Project description:<p>PD-1 is an important immune checkpoint inhibitor that shows great promise in the clinic, particularly for melanoma and lung cancers. Since PD-1 is also expressed on infiltrating CD4+ Treg and Teffector cells in glioblastoma, we sought to better understand the role of PD-1 on these infiltrating CD4+ Treg and Teffector cells. To this end, we performed functional and transcriptional profiling using CD4+ Treg and Teffector cells isolated from healthy donors and glioblastoma patients (from both tumors and blood).</p>
Project description:CD4+ helper T (Th) cells are critical regulators of immune responses but their role in breast cancer is currently unknown. This work aims to characterize Th cells infiltrating invasive primary human breast tumors, analyze the influence by the tumor microenvironment and identify Th cell specific prognostic gene signatures. CD4+ T cells isolated from the tumor (TIL), axillary lymph node (LN) and blood (PB) of 10 patients were analyzed on Affymetrix U133 Plus 2.0 arrays. A confirmation set of 60 patients were studied by flow cytometry, qRT-PCR or immunohistochemistry and analyzed according to the extent of the tumor immune infiltrate. Gene expression profiles of freshly isolated TIL were also compared with TIL that had been rested overnight or with CD4+ T cells [non-stimulated (NS) or stimulated (S)] from healthy donor PB treated with tumor supernatant (SN). Comparing gene expression profiles of donor blood derived total CD4+ T cells [non-stimulated (NS)] with and without tumor supernatant (SN) treatment Total CD4+ T cells from a healthy donor blood (NS) were treated (and as control: untreated samples in biological triplicate) with SN from fresh breast tumor homogenates of 3 patients and analyzed on Affymetrix U133 Plus 2.0 arrays
Project description:CD4+ helper T (Th) cells are critical regulators of immune responses but their role in breast cancer is currently unknown. This work aims to characterize Th cells infiltrating invasive primary human breast tumors, analyze the influence by the tumor microenvironment and identify Th cell specific prognostic gene signatures. CD4+ T cells isolated from the tumor (TIL), axillary lymph node (LN) and blood (PB) of 10 patients were analyzed on Affymetrix U133 Plus 2.0 arrays. A confirmation set of 60 patients were studied by flow cytometry, qRT-PCR or immunohistochemistry and analyzed according to the extent of the tumor immune infiltrate. Gene expression profiles of freshly isolated TIL were also compared with TIL that had been rested overnight or with CD4+ T cells [non-stimulated (NS) or stimulated (S)] from healthy donor PB treated with tumor supernatant (SN). Analysis of CD4+ TIL by comparing their expression profiles to those of their conterparts from patient axillary lymph nodes and peripheral blood and healthy donor blood CD4+ T cells were isolated from primary tumors, axillary lymph nodes and peripheral blood of 10 patients with invasive breast carcinomas and blood of 4 healthy donors and analyzed on Affymetrix U133 Plus 2.0 arrays
Project description:Lymph node involvement is a major prognostic variable in breast cancer. Whether the molecular mechanisms that drive breast cancer cells to colonize lymph nodes are shared with their capacity to form distant metastases is yet to be established. In a transcriptomic survey aimed at identifying molecular factors associated with lymph node involvement of ductal breast cancer, we found that luminal differentiation, assessed by the expression of estrogen receptor (ER) and/or progesterone receptor (PR) and GATA3, was only infrequently lost in node-positive primary tumors and in matched lymph node metastases. The transcription factor GATA3 critically determines luminal lineage specification of mammary epithelium and is widely considered a tumor and metastasis suppressor in breast cancer. Strong expression of GATA3 and ER in a majority of primary node-positive ductal breast cancer was corroborated by quantitative RT-PCR and immunohistochemistry in the initial sample set, and by immunohistochemistry in an additional set from 167 patients diagnosed of node-negative and positive primary infiltrating ductal breast cancer, including 102 samples from loco-regional lymph node metastases matched to their primary tumors, as well as 37 distant metastases. These observations suggest that loss of luminal differentiation is not a major factor driving the ability of breast cancer cells to colonize regional lymph nodes. The transcriptomic study comprises 16 samples from Lymph node metastasis from infiltrating ductal breast carcinoma, 18 samples from Primary node-positive infiltrating ductal,7 samples from Primary node-negative infiltrating ductal and 3 samples from Unaffected lymph node were included. Their RNA was isolated and prepared for hybridization to human Affymetrix GeneChip arrays.
Project description:CD4+ helper T (Th) cells are critical regulators of immune responses but their role in breast cancer is currently unknown. This work aims to characterize Th cells infiltrating invasive primary human breast tumors, analyze the influence by the tumor microenvironment and identify Th cell specific prognostic gene signatures. CD4+ T cells isolated from the tumor (TIL), axillary lymph node (LN) and blood (PB) of 10 patients were analyzed on Affymetrix U133 Plus 2.0 arrays. A confirmation set of 60 patients were studied by flow cytometry, qRT-PCR or immunohistochemistry and analyzed according to the extent of the tumor immune infiltrate. Gene expression profiles of freshly isolated TIL were also compared with TIL that had been rested overnight or with CD4+ T cells [non-stimulated (NS) or stimulated (S)] from healthy donor PB treated with tumor supernatant (SN). Comparing gene expression profiles of donor blood derived memory CD4+ T cells [non-stimulated (NS) or stimulated (S)] with and without tumor supernatant (SN) treatment Memory CD4+ T cells isolated from a healthy donor blood (NS or S) were treated (and as control: untreated samples in biological triplicate) with SN obtained from fresh breast tumor homogenates of 4 patients and analyzed on Affymetrix U133 Plus 2.0 arrays
Project description:CD4+ helper T (Th) cells are critical regulators of immune responses but their role in breast cancer is currently unknown. This work aims to characterize Th cells infiltrating invasive primary human breast tumors, analyze the influence by the tumor microenvironment and identify Th cell specific prognostic gene signatures. CD4+ T cells isolated from the tumor (TIL), axillary lymph node (LN) and blood (PB) of 10 patients were analyzed on Affymetrix U133 Plus 2.0 arrays. A confirmation set of 60 patients were studied by flow cytometry, qRT-PCR or immunohistochemistry and analyzed according to the extent of the tumor immune infiltrate. Gene expression profiles of freshly isolated TIL were also compared with TIL that had been rested overnight or with CD4+ T cells [non-stimulated (NS) or stimulated (S)] from healthy donor PB treated with tumor supernatant (SN). Analysis of CD4+ TIL with or without 24h ex-vivo rest, including donor blood memory CD4+ T cells treated in the same conditions as control CD4+ T cells isolated from primary tumors of 2 patients and memory CD4+ T cells from a healthy donor blood were immediately analyzed or incubated for 24h without stimulation before being analyzed on Affymetrix U133 Plus 2.0 arrays
Project description:The growing tumor avoids recognition and destruction by immune system. During continuous stimulation of tumor infiltrating lymphocytes (TILs) by tumor, TILs become functionally exhausted. Thus, they become unable to kill tumor cells and to produce some cytokines, and lose their ability to proliferate. It collectively results in the immune escape of cancer cells. Here, we show that breast cancer cells expressing PD-L1 can accelerate exhaustion of persistently activated human effector CD4+ T cells, manifesting in high PD-1 and PD-L1 expression level on cell surface, decreased glucose metabolism genes, strong downregulation of SWI/SNF chromatin remodeling complex subunits and p21 cell cycle inhibitor upregulation. This results in inhibition of T cell proliferation and reduction of T cells number. The RNAseq analysis on exhausted CD4+ T cells indicated strong overexpression of IDO1 and genes encoding pro-inflammatory cytokines and chemokines. The PD-L1 overexpression was also observed in CD4+ T cells after co-cultivation with other cell line overexpressing PD-L1 that suggested the existence of general mechanism of CD4+ T cell exhaustion induced by cancer cells. The ChIP analysis on PD-L1 promoter region indicated that the strong BRM recruitment in control CD4+ T cells is replaced by BRG1 and EZH2 in CD4+ T cells strongly exhausted by cancer cells. These findings suggest that such epi-drugs as EZH2 inhibitors may be used as immunomodulators in cancer treatment.
Project description:CD4+ helper T (Th) cells are critical regulators of immune responses but their role in breast cancer is currently unknown. This work aims to characterize Th cells infiltrating invasive primary human breast tumors, analyze the influence by the tumor microenvironment and identify Th cell specific prognostic gene signatures. CD4+ T cells isolated from the tumor (TIL), axillary lymph node (LN) and blood (PB) of 10 patients were analyzed on Affymetrix U133 Plus 2.0 arrays. A confirmation set of 60 patients were studied by flow cytometry, qRT-PCR or immunohistochemistry and analyzed according to the extent of the tumor immune infiltrate. Gene expression profiles of freshly isolated TIL were also compared with TIL that had been rested overnight or with CD4+ T cells [non-stimulated (NS) or stimulated (S)] from healthy donor PB treated with tumor supernatant (SN). Analysis of CD4+ TIL with or without 24h ex-vivo rest, including donor blood memory CD4+ T cells treated in the same conditions as control