The mainstay of treatment for metastatic Renal Cell Carcinoma (RCC) is IL-2-based immunotherapy. We recently reported on a Phase II trial of autologous Dendritic Cells (DC) injected intranodally in combination with IL-2 and Interferon-alpha. This trial showed very impressive clinical results. CD4⁺CD25⁺FoxP3⁺ Regulatory T cells (nTreg) are key regulators of self-tolerance and play a pivotal role in the suppression of anti-tumor immune responses. In this study, we analyzed the subpopulation of nTreg in patients enrolled in this vaccine trial.
Samples were collected via leukapheresis at baseline, after the induction cycle and after completion of therapy. Five-color flow cytometry was performed on lymphocytes utilizing a comprehensive panel of cell surface and intracellular T cell markers. Patients were divided into responders (complete n=8) and non-responders (stable and progressive disease, n=10) and compared with healthy donors. Statistical analyses were performed using Student’s t-test and P-values of <0.05 were considered significant.
18 patients were treated in this trial and samples were available for all patients. At baseline, cancer patients had significantly more CD4⁺CD25⁺FoxP3⁺ cells than healthy donors (mean: 2.8% (StD 1.36) vs. 1.3% (StD 0.69) p=0.002). The same was evident for CD4⁺CD25^high (3.0 % (StD 2.2) vs. 1.28 (StD 0.8) p=0.003) and CD4⁺CD25^highFoxP3⁺ (1.96% (StD 1.0) vs. 0.84% (StD 0.56) p=0.003). After the two induction cycles, we observed a significant increase in CD4⁺CD25⁺FoxP3⁺ Treg cells (pre:2.8% (StD 1.36) post: 7.8% (StD 4.7) p=0.003), an increase in CD3⁺CD4⁺CD25^high (pre: 3.0 (StD 2.2) post: 6.3% (StD 4.4) p=<0.001) and an increase in CD3⁺CD4⁺CD25^high FoxP3⁺ (pre: 1.96% (StD 1.0) post: 4.2% (StD 1.1) p=0.012). The numbers of Treg did not show any difference in responders vs. non-responders at baseline. However, after induction, significantly higher frequencies of Treg were detected in non-responders: CD4⁺CD25⁺FoxP3⁺ : 8.6% (StD 5.4) vs. 4.6% (StD 1.7) p=0.026. CD4⁺CD25⁺FoxP3⁺ cells were increased in the total lymphocyte population: 6.1% (StD 2.7) vs. 2.7% (StD 1.0) p=0.011.
This study represents the most extensive phenotypic analysis of Treg subpopulations to date in cancer patients. Significant differences between healthy donors and RCC patients were demonstrated, as were differences between responding and non-responding patients. This data will have significant impact on future immunotherapeutic trials.