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: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: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). 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
Project description:Breast cancer, categorised into hormone receptor-positive (HR+), HER2-positive (HER2+), and triple-negative (TNBC) subtypes, exhibits varied outcomes based on the number of tumour-infiltrating lymphocytes (TILs). Increased TIL levels are associated with better outcomes in HER2+ and TNBC, while HR+ individuals with high TIL levels show shorter survival but greater neoadjuvant chemotherapy response. To explore the divergent roles of TIL levels across various subtypes and their effect on immune cell composition, we employed single-cell RNA sequencing on 31 patients with breast cancer. HR+ breast cancer with high TIL levels (TIL-high) revealed increased SPP1+ macrophages, increased SPP1 expression in other monocytes/macrophages (mono/macro) subgroups, and enriched pathways associated with extracellular matrix (ECM) remodelling in mono/macro. Moreover, cell–cell interaction analyses revealed enhanced SPP1, MIF, and FN1 signalling in the interaction between SPP1+ macrophages and T-cells in TIL-high HR+ breast cancer. Spatial transcriptomics data highlighted the close proximity of SPP1+ macrophages, CD8+ T-cells, and CD4+ T-cells in TIL-high HR+ breast cancer. Our findings unveil the novel influence of SPP1+ macrophages on T-cells in TIL-high HR+ breast cancer, potentially explaining the poor prognosis and offering insights for targeted interventions.
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
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
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
Project description:Background: Adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TIL) is effective in treating PD-1 refractory melanoma, but requires adequate ex vivo expansion of TIL. Methods: CD4+ and CD8+ TIL from metastatic melanoma patients treated with TIL ACT were analyzed by RNA-seq (n=12) and ChIP-seq of acetylated histone 3 (n=19). Patients were grouped into “TIL high” and “TIL low” based on division at the median number of TIL infused. The number of TIL infused and CD4+ TIL frequency were correlated with overall survival (OS). Results: The number of TIL infused correlated with longer OS (R2=0.57, p=0.00076), and the percent of CD4+ infused was negatively correlated with the total number of TIL infused (R2=0.64, p=0.00047). RNA-seq analysis of CD4+ TIL showed increases in Th2/Th17/Treg transcripts and pathways in the TIL low group. ChIP-seq analysis of CD8+ TIL showed decreased acetylation in the TIL low group in genes upregulated during CD8+ activation. Conclusion: The numbers of TIL infused were associated with increased overall survival, while RNA-seq suggested that polarized CD4+ cells in the transferred TIL were associated with decreased overall expansion. These data suggest that improper CD4+ TIL polarization may reduce expansion and treatment efficacy.