scholarly journals Comprehensive Analysis of the Immune Microenvironment in Checkpoint Inhibitor Pneumonitis

2022 ◽  
Vol 12 ◽  
Author(s):  
Xinqing Lin ◽  
Jiaxi Deng ◽  
Haiyi Deng ◽  
Yilin Yang ◽  
Ni Sun ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Immune Cells ◽  
Checkpoint Inhibitor ◽  
Memory B Cells ◽  
Gene Set Enrichment Analysis ◽  
Control Group ◽  
Immune Microenvironment ◽  
M1 Macrophages ◽  
Nsclc Patients

BackgroundWhile immune checkpoint inhibitors (ICIs) are a beacon of hope for non-small cell lung cancer (NSCLC) patients, they can also cause adverse events, including checkpoint inhibitor pneumonitis (CIP). Research shows that the inflammatory immune microenvironment plays a vital role in the development of CIP. However, the role of the immune microenvironment (IME) in CIP is still unclear.MethodsWe collected a cohort of NSCLC patients treated with ICIs that included eight individuals with CIP (CIP group) and 29 individuals without CIP (Control group). CIBERSORT and the xCell algorithm were used to evaluate the proportion of immune cells. Gene set enrichment analysis (GSEA) and single-sample GSEA (ssGSEA) were used to evaluate pathway activity. The ridge regression algorithm was used to analyze drug sensitivity.ResultsCIBERSORT showed significantly upregulated memory B cells, CD8+ T cells, and M1 Macrophages in the CIP group. The number of memory resting CD4+ T cells and resting NK cells in the CIP group was also significantly lower than in the Control group. The XCell analysis showed a higher proportion of Class-switched memory B-cells and M1 Macrophages in the CIP group. Pathway analysis showed that the CIP group had high activity in their immune and inflammatory response pathways and low activity in their immune exhaustion related pathway.ConclusionsIn this study, we researched CIP patients who after ICIs treatment developed an inflammatory IME, which is characterized by significantly increased activated immune cells and expression of inflammatory molecules, as well as downregulated immunosuppressive lymphocytes and signaling pathways. The goal was to develop theoretical guidance for clinical guidelines for the treatment of CIP in the future.

scholarly journals Immune Tolerance Developed in Platelet-Targeted FVIII Gene Therapy in Hemophilia Mice Is CD4+ T Cell-Mediated

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1071-1071
Author(s):  
Yingyu Chen ◽  
Xiaofeng Luo ◽  
Juan Chen ◽  
Jocelyn Schroeder ◽  
Christina K Baumgartner ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Immune Tolerance ◽  
Cd4 T Cells ◽  
Memory B Cells ◽  
Cd4 T Cell ◽  
Control Group

Abstract Immune response to factor VIII (FVIII) is not only a severe complication in protein replacement therapy, but also a major concern in gene therapy of hemophilia A. Our previous studies have demonstrated that platelet-targeted FVIII (2bF8) gene therapy together with in vivo drug-selection of transduced cells can not only rescue the bleeding diathesis but also induce anti-FVIII specific immune tolerance in FVIIInull mice. In the current study, we investigated 1) whether our non-selectable lentiviral vector (LV) for the induction of platelet-FVIII expression is sufficient to induce immune tolerance and 2) which cell compartment is tolerized after platelet gene therapy. Platelet-specific FVIII expression was introduced by 2bF8LV-transduction of hematopoietic stem cells followed by syngeneic transplantation into FVIIInull mice preconditioned with 660 cGy total body irradiation (TBI) or Busulfan (Bu) plus ATG (anti-thymocyte globulin). After bone marrow transplantation and reconstitution, animals were analyzed by PCR, qPCR, FVIII:C assay, and tail clipping test to confirm the success of 2bF8 gene therapy. Sixteen weeks after transplantation, animals were challenged with recombinant human FVIII (rhF8) via retro-orbital venous administration at a dose of 50 U/kg weekly for 4 weeks. The titers of anti-FVIII inhibitory antibodies (inhibitors) were determined by Bethesda assay. The CFSE-labeled CD4 T cell proliferation assay and ELISPOT-based memory B cell maturation assay were used to determine which cell compartment is tolerized to FVIII after 2bF8 gene therapy. The level of platelet-FVIII expression was 1.44 ± 0.39 mU/108 platelets (n = 6) in the 660 cGy group, which is not significantly different from the level obtained from the Bu+ATG group [3.04 ± 1.19 mU/108 platelets (n = 6)]. Even after rhF8 challenge, no antibodies were detected in 2bF8LV-transduced recipients in either group. In contrast, all animals in the control group that did not undergo gene therapy developed various levels of inhibitors (204±97 BU/ml, n=7). The frequency of regulatory T cells in both 660 cGy TBI (11.01±0.52%) and Bu+ATG (11.02±0.68%) groups were significantly higher than the control group (8.05±0.57%). T cell proliferation assay demonstrated that CD4+ T cells from 2bF8 LV-transduced recipients that had been challenged with rhF8 did not proliferate when restimulated with rhF8 in vitro. The daughter CD4+ T cells in the group with 10 U/ml of rhF8 were 5.84 ± 2.49% (n = 6), which was not significantly different from the control group without no rhF8 stimulation (0 U/ml) (5.33 ± 1.72%). CD4+ T cells from primed FVIIInull mice did proliferate after rhF8 restimulation. The proliferated daughter cell was 13.12 ± 6.76% (n = 7) in the group with rhF8 (10 U/ml) re-stimulation, which is significantly higher than the group without rhF8 co-culture (4.99 ± 1.16%). Since FVIII-specific memory B cell maturation is CD4+ T cell dependent, we isolated CD4+ T and memory B cells from 2bF8LV-transduced or FVIIInull mice after rhF8 immunization and co-cultured with rhF8 followed by ELISPOT assay to examine the antibody secreting cells. No spots were detected when memory B cells from rhF8-primed FVIIInull mice were co-cultured with CD4+ T cells from 2bF8LV-transduced recipients. In contrast, when memory B cells from either rhF8 immunized 2bF8LV-transduced or untreated FVIIInull mice were cultured with CD4+ T cells from rhF8-primed FVIIInull mice, there were 142 and 205 anti-FVIII antibody secreting cells, respectively, detected per 106 cells seeded. These results indicate that CD4+ T cells from 2bF8LV-transduced mice are tolerized to rhF8 stimulation. In conclusion, 2bF8 lentiviral gene transfer without in vivo selection of genetically manipulated cells can introduce FVIII-specific immune tolerance in hemophilia A mice and this immune tolerance is CD4+ T cell-mediated. Disclosures Baumgartner: Novo Nordisk: Research Funding. Shi:BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETTED GENE EXPRESSION..

Association of Th2 high tumors with aggressive features of breast cancer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e12584-e12584
Author(s):  
Yoshihisa Tokumaru ◽  
Lan Le ◽  
Masanori Oshi ◽  
Eriko Katsuta ◽  
Nobuhisa Matsuhashi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Cancer Progression ◽  
Immune Cells ◽  
Gene Set Enrichment Analysis ◽  
The Cancer Genome Atlas ◽  
Th2 Cells ◽  
Immune Microenvironment ◽  
Adaptive Immune Cells ◽  
Tumor Immune Microenvironment

e12584 Background: Recent studies have shown that infiltrating T-lymphocytes have been implicated in the promotion of breast cancer progression. Upon activation, these antigen-presenting cells then recruit adaptive immune cells. It has been proposed that polarization of CD4+ effector T-cells towards the immunosuppressive Th2 cells induce cytokine release and T-cell anergy, which lead to polarization of M2 tumor-associated macrophages (TAM’s), providing a protumorigenic microenvironment. We hypothesized that there is a correlation between high levels of Th2 cells and aggressive features of breast cancer and unfavorable tumor immune environment. Methods: Clinicopathological data and overall survival information was obtained on 1069 breast cancer patients from The Cancer Genome Atlas (TCGA) database. We defined Th2 high and low levels with the median cutoff. Results: Analysis of cell composition of the immune cells within tumor immune microenvironment demonstrated that Th2 high tumors did not consistently associated with unfavorable tumor immune microenvironment. Pro-cancer immune cells, such as macrophage M2 cells were increased with Th2 high tumors whereas, regulatory T cells were decreased with Th2 high tumors (p < 0.01 and p < 0.001 respectively). On the contrary, infiltration of anti-cancer cells, such as macrophage M1 was increased whereas CD8 T cells were decreased with Th2 high tumors (p < 0.05 and p < 0.001 respectively). Th2 was not shown to have correlation with IL-4, IL-6, IL-10 and IL-13, all of which has been reported to associate with Th2 cells. Th2 levels were associated with advanced grades. Also, correlation analysis demonstrated that there was a strong correlation between Th2 levels and Ki-67. These results were further validated with gene set enrichment analysis (GSEA). GSEA revealed that in Th2 high tumors enriched the gene sets associated with cell proliferation and cell cycle. Conclusions: High expression of immunosuppressive Th2 cells was associated with highly proliferative features of breast cancer, but not with unfavorable tumor immune microenvironment.

scholarly journals CLEC10A is a Potential Prognostic Biomarker and Immune Regulator of Tumor Microenvironment in Breast Cancer

2021 ◽  
Author(s):  
Jincheng He ◽  
Lei Jiang ◽  
Jun Wang ◽  
Guangtao Min ◽  
Xiangwen Wang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
B Cells ◽  
Tumor Microenvironment ◽  
Immune Cells ◽  
Cox Regression ◽  
Clinical Stage ◽  
Gene Set Enrichment Analysis ◽  
The Cancer Genome Atlas ◽  
Cox Regression Analysis

Abstract The communication between tumor cells and immune cells influences the ecology of the tumor microenvironment in breast cancer, as well as the disease progression and clinical outcome. The aim of this study was to investigate the prognostic value of the immunomodulatory factor CLEC10A in breast cancer. We applied the CIBERSORT and ESTIMATE calculation methods to calculate the proportion of tumor-infiltrating immune cells (TICs) and the amount of immune and stromal components in 1053 BRCA cases from The Cancer Genome Atlas (TCGA) database. The differentially expressed genes (DEGs) were analyzed by COX regression analysis and protein-protein interaction (PPI) network construction. Then, CLEC10A was identified as a prognostic factor by the intersection analysis of univariate COX and PPI. Further analysis revealed that CLEC10A expression was negatively correlated with the clinical pathologic characteristics (age, clinical stage) and positively correlated with survival of BRCA patients. Gene set enrichment analysis (GSEA) showed that genes in the high CLEC10A expression group were mainly enriched in immune-related activities. Genes in the low CLEC10A expression group were enriched in biochemical functions. CIBERSORT analysis of the proportion of TICs revealed that Macrophages M1, B cells memory, B cells naive, T cells CD4+ memory activated, T cells CD8+, and T cells gamma delta were positively correlated with CLEC10A expression, and Macrophages M0, Macrophages M2, Neutrophils, and NK cells resting were positively correlated with CLEC10A expression was negatively correlated, suggesting that CLEC10A may be an important factor in the immune regulation of the tumor microenvironment, especially in mediating the anti-tumor immune response of tumor-infiltrating immune cells at the tumor initiation stage. Therefore, CLEC10A expression may contribute to the prognosis of BRCA patients and provide a new idea for the immunotherapy of BRCA.

scholarly journals Effect of Autoimmune Cell Therapy on Immune Cell Content in Patients with COPD: A Randomized Controlled Trial

2022 ◽  
Vol 2022 ◽  
pp. 1-11
Author(s):  
Wen Li ◽  
Guanhong Li ◽  
Wei Zhou ◽  
Hui Wang ◽  
Yuqiong Zheng
Keyword(s):  
T Cells ◽  
B Cells ◽  
Treatment Group ◽  
Cell Therapy ◽  
Nk Cells ◽  
Immune Cells ◽  
Cd4 T Cells ◽  
Conventional Treatment ◽  
Control Group ◽  
Cd8 Cells

Objective. To explore the effect of autoimmune cell therapy on immune cells in patients with chronic obstructive pulmonary disease (COPD) and to provide a reference for clinical treatment of COPD. Methods. Sixty patients with stable COPD were randomly divided into control group and treatment group ( n = 30 ). The control group was given conventional treatment, and the treatment group was given one autoimmune cell therapy on the basis of conventional treatment. The serum levels of CD3+ T cells, CD4+ T cells, CD8+ cells, B cells, and NK cells in the peripheral blood were detected by flow cytometry. Possible adverse reactions were detected at any time during treatment. Results. There were no significant differences in the contents of CD3+ T cells, CD4+ T cells, CD8+ cells, B cells, and NK cells in the serum of the control group ( P > 0.05 ). Compared with before treatment, the contents of CD3+ T cells, CD4+ T cells, CD8+ cells, B cells, and NK cells in the serum of the treatment group were significantly increased ( P < 0.05 ). The ratio of CD 4 + / CD 8 + T cells in both control and treatment groups did not change significantly during treatment ( P > 0.05 ). There were no significant differences in serum CD3+ T cells, CD4+ T cells, CD8+ cells, B cells, and NK cells in the treatment group at 30 days and 90 days after treatment ( P > 0.05 ), but they were significantly higher than those in the control group ( P < 0.05 ). Conclusion. Autoimmune cell therapy can significantly increase the level of immune cells in the body and can be maintained for a long period of time, which has certain clinical benefits for recurrent respiratory tract infections and acute exacerbation in patients with COPD.

scholarly journals The Immune Subtypes and Landscape of Gastric Cancer and to Predict Based on the Whole-Slide Images Using Deep Learning

2021 ◽  
Vol 12 ◽  
Author(s):  
Yan Chen ◽  
Zepang Sun ◽  
Wanlan Chen ◽  
Changyan Liu ◽  
Ruoyang Chai ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
T Cells ◽  
Deep Learning ◽  
Immune Cells ◽  
Gene Set Enrichment Analysis ◽  
Functional Enrichment ◽  
Resting Cells ◽  
M1 Macrophages ◽  
Immune Score ◽  
Subtype 3

BackgroundGastric cancer (GC) is a highly heterogeneous tumor with different responses to immunotherapy. Identifying immune subtypes and landscape of GC could improve immunotherapeutic strategies.MethodsBased on the abundance of tumor-infiltrating immune cells in GC patients from The Cancer Genome Atlas, we used unsupervised consensus clustering algorithm to identify robust clusters of patients, and assessed their reproducibility in an independent cohort from Gene Expression Omnibus. We further confirmed the feasibility of our immune subtypes in five independent pan-cancer cohorts. Finally, functional enrichment analyses were provided, and a deep learning model studying the pathological images was constructed to identify the immune subtypes.ResultsWe identified and validated three reproducible immune subtypes presented with diverse components of tumor-infiltrating immune cells, molecular features, and clinical characteristics. An immune-inflamed subtype 3, with better prognosis and the highest immune score, had the highest abundance of CD8+ T cells, CD4+ T–activated cells, follicular helper T cells, M1 macrophages, and NK cells among three subtypes. By contrast, an immune-excluded subtype 1, with the worst prognosis and the highest stromal score, demonstrated the highest infiltration of CD4+ T resting cells, regulatory T cells, B cells, and dendritic cells, while an immune-desert subtype 2, with an intermediate prognosis and the lowest immune score, demonstrated the highest infiltration of M2 macrophages and mast cells, and the lowest infiltration of M1 macrophages. Besides, higher proportion of EVB and MSI of TCGA molecular subtyping, over expression of CTLA4, PD1, PDL1, and TP53, and low expression of JAK1 were observed in immune subtype 3, which consisted with the results from Gene Set Enrichment Analysis. These subtypes may suggest different immunotherapy strategies. Finally, deep learning can predict the immune subtypes well.ConclusionThis study offers a conceptual frame to better understand the tumor immune microenvironment of GC. Future work is required to estimate its reference value for the design of immune-related studies and immunotherapy selection.

scholarly journals Tumor Immune Microenvironment Characteristics and Their Prognostic Value in Non-Small-Cell Lung Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Dan Su ◽  
Gao Wu ◽  
Ran Xiong ◽  
Xiangxiang Sun ◽  
Meiqing Xu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
Immune Cells ◽  
Small Cell ◽  
Normal Lung ◽  
Small Cell Lung ◽  
Immune Microenvironment ◽  
Tumor Immune Microenvironment ◽  
Nsclc Patients

IntroductionCancer progression is determined not only by the malignant behavior of tumors but also by the immune microenvironment. The tumor immune microenvironment also plays a pivotal role in determining the clinical response of non-small-cell lung cancer (NSCLC) to immunotherapies. To understand the possible mechanisms and explore new targets in lung cancer immunotherapy, we characterized the immune profiles in NSCLC patients.MethodsSeventy-one NSCLC patients who underwent radical resection were selected. The immune cell composition in paired tumor and adjacent normal lung tissues was tested by flow cytometry. The associations of tumor immune microenvironment characteristics with clinicopathological factors and overall survival were analyzed. Kaplan–Meier curves and Cox proportional hazards models were used to determine differences in survival.ResultsCompared with adjacent normal lung tissues, an increased proportion of CD45+ hematopoietic-derived cells, CD4+ T cell subtypes, Tregs and B cells was observed in tumor samples with a reduced frequency of myeloid cell populations. There was no significant increase in total CD8+ T cells, but both PD1+ and CD38+ CD8+ T cells were significantly enriched in tumor samples and statistically significantly associated with tumor size. In addition, positive CD38 expression was highly correlated with PD1 positivity. A high proportion of CD8+ T cells and a low percentage of PD1+ CD8+ T cells were statistically significantly associated with better survival in stage II and III patients, whereas a low frequency of CD38+ CD8+ T cells was statistically significantly associated with better survival in all patients and identified as an independent prognostic factor (p=0.049).ConclusionWe profiled the immune cells in the tumor tissues of NSCLC patients using flow cytometry. The results revealed significant enrichment of infiltrating immune cells. A strong correlation was identified between CD38 and PD-1 expression on CD8+ T cells in tumors. CD8+ T cells and their subtypes play a critical role in the prediction of prognosis.

scholarly journals THU0035 OX40L EXPRESSING NEUTROPHILS INDUCE CD4 T FOLLICULAR AND PERIPHERAL HELPER CELL DIFFERENTIATION IN SYSTEMIC LUPUS ERYTHEMATOSUS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 230.2-231
Author(s):  
A. Pappalardo ◽  
E. Wojciechowski ◽  
I. Odriozola ◽  
I. Douchet ◽  
N. Merillon ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
T Cells ◽  
B Cells ◽  
Lupus Erythematosus ◽  
Cd4 T Cells ◽  
Close Contact ◽  
Memory B Cells ◽  
Systemic Lupus ◽  
Research Support

Background:Neutrophils have been described as potent antigen-presenting cells able to activate T cells by MHC/TCR interaction and costimulatory molecules in tumor immunity. However, little is known about the direct interaction between neutrophils and CD4 T cells with respect to systemic lupus erythematosus (SLE). We have previously shown that OX40L expressed by monocytes from SLE patients promote the differentiation of naïve and memory cells into IL21 secreting T cells that are able to help B cells1,2.Objectives:In this study, we investigate OX40L expression on neutrophils from SLE patients and contribution of these OX40L+neutrophils in SLE pathogenesis to modulation of the B cell helper role of CD4 T cells.Methods:Surface expression of co-stimulatory molecules (OX40L, ICOSL, GITRL, 4-1BBL) on neutrophils from SLE patients and healthy donors (HD) was measured by flow cytometry (FC). Neutrophils from HD were stimulated with TLR7 or TLR8 agonists and IFNα after 5 hours of culture, OX40L expression was measured by FC and Western Blotting. CD4 T cells were cultured with the stimulated neutrophils for 3 days. At the end of the co-culture, percentages of IL21-expressing T follicular (Tfh) and peripheral helper (Tph) cells measured by FC. These generated T cells were also cultured in the presence of memory B cells. After 5 days of co-culture, plasmablast generation and Ig levels were assessed by FC and ELISA, respectively. Inhibition of OX40-OX40L interaction in vitro was achieved using ISB 830, a novel anti-OX40 mAb currently used in clinical trials.Results:Among the co-stimulatory molecules tested, percentages of OX40L+neutrophils in SLE (n=54) were increased compared to HD (n=25)(mean + SD: HD = 1,34%±1.62 vs SLE = 4,53%±8.1; p=0.29). OX40L expression positively correlated with SLE disease activity score (SLEDAI) (p = 0,04; r = 0,31) and with anti-DNA antibodies (p= 0,04, r = 0,33). Of note, the percentage of OX40L+neutrophils was higher in anti-sm-RNP+patients (n=16, mean= 9%±9.8), compared to anti-sm-RNP-patients (n=27, mean = 1,4%±2.5; p = 0,02). The percentage of OX40L+neutrophils was higher in patients with class III or IV lupus nephritis, and inflammatory infiltrate within the kidney biopsy disclosed OX40L+neutrophils, in close contact with T cells. Neutrophils from HD express OX40L with TLR8 agonist, or IFNα priming followed by TLR7 agonist. When memory CD4 T cells were cultured in the presence of TLR8-stimulated neutrophils, the proportion of IL21-expressing Tfh (CXCR5+PD1+) and Tph (CXCR5-PD1hi) were increased, compared to culture with unstimulated neutrophils. This process was dependent on OX40-OX40L interactions, since in vitro treatment with the anti-OX40 blocking antibody ISB 830, inhibited the differentiation of memory T cells into Tfh and Tph. Both generated Tfh and Tph were able to promote the differentiation of memory B cells into Ig-secreting plasmablasts.Conclusion:Our results disclose an unprecedented phenomenon where cross-talk between TLR7/8-activated neutrophils and CD4 lymphocytes operates through OX40L-OX40 costimulation, and neutrophils promote the differentiation of pro-inflammatory Tfh and Tph, as well as IL21 production. Therefore, OX40L/OX40 should be considered as a potentially therapeutic axis in SLE patients.References:[1]Jacquemin et al. Immunity 2015;[2]Jacquemin et al. JCI Insight 2018Disclosure of Interests:Angela Pappalardo Grant/research support from: Ichnos Sciences, Elodie Wojciechowski: None declared, Itsaso Odriozola: None declared, Isabelle Douchet: None declared, Nathalie Merillon: None declared, Andrea Boizard-Moracchini: None declared, Pierre Duffau: None declared, Estibaliz Lazaro: None declared, Marie-Agnes Doucey Employee of: Ichnos Sciences, Lamine Mbow Employee of: Ichnos Sciences, Christophe Richez Consultant of: Abbvie, Amgen, Mylan, Pfizer, Sandoz and UCB., Patrick Blanco Grant/research support from: Ichnos Sciences

Engineered immunostimulatory cells can convert PBMCs from chronic lymphocytic leukemia (CLL) patients into potent tumor killing immune cells.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 7517-7517
Author(s):  
Joshua W. Keegan ◽  
Frank Borriello ◽  
Stacey M. Fernandes ◽  
Jennifer R. Brown ◽  
James A. Lederer
Keyword(s):  
T Cells ◽  
B Cells ◽  
Tumor Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Killing Activity ◽  
Tumor Killing ◽  
Tumor Cell Killing ◽  
Potent Tumor

7517 Background: Alloplex Biotherapeutics has developed a cellular therapeutic that uses ENgineered Leukocyte ImmunoSTimulatory cell lines called ENLIST cells to activate and expand populations of tumor killing effector cells from human peripheral blood mononuclear cells (PBMCs). This process leads to a 300-fold expansion of NK cells, CD8+ T cells, NKT cells, and TCRγδ T cells that are called SUPLEXA cells, which will be cryopreserved and transferred back into patients as an autologous immune cell therapy for cancer. In this study, PBMCs from CLL patients were used to generate SUPLEXA cells as a first approach to comparatively profile SUPLEXA cells from cancer patients and normal healthy volunteers (NHVs). Methods: ENLIST cell lines were engineered by expressing curated immunomodulatory proteins in the SK-MEL-2 melanoma cell line. Two million (M) PBMCs from 10 CLL patients or 2 NHVs were incubated with 0.4 M freeze/thaw killed ENLIST cells for 5 days in XVIVO-15 medium with 2% heat-inactivated human AB serum (XAB2) and then split 1:15 in XAB2 containing IL-7 and IL-15 to expand. After 9 days, SUPLEXA cells were harvested and cryopreserved. Results: Original PBMCs and matched SUPLEXA cells from each donor were thawed and characterized by mass cytometry (CyTOF) using a 47-marker antibody panel. CyTOF staining results of PBMCs from CLL patients demonstrated approximately 95% leukemia cells and few T cells, NK cells, B cells, and monocytes. CyTOF staining of SUPLEXA cells from all 10 CLL patients showed expansion of NK cells (17%), CD8 T cells (11%), and CD4 T cells (7.5%) that were similar in phenotype to SUPLEXA cells from NHVs showing high expression of granzymes and perforin that are indicative of potent tumor cell killing activity. Cancer cells in the original CLL PBMC samples were reduced to 0.78%. However, a population of non-T/non-B cells (60% ± 9.5%) was detected in SUPLEXA cells from all CLL patients that require further characterization. Next, SUPLEXA cells from CLL and NHV patients were comparatively tested for tumor cell killing activity at 2:1, 1:1, and 1:2 effector to target cell (MEL-14 melanoma cells expressing RFP) ratios. Percent killing of tumor cells by SUPLEXA cells prepared from CLL patients (77.8% ± 2.6% at 2:1) and NHVs (81.5% ± 0.3% at 2:1) were nearly identical at all effector to target ratios. Conclusions: We demonstrate for the first time that PBMCs from CLL patients can be converted into SUPLEXA cells despite low numbers of normal immune cells at baseline and the known immunologic impairment present in CLL patients. Importantly, SUPLEXA cells derived from CLL patients acquire potent tumor killing activity that is indistinguishable from SUPLEXA cells prepared from NHVs. Taken together, these findings support the feasibility of converting PBMCs from CLL patients with low percentages of NK and T cells into an autologous cellular therapy for cancer.

Can Intratumoral neutrophil lymphocyte ratio (NLR) be a prognostic biomarker in breast cancer patients?

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e12573-e12573
Author(s):  
Yoshihisa Tokumaru ◽  
Masanori Oshi ◽  
Vijayashree Murthy ◽  
Eriko Katsuta ◽  
Nobuhisa Matsuhashi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Cancer Patients ◽  
Immune Cells ◽  
Breast Cancer Patients ◽  
Immune Microenvironment ◽  
High Group ◽  
Anti Cancer ◽  
Tumor Immune Microenvironment ◽  
Er Positive

e12573 Background: In breast cancer patients, it is well known that the elevation of neutrophil lymphocyte ratio (NLR) in the blood are reported to associate with poor prognosis based on the notion that neutrophils represent pro-cancer, and lymphocytes represent anti-cancer immune cells. Tumor immune microenvironment has been demonstrated to play critical roles in the outcome of breast cancer patients. However, there is scarce evidence on the clinical relevance of intratumoral NLR in breast cancer patients. In the current study, we hypothesized that intratumoral NLR high tumors are associated with worse survival particularly in TNBC that is known to have high immune cell infiltration. Methods: A total of 1904 breast cancer patients’ data from METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) and analyzed. NLR was calculated by the gene expressions of CD66b (CEACAM8) and CD8 (CD8A). NLR high and low were divided by the median. Overall Survival (OS) and Disease-Free Survival were calculated utilizing Kaplan Meier method between intratumoral NLR high and low groups. xCell algorithm was used to analyze the infiltrated immune cells within the tumor immune microenvironment as we have previously published. Results: Intratumoral NLR high group was associated with worse OS in whole, ER-positive/HER2-negative, and triple negative (TN) subtypes, in agreement with the previous studies. TN subtype alone demonstrated worse DFS of NLR high group. Surprisingly, gene set enrichment analysis (GSEA) demonstrated no gene set enrichment to NLR high group, which implicates that there is no distinctive mechanism that associate with worse survival. Whereas, immune response-related gene sets significantly enriched to NLR low group in TN subtype. This enrichment was consistent in ER-positive/HER2-negative. Compared with ER-positive/HER2-negative subtype, anti-cancer immune cells such as CD4+ T cells, CD8+ T cells, M1 macrophage, and helper T helper type 1 cells were significantly infiltrated in TN patients (p < 0.001 for all genes), where M2 macrophages and neutrophils were less and regulatory T cells and T helper type 2 cells were more infiltrated in TN subtype. Furthermore, intratumoral NLR was significantly lower in TN compared with ER-positive/HER2-negative subtype (p < 0.001). These results suggest that intratumoral NLR low group is associated with better survival due to favorable tumor immune microenvironment in TN subtype rather than NLR high group has worse survival. Conclusions: Intratumoral NLR low tumor demonstrated more favorable OS and more favorable DFS in TN patients. Intratumoral NLR low breast cancer was associated with enhanced immune response and higher infiltration of anti-cancer immune cells were observed in TN subtype compared to ER-positive/HER2-negative which may contribute to the favorable outcome of in TN breast cancer.

Single Cell Profiling Reveals Unique CXCL13 Positive T Cell Subsets in the Tumor Microenvironment of Lymphocyte Rich Classic Hodgkin Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-33
Author(s):  
Tomohiro Aoki ◽  
Lauren C. Chong ◽  
Katsuyoshi Takata ◽  
Katy Milne ◽  
Elizabeth Chavez ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Single Cell ◽  
Immune Cells ◽  
Research Funding ◽  
Immune Cell ◽  
Expression Patterns ◽  
The Other ◽  
Tfh Cells

Introduction: Classic Hodgkin lymphoma (CHL) features a unique crosstalk between malignant cells and different types of normal immune cells in the tumor-microenvironment (TME). On the basis of histomorphologic and immunophenotypic features of the malignant Hodgkin and Reed-Sternberg (HRS) cells and infiltrating immune cells, four histological subtypes of CHL are recognized: Nodular sclerosing (NS), Mixed cellularity, Lymphocyte-rich (LR) and Lymphocyte-depleted CHL. Recently, our group described the high abundance of various types of immunosuppressive CD4+ T cells including LAG3+ and/or CTLA4+ cells in the TME of CHL using single cell RNA sequencing (scRNAseq). However, the TME of LR-CHL has not been well characterized due to the rarity of the disease. In this study, we aimed at characterizing the immune cell profile of LR-CHL at single cell resolution. METHODS: We performed scRNAseq on cell suspensions collected from lymph nodes of 28 primary CHL patients, including 11 NS, 9 MC and 8 LR samples, with 5 reactive lymph nodes (RLN) serving as normal controls. We merged the expression data from all cells (CHL and RLN) and performed batch correction and normalization. We also performed single- and multi-color immunohistochemistry (IHC) on tissue microarray (TMA) slides from the same patients. In addition, an independent validation cohort of 31 pre-treatment LR-CHL samples assembled on a TMA, were also evaluated by IHC. Results: A total of 23 phenotypic cell clusters were identified using unsupervised clustering (PhenoGraph). We assigned each cluster to a cell type based on the expression of genes described in published transcriptome data of sorted immune cells and known canonical markers. While most immune cell phenotypes were present in all pathological subtypes, we observed a lower abundance of regulatory T cells (Tregs) in LR-CHL in comparison to the other CHL subtypes. Conversely, we found that B cells were enriched in LR-CHL when compared to the other subtypes and specifically, all four naïve B-cell clusters were quantitatively dominated by cells derived from the LR-CHL samples. T follicular helper (TFH) cells support antibody response and differentiation of B cells. Our data show the preferential enrichment of TFH in LR-CHL as compared to other CHL subtypes, but TFH cells were still less frequent compared to RLN. Of note, Chemokine C-X-C motif ligand 13 (CXCL13) was identified as the most up-regulated gene in LR compared to RLN. CXCL13, which is a ligand of C-X-C motif receptor 5 (CXCR5) is well known as a B-cell attractant via the CXCR5-CXCL13 axis. Analyzing co-expression patterns on the single cell level revealed that the majority of CXCL13+ T cells co-expressed PD-1 and ICOS, which is known as a universal TFH marker, but co-expression of CXCR5, another common TFH marker, was variable. Notably, classical TFH cells co-expressing CXCR5 and PD-1 were significantly enriched in RLN, whereas PD-1+ CXCL13+ CXCR5- CD4+ T cells were significantly enriched in LR-CHL. These co-expression patterns were validated using flow cytometry. Moreover, the expression of CXCR5 on naïve B cells in the TME was increased in LR-CHL compared to the other CHL subtypes We next sought to understand the spatial relationship between CXCL13+ T cells and malignant HRS cells. IHC of all cases revealed that CXCL13+ T cells were significantly enriched in the LR-CHL TME compared to other subtypes of CHL, and 46% of the LR-CHL cases showed CXCL13+ T cell rosettes closely surrounding HRS cells. Since PD-1+ T cell rosettes are known as a specific feature of LR-CHL, we confirmed co-expression of PD-1 in the rosetting cells by IHC in these cases. Conclusions: Our results reveal a unique TME composition in LR-CHL. LR-CHL seems to be distinctly characterized among the CHL subtypes by enrichment of CXCR5+ naïve B cells and CD4+ CXCL13+ PD-1+ T cells, indicating the importance of the CXCR5-CXCL13 axis in the pathogenesis of LR-CHL. Figure Disclosures Savage: BeiGene: Other: Steering Committee; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie: Honoraria; Roche (institutional): Research Funding; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie, Servier: Consultancy. Scott:Janssen: Consultancy, Research Funding; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding; NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy. Steidl:AbbVie: Consultancy; Roche: Consultancy; Curis Inc: Consultancy; Juno Therapeutics: Consultancy; Bayer: Consultancy; Seattle Genetics: Consultancy; Bristol-Myers Squibb: Research Funding.

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