Prophylactic Use of Flagellin: A Novel Method to Boost Immune Reconstitution in Allogeneic HSCT Recipients with Limited GvHD.

Graft-vs-host disease (GvHD) is a major complication in allogeneic Hematopoietic Stem Cell Transplant (HSCT) recipients. Immunosuppressive drugs limit clinical GvHD but increase relapse and susceptibility to opportunistic infections and also result in drug related toxicities. To develop an alternative approach to control GvHD, we tested the immunomodulatory immune properties of flagellin, a bacterial protein that agonistically binds with TLR5 and protects mice from radiation-induced gut injury, in murine allo-BMT models.

We used established BA.B10 (H-2K) → C57BL/6 (H-2b) MHC mismatched experimental models of allogeneic HSCT in which GvHD is a major complication. 50 μg LPS-free purified flagellin in PBS or PBS alone were administered intraperitoneally in two doses: 3 hours before fractionated irradiation (5.5Gy X 2 fractions) and 1 day post-transplant. Allografts were performed 1 day after irradiation and contained 5 million (M) T-cell depleted bone marrow (BM) cells and 5 M plastic non-adherent splenocytes from naïve BA.B10 donors. The primary end-points was survival; HSCT recipients were monitored twice a day for mortality and GvHD signs and recipients having more than 25% weight loss were sacrificed. Blood, spleen, thymus and BM were collected from surviving mice on day 132 post transplant, live cells counted, and immune phenotypes were analyzed by FACS. The numbers and phenotype of immune cells in organs from flagellin-treated HSCT recipients were compared to the similar immune cells per organs analyzed from a normal B6 mouse having similar age of HSCT recipients.

Flagellin treated recipients had 15% weight-loss and 33% transplant-related death by 132 days post transplant versus severe acute GvHD and 100% early post-transplant mortality among control HSCT recipients that received PBS. Flagellin-treated recipients had 100% donor chimerism with limited clinical signs of GvHD. While total cell numbers per spleen (8.2 ± 5.4M) and thymus (7.1 ± 4.9M) were very low in flagellin-treated recipients compared to normal B6 mice (>100M/organ), the cell numbers isolated from blood (8.9 ± 2.6 M/ml) and BM (104.5 ± 37.4 M) were similar to non-transplanted B6 mice (11.4M/ml blood and 108 M/BM, respectively). BM of flagellin-treated HSCT recipients contained similar numbers of CD4+ T cells (4.6 ± 2.7 M) and CD8+ T cells (2.5 ± 1.4 M) as normal B6 mice (4.03M and 1.3M, respectively). Numbers of naïve and memory CD4+ T-cells in the BM were similar between flagellin-treated and control mice: CD4+CD62L+(0.7 ± 0.2 versus 0.5M); CD4+CD62L- (3.9 ± 2.5 versus 3.5 M); CD4+ CD44hi (2.8 ± 1.4 versus B6 3.6M); and CD44lo (1.7 ± 1.3 M versus 0.44M). In contrast, flagellin-treated HSCT recipients had more naïve CD8+ T-cells but similar memory CD8+ T-cells in their BM compared with control mice: CD8+CD62L+(2.6 ± 1.4 versus 1.0M); CD8+CD62L- (1.7 ± 1.2M versus 0.3 M); CD8+CD44hi(0.8 ± 1.1 versus 0.7M); and CD8+CD44lo (0.7 ± 0.3M versus 0.6 M). The numbers of total CD3+ T cells, NK cells, and lin-CD11b-Sca-1+Ckit+ Stem cells in the BM were also similar comparing flagellin-treated recipients with non-transplanted B6 control mice. The number of CD3-B220+ B cells in the BM were lower in flagellin-treated recipients compared to B6 mouse (18.1 ± 3.2M versus 43.1M) as were the numbers of T-cells and B-cells per mL blood of flagellin-treated mice were found lower compared with the blood of normal B6 mouse: 0.8 ± 0.2M T-cells/mL versus 2.1M/mL; 5.5 ± 2.5M B cells/mL versus 9.1M/mL. Although the cellularity of the thymus in flagellin-treated animals was very low compared to normal B6 mice, a usual percentage (62.5 ± 10.5%) of thymocytes were of CD4/CD8 double positive, indicating functional thymopoiesis in these recipients.

Flagellin protected allogeneic HSCT recipients from irradiation-induced BM damage and prevented lethal GvHD in a major MHC mis-matched model of GvHD. Flagellin and other TLR5 agonists may be novel therapeutic approaches to prevent or reduce GvHD in allogeneic HSCT recipients.

No relevant conflicts of interest to declare.