Characterization of CD4⁺ T Cell Response Induced by p210-Derived Peptide Vaccinations in CML Patients.

We have previously shown that vaccination of chronic myelogenous leukaemia (CML) patients with CMLVAX100 (a mixture of 5 p210-b3a2 breakpoint derived peptides) plus GM-CSF was able to induce an evident and durable peptide-specific CD4⁺ T cell response in the majority of patients. Peptide-specific CD4⁺ T cell proliferation was measured by standard [3H] thymidine incorporation assay and this response was mainly mediated by a 25 mer long breakpoint peptide (b3a2-25) included in the vaccine. In this pilot clinical trial we showed that about 60% of 28 CML patients vaccinated while on imatinib, showed a reduction of their long lasting molecular residual disease after immunization (first 6 vaccinations) and about 25% of them achieved in addition a complete molecular response. To investigate the contribute of this peptide specific CD4⁺ T cell response in antitumor activity, we further characterize peptide-induced proliferating CD4⁺ cells. Briefly, in 10 CMLVAX100 vaccinated patients in which a strong b3a2-25 specific CD4⁺ proliferation was previously observed, we freshly isolated peripheral blood CD4⁺ T cells and we cultured them 4 days only in the presence or not of b3a2-25 vaccine peptide or control PR-25 peptide. Afterwards, each culture condition was analyzed both for the co-expression of CD25, perforin and FOXP3 molecules and for standard [3H] thymidine incorporation assay. As expected all patients showed a strong b3a2-25 specific CD4⁺ proliferation (mean stimulation index 43 -range 19–81-). When flow cytometric analysis was performed, we observed a consistent increase of two main CD4⁺ T cells subsets only in the b3a2-25 stimulated CD4⁺ T cells, not in “no peptide” or “control peptide” stimulated CD4⁺ T cells.

1. a “small-size” CD4⁺/perforin⁺ T cell population (raising from a median of 1.06% (range 0.21–5.2) to 2.32% (range 0.49–6.2) with very likely cytotoxic activity

2. a “large-size” CD4⁺/CD25⁺/FOXP3⁺ T cell population (raising from 0.29% (range 0.03–0.66) to 5.65% (range 0.71–9.5) with potential regulatory function.

Since the large size of these cells and their high proliferative activity would argue against a regulatory features and considering that the immunophenotypic profile of Tregs in human is not yet fully defined we performed a functional test to measure a potential regulatory activity of these vaccine-induced T cell population. Thus, we evaluated the ability of b3a2-25 specific CD4⁺ T cells to inhibit the growth of CFSE labelled normal subjects naïve CD4⁺ T cells stimulated with autologous CD3-depleted APCs and antiCD3 MoAb. In our experimental conditions, naïve CFSE CD4⁺ T cells equally proliferated in the presence of b3a2-25 specific CD4⁺ cells, “no peptide” CD4⁺ cells or PR-25 control peptide CD4⁺ cells from vaccinated patients and the rate of proliferation was similar to the one observed in co-culture. experiments with allogeneic normal CD4⁺ cells. Our data suggest that the immune response induced in CML patients by CMLVAX100 vaccinations, consists of an increase of peptide-specific cytotoxic CD4⁺ T cells and a much more evident augment of CD4⁺/CD25⁺/FOXP3⁺ T cells that despite resembling a Treg phenotype display apparently no suppressive activity. The exact role of this peptide-specific CD4⁺ T cells subset in the context of CMLVAX100 mediated immune response needs to be further elucidated.