of IL-6 and IL-10 in response to herpes simplex virus type 1

Objective We hypothesized that autoaggressive immune responses observed in multiple sclerosis (MS) could be associated with an imbalance in proportion of immune cell subsets and in cytokine production in response to infection, including viruses. Methods We collected blood mononuclear cells (MNC) from 23 patients with MS and 23 sex- and age-matched healthy controls (HC) from the island of Sardinia, Italy, where the prevalence of MS is extraordinarily high. Using flow cytometry, we studied MNC for expression of blood dendritic cell antigens (BDCA)-2 and BDCA-4 surface markers reflecting the proportion of plasmacytoid dendritic cells (pDC) that produce type I interferons (IFNs) after virus challenge and promote Th2/anti-inflammtory cytokine production. In parallel, pro-inflammatory (interleukin [IL]-2, IL-12, IFN-γ), anti-inflammatory (IL-4, IL-10), and immuno-regulatory/pleiotropic cytokines (type I IFNs including IFN-α and β, IL-6) were measured before and after an in vitro exposure to herpes simplex virus type 1 (HSV-1). Results The subset of lineage negative (lin−), BDCA-2+ cells was lower in patients with MS compared with HC (0.08 ± 0.02% vs 0.24 ± 0.02%; P < 0.001). A similar pattern was observed for lin−BDCA-4+ cells (0.08 ± 0.02% vs 0.17% ± 0.03; P < 0.01). Spontaneous productions of IL-6 (45 ± 10 pg/mL vs 140 ± 26 pg/mL; P < 0.01) and IL-10 (17 ± 0.4 pg/mL vs 21 ± 1 pg/mL; P < 0.05) by MNC were lower in patients with MS compared with HC. Spontaneous production of IL-6 (6.5 ± 0.15 pg/mL vs 21 ± 5 pg/mL; P < 0.01 and IL-10 (11 ± 1 pg/mL vs 14 ± 3 pg/mL; P < 0.05) by pDC was also lower in patients with MS compared with HC. Exposure of MNC to HSV-1 showed, in both patients with MS and HC, increased production of IFN-α, IL-6, and IL-10 but decreased production of IL-4. In response to HSV-1 exposure, productions of IL-6 (165 ± 28 pg/mL vs 325 ± 35 pg/mL; P < 0.01) and IL-10 (27 ± 3 vs 33 ± 3 P < 0.05) by MNC as well as by pDC (IL-6: 28 ± 7 vs 39 ± 12 P < 0.05; IL-10: 14 ± 1 vs 16 ± 3 P < 0.05) were lower in patients with MS compared with HC. Conclusion The results implicate a new evidence for altered immune cells and reduced immune responses in response to viral challenge in MS.