Host and Donor Immune Responses Contribute to Anti-Viral Effects of Amotosalen-Treated Donor Lymphocytes Following Early Post-Transplant CMV Infection.

Background: GvHD and opportunistic infections are the major causes of morbidity and mortality in cancer patients treated with allogeneic BMT. In allogeneic BMT patients, donor derived T-cells help eradicate residual cancer and fight against opportunistic infections but they also cause the major deleterious effects, including GvHD which is the result of host allo-antigens recognition by the donor T-cells. Moreover, donor T-cells also play a critical role in promoting stem cell engraftment, encouraging rapid recovery of cellular immunity, and decreasing the probability of disease relapse. Thus, to establish a therapeutically useful adoptive immunotherapy using donor T-cells, separation of the beneficial anti-opportunistic infection and anti-tumor effects of donor T-cells from the deleterious GvHD effect are highly desirable. We previously showed that amotosalen-treated splenocytes rescued recipients from a lethal dose of MCMV administered on day 0 in experimental parent to F1 allogeneic bone marrow transplant (BMT). To model early post-transplant CMV reaction, in this study, we investigated the anti-viral immune responses and GvHD activity of treated donor T-cells after infecting allogeneic BMT recipients with a lethal dose of MCMV on 7 days post transplant.

Methods: Using a parent to F1 mouse BMT model, splenocytes (3×10⁶ untreated or 10×10⁶ amotosalen-treated) harvested from the MCMV immunized C57BL/6 donors were transplanted along with 5×106 T-cell depleted bone marrow (TCD BM) from naïve congeneic mice into lethally irradiated (11Gy) CB6F1 (C57BL/6 × Balb/C) recipients. Recipient mice were infected i.p. with a sublethal dose (5×104 pfu per mouse) of MCMV 7 days after transplant. Flow cytometry was used to quantitate T cell chimerism (in recipient spleen and thymus) and MCMV-peptide specific tetramer+ CD8+ T-cells. Serum IFN-γ and TNF-α were determined by ELISA. Liver viral load was determined by counting PFU in tissue homogenates plated onto 3T3 confluent monolayers.

Results: MCMV infection in recipients of amotosalen-treated splenocytes did not cause any mortality whereas recipients of untreated splenocytes had 40% early mortality due to acute GvHD. Like the recipients of untreated splenocytes, recipients of amotosalen-treated splenocytes effectively cleared MCMV from their liver within 10 days of infection. In contrast to full donor chimerism in recipients of untreated splenocytes, recipients of amotosalen-treated splenocytes showed mixed chimerism with donor spleen- and host-derived MCMV peptide specific tetramer+ CD8+ T cells that proliferated following day 7 post MCMV infection. Significantly higher numbers of host derived CD4−CD8− (DN) TCRαβT-cells appeared in the spleen with peak on day 3 post MCMV infection among recipients of amotosalen-treated splenocytes compared with the recipients of untreated splenocytes. Lower levels of serum IFN-γ and TNF-α and preservation of thymic function were also noted in the recipients of amotosalen-treated splenocytes compared with the recipients of untreated splenocytes following MCMV infection.

Conclusion: Adoptive immunotherapy with amotosalen-treated T cells is an ideal therapeutic approach that facilitates early hematopoietic engraftment, anti-viral donor immune reconstitution and preserves early post-transplant host immunity leading to protection from lethal viral infection without causing aGvHD.