CMLVAX100 Peptide Vaccinations induce Peptide Specific “cytotoxic” CD4+ T Cells (CD4+CTLs) in Chronic Myeloid Leukemia Patients: New Insights about Peptide Vaccine-Mediated Antitumor Response

We have shown that vaccinations with a p210-derived peptide vaccine (CMLVAX100) induced a strong peptide-specific CD4+T cell proliferation in the majority of vaccinated chronic myeloid leukemia (CML) patients. CD4+ T cell response was strictly mediated by the longest peptide included in the vaccine (b3a2-25) and correlated with disease response as about 65% of vaccinated patients showed reduction of minimal residual disease still persisting during imatinib treatment. The role of b3a2-25-peptide specific CD4+ T cells as potential mediator of antitumor response has not yet been fully elucidated. The majority of vaccine-induced T cells resulted to be CD4+/CD25+/Foxp3+, but despite this phenotype they showed no regulatory/inhibitory activity on naïve T cells. A smaller proportion resulted instead to be perforin+, thus potentially cytotoxic. To evaluate if b3a2-25-peptide specific CD4+T cells could exert a direct cytotoxic effect, we used CML-derived JURL-MK2 cell line expressing b3a2-p210 as target. Effector cells were b3a2-25-specific CD4+ T cells freshly isolated from 3 vaccinated patients and further in vitro expanded in the presence of IL-2 and b3a2-25 peptide. After in vitro expansion, peptide-specific CD4+/CD25+/Foxp3+ population represented about 60% of total CD4+ isolated cells, while peptide-specific CD4+/perforin+ cells counted for about 9%. Cellular cytotoxicity measurement was then carried out by flow cytometry using a fluorescent dye to label target cells and a fluorescent-DNA dye to determine the dead cells. Briefly, 1×10⁴ JURL target cells labelled with DIOC18 green fluorescent dye were mixed with b3a2-25-specific CD4+ T (effector cells) at various E:T ratio (5:1, 10:1, 20:1, 35:1) and incubated 4 hours at 37°C. After co-incubation, Propidium Iodide (PI) red fluorescent was applied and the samples were run on flow cytometer for the determination of DIOC+/PI+ dead cells. Spontaneous cell death was determined by incubation at 37°C of target cells alone and maximum cell death was determined by incubation of target cells with 2% paraphormaldeide. Control experiments included different effector cells:

1. freshly isolated CD4+ T cells from the same 3 vaccinated patients not further in vitro expanded (in this experimental condition the percentage of b3a2-25-specific CD4+/CD25+/Foxp3+ is about 10% of total CD4+ cells while b3a2-specific CD4+/perforin+ is about 2%);

2. CD4+ T cells in vitro stimulated with IL-2 (without b3a2-25 peptide) from healthy subjects or from not previously vaccinated CML patients.

Our results showed a specific killing of JURL-MK2 cells only in the presence of expanded peptide-specific CD4+ T cells from all 3 vaccinated patients with a linear increase of DIOC+/PI+ target cells from 5.3% (E:T 5:1) to 33% (E:T 35:1). No cytotoxicity was observed when CD4+ cells were expanded from healthy donors of from not vaccinated patients ruling out the possibility of killing mediated by a “non specific” activation of CD4+ cells due to IL-2 exposure. On the contrary, specific cytotoxicity appeared to correlate to the increased percentage of peptidespecific CD4+ cells obtained after in vitro re-stimulation with b3a2-25 peptide, as only background killing was observed when freshly isolated CD4+ T cells from all 3 vaccinated patients were cultured with JURL-MK2 cells. In conclusion, CMLVAX100 induced b3a2- 25-peptide specific CD4+ T cells appear to exert direct cytotoxity toward b3a2-CML JURL-MK2 cells. To our knowledge this is the first time that CML-peptide specific CD4+/cytotoxic T cells are induced in vivo and they could mediate the minimal disease reduction observed after CMLVAX100 vaccinations in CML patients. Experiments focused on determining which subtype, CD4+/CD25/Foxp3 or CD4+/perforin+, is the main effector of cytotoxicity as well as experiments clarifying if CD4+ cytotoxicity is mediated by HLA-DR molecules presentation of b3a2-p210 derived peptides are ongoing.