Flagellin, a TLR5 Agonist, Down-Regulate CD62L on Donor T Cells and Limit GvHD in Allogeneic Hematopoietic Stem Cell Transplantation

Bacground: Graft-vs-host disease (GvHD) is a major complication in allogeneic Hematopoietic Stem Cell Transplant (HSCT) recipients. Flagellin is a bacterial protein and a TLR5 agonist that showed diverse immunological responses in both human and animal including both activation of dendritic cells and immuno-suppression. We recently observed that prophylactic use of flagellin protected allogeneic HSCT recipient from GvHD without affecting host immune reconstitution. Acute GvHD has been reported to be mediated by allo-reactive CD62L+ T cells, and over 80% of murine naïve splenic CD4+ and CD8+ T cells express CD62L. In order to test the effect of flagellin on GvHD mediated by the CD62L+ CD4+ and CD62L+CD8+ donor T cells, we investigated clinical manifestation of GvHD as well as the in vivo expression of CD62L on donor T cells in flagellin treated versus control treated allogeneic HSCT recipients.

Methods: We established a parent →F1 MHC major mismatched model (C57BL/6 → C57BL/6 × BALB/c) for allogeneic HSCT for which GvHD is the major complication. Recipient mice received 5 × 10^6 T cell depleted (TCD) bone marrow cells and 5×10^6 or 10×10^6 CFSE labeled donor splenocytes from naïve C57Bl/6 congenic donors. 50 μg flagellin per recipient was administered intraperitoneally 3 hours before irradiation and 24 hours after allogeneic HSCT (treated). CB6F1 recipients that received no flagellin (untreated) and recipients of syngeneic HSCT were used as control. Recipients were sacrificed on day 66+ transplant and the numbers of CD62L+ T cells and foxp3+CD4+CD25+ T cells were determined by FACS. Recipients of CFSE treated donor splenocytes were sacrificed on day 4 post HSCT, splenocytes were harvested and analyzed for CD62L expression on T cell subsets undergone in vivo cell division by Flow cytometry. 5 mice were used per group.

Results: Flagellin treated recipients did not have GvHD and had no mortality. Untreated control recipients had 87% survival at 30 days post transplant and had signs of chronic GvHD. While total cell number and also donor spleen- and BM-derived CD4+ and CD8+ T cells per spleen in untreated recipients were significantly lower compared to flagellin treated recipients (p=0.0006) on day 66 post transplant, persistent of donor spleen-derived CD62L+CD4+ T cells and CD62L+CD8+ T cells per spleen were not significantly different (p=0.13 and p=0.07, respectively). Moreover, higher number of foxp3+CD25+CD4+ regulatory T cells were found in the spleen and thymus in treated recipients compared to untreated recipients. Within day 4 post transplant, the number of CD4+ T cells per spleen of treated and untreated recipients increased significantly compared to syngeneic recipients (p=0.001 and p=0.03, respectively). Although equivalent numbers of CD62L+CD4+ T cells were observed in both treated and untreated recipients (p=0.3), significantly increased numbers of CD62L+CD8+ T cells was found in treated recipients compare to untreated recipients (p=0.02). Moreover, significantly higher numbers of divided (far left CFSE staining population) CD62L+CD4+ and CD62L+CD8+ T cells were found in recipients of treated splenocytes within day 4 post transplant followed by down regulation of CD62L surface marker compared to untreated recipients (p=0.02 and p=0.01, respectively).

Conclusion: Flagellin treated recipients had limited GvHD and had rapid increased divided CD4+CD62L+ T cells followed by CD62L-ve activated CD4+ T cells per spleen in treated recipients compared to untreated recipients may be one of the major affect mediated by flagellin. Flagellin-TLR5 receptor agonistic effect may

1. reduce production of biological factor(s) essential to generate allo-reactive T cells or directly stimulate CD62L+CD4+ and CD62L+CD8+ T cells in different activation status other than allo-reactive T cells;

2. maintain a balanced immune reconstitution in lymphoid organs by producing regulatory T cells through their thymus.

Therefore, use of flagellin may be a novel therapeutic approach to treat blood cancer patients with allogeneic HSCT without GvHD and toxicity.