Comparing CD4⁺CD25⁺FoxP3⁺ regulatory T cell levels with ex vivo immune assays and clinical delayed type hypersensitivity responses in a novel HLA Class II HER2/neu peptide vaccine clinical trial in breast cancer patients Free

1861

Introduction: CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) have been implicated in the suppression of immune responses against various tumors. To monitor the potential induction of Tregs in breast cancer (BrCa) patients receiving a novel HLA Class II HER2/neu peptide (AE37) vaccine in a clinical trial, we have analyzed peripheral blood lymphocytes (PBL) from vaccinated patients for the presence of Tregs and correlated our findings with ex vivo immune assays and clinical delayed type hypersensitivity (DTH) responses to the vaccine.

Methods: Six BrCa patients have completed 6 monthly injections of the AE37 vaccine in a dose escalation safety study. PBL obtained pre- and post-vaccination were stained with anti-CD4, CD25 and FoxP3 antibodies (PCH101 and 236A/E7) and analyzed by flow cytometry. Cells were also cultured ex vivo with AE37 peptide to measure proliferation (3H-thymidine-cpm) and cytokine secretion (IFN-gamma ELISPOT). Clinical DTH responses to the AE37 peptide were monitored.

Results: Tregs were reduced in all 6 patients post-vaccination with pre- and post-vaccination levels of 1.68-2.93% (pre-) vs. 0.50-1.57% (post-) with an average significant decrease of 2.24±0.55% (pre-) vs. 1.0±0.46% (post-), P=0.002. Clinical DTH responses were increased in all patients post-vaccination. An inverse relationship was demonstrated between the level of post-vaccination Tregs and post-vaccination DTH response to AE37. The largest DTH responses (106 and 113 mm) were seen in the two patients with the lowest levels of Tregs (0.52 and 0.50%) while all other patients had smaller DTH responses (20-71 mm) with higher levels of Tregs (0.80 to 1.57%) including the smallest DTH response of 20 mm being observed in the patient with the highest level of Tregs (1.57%). The decrease in levels of Tregs also correlated with increased AE37 vaccine-specific proliferative responses in all 6 patients, 0-6645cpm (pre-) vs. 1231-19078cpm (post-) with an average significant increase of 3169±2500cpm (pre-) vs. 6047±8453cpm (post-), P=0.01. Increased IFN-gamma secreting cells was seen in 4/6 patient samples analyzed.

Conclusion: Based on our analysis, the novel AE37 HER2/neu peptide vaccine does not result in increased levels of Tregs in patients who have completed the vaccination schedule. Furthermore, the reduced levels of Tregs in vaccinated patients could be associated with more robust responses in ex vivo immune assays and in vivo clinical DTH reactions suggesting that the AE37 vaccine may be clinically useful.