Flagellin, a TLR5 agonist, reduces graft-versus-host disease in allogeneic hematopoietic stem cell transplantation recipients while enhancing antiviral immunity

Abstract

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). Posttransplant immunosuppressive drugs incompletely control GVHD and increase susceptibility to opportunistic infections. In this study, we used flagellin, a TLR5 agonist protein (∼50 kDa) extracted from bacterial flagella, as a novel experimental treatment strategy to reduce both acute and chronic GVHD in allogeneic HSCT recipients. On the basis of the radioprotective effects of flagellin, we hypothesized that flagellin could ameliorate GVHD in lethally irradiated murine models of allogeneic HSCT. Two doses of highly purified flagellin (administered 3 h before irradiation and 24 h after HSCT) reduced GVHD and led to better survival in both H-2(b) → CB6F1 and H-2(K) → B6 allogeneic HSCT models while preserving >99% donor T cell chimerism. Flagellin treatment preserved long-term posttransplant immune reconstitution characterized by more donor thymic-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells and significantly enhanced antiviral immunity after murine CMV infection. The proliferation index and activation status of donor spleen-derived T cells and serum concentration of proinflammatory cytokines in flagellin-treated recipients were reduced significantly within 4 d posttransplant compared with those of the PBS-treated control recipients. Allogeneic transplantation of radiation chimeras previously engrafted with TLR5 knockout hematopoietic cells showed that interactions between flagellin and TLR5 expressed on both donor hematopoietic and host nonhematopoietic cells were required to reduce GVHD. Thus, the peritransplant administration of flagellin is a novel therapeutic approach to control GVHD while preserving posttransplant donor immunity.

Figure 1. Flagellin protected allogeneic HSCT recipients from GvHD

A. Survival and %weight loss for flagellin(Fla)- and PBS-treated B6 recipients transplanted with 5 × 10⁶ splenocytes and 5 × 10⁶ TCD BM from B10.BR and BA.B10 congenic donors respectively in H-2^K → H-2^b model. B. Survival and %weight loss of the flagellin- and PBS-treated CB6F1 recipients transplanted with 5 × 10⁶ splenocytes and 5 × 10⁶ TCD BM from PepBoy and BA congenic donors, respectively in H-2^b → H-2^b/d model. Survival data were derived from daily monitoring of HSCT recipients for 3 weeks after transplant, and twice per week thereafter. Body weights of individual recipients were measured twice/week during the first 3 weeks after HSCT, and once a week after that. 6 to 10 mice were used per group. The * represents the p values <0.5 obtained from the Student’s t-Test analysis. The data are the representative of three similar experiments.

Figure 2. Flagellin-treated recipients had improved immune reconstitution with complete donor chimerism

Flagellin- and PBS-treated control recipients of H-2^b→H-2^b/d HSCT model were sacrificed on day 66 after HSCT. PBMC, splenocytes and thymocytes were harvested from the blood, spleen and thymus, respectively. Nucleated cells per organ were counted microscopically and donor spleen- and BM-derived T cells were determined by FACS analysis as described in materials and methods. A. Total nucleated cells per ml blood. B. Total donor spleen derived T cells per mL of blood obtained by the FACS analysis. C. Total nucleated cells per spleen. D. Total donor spleen- derived T cells per spleen. E. FACS data for T cells chimerism obtained from the CD3 gated population of the spleen of one mouse per group. F. Donor spleen- and BM-derived CD4+ and CD8+ T cells per spleen. G. Donor spleen- and donor BM-derived CD62L+CD4+ T cells per spleen. H. Absolute numbers of thymocytes obtained from the thymus of flagellin- and PBS-treated recipients. I. Absolute numbers of total Tregs per thymus. J and K. FACS data from blood and spleen, respectively, of one mouse from each group of flagellin- and PBS-treated recipients. Side scatter data of CD45.2+ve cells are for the donor BM-derived and CD45.2-ve cells are for the donor spleen-derived populations. B220+CD3- cells from the donor BM- and donor spleen-derived gated cells are presented as B cells. 4 to 5 mice were used per group. The * and ** represent the p values <0.5 and <0.005 respectively, obtained from the Student’s t-Test analysis.

Figure 3. Flagellin-treated recipients had higher antiviral T cells response than the PBS-treated recipients

A. Flagellin- and PBS-treated recipients of H-2^b→H-2^b/d allogeneic HSCT model were infected with 2 × 10⁴ PFU mCMV i.p after 100+ days post transplant. Infected mice were sacrificed on 10 days post mCMV infection, and blood and splenic anti-mCMV immunity were determined. A. Total numbers of nucleated cells per ml blood. B. Total numbers of donor spleen-derived T cells per ml blood. C. Representative FACS data for the donor spleen- and BM-derived mCMV specific CD8+ T cells in blood obtained from one mouse from each group of flagellin- and PBS-treated recipients. D. Absolute numbers of donor spleen- and donor BM-derived mCMV peptide specific tetramer+ CD8+ T cells per ml blood. E. Frequencies of intracellular Granzyme B and IFN-γ producing donor spleen- and BM-derived CD8+ T cells after in vitro stimulation with PMA-ionomycin. F. Chronic GvHD scores of the small intestinal histological tissue sections obtained from the flagellin- and PBS-treated recipients on 10 days post mCMV infection. G. GvHD scores obtained from the formalin-fixed histological small intestinal tissue section of flagellin- and PBS-treated recipients on 10 days post mCMV infection. H. Mean skin dermal thickness obtained from the flagellin- and PBS-treated recipients on 10 days post mCMV infection. I. Skin pictures for the dermal thickness (black arrow) of one mouse of each group of flagellin- and PBS-treated recipients. The * and ** represent the p values <0.05 and <0.005, respectively, obtained from the Student’s t-Test. 5 mice were used per group and the data represent one of two similar experiments.

Figure 4. Flagellin reduced acute GvHD by reducing early donor T cells proliferation and activation

Flagellin- and PBS-treated recipients transplanted with 3 × 10⁶ splenocytes from naïve B10.BR and 5 × 10⁶ TCD BM from naïve BA.B10 congenic donors of H-2^K → H-2^b allogeneic HSCT model were sacrificed on 4 and 10 days post HSCT and splenocytes were analyzed. A. Absolute numbers of nucleated cells per spleen determined microscopically. B. Absolute numbers of donor spleen-derived T cells per spleen determined by FACS. C. Acute GvHD scores obtained from the small intestine histological tissue sections of flagellin- and PBS-treated recipients on 4 days post HSCT. D. Activation status of donor spleen-derived CD4+ and CD8+ T cells were determined by co-staining the cells with the antibodies to CD62L, ICOS-1, PD-1, CD25 and CD69 along with the antibodies to CD4, CD8 and CD45.2 and Thy1.2. Closed circles represent the data obtained from the flagellin-treated recipients; open circles represent PBS-treated recipients. E. FACS data representing the KI-67+ and CFSE stained donor spleen-derived CD4+ and CD8+ T cells, and Ki-67+ Thy1.2-CD11b+ myloid populations in the spleen of flagellin- and PBS-treated allogeneic HSCT recipients on day 4 post-transplant. F. Average percentages of Ki-67+ donor spleen-derived CD4+, CD8+ T cells and Thy1.2-CD11b+ myloid cells in the spleen of flagellin-and PBS-treated recipients on day 4 post-transplant. G. Proliferation index calculated from the CFSE stained donor spleen-derived CD4+ and CD8+ T cells in the spleen of flagellin-and PBS-treated recipients on day 4 post-transplant. 5 to 6 mice were used per group in each time point. The * and ** represent the p values of <0.05 and <0.005, respectively obtained using Student’s t-Test.

Figure 5. Flagellin reduced acute GvHD by producing reduced levels of proinflammatory cytokines at the early days of transplantation

Flagellin- (closed squire) and PBS (opened squire) -treated recipients transplanted with 3 × 10⁶ splenocytes from naïve B10.BR and 5 × 10⁶ TCD BM from naïve BA.B10 congenic donors of H-2^K → H-2^b allogeneic HSCT model were sacrificed on 4 and 10 days post HSCT. Serum was harvested and concentrations of IFN-γ, TNF-α and IL-6 per ml serum were determined by ELISA as described in Materials and Methods. Serum concentration of IFN-γ (left panel), TNF-α (middle panel) and IL-6 (right panel) are presented. The “ND” is for the non-detectable serum cytokines concentration. The * represents the p<0.05 obtained from Student’s t-Test. 5 to 6 mice were used per group in each time point.

Figure 6. Flagellin-TLR5 immune interaction is required to reduce GvHD in allogeneic HSCT recipients

B6 → TLR5 KO (KO) and KO→B6 radiation chimeras were generated by transplanting 10 × 10⁶ BM cells from wild-type (WT) B6 or TLR5 KO donors into congenic CD45.1 recipients. Radiation chimeras of were irradiated again with 9.0Gy (4.5Gy × 2) after 60+ days post transplant and transplanted with 3 × 10⁶ splenocytes from naïve B10.BR and 5 × 10⁶ TCD BM from naïve BA.B10 congenic donors. 50% of the radiation chimeras were treated with 50µg flagellin/mouse i.p 3 hours before irradiation and 24 hours after HSCT. 50% radiation chimeras were treated with 0.2ml PBS i.p. Transplanted mice were observed everyday for mortality until 3 weeks and once per week after that. 6 mice were used per group. The data are representative of two similar experiments.