Purification of Allogeneic Idiotype-Specific T Lymphocytes According to IFN-γ Production for Adoptive Immunotherapy in Multiple Myeloma Patients.

In multiple myeloma (MM) adoptive immunotherapy through allogeneic donor lymphocyte infusions results in a well-documented graft versus myeloma effect, but it is associated with high incidence of graft versus host disease. The idiotype (Id) expressed by MM cells can be regarded as a tumor-specific antigen and it has been used for immunotherapy. To enhance antitumor immunity and reduce alloreactivity, allogeneic T cells were activated against tumor-derived Id and then purified according to IFN-γ production. Total peripheral blood mononuclear cells from healthy donors were incubated with autologous monocyte-derived dendritic cells and pulsed with patient-derived Id protein. Cells were maintained in serum-free medium and supplemented during the priming phase with IL-7 and IL-12. Subsequently, the T-cell culture was restimulated every 7 days with pulsed DCs in the presence of low doses of IL-2. After 2 or 3 stimulations, the percentage of IFN-γ-producing T cells was as high as 5–10%, whereas that observed in presence of not stimulated T-cells was undetectable. Based on their IFN-γ production, T cells were isolated by using a commercial immunomagnetic IFN-γ capture assay. The purity of enriched IFN-γ-producing T cells ranged between 60 and 90% as evaluated by flow cytometry. The yield was 60% of pre-selection IFN-γ positive T cells and cell viability after selection was 80%. Functionally, IFN-γ purified T cells showed better Id-specific proliferative response when compared both to not-stimulated T cells and to stimulated but not purified T cells. IFN-γ purified T cells were shown to be memory CD45RO⁺ T cells expressing HLA-DR, CD25, CD95 at high level, whereas the negative fraction was mainly composed of naive, poorly activated T cells. T-cell receptor spectratyping analysis demonstrated that IFN-γ positive T cells were represented by a pauciclonal cell population as compared to policlonal T cells obtained before cell culture. Moreover, addition of not-stimulated T cells resulted in a significant reduction of allogeneic CD34⁺-derived colony-forming capacity. Conversely, the absolute number of allogeneic total colony-forming units-cells was not affected by IFN-γ purified T cells.These data demonstrate that Id-specific T cells may be generated from healthy donors and significantly enriched on the basis of their IFN-γ production. Id-specific IFN-γ - purified T cells have better anti-Id response and reduced alloreactivity than unselected T cells.