CD8+/PBN cross-talk is contact dependent. (A) Cumulative flow-cytometry histograms documenting CD69 expression in CD8+ T cells activated by using different concentrations of surface bound anti-CD3 (TR66, 0.5 and 2μg/ml) and soluble anti CD28 (1μg/ml) upon overnight co-culture in the presence or absence of neutrophils. (B) Representative dot plots showing CD69 expression in peripheral blood CD8+ cells stimulated by suboptimal concentrations of anti-CD3/ anti-CD28 in presence or absence of autologous PBN and in control and transwell conditions, preventing cell-cell contact. (C) Representative plots documenting the effects of anti CD11a mAb on CD69 expression in CD8+ T cells activated by a suboptimal concentration of anti-CD3/anti-CD28 in the presence of autologous PBNs. (D) CFSE dilution was used to assess CD8+ T cell proliferation in cultured stimulated with suboptimal concentrations of anti CD3 mAbs and anti CD28 in the presence or absence of PBN.
ARTICLE ABSTRACT
Purpose: Tumor infiltration by different T lymphocyte subsets is known to be associated with favorable prognosis in colorectal cancer. Still debated is the role of innate immune system. We investigated clinical relevance, phenotypes, and functional features of colorectal cancer–infiltrating CD66b+ neutrophils and their crosstalk with CD8+ T cells.Experimental Design: CD66b+ and CD8+ cell infiltration was analyzed by IHC on a tissue microarray including >650 evaluable colorectal cancer samples. Phenotypic profiles of tissue-infiltrating and peripheral blood CD66b+ cells were evaluated by flow cytometry. CD66b+/CD8+ cells crosstalk was investigated by in vitro experiments.Results: CD66b+ cell infiltration in colorectal cancer is significantly associated with increased survival. Interestingly, neutrophils frequently colocalize with CD8+ T cells in colorectal cancer. Functional studies indicate that although neutrophils are devoid of direct antitumor potential, coculture with peripheral blood or tumor-associated neutrophils (TAN) enhances CD8+ T-cell activation, proliferation, and cytokine release induced by suboptimal concentrations of anti-CD3 mAb. Moreover, under optimal activation conditions, CD8+ cell stimulation in the presence of CD66b+ cells results in increasing numbers of cells expressing CD45RO/CD62L “central memory” phenotype. Importantly, combined tumor infiltration by CD66b+ and CD8+ T lymphocytes is associated with significantly better prognosis, as compared with CD8+ T-cell infiltration alone.Conclusions: Neutrophils enhance the responsiveness of CD8+ T cells to T-cell receptor triggering. Accordingly, infiltration by neutrophils enhances the prognostic significance of colorectal cancer infiltration by CD8+ T cells, suggesting that they might effectively promote antitumor immunity. Clin Cancer Res; 23(14); 3847–58. ©2017 AACR.
