Carole Equeter

CD4+ follicular helper T cell infiltration predicts breast cancer survival

CD4⁺ T cells are critical regulators of immune responses, but their functional role in human breast cancer is relatively unknown. The goal of this study was to produce an image of CD4⁺ T cells infiltrating breast tumors using limited ex vivo manipulation to better understand the in vivo differences associated with patient prognosis. We performed comprehensive molecular profiling of infiltrating CD4⁺ T cells isolated from untreated invasive primary tumors and found that the infiltrating T cell subpopulations included follicular helper T (Tfh) cells, which have not previously been found in solid tumors, as well as Th1, Th2, and Th17 effector memory cells and Tregs. T cell signaling pathway alterations included a mixture of activation and suppression characterized by restricted cytokine/chemokine production, which inversely paralleled lymphoid infiltration levels and could be reproduced in activated donor CD4⁺ T cells treated with primary tumor supernatant. A comparison of extensively versus minimally infiltrated tumors showed that CXCL13-producing CD4⁺ Tfh cells distinguish extensive immune infiltrates, principally located in tertiary lymphoid structure germinal centers. An 8-gene Tfh signature, signifying organized antitumor immunity, robustly predicted survival or preoperative response to chemotherapy. Our identification of CD4⁺ Tfh cells in breast cancer suggests that they are an important immune element whose presence in the tumor is a prognostic factor.

Gene expression profiling reveals differences in microenvironment interaction between patients with chronic lymphocytic leukemia expressing high versus low ZAP70 mRNA

ZAP70 is a strong indicator of poor prognosis in chronic lymphocytic leukemia. In this study, using gene expression profiles associated with ZAP70 expression, the importance of microenvironmental interaction for tumor behavior is revealed. See related perspective article on page 752. Background Zeta-associated protein 70 (ZAP70) is a widely recognized prognostic factor in chronic lymphocytic leukemia, but mechanisms by which its higher expression leads to a poor outcome must still be fully explained. Design and Methods In an attempt to unveil unfavorable cellular properties linked to high ZAP70 expression, we used gene expression profiling to identify genes associated with disparities in B cells from chronic lymphocytic leukemia patients expressing high versus low ZAP70 mRNA, measured by quantitative real-time PCR. Two groups of 7 patients were compared, selected on the basis of either high or low ZAP70 mRNA expression. Results Twenty-seven genes were differentially expressed with an FDR<10%, and several genes were significant predictors of treatment-free survival (TFS) and/or overall survival; PDE8A and FCRL family genes (down-regulated in ZAP70+ patients) could predict TFS and overall survival; ITGA4 mRNA (up-regulated in ZAP70+ patients) could significantly predict overall survival. Importantly, gene set enrichment analysis revealed overrepresentation of adhesion/migration genes. We therefore investigated in vitro adhesion/migration capacity of chronic lymphocytic leukemia cells into a stromal microenvironment or in response to conditioned medium. We showed that ZAP70+ cells had better adhesion/migration capacities and only ZAP70+ patient cells responded to microenvironment contact by CXCR4 downregulation. Conclusions We concluded that several prognostic factors are the reflection of microenvironment interactions and that the increased adhesion/migratory capacity of ZAP70+ cells in their microenvironment can explain their better survival and thus the aggressiveness of the disease.

Abstract 3541: CD4+ T cells infiltrating human breast cancer are critical for an effective anti-tumor immune response

CD4+ helper T (Th) cells are critical regulators of immune responses but their role in breast cancer is currently unknown. The goal of this project was to characterize CD4+ T lymphocytes infiltrating primary human breast tumors (TIL), analyze T cell signaling pathways influenced by the tumor microenvironment, correlate differential gene expression with the presence of a specific CD4+ T cell subset(s) and identify a profile predictive of positive patient outcome. Affymetrix U133 Plus 2.0 arrays were used to analyze CD4+ T cells freshly isolated from the primary tumor, axillary lymph node (LN) and blood (PB) of patients with ER+ and ER- invasive breast carcinoma in the discovery set (10 patients). Microarrays, flow cytometry and/or qRT-PCR were used to extend and confirm the data in CD4+ TIL isolated from >60 primary breast tumors as well as purified CD4+ T cells from healthy donors PB treated with supernatant (SN) derived from fresh breast tumor tissue. Assessment of the relative distribution of infiltrating mononuclear cells revealed that the CD4+ T cell subset was consistently the principal component. We detected gene expression changes in the CD4+ TIL compared to patient9s LN and PB counterparts that reflected altered cellular signaling pathways, including significant suppression of the TCR/CD3 complex and downstream signaling molecules along with a highly restricted pattern of Th cytokine and chemokine expression. A comparison with publically available Th profiling microarray data revealed that the TIL have characteristics of an activated memory effector CD4+ T cell phenotype, with all the major subsets (Th1, Th2, Th17, Treg, Tfh) represented. Although very few differences were detected in a direct comparison between CD4+ TIL from ER+ and ER- tumors, important changes in TIL (including a Th subset skew and differential cytokine expression) from extensive versus minimally infiltrated tumors were observed, with those containing an extensive CD4+ T cell infiltrate associated with a better clinical outcome. Gene expression profiles of freshly isolated TIL were also compared with TIL that had been rested overnight. These experiments revealed that the rested TIL reverse expression in a number of critical cytokine and signaling genes. PB CD4+ T cells from healthy donors treated with primary tumor SN also displayed significant changes in gene expression, gaining >75% similarity to freshly isolated CD4+ TIL. These experiments further found that activation of CD4+ T cells in conjunction with tumor SN treatment rapidly suppressed stimulation-induced changes in gene expression. Together our data suggest that although activated CD4+ T cells infiltrating breast tumors are quickly suppressed, tumors that recruit higher levels of CD4+ T cells, even in the face of rapid immunosuppression, produce a better memory T cell pool that is ultimately capable of maintaining effective anti-tumor immunity in patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3541. doi:1538-7445.AM2012-3541

The human CD3 g gene promoter is controlled by an RNA element that binds P-TEFb and is negatively regulated by HIV-1 Tat

Our studies show that HIV-1 infection provokes a progressive defect in surface TCR/CD3 receptors due to the specific and escalating loss of CD3 g mRNA. The human CD3 g gene is transcribed from a weak, lymphoid-specific promoter with significant transcription initiation site heterogeneity in normal T cells. However, early after HIV-1 infection CD3 g transcripts preferentially initiate at the +1 and +13 with further focusing over time as +1 transcripts are lost first. Mutant and deletion analysis delimited a 43 bp sequence from the +1 as critical for positive gene expression. DNA probes covering this sequence do not bind nuclear proteins. RNA probes from the +1 or +13 specifically bind the cellular proteins Cyclin T1 and cdk9 (P-TEFb) as well as HIV-1 Tat. The +1 to +43 sequence, defined as the CD3 g RCE (RNA control element), forms a secondary structure containing a uridine bulge, a side loop and a double apical loop with sequence similarity to HIV-1 TAR. Five GGCU repeats present from +18 to +37 form a similar double apical loop structure for +13 transcripts that lack the bulge and side loop. Deletion or mutation in the CD3 g RCE dramatically affects function with a 4 nt apical loop mutation abrogating both promoter activity and nuclear protein binding. Co-transfection of Tat with CD3 g promoter constructs suppresses promoter activity. In infected cells, knocking down tat gene expression restores surface TCR/CD3 whereas treatment with Tat protein accelerates TCR/CD3 downmodulation. Thus, P-TEFb binding to the CD3 g RCE in the presence of Tat is associated with negative transcriptional regulation.