Abstract LB-329: Defining rgulatory T-cells as CD4+CD25hiCD127- or as CD4+CD25hiFoxP3+ in immune monitoring of cancer vaccine clinical trials

Abstract

Abstract Background: Regulatory T cells (Treg) are widely accepted to play a key role in the immune tolerance and escape of cancer cells. Originally Tregs were identified as being CD4+ T cells expressing high levels of CD25 (IL2 receptor). The identification of the transcription factor FoxP3 resulted in defining Tregs as CD4CD25hiFoxP3+ T cells by staining permeablized cells. Recently Tregs have been identified as CD4CD25hi T cells with low levels of CD127 (IL7 receptor) expressing a CD4+CD25hiCD127- cell surface phenotype. In an ongoing clinical trial with a MHC class II HER2/neu pep tide vaccine we have evaluated these various definitions of Treg phenotypes for immunological monitoring. Methods: Tregs were quantified in disease-free, high risk BCa patients enrolled in adjuvant peptide-based breast cancer vaccine trials. Peripheral blood mononuclear cells (PBMCs) were both permeabilized and stained with CD4, CD25, and FoxP3 monoclonal antibodies and surface stained with CD4, CD25, and CD127. Samples were then analyzed by flow cytometry. The means are compared with t-test and correlation is determined with Pearson's correlation coefficient. Results: 196 PBMC samples from 72 patients were available for Treg quantification. There was a greater mean percentage of cells identified as Treg in permeabilized than nonpermeabilized cells by staining with CD4+CD25hi (5.9±0.1% vs 4.8 ±0.1%, p<0.01) and excellent correlation between the tests (r=0.78, p<0.01). There was a greater mean percentage of cells identified as Tregs by CD4+CD25hi than CD4+CD25hiFoxP3+ (5.9±0.1% vs 3.6±0.1%, p<0.01) with good correlation (r=0.68, p<0.01). There was a greater mean percentage of Tregs by CD4+CD25+ than CD4+CD25+CD127- (4.8±0.1 vs 4.0±0.1, p<0.01) with excellent correlation (r=0.89, p<0.01). There was a greater mean percentage of Tregs by CD4+CD25hiCD127- than Tregs by CD4+ CD25hiFoxP3+ although levels were similar (4.0±0.1 vs 3.6±0.1, p<0.01) and the two tests had good correlation (r=0.58, p<0.01). Conclusion: There was good correlation between various methods of defining Tregs with and without cell membrane permeabilization. Defining Treg cells as either CD4+CD25hiFoxP3+ or CD4+CD25+CD127- appear to be more specific than defining Tregs as CD4+CD25hi alone. Furthermore, defining Treg populations by surface staining as CD4+CD25hiCD127- yields similar levels with good correlation as CD4+CD25hiFoxP3+ and may be the preferred method for immune monitoring. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-329.