839 Transcriptomic profiling of T-cell populations in non-muscle invasive and muscle invasive bladder cancer

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A891-A891
Author(s):  
Viktor Sincic ◽  
Milad Abolhalaj ◽  
Henrik Lilljebjörn ◽  
Alar Aab ◽  
Karin Hagerbrand ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bladder Cancer ◽  
T Cells ◽  
T Cell ◽  
Cell Populations ◽  
Bladder Tissue ◽  
Transcriptomic Profiling ◽  
Muscle Invasive ◽  
And Control ◽  
Biological Differences

BackgroundBladder cancer is categorized as non-muscle invasive (NMIBC) or muscle invasive (MIBC). NIMBC makes up around 70% of the cases and although it is less aggressive, the recurrence rate is 50-70%, thus requiring extensive monitoring. Additionally, there is a risk of progression into MIBC with a 5-year survival of only 50% even when treated with radical cystectomy. Immune checkpoint inhibitors have shown promising results for treatment of bladder cancer; however, only around 30% of patients have a therapeutic effect and novel therapies are thus required. With the aim of pinpointing novel targets for T-cell based therapy, we have performed transcriptomic profiling of specific T cell populations in MIBC and NMIBC, as well as in control bladder tissue.MethodsMuscle-invasive (n=7) as well as non-muscle invasive (n=13) bladder tumor biopsies were obtained from untreated patients and control bladder tissue (n=7). Upon digestion, cells were stained with an antibody panel to enable sorting of CD8+ cytotoxic T-cells (CD8T), CD4+ T-helper cells (Th) and regulatory T-cells (Treg) using fluorescence activated cell sorting. RNA was extracted and subject to sequencing. Differential gene expression analysis was performed, using DESeq2 (genes with padjResultsPrincipal component analysis demonstrated that CD8T, unlike Th and Tregs, cluster according to the invasiveness of the disease. Accordingly, many genes were significantly differentially expressed between CD8T in MIBC and NMIBC compared to control, and also between CD8T in MIBC compared to NMIBC. Several genes associated with CD8 T-cell exhaustion were significantly upregulated in MIBC compared to both NMIBC and control. Further, GSEA results indicated biological differences of the CD8T compartment between different tumor stages.ConclusionsThe gene expression profiles of CD8 T-cells were significantly different in NMIBC, MIBC and control. The transcriptional profiles give clues on biological differences and disease progression and can be relevant for development of novel treatment strategies.Ethics ApprovalThe study was approved by the Regional Ethics Committee (EPN - Regionala Etikprövningsnämnden i Lund), approval number 2017/34.ConsentWritten informed consent was obtained from all patients included in the study.

scholarly journals Intratumoral TIGIT+ CD8+ T-cell infiltration determines poor prognosis and immune evasion in patients with muscle-invasive bladder cancer

2020 ◽  
Vol 8 (2) ◽  
pp. e000978
Author(s):  
Zhaopei Liu ◽  
Quan Zhou ◽  
Zewei Wang ◽  
Hongyu Zhang ◽  
Han Zeng ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
T Cells ◽  
T Cell ◽  
Invasive Bladder Cancer ◽  
Th2 Cells ◽  
Cancer Center ◽  
Muscle Invasive Bladder Cancer ◽  
Cell Abundance ◽  
Immune Contexture ◽  
Muscle Invasive

BackgroundT-cell immunoglobulin and ITIM domain (TIGIT) is identified as a novel checkpoint receptor that can facilitate immune escape via mediating T-cell exhaustion in tumors. However, the clinical significance and immune contexture correlation of intratumoral TIGIT+ CD8+ T-cells remain to be further explored in muscle-invasive bladder cancer (MIBC).Methods259 patients with MIBC from two clinical centers (Zhongshan Hospital, n=141; Shanghai Cancer Center, n=118) were analyzed to evaluate the prognostic value and immune contexture association of TIGIT+ CD8+ T-cells through immunohistochemistry. Fresh tumor tissue samples from 26 patients with MIBC were examined to discover the phenotype of this CD8 subpopulation by flow cytometry.ResultsHigh infiltration of intratumoral TIGIT+ CD8+ T-cells predicted poor overall survival (OS) and recurrence-free survival (RFS) in MIBC. For patients with stage II MIBC with low infiltration of TIGIT+ CD8+ cells, adjuvant chemotherapy (ACT) could significantly prolong their OS and RFS. Intratumoral TIGIT+ CD8+ T-cell abundance was correlated with impaired CD8+ T-cell cytotoxicity and exhibited production of immunosuppressive cytokine IL-10. Further analysis of tumor-infiltrating immune cell landscape revealed TIGIT+ CD8+ T-cells were associated with suppressive immune contexture, including Th2 cells, regulatory T-cells, mast cells and neutrophils.ConclusionIntratumoral TIGIT+ CD8+ T-cell abundance could serve as an independent prognosticator for clinical outcome and a predictive biomarker for inferior ACT responsiveness. Intratumoral TIGIT+ CD8+ T-cell abundance correlated with dampened CD8+ T-cell antitumor immunity and immunosuppressive contexture abundance, highlighting a tumor-promoting role of TIGIT+ CD8+ T-cells.

scholarly journals EPEN-23. A COMPUTATIONAL ANALYSIS OF THE TUMOUR IMMUNE MICROENVIRONMENT IN PAEDIATRIC EPENDYMOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii312-iii312
Author(s):  
Timothy Ritzmann ◽  
Anbarasu Lourdusamy ◽  
Andrew Jackson ◽  
Lisa Storer ◽  
Andrew Donson ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Computational Analysis ◽  
Immune Cell ◽  
Cell Populations ◽  
Immune Microenvironment ◽  
Tumour Immunity ◽  
Follicular Helper

Abstract Ependymoma is the third commonest childhood brain tumour. Relapse is frequent, often fatal and current therapeutic strategies are inadequate. Previous ependymoma research describes an immunosuppressive environment with T-cell exhaustion, indicating a lack of response to T-cell directed immunotherapy. Understanding the immune microenvironment is therefore critical. We present a computational analysis of ependymoma, gene expression derived, immune profiles. Using 465 ependymoma samples from gene expression datasets (GSE64415, GSE50385, GSE100240) and two RNA-seq databases from UK ependymomas, we applied bulk tumour deconvolution methods (CIBERSORT and xCell) to infer immune cell populations. Additionally, we measured checkpoint blockade related mRNAs and used immunohistochemistry to investigate cell populations in ependymoma sections. CIBERSORT indicated high proportions of M2-like macrophages and smaller proportions of activated natural killer (NK) cells, T follicular helper cells, CD4+ memory T-cells and B-cells. xCell overlapped with the M2-like macrophage and CD4+ memory T-cell signatures seen in CIBERSORT. On immunohistochemistry, T and B cells were scarce, with small numbers of CD8+, CD4+ and CD20+ cells in the parenchyma but greater numbers in surrounding regions. CD68 was more highly expressed in the parenchyma. Analysis of nine checkpoint ligands and receptors demonstrated only the TIM3/GAL9 combination was reliably detectable. GAL9 is implicated in tumour interactions with T-cells and macrophages elsewhere, possibly contributing to poorer outcomes. Our study supports the presence of myeloid cells being leading contributors to the ependymoma immune microenvironment. Further work will delineate the extent of myeloid contribution to immunosuppression across molecular subtypes. Modulation of tumour immunity may contribute to better clinical outcomes.

scholarly journals Avadomide (CC-122) Alters T Cell Repertoire and Enhances Infiltration of Lymphocytes into Tumor Microenvironment in DLBCL Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4211-4211
Author(s):  
Patrick R. Hagner ◽  
Fadi Towfic ◽  
Frank Schmitz ◽  
Xuehai Wang ◽  
Andrew P. Weng ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Populations ◽  
Employment Equity ◽  
Equity Ownership

Abstract Background : Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, constituting 30-40% of all new cases. Avadomide, a small molecule cereblon modulator currently being developed in DLBCL, binds to cereblon in the CRL4CRBN E3 ligase, leading to ubiquitination and subsequent proteasomal degradation of transcription factors Aiolos and Ikaros. This results in decreased proliferation and increased apoptosis of DLBCL cells, independent of cell-of-origin, and immunostimulatory effects in T and NK cells, as measured by increased cytokine production, cell surface activation markers, and enhanced antibody-dependent cellular cytotoxicity. A novel gene expression-based classifier, which detects DLBCL patients with T cell and macrophage infiltration within the tumor microenvironment, has been shown to enrich for responders to avadomide. Avadomide, as a single agent and in combination with rituximab, is currently being investigated in relapsed/refractory DLBCL (NCT01421524 and NCT02031419). Methods : Eighty-one DLBCL patients were enrolled in the expansion phase of the CC-122-ST-001 study (NCT01421524). Peripheral blood T cell subsets were enumerated at screening (baseline), cycle 1 day 15 (C1D15) and cycle 2 day 15 (C2D15) by flow cytometric immunophenotyping. Ex vivo production of IL-2 and IFNγ, as a measure of T cell activation, was determined using the α-CD3 TruCulture Assay. Changes from baseline were evaluated using the t-test with P<0.05 considered significant. T cell receptor (TCR) repertoire analysis through TCRB CDR3 region sequencing was done to derive metrics of population diversity and composition. RNAseq was performed on screening and on-treatment (C1D10/15) biopsies; gene expression deconvolution analyses were used to identify immune cell populations within the tumor microenvironment. Results : Avadomide treatment results in decreased peripheral CD4+ and CD8+ naïve (CD45RA+/CD45RO-) T cells and increased memory (CD45RA-/CD45RO+) and activated (HLA-DR+) T cells, without significantly affecting the absolute numbers of total CD3+, CD4+ or CD8+ populations (Table). High-dimensional single-cell mass cytometry of longitudinally collected peripheral blood samples confirmed the significant increase in CD8+ memory T cells and identified an increase in Treg populations and decreases in CD16+ monocytes and dendritic cells (adj. P<0.02). A single dose of avadomide on C1D1 significantly activated T cells, as indicated by a 300% increase in IL-2 (P=0.018) and 185% increase in IFNγ (P=0.003) secretion. Assessment of TCR B clonotypes revealed that avadomide increases the TCRB repertoire breadth, while reducing its clonality. To understand the influence of avadomide treatment on the tumor microenvironment, we performed RNA sequencing on tumor biopsies collected at screening and two weeks after initiating avadomide treatment (n=18 patients). Deconvolution analyses identified an increase in the expression of genes indicative of various T cell populations, dendritic cells and macrophages, while B cell associated gene expression decreased in on-treatment biopsies compared to screening biopsies. Gene set enrichment analysis (GSEA) revealed significantly increased expression of genes associated with "HALLMARK Interferon Alpha Response" (adj. P=0.04), indicative of an increase in Type I/II interferon production by cells such as T and NK cells. Buttressing the in vitro observations of avadomide-mediated inhibition of DLBCL cell proliferation, GSEA identified a decrease in "E2F targets" (adj. P=0.007) consistent with decreased proliferation of malignant B cells. Conclusion : Avadomide is a potent immunomodulating agent with multiple immune activating properties, including positive effects on T cell activation, as well as a broad expansion of T cell populations as defined by an increase in the richness of the T cell repertoire in blood. In addition, our data demonstrate decreased proliferation of malignant B cells in the tumor, with concomitant increased trafficking of immune cells, such as dendritic cells and macrophages, to the tumor microenvironment. These data further delineate the immune enhancing activity of avadomide in DLBCL patients beyond T-cell activation and provide rational combination strategies. Table. Table. Disclosures Hagner: Celgene Corporation: Employment, Equity Ownership. Towfic:Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment, Equity Ownership. Pourdehnad:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership.

HLA-Peptidomics and the Identification of Clinical Relevant Minor Histocompatibility Antigens,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4038-4038
Author(s):  
Pleun Hombrink ◽  
Chopie Hassan ◽  
Michel G.D. Kester ◽  
Cornelis A.M. van Bergen ◽  
J.H.F. Falkenburg ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
High Throughput ◽  
Cell Populations ◽  
Histocompatibility Antigens ◽  
Minor Histocompatibility Antigens ◽  
Cell Clones ◽  
Combined Use ◽  
Peptide Affinity

Abstract Abstract 4038 T-cell recognition of minor histocompatibility antigens (MiHA) plays an important role in the graft-versus-tumor (GVT) effect of allogeneic stem cell transplantation (allo-SCT). However, MiHA recognition is also associated with graft-versus-host disease (GVHD). It is assumed that the selective infusion of T-cells reactive with hematopoiesis-restricted MiHA may help to separate the GVT and GVHD effects of allo-SCT. However, the number of attractive MiHA identified to date remains limited. In this study we aimed to determine whether it is feasible to identify MiHA using HLA-peptidomics in a reverse-immunology approach, based on bona fide eluted MiHA epitopes. Successful development of such a technology could allow the rapid identification of new MiHA, required to make antigen-selective adoptive T-cell therapy a realistic option. In addition, when compared to classical forward approaches, this strategy may provide tools to efficiently identify favorable GVT-involved MiHA, rather than random identifying targets of activated T-cells isolated during a GVT-response. To identify biological relevant MiHA candidates, HLA class I peptides were isolated from lysed EBV-transformed B-cells (EBV-LCL), analyzed by mass spectrometry (MS) and matched with a human protein database (IPI). This effort resulted in a set of fifteen thousand peptides, encoded in the normal reading frame with high probability MS scores. To identify potential MiHA candidates, the total set was matched with our newly developed public available Human Short Peptide Variation Database (http://srs.bioinformatics.nl/hspv), dedicated to polymorphic peptides. The quality of this peptide set was demonstrated by a detection efficiency of fifty percent of known MiHA including various length variants and eluted MiHA counterparts. Subsequently the combined use of gene expression databases, validated single nucleotide polymorphism (SNP) arrays and HLA-peptide binding assays resulted in a further selection of 27 high potential HLA-A*0201 and B*0701 MiHA candidates. This set was used for the generation of pMHC tetramers by UV-mediated exchange technology. Next, pMHC tetramer positive specific T-cell lines were generated from eighteen healthy SNP-typed PBMC donors following MACS isolation. To decrease the incidence of isolating low affinity T-cells, due to self-tolerance induction, pMHC tetramer isolations were only performed using donors homozygous negative for the specific SNP. After repeated pMHC tetramer pull down, in vitro expanded cell samples were analyzed on a multi-color FACS LSRII flow cytometer and clonally expanded following FACS cell sorting. Using this approach we were able to detect 16 unique pMHC tetramer positive T-cell populations corresponding with 70% of eluted MiHA candidates. Most of these pMHC tetramer positive T-cell populations were detected in multiple individuals, and appeared to be oligoclonal. Although most T-cell clones produced IFN-γ when co-cultured with peptide-pulsed target cells, there appeared to be a wide variety of peptide affinity among the pMHC tetramer positive T-cell clones. High throughput screening of all clones for MiHA specific recognition patterns of SNP-typed EBV-LCL panels revealed a clear correlation between the peptide-affinity of the T-cell clone and its capacity to recognize endogenously processed and presented peptide. Collectively these efforts resulted in the validation of two previously described MiHA and the identification of three new biological relevant MiHA. In summary, this study resulted in the establishment of an algorithm for the high-throughput identification of MiHA based on the combined use of HLA-peptidomics and reverse-immunology by pMHC tetramers. Our data indicate that the technology developed within this project can be of great value to the efficient identification of novel MiHA with potential clinical value especially when epitope selection criteria are supplemented with gene expression data, allowing pre-selection for those MiHA candidates with a hematopoiesis restricted gene expression patterns that may direct reactivity towards GVT. Disclosures: No relevant conflicts of interest to declare.

The intratumoral CXCR3 chemokine system is predictive of chemotherapy response in human bladder cancer

2021 ◽  
Vol 13 (576) ◽  
pp. eabb3735
Author(s):  
Tino Vollmer ◽  
Stephan Schlickeiser ◽  
Leila Amini ◽  
Sarah Schulenberg ◽  
Desiree J. Wendering ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
T Cells ◽  
T Cell ◽  
Splice Variants ◽  
Cancer Control ◽  
Chemotherapy Response ◽  
Variant Form ◽  
Muscle Invasive ◽  
T Cell Subpopulations

Chemotherapy has direct toxic effects on cancer cells; however, long-term cancer control and complete remission are likely to involve CD8+ T cell immune responses. To study the role of CD8+ T cell infiltration in the success of chemotherapy, we examined patients with muscle invasive bladder cancer (MIBC) who were categorized on the basis of the response to neoadjuvant chemotherapy (NAC). We identified the intratumoral CXCR3 chemokine system (ligands and receptor splice variants) as a critical component for tumor eradication upon NAC in MIBC. Through characterization of CD8+ T cells, we found that stem-like T cell subpopulations with abundant CXCR3alt, a variant form of the CXCL11 receptor, responded to CXCL11 in culture as demonstrated by migration and enhanced effector function. In tumor biopsies of patients with MIBC accessed before treatment, CXCL11 abundance correlated with high numbers of tumor-infiltrating T cells and response to NAC. The presence of CXCR3alt and CXCL11 was associated with improved overall survival in MIBC. Evaluation of both CXCR3alt and CXCL11 enabled discrimination between responder and nonresponder patients with MIBC before treatment. We validated the prognostic role of the CXCR3-CXCL11 chemokine system in an independent cohort of chemotherapy-treated and chemotherapy-naïve patients with MIBC from data in TCGA. In summary, our data revealed stimulatory activity of the CXCR3alt-CXCL11 chemokine system on CD8+ T cells that is predictive of chemotherapy responsiveness in MIBC. This may offer immunotherapeutic options for targeted activation of intratumoral stem-like T cells in solid tumors.

scholarly journals Identification of Signature Genes Associated With Invasiveness and the Construction of a Prognostic Model That Predicts the Overall Survival of Bladder Cancer

2021 ◽  
Vol 12 ◽  
Author(s):  
Yang He ◽  
Yongxin Wu ◽  
Zhe Liu ◽  
Boping Li ◽  
Ning Jiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Overall Survival ◽  
Bladder Cancer ◽  
T Cells ◽  
Nk Cells ◽  
Risk Scores ◽  
Hub Genes ◽  
Muscle Invasive Bladder Cancer ◽  
Signature Genes ◽  
Muscle Invasive

Background: Bladder cancer has become the tenth most diagnosed cancer worldwide. The prognosis has been shown to differ between non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). We aimed to identify signature genes that are associated with the invasiveness and survival of bladder cancer and to identify potential treatments.Methods: We downloaded gene expression profiles of bladder cancer from the Gene Expression Omnibus database to identify differentially expressed genes and perform weighted gene co-expression network analysis. Functional enrichment was analyzed by GO and KEGG analyses. Hub genes were identified from the significant module. Another dataset was also acquired to verify the expression of hub genes. Univariate and multivariate Cox regression analyses were applied to the dataset downloaded from The Cancer Genome Atlas database. Risk scores were calculated and the effect was evaluated by Kaplan-Meier survival analysis. A nomogram was constructed and validated using training and testing samples, respectively. Analysis of the tumor immune microenvironment was conducted with the CIBERSORT algorithm.Results: In total, 1,245 differentially expressed genes (DEGs) were identified. A distinct module was identified that was significantly correlated to invasiveness. The genes within this module were found to be significantly associated with extracellular exosomes, GTPase activity, metabolic pathways, etc. Three hub genes (VSIG2, PPFIBP2, and DENND2D) were identified as biomarkers of invasiveness; two of these (PPFIBP2 and DENND2D) were closely associated with prognosis. The risk score was regarded as an independent prognostic factor. The nomogram was associated with acceptable accuracy for predicting 1- and 5-year overall survival. The infiltrating levels of resting NK cells, activated natural killer (NK) cells, CD8+ T cells, activated memory CD4+ T cells, and T follicular helper cells, were significantly higher in the group with lower risk scores. The group with higher risk scores showed predominant infiltration by regulatory T cells (Tregs).Conclusion: We successfully identified three signature genes related to invasiveness and constructed a nomogram of bladder cancer with acceptable performance. Differences suggested by risk scores between groups of patients showing diverse patterns of immune cell infiltration may be beneficial for selecting therapeutic approaches and predicting prognosis.

scholarly journals Immunological Hallmarks for Clinical Response to BCG in Bladder Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Chun Jye Lim ◽  
Phuong Hoang Diem Nguyen ◽  
Martin Wasser ◽  
Pavanish Kumar ◽  
Yun Hua Lee ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Predictive Biomarkers ◽  
Cell Receptor ◽  
Receptor Repertoire ◽  
Muscle Invasive ◽  
Anti Tumor Activity

Intravesical Bacillus Calmette-Guerin (BCG) is an effective immunotherapy for non-muscle invasive bladder cancer (NMIBC). However, recurrence and progression remain frequent warranting deeper insights into its mechanism. We herein comprehensively profiled blood and tissues obtained from NMIBC patients before, during and after BCG treatment using cytometry by time-of-flight (CyTOF) and RNA sequencing to identify the key immune subsets crucial for anti-tumor activity. We observed the temporal changes of peripheral immune subsets including NKT cells, central memory CD4+ T cells, CD8+ T cells and regulatory T cells (Treg) during the course of BCG. Gene expression analysis revealed enriched immune pathways involving in T cell activation and chemotaxis, as well as a more diversified T cell receptor repertoire in post-BCG tissues. Moreover, tissue multiplexed-immunofluorescence (mIF) showed baseline densities of non-Treg and CD8+PD-1+ T cells were predictive of response and better recurrence-free survival after BCG. Remarkably, post-BCG tissues from responders were found to be infiltrated with more active CD8+PD-1- T cells and non-Treg CD4+FOXP3- T cells; but increased exhausted CD8+PD-1+ T cells were found in non-responders. Taken together, we identified predictive biomarkers for response and uncovered the post-treatment expansion of exhausted PD-1+CD8+ T cells as key to BCG resistance, which could potentially be restored by combining with anti-PD-1 immunotherapy.

Regulatory T-cells Infiltrate Periodontal Disease Tissues

2005 ◽  
Vol 84 (7) ◽  
pp. 639-643 ◽  
Author(s):  
T. Nakajima ◽  
K. Ueki-Maruyama ◽  
T. Oda ◽  
Y. Ohsawa ◽  
H. Ito ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
T Cells ◽  
Regulatory T Cell ◽  
Gene Expression Analysis ◽  
Periodontal Diseases ◽  
Cell Populations ◽  
And Control ◽  
Immunohistological Analysis ◽  
Tgf Β1

CD4+CD25+ regulatory T (Tr) cells are critical in regulating the immune response and thereby play an important role in the defense against infection and control of autoimmune diseases. Our previous studies demonstrated the involvement of autoimmune responses in periodontitis. The aim of this study was to identify CD4+CD25+ Tr cells in periodontitis tissues and compare them with those in gingivitis tissues. Immunohistological analysis of CD4, CD25, and CTLA-4 and the gene expression analysis of FOXP3, TGF-β1, and IL-10 on gingival biopsies revealed the presence of CD4+CD25+ Tr cells in all tissues. In periodontitis, the percentage of CD4+CD25+ Tr cells increased with increasing proportions of B-cells relative to T-cells. FOXP3, a characteristic marker for CD4+CD25+ Tr cells, TGF-β1 and IL-10 were expressed more highly in periodontitis compared with gingivitis. These findings suggest that CD4+CD25+ Tr cells and possibly other regulatory T-cell populations do exist and may play regulatory roles in periodontal diseases.

scholarly journals 49 In situ phenotypic analysis of T cells in the tumor microenvironment of a pre-clinical model of non-small cell lung cancer (NSCLC) by tissue sectioning and whole-mount immunofluorescence imaging

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A52-A52
Author(s):  
Elen Torres ◽  
Stefani Spranger
Keyword(s):  
T Cells ◽  
Spatial Distribution ◽  
T Cell ◽  
Spatial Location ◽  
Cell Infiltration ◽  
Cell Populations ◽  
Tumor Bearing ◽  
T Cell Infiltration ◽  
Volume Imaging ◽  
Whole Mount

BackgroundUnderstanding the interactions between tumor and immune cells is critical for improving current immunotherapies. Pre-clinical and clinical evidence has shown that failed T cell infiltration into lung cancer lesions might be associated with low responsiveness towards checkpoint blockade.1 For this reason, it is necessary to characterize not only the phenotype of T cells in tumor-bearing lungs but also their spatial location in the tumor microenvironment (TME). Multiplex immunofluorescence staining allows the simultaneous use of several cell markers to study the state and the spatial location of cell populations in the tissue of interest. Although this technique is usually applied to thin tissue sections (5 to 12 µm), the analysis of large tissue volumes may provide a better understanding of the spatial distribution of cells in relation to the TME. Here, we analyzed the number and spatial distribution of cytotoxic T cells and other immune cells in the TME of tumor-bearing lungs, using both 12 µm sections and whole-mount preparations imaged by confocal microscopy.MethodsLung tumors were induced in C57BL/6 mice by tail vein injection of a cancer cell line derived from KrasG12D/+ and Tp53-/- mice. Lung tissue with a diverse degree of T cell infiltration was collected after 21 days post tumor induction. Tissue was fixed in 4% PFA, followed by snap-frozen for sectioning. Whole-mount preparations were processed according to Weizhe Li et al. (2019) 2 for tissue clearing and multiplex volume imaging. T cells were labeled with CD8 and FOXP3 antibodies to identify cytotoxic or regulatory T cells, respectively. Tumor cells were labeled with a pan-Keratin antibody. Images were acquired using a Leica SP8 confocal microscope. FIJI3 and IMARIS were used for image processing.ResultsWe identified both cytotoxic and regulatory T cell populations in the TME using thin sections and whole-mount. However, using whole-mount after tissue clearing allowed us to better evaluate the spatial distribution of the T cell populations in relation to the tumor structure. Furthermore, tissue clearance facilitates the imaging of larger volumes using multiplex immunofluorescence.ConclusionsAnalysis of large lung tissue volumes provides a better understanding of the location of immune cell populations in relation to the TME and allows to study heterogeneous immune infiltration on a per-lesion base. This valuable information will improve the characterization of the TME and the definition of cancer-immune phenotypes in NSCLC.ReferencesTeng MW, et al., Classifying cancers based on T-cell infiltration and PD-L1. Cancer Res 2015;75(11): p. 2139–45.Li W, Germain RN, and Gerner MY. High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging. Nat Protoc 2019;14(6): p. 1708–1733.Schindelin J, et al, Fiji: an open-source platform for biological-image analysis. Nat Methods 2012;9(7): p. 676–82.

scholarly journals Rapamycin enhances BCG-specific γδ T cells during intravesical BCG therapy for non-muscle invasive bladder cancer: a randomized, double-blind study

2021 ◽  
Vol 9 (3) ◽  
pp. e001941
Author(s):  
Niannian Ji ◽  
Neelam Mukherjee ◽  
Ryan M Reyes ◽  
Jonathan Gelfond ◽  
Martin Javors ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
T Cells ◽  
Γδ T Cells ◽  
Percentage Change ◽  
Invasive Bladder Cancer ◽  
Muscle Invasive Bladder Cancer ◽  
Double Blind ◽  
Bcg Therapy ◽  
Intravesical Bcg ◽  
Muscle Invasive

BackgroundAlthough intravesical BCG is the standard treatment of high-grade non-muscle invasive bladder cancer (NMIBC), response rates remain unsatisfactory. In preclinical models, rapamycin enhances BCG vaccine efficacy against tuberculosis and the killing capacity of γδ T cells, which are critical for BCG’s antitumor effects. Here, we monitored immunity, safety, and tolerability of rapamycin combined with BCG in patients with NMIBC.MethodsA randomized double-blind trial of oral rapamycin (0.5 or 2.0 mg daily) versus placebo for 1 month was conducted in patients with NMIBC concurrently receiving 3 weekly BCG instillations (NCT02753309). The primary outcome was induction of BCG-specific γδ T cells, measured as a percentage change from baseline. Post-BCG urinary cytokines and immune cells were examined as surrogates for local immune response in the bladder. Secondary outcomes measured were adverse events (AEs) and tolerability using validated patient-reported questionnaires.ResultsThirty-one patients were randomized (11 placebo, 8 rapamycin 2.0 mg, and 12 rapamycin 0.5 mg). AEs were similar across groups and most were grade 1–2. One (12.5%) patient randomized to 2.0 mg rapamycin was taken off treatment due to stomatitis. No significant differences in urinary symptoms, bowel function, or bother were observed between groups. The median (IQR) percentage change in BCG-specific γδ T cells from baseline per group was as follows: −26% (−51% to 24%) for placebo, 9.6% (−59% to 117%) for rapamycin 0.5 mg (versus placebo, p=0.18), and 78.8% (−31% to 115%) for rapamycin 2.0 mg (versus placebo, p=0.03). BCG-induced cytokines showed a progressive increase in IL-8 (p=0.02) and TNF-α (p=0.04) over time for patients on rapamycin 2.0 mg, whereas patients receiving placebo had no significant change in urinary cytokines. Compared with placebo, patients receiving 2.0 mg rapamycin had increased urinary γδ T cells at the first week of BCG (p=0.02).ConclusionsFour weeks of 0.5 and 2.0 mg oral rapamycin daily is safe and tolerable in combination with BCG for patients with NMIBC. Rapamycin enhances BCG-specific γδ T cell immunity and boosts urinary cytokines during BCG treatment. Further study is needed to determine long-term rapamycin safety, tolerability and effects on BCG efficacy.

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