gp 100209 – 2 M Peptide Immunization of Human Lymphocyte Antigen-A 2 Stage I-III Melanoma Patients Induces Significant Increase in Antigen-Specific Effector and Long-Term Memory CD 8 T Cells

Thirty-five HLA-A2 patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100209–2M, emulsified in Montanide adjuvant. Direct ex vivo gp100209–2M tetramer analysis of preand postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer CD8 T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05–8.9%). Ex vivo IFNcytokine flow cytometry analysis of postvaccine PBMCs after brief gp100209–2M in vitro activation showed that for all of the patients studied tetramer CD8 T cells produced IFN; however, some patients had significant numbers of tetramer IFNCD8 T cells suggesting functional anergy. Additionally, 8 day gp100209–2M in vitro stimulation (IVS) of preand postvaccine PBMCs resulted in significant expansion of tetramer CD8 T cells from postvaccine cells for 34 patients, and these IVS tetramer CD8 T cells were functionally responsive by IFNcytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8 T cells demonstrated the proliferation of functionally anergic gp100209–2Mtetramer CD8 T cells in several patients and also indicated interpatient variability of gp100209–2M stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer CD8 T cells (78.1 4.2%) had either an “effector” (CD45 RA /CCR7 ) or an “effector-memory” phenotype (CD45RA /CCR7 ). Notably, analysis of PBMCs collected 12–24 months after vaccine therapy demonstrated the durable presence of gp100209–2M-specific memory CD8 T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8 T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.