MCMV Infection Lowers the Threshold for the Development of Clinical GvHD after Allogeneic Bone Marrow Transplantation.

Abstract CMV infection is reported to increase the incidence and severity of chronic GvHD and clinical data have shown that preemptive antiviral therapy decreased the risk of extensive chronic GvHD. Using mouse model of Allogeneic BMT, we investigated the mechanism for the association of MCMV infection and GvHD. We hypothesize that MCMV infection leads to generalized immune activation and increases the number of donor derived allo-reactive T cells and GvHD activity. Methods: A parenteral to F1 mouse BMT model was used to study anti-CMV immunity and GvHD. Low dose splenocytes (3x106) from MCMV immunized C57BL/6 donors (H-2b, Thy1.2+, CD45.1+) were transplanted with 5x106 T cell depleted bone marrow (TCD BM) from congeneic mice (H-2b, Thy1.1+, CD45.2+) into lethally irradiated (11Gy) CB6F1 recipients (C57BL/6 x Balb/C, H-2b/d, Thy1.2+, CD45.2+). Previous work has established this as a dose that protects against CMV without immediate lethality from GvHD. Non-GvHD control mice received a dose of Amotosalen treated splenocytes (10x106) and 5x106 TCD BM that protects against CMV without GvHD. Recipient mice were infected i.p. with a supralethal dose (2.5x104 pfu) of MCMV 7 days post transplant. Clinical GvHD was monitored twice weekly by weight loss, hair loss, ruffled fur, diarrhea, and decreased activity. T cell chimerism in recipient spleen and thymus, and MCMV peptide specific tetramer+CD8+ T cells were determined by flow cytometry. Liver and lung viral loads were determined by counting PFU in tissue homogenates plated onto 3T3 confluent monolayers. Results: During the acute phase of MCMV infection (day 3 to 14 post infection), recipient mice that received 3x106 untreated donor splenocytes developed GvHD characterized by weight loss and higher mortality than the non-GvHD control mice. Although both GvHD+ and control mice effectively cleared the virus from their liver, delayed viral clearance from the lung was found in non-GvHD recipients. Viral clearance was associated with expansion of donor spleen-derived MCMV peptide specific tetramer+ CD8+ T cells. The kinetics of donor T-cell expansion varied significantly between the two treatment groups, with GvHD+ recipients showed rapid early expansion of donor derived T-cells followed by the development of GvHD with subsequent lymphopenia. Recipients of Amotosalen-treated splenocytes had more gradual expansion of total and 400-fold expansion of antigen specific T-cells with sustained lymphoid reconstitution. GvHD+ recipients of untreated splenocytes had complete chimerism comprised of >80% of CD8+ donor T cells whereas non-GvHD controls had significant expansion of host derived T cells following MCMV infection and lacked allo-reactive of donor- spleen-derived T cells. Thymic function was inhibited among animals that developed GvHD and preserved among control mice throughout the infectious phase. Very delayed CMV infection (on day 60 after transplant) in mice with established chronic GvHD exacerbated GvHD and was associated with delayed lung viral clearance. Conclusion: After CMV infection there is extensive expansion of allo-reactive T cells in GvHD+ mice with associated damage to the microenvironment in the spleen and thymus. Amelioration of the immuno-suppressive effect of CMV infection (in clinical transplantation) will likely require more effective prophylaxis and treatment of GvHD in allotransplant recipients.