Introduction; Abundant FOXP3+CD4+ regulatory T cell (Tregs) infiltration into tumors is significantly associated with poor clinical outcomes in various types of cancer. However, the role of Tregs is controversial in colorectal cancers (CRCs), in which FOXP3+ T-cell infiltration indicated better prognosis in some studies. We previously reported that FOXP3+ T cells are composed of three functionally distinct subpopulations (Miyara et al, Immunity 2009): CD45RA+FOXP3lo+ naïve Tregs, CD45RA-FOXP3hi+ effector Tregs, and CD45RA-FOXP3lo+ non-Tregs. By analyzing the Treg subpopulation in tumor infiltrating lymphocytes (TILs) of CRCs, we aimed to precisely assess the impact of Treg infiltration on CRC prognosis.
Procedures; TILs from 35 CRCs were analyzed by flow cytometry. CRCs were thus divided into two groups: Type A (low % of non-Tregs in CD4+ T cells) and Type B (high % of non-Tregs in CD4+ T cells) CRCs. mRNA expression of tumor tissues was analyzed with microarray analysis and quantitative polymerase chain reaction (PCR) analysis to assess the difference between Type A and Type B tumors. In-vitro naïve T cell culture was performed to confirm the effect of observed cytokines to induce FOXP3+ T cells. Shorty naïve T cells were sorted by flow cytometry and cultured with anti-CD3/CD28 TCR stimulation with or without the observed cytokines. Based on observed differences in gene expression, the impact of FOXP3 mRNA levels on survival was evaluated using mRNA from another set of 109 CRCs. Finally, to clarify the association of bacteria existence with the difference of the CRC type, bacteria infiltration was assessed by fluorescence in situ hybridization (FISH) analysis with Formalin-Fixed Paraffin-Embedded (FFPE) tumor tissues.
Results; More than half of 35 CRCs were classified into Type B CRCs. Inflammatory and immune response related genes, especially IL12A, TGFB1 and TNF mRNA expression levels were significantly upregulated in Type B relative to Type A CRCs. Of these cytokines, TGF-β effectively induced FOXP3hi+ T cells from naïve T cells in vitro and the combination of IL12 and TGF-β mostly induced FOXP3lo+ T cells. Tumor tissue mRNA gene expression showed that Type A CRCs were largely IL12Alo and TGFB1lo, whereas Type B CRCs were either IL12Ahi or TGFB1hi, or both. With this cytokine-dependent classification of CRC tissues, we evaluated the impact of FOXP3+ T cells on prognosis with another cohort of 109 CRC patients. High FOXP3 mRNA expression was associated with poor prognosis in Type A where FOXP3+ T cells were mostly Tregs. In contrast, high FOXP3 mRNA expression was not associated with prognosis in Type B as these tumors contained both non-Tregs and effecter Tregs. As regard to bacterial existence, intestinal bacteria deeply invading tumor tissues were found at a significant frequency by the FISH method in Type B CRC tissues relative to Type A CRC tissues.
Conclusion; We hypothesize that functionally distinct subpopulations of tumor-infiltrating FOXP3+ T cells oppositely contribute to the prognosis of CRCs. Non-Treg infiltration is caused by inflammatory response of CRCs possibly induced by bacteria infiltration. Our results also indicate that Treg-cell depletion may augment anti-tumor immunity and provide clinical benefits in Type A CRCs and also other cancers with predominant FOXP3hi Treg-cell infiltration.
Citation Format: Takuro Saito, Hiroyoshi Nishikawa, Hisashi Wada, Masaki Mori, Yuichiro Doki, Shimon Sakaguchi. FOXP3+CD4+ T-cell subpopulations distinctly control the prognosis of colorectal cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B174.
- ©2016 American Association for Cancer Research.