Objective Analysis of tumor-infiltrating lymphocytes (TILs) is one of the cornerstones for the understanding of immune responses prevailing in the tumor microenvironment. We studied TILs from squamous cell carcinoma of the cervix ex vivo without proliferating them in vitro before analysis.

Methods Whereas TILs were magnetic activated cell separation enriched and flow sorted into CD4⁺ CD25^hi (regulatory T cells [Tregs]), CD4⁺ CD25^int (effector T cells [Teffs]) were directly purified by flow cytometry, and both these subsets were characterized phenotypically and functionally. Tissue sections were probed for interleukin 4 (IL-4) and interferon γ.

Results Effector T cells constitutively expressed both interferon γ and IL-4 prototypical cytokines of T_H1 and T_H2, respectively, and were able to proliferate and secrete higher quantities of both cytokines in response to anti-CD3/anti-CD28 and autologous tumor lysates. Only 53% of cervical cancer Tregs were FOXP3⁺, elaborated transforming growth factor β1, and IL-10 and were able to inhibit both T helper subsets.

Conclusions Intratumoral Teffs represented functionally active subsets of both T_H1 and T_H2 that were not anergic but were suppressed by multiple Treg subsets, which comprised FOXP3 + Tregs and Tregs secreting transforming growth factor β1 and IL-10. These results imply that the microenvironment of cervical carcinomas harbored both T_H1 and T_H2 subsets of CD4⁺ Teffs that were functionally active but were perhaps unable to perform because of the overpowering effect of Tregs.