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. 2021 Sep;9(3):721-733.
doi: 10.1002/iid3.425. Epub 2021 May 4.

Donor Treg expansion by liposomal α-galactosylceramide modulates Tfh cells and prevents sclerodermatous chronic graft-versus-host disease

Hiroyuki Sugiura  1 , Ken-Ichi Matsuoka  1 , Takuya Fukumi  1 , Yuichi Sumii  1 , Takumi Kondo  1 , Shuntaro Ikegawa  1 , Yusuke Meguri  1 , Miki Iwamoto  1 , Yasuhisa Sando  1 , Makoto Nakamura  1 , Tomohiro Toji  2 , Yasuyuki Ishii  3   4 , Yoshinobu Maeda  1
Affiliations

Donor Treg expansion by liposomal α-galactosylceramide modulates Tfh cells and prevents sclerodermatous chronic graft-versus-host disease

Hiroyuki Sugiura et al. Immun Inflamm Dis. 2021 Sep.

Abstract

Background and aim: Chronic graft-versus-host disease (cGVHD) is a major cause of nonrelapse morbidity and mortality following hematopoietic stem cell transplantation (HSCT) VSports手机版. α-Galactosylceramide (α-GC) is a synthetic glycolipid that is recognized by the invariant T-cell receptor of invariant natural killer T (iNKT) cells in a CD1d-restricted manner. Stimulation of iNKT cells by α-GC leads to the production of not only immune-stimulatory cytokines but also immune-regulatory cytokines followed by regulatory T-cell (Treg) expansion in vivo. .

Methods: We investigated the effect of iNKT stimulation by liposomal α-GC just after transplant on the subsequent immune reconstitution and the development of sclerodermatous cGVHD. V体育安卓版.

Results: Our study showed that multiple administrations of liposomal α-GC modulated both host- and donor-derived iNKT cell homeostasis and induced an early expansion of donor Tregs. We also demonstrated that the immune modulation of the acute phase was followed by the decreased levels of CXCL13 in plasma and follicular helper T cells in lymph nodes, which inhibited germinal center formation, resulting in the efficient prevention of sclerodermatous cGVHD. V体育ios版.

Conclusions: These data demonstrated an important coordination of T- and B-cell immunity in the pathogenesis of cGVHD and may provide a novel clinical strategy for the induction of immune tolerance after allogeneic HSCT. VSports最新版本.

Keywords: Tfh cells; chronic graft-versus-host disease; hematopoietic stem cell transplantation; iNKT cells; regulatory T cells; α-galactosylceramide V体育平台登录. .

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Conflict of interest statement

Yasuyuki Ishii is the CEO of REGiMMUNE V体育官网入口. The remaining authors declare that there are no conflicts of interests. .

Figures (V体育平台登录)

Figure 1
Figure 1
Lipo α‐GC prevents cGVHD in a sclerodermatous cGVHD mouse model. (A) Experimental scheme. BALB/c recipient mice received 6 Gy of TBI; then, 8 × 106 BM cells and 10 × 106 spleen cells were administered from B10.D2 donor mice. Lipo α‐GC was administered at a dose of 1 μg/kg via tail vein injection on Days 0, 3, and 5 after BMT. (B) Time courses of skin cGVHD score and percent body weight changes from baseline after BMT in syngeneic/vehicle (n = 3), syngeneic/lipo α‐GC (n = 3), allogeneic/vehicle (n = 8), and allogeneic/lipo α‐GC (n = 8) groups. Representative data from one of three independently performed experiments are expressed as the means ± SEM. p‐values were determined by the Kruskal–Wallis test; *p < .05. (C) Representative photographs of mice from syngeneic/vehicle (n = 3), syngeneic/lipo α‐GC (n = 3), allogeneic/vehicle (n = 8), and allogeneic/lipo α‐GC (n = 8) groups on Day 56 after BMT from one of three independently performed experiments. BMT, bone marrow transplantation; BM, bone marrow; cGVHD, chronic graft‐versus‐host disease; α‐GC, α‐Galactosylceramide
Figure 2
Figure 2
Lipo α‐GC reduces pathological scores in a sclerodermatous cGVHD mouse model. (A) Representative photomicrographs of hematoxylin‐ and eosin‐stained skin sections from syngeneic/vehicle, syngeneic/lipo α‐GC, allogeneic/vehicle, and allogeneic/lipo α‐GC mice on Day 56 after BMT. Scale bar = 100 μm. ×20. (B) Pathological scores of skin cGVHD in syngeneic/vehicle (n = 2, one experiment), syngeneic/lipo α‐GC (n = 2, one experiment), allogeneic/vehicle (n = 8, data combined from two independently performed experiments with four mice per experiments), and allogeneic/lipo α‐GC (n = 8, data combined from two independently performed experiments with four mice per experiments) groups on Day 56. Data are expressed as the means ± SEM. p‐values were determined by the Mann–Whitney U‐test (allogeneic/vehicle vs. allogeneic/lipo α‐GC); *p < .05. BMT, bone marrow transplantation; cGVHD, chronic graft‐versus‐host disease; α‐GC, α‐Galactosylceramide
Figure 3
Figure 3
Lipo α‐GC increases donor iNKT cells, decreases host iNKT cells, and expands donor Tregs in an early phase after BMT. Tregs were defined as CD4+FoxP3+CD25+ cells and iNKT cells were defined as CD19−TCR‐β+CD1d tetramer+ cells. Chimerism was evaluated in the host (Ly9.1+) or donor (Ly9.1−). (A) The number of donor iNKT cells and host iNKT cells in spleen cells after BMT in vehicle (n = 5, one experiment) and lipo α‐GC (n = 5, one experiment) mice on Day 7. Data are expressed as means ± SEM. p‐values were determined using an unpaired, two‐tailed Student's t test. *p < .05. (B) Percentages of donor Tregs, and host Tregs among CD4+ T cells in spleen cells in vehicle (n = 5, one experiment) and lipo α‐GC (n = 5, one experiment) mice on Day 7. Data are expressed as means ± SEM. p‐values were determined using an unpaired, two‐tailed Student's t test. *p < .05. (C) Percentages of total Tregs among CD4+ T cells in spleen cells in syngeneic/vehicle (n = 3, one experiment), syngeneic/lipo α‐GC (n = 3, one experiment), allogeneic/vehicle (n = 5, one experiment) and allogeneic/lipo α‐GC (n = 5, one experiment) mice on Day 7. Data are expressed as means ± SEM. P‐values were determined using an unpaired, two‐tailed Student's t test. *p < .05. BMT, bone marrow transplantation; iNKT, invariant natural killer T; α‐GC, α‐Galactosylceramide
Figure 4
Figure 4
CD25 depletion inhibits donor Treg expansion and exacerbates cGVHD scores with lipo α‐GC treatment. (A) Representative flow cytometry images of donor Tregs (CD4+Ly9.1−FoxP3+CD25+) in whole spleen/vehicle, whole spleen/lipo α‐GC, and CD25‐depleted spleen/lipo α‐GC mice on Day 7. (B) Tregs and donor Tregs in whole spleen/vehicle (n = 5, one experiment), whole spleen/lipo α‐GC (n = 5, one experiment), and CD25‐depleted spleen/lipo α‐GC (n = 5, one experiment) groups. Data are expressed as means ± SEM. P‐values were determined by one‐way analysis of variance with Tukey's post hoc test. ***p < .001; ****p < .0001. (C) Skin cGVHD scores in syngeneic (n = 3, one experiment), whole allogeneic spleen/vehicle (n = 11, data combined from two independently performed experiments with five to six mice per experiments), whole allogeneic spleen/lipo α‐GC (n = 11, data combined from two independently performed experiments with five to six mice per experiments), and allogeneic CD25‐depleted spleen/lipo α‐GC (n = 11, data combined from two independently performed experiments with five to six mice per experiments) groups. Data are expressed as means ± SEM. p‐values were determined by the Kruskal–Wallis test. *p < .05. cGVHD,chronic graft‐versus‐host disease; α‐GC, α‐Galactosylceramide
Figure 5
Figure 5
Lipo α‐GC inhibits Tfh and GCB cells in a late phase after BMT. Tfh cells were defined as CD4+CXCR5+PD‐1+ cells and GCB cells were defined as B220+GL7 +Fas+ cells. (A) Proportions of Tfh cells in MLNs of syngeneic/vehicle (n = 3), syngeneic/lipo α‐GC (n = 3), allogeneic/vehicle (n = 8) and allogeneic/lipo α‐GC (n = 8) groups on Days 28 and 56. Data were combined from two independently performed experiments with four mice per experiments and are expressed as the means ± SEM. P‐values were determined using an unpaired, two‐tailed Student's t test. *p < .05. (B) Proportions of GCB cells in MLNs of vehicle (n = 8), and lipo α‐GC (n = 8) groups on Days 28 and 56. Data were combined from two independently performed experiments with four mice per experiments and are expressed as the means ± SEM. P‐values were determined using an unpaired, two‐tailed Student's t test. *p < .05. GCB, germinal center B cell; MLN, mesenteric lymph nodes; Tfh, follicular helper T cell; α‐GC, α‐Galactosylceramide
Figure 6
Figure 6
CXCL13 concentration increases in the allo lipo α‐GC group and is correlated with the proportion of Tfh cells. (A) Concentrations of CXCL13 in the plasma of syngeneic/vehicle (n = 2, one experiment), syngeneic/lipo α‐GC (n = 2, one experiment), allogeneic/vehicle (n = 8, data combined from two independently performed experiments with four mice per experiments), and allogeneic/lipo α‐GC (n = 8, data combined from two independently performed experiments with four mice per experiments) mice on Day 56. Data are expressed as means ± SEM. (B–D) Each orange, green, red, and blue marker indicates the result from syngeneic/vehicle, syngeneic/lipo α‐GC, allogeneic/vehicle, and allogeneic/lipo α‐GC, respectively. (B) Spearman's rank correlation between the percentage of Tfh cells in MLNs and the plasma concentration of CXCL13 on Day 56 (r = .853, p < 10−6). (C) Spearman's rank correlation between the percentage of Tfh cells in MLNs and the skin cGVHD score on Day 56 (r = .775, p < 10−5). (D) Spearman's rank correlation between the percentage of Tfh cells in MLNs and the skin pathological score on Day 56 (r = .766, p < 10−5). cGVHD, chronic graft‐versus‐host disease; MLN, mesenteric lymph nodes; Tfh, follicular helper T cell; α‐GC, α‐Galactosylceramide
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