Analysis of Antigen-Specific T-Cell Responses and Regulatory T Cells Following Sibling Donor Immunization with Patient-Derived Idiotype Vaccine Prior to Non-Myeloablative Allogeneic Stem Cell Transplantation for Multiple Myeloma.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative treatment for multiple myeloma (MM) due to its graft-versus-myeloma effect. The use of reduced-intensity conditioning regimens with lower transplant related mortality has extended applicability of allo-HCT but is associated with a high risk of relapse. Active immunization of donors with a tumor-specific antigen prior to stem cell harvest and recipients following transplantation may enhance graft-versus myeloma effect without increasing the risk of graft-versus-host-disease (GVHD). Here, we evaluated the magnitude and quality of the vaccine-induced T-cell immunity in the donors and its transfer to recipients.

Patient-specific idiotype (Id) protein was isolated from the plasma of the MM patient, conjugated to keyhole limpet hemocyanin (Id-KLH) and administered with GM-CSF as an adjuvant. Donors received 3 vaccine doses subcutaneously at 10, 8, and 4 weeks before the stem cell harvest. MM pts received 3 booster subcutaneous injections at 3, 4, and 6 months after allo-HCT following reduced-intensity conditioning regimen (Fludarabine/Cyclophosphamide) and wash out of GVHD prophylaxis. Th1 (IL-2, IFN-γ, TNF-β, and GM-CSF) and Th2 (IL-4, IL-5, IL-10, and IL-13) cellular immune responses against Id and the carrier molecule KLH were determined by multiplex cytokine assay on a Luminex platform. Th1 responses (IL-2, IFN-γ, TNF-β) were further quantified and characterized at the single cell level by intracellular cytokine assay (ICCA) in CD4⁺ T cells from unfractionated cryopreserved pre and postvaccine PBMC. Multicolor intracellular flow cytometry analysis was also used to determine the frequency of circulating regulatory T cells (Tregs), identified as CD4⁺CD25⁺CD127^lowFOXP3⁺, at various time points in both donors and recipients.

Ten MM pts (median age: 54 years; IgG = 8, IgA = 2) and their respective donors were enrolled in this study. All 10 donors completed vaccinations without significant toxicity. Following vaccination, KLH-specific Th1/Th2 responses were observed in all 10 donors by multiplex cytokine assay and confirmed by intracellular cytokine assay (TNFβ and IL-2). Id-specific Th1 and Th2 responses were noted in 5 and 6 donors, respectively. All 10 MM pts underwent reduced intensity allo-HCT and 9 of these completed their post-transplant vaccinations. KLH-specific Th1 responses were detected in 6/7 evaluable recipients prior to post-transplant immunization and in 7/7 following booster immunizations by ICCA. Phenotypic characterization revealed that the transferred donor-derived KLH-specific CD4⁺TNF-a producing T cells mainly consisted of intermediate or late effectors (CD62L^-CD27⁺ or CD62L^-CD27^-). Id-specific Th1 and Th2 responses were measured in 5 and 3 pts, respectively, prior to post-transplant booster injections. Two pts had Id-specific Th1 responses (IFNγ or TNFβ) detectable by ICCA after in vitro expansion from post-transplant immunizations PBMC with a frequency of 2.1% and 2.5% respectively. Since the use of GM-CSF has been associated with increased Tregs in mouse models, we determined the effects of Id-KLH+GM-CSF vaccination on the number of Tregs in the peripheral blood. The percentage of circulating Tregs did not change significantly following vaccination in the donors. In contrast, we observed a progressive increase in the number of Tregs in recipients reaching statistical significance at 7 months post transplant as compared to day 100 (median values 12.8% and 6.1 respectively, p<0.01). After a median follow-up post-transplant of 43.8 months, median progression-free survival is 27.3 months; median survival has not been reached.

Our results suggest that Id-specific and KLH-specific T cell immunity can be induced in sibling donors and transferred to recipients and may represent a strategy to enhance the graft-versus-myeloma effect. Regulatory T cells increased progressively in number in the recipients following transplantation and immunizations and may affect the quality and the magnitude of the vaccine-induced immune responses. Further investigations are needed to determine the reasons for the increased number of Tregs and to determine the optimal formulation and timing of booster immunizations in the recipients.

No relevant conflicts of interest to declare.