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. 2021 Sep 3:12:720354.
doi: 10.3389/fimmu.2021.720354. eCollection 2021.

The Potential Roles of Mucosa-Associated Invariant T Cells in the Pathogenesis of Gut Graft-Versus-Host Disease After Hematopoietic Stem Cell Transplantation

Meng-Ge Gao  1 , Yan Hong  1 , Xiang-Yu Zhao  1   2 , Xin-An Pan  1 , Yu-Qian Sun  1 , Jun Kong  1 , Zhi-Dong Wang  1 , Feng-Rong Wang  1 , Jing-Zhi Wang  1 , Chen-Hua Yan  1   3 , Yu Wang  1   2 , Xiao-Jun Huang  1   2   3   4 , Xiao-Su Zhao  1   2   3
Affiliations

The Potential Roles of Mucosa-Associated Invariant T Cells in the Pathogenesis of Gut Graft-Versus-Host Disease After Hematopoietic Stem Cell Transplantation (V体育官网入口)

Meng-Ge Gao et al. Front Immunol. .

Abstract

Gut acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is associated with high mortality. Mucosa-associated invariant T (MAIT) cells are a group of innate-like T cells enriched in the intestine that can be activated by riboflavin metabolites from various microorganisms. However, little is known about the function or mechanism of action of MAIT cells in the occurrence of gut aGVHD in humans. In our study, multiparameter flow cytometry (FCM) was used to evaluate the number of MAIT cells and functional cytokines. 16S V34 region amplicon sequencing analysis was used to analyze the intestinal flora of transplant patients. In vitro stimulation and coculture assays were used to study the activation and function of MAIT cells. The number and distribution of MAIT cells in intestinal tissues were analyzed by immunofluorescence technology. Our study showed that the number and frequency of MAIT cells in infused grafts in gut aGVHD patients were lower than those in no-gut aGVHD patients VSports手机版. Recipients with a high number of MAITs in infused grafts had a higher abundance of intestinal flora in the early posttransplantation period (+14 days). At the onset of gut aGVHD, the number of MAIT cells decreased in peripheral blood, and the activation marker CD69, chemokine receptors CXCR3 and CXCR4, and transcription factors Rorγt and T-bet tended to increase. Furthermore, when gut aGVHD occurred, the proportion of MAIT17 was higher than that of MAIT1. The abundance of intestinal flora with non-riboflavin metabolic pathways tended to increase in gut aGVHD patients. MAIT cells secreted more granzyme B, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ under the interleukin (IL)-12/IL-18 stimulation [non-T-cell receptor (TCR) signal] and secreted most of the IL-17 under the cluster of differentiation (CD)3/CD28 stimulation (TCR signal). MAIT cells inhibited the proliferation of CD4+ T cells in vitro. In conclusion, the lower number of MAIT cells in infused grafts was related to the higher incidence of gut aGVHD, and the number of MAIT cells in grafts may affect the composition of the intestinal flora of recipients early after transplantation. The flora of the riboflavin metabolism pathway activated MAIT cells and promoted the expression of intestinal protective factors to affect the occurrence of gut aGVHD in humans. .

Keywords: allo-HSCT; gut acute graft-versus-host disease; immunomodulatory; intestinal flora; mucosa-associated invariant T cell V体育安卓版. .

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Comparison of the frequency and number of mucosa-associated invariant T (MAIT) cells in the grafts from the recombinant human granulocyte colony-stimulating factor (rhG-CSF) mobilized donors and healthy person. (A) Flow cytometry of MAIT cells in the grafts of four patient groups. (B) The frequency of MAIT cells (% of CD3+ cells) in graft-bone marrow (BM) [Healthy, n = 4; No acute graft-versus-host disease (aGVHD), n = 21; Skin aGVHD, n = 28; Gut aGVHD, n = 12] and graft-peripheral blood (PB) (Healthy, n = 8; No aGVHD, n = 24; Skin aGVHD, n = 27; Gut aGVHD, n = 15; Gut aGVHD vs. Healthy, p = 0.029). (C) The number of MAIT cells in graft-BM, graft-PB (Gut aGVHD vs. no aGVHD, p = 0.028; Skin aGVHD vs. no aGVHD, p = 0.042), and the total grafts (No aGVHD, n = 16; Skin aGVHD, n = 13; Gut aGVHD, n = 13). (D) The influence of the high MAIT (>5.3 × 106/kg) and low MAIT (<5.3 × 106/kg) group level in the graft on the occurrence of aGVHD (I aGVHD and II–IV aGVHD). (E) The influence of the high MAIT (>5.3 × 106/kg) and low MAIT (<5.3×106/kg) groups in infused graft on gut aGVHD (p = 0.018) or skin aGVHD. (F) The influence of the high MAIT (>5.3 × 106/kg) and low MAIT (<5.3 × 106/kg) groups in infused graft on the prognosis after transplantation. Levels of significances are given as p-values with **≤0.05 and *≤0.1.
Figure 2
Figure 2
The comparison of the intestinal flora abundance of recipients on posttransplant 14 days between high number of mucosa-associated invariant T (MAIT) cells (>5.3 × 106/kg, H group, n = 12) and low number of MAIT cells (<5.3 × 106/kg, L group, n = 10) in infused grafts. (A) Venn diagram, each circle represents a group, the number in the overlapped part of the circle and circle represent the number of operational taxonomic units (OTUs) shared between the two groups, and the number without overlap represents the unique OTUs of the group. (B) The column chart of relative abundance of species at phylum level. The abscissa is the group name. The ordinate (Relative Abundance) represents the relative abundance. Others represents the sum of the relative abundances of all the phyla except these 10 phyla in the figure. (C) Cluster heat map of species abundance at the genus level. According to the species annotation and abundance information of all samples at the genus level, select the top 35 abundant genera. (D) T-test species difference analysis diagram at the genus and family levels between H and L groups. (E) Latent Dirichlet Allocation (LDA) score distribution histogram and cladogram. The LDA score distribution histogram shows the Biomarker species with statistical differences between the groups whose LDA Score is greater than the set value (the default setting is 4). The length of the histogram represents the impact of different species (that is, LDA Score). In the cladogram, the circles radiating from the inside to the outside represent the taxonomic level from the phylum to the genus (or species).
Figure 3
Figure 3
Reconstitution of mucosa-associated invariant T (MAIT) cells and their different subgroups in the peripheral blood after transplantation. (A) Posttransplant number of MAIT cells reconstituted under haplo-hematopoietic stem cell transplantation (HSCT) and sibling-identical HSCT. (B) Posttransplant number of MAIT cells reconstituted in gut acute graft-versus-host disease (aGVHD) patients, skin aGVHD and no gut aGVHD patients. (C) Posttransplant reconstitution of MAIT1 and MAIT17 subsets based on the expression of transcription factors T-bet and Rorgt respectively in no gut aGVHD patients. (D) Posttransplant reconstitution of MAIT1 and MAIT17 subsets based on the expression of transcription factors T-bet and Rorgt respectively in skin aGVHD patients. (E) Posttransplant reconstitution of MAIT1 and MAIT17 subsets based on the expression of transcription factors T-bet and Rorgt respectively in gut aGVHD patients.
Figure 4
Figure 4
The comparison of the cytokine expression of mucosa-associated invariant T (MAIT) cells in peripheral blood under different stimulation signals in vitro among the gut acute graft-versus-host disease (aGVHD) group (n = 8), infection or fever group (n = 10), and no-event group (n = 7). (A) The comparison of the cytokine expression of MAIT cells under different stimulation signals in vitro. (B) The comparison of the cytokine expression of MAIT cells under interleukin (IL)-12/IL18 stimulation in vitro among the gut aGVHD group (n = 8), infection or fever group (n = 10), and no-event group (n = 7). (C) The comparison of the cytokine expression of MAIT cells under cluster of differentiation (CD)3/CD28 stimulation signals in vitro among the gut aGVHD group (n = 8), infection or fever group (n = 10), and no-event group (n = 7). (D) The comparison of cytokine expression under IL-12/IL18 stimulation and CD3/CD28 stimulation.
Figure 5
Figure 5
Mucosa-associated invariant T (MAIT) cell functional changes in 16 gut acute graft-versus-host disease (aGVHD) patients before and after the onset of gut aGVHD. (A) The changes of MAIT cell number at three posttransplant time points, namely, at the time of neutrophil engraftment, at the onset of gut aGVHD, and at the CR of gut aGVHD. (B) The changes of transcription factors PLZF, T-bet, and Rorγt in patients with gut aGVHD at different points. (C) Posttransplant comparison of MAIT1 and MAIT17 subsets based on the expression of transcription factors T-bet and Rorγt respectively in gut aGVHD patients. (D) The changes of activation markers and chemokine receptors in patients with gut aGVHD at different points. (E) Cluster heat map of species abundance at the genus level. (F) LDA score distribution histogram and cladogram. Levels of significances are given as p-values with ***≤0.01 and **≤0.5.
Figure 6
Figure 6
The comparison of the number and function of mucosa-associated invariant T (MAIT) cells among patients with gut acute graft-versus-host disease (aGVHD) (n = 16), patients with infection or fever (n = 38) at the same time, and patients with no-event (n = 19) at the same time. (A) The comparison of the frequency of MAIT cells among gut aGVHD group, infection or fever group, and no-event group (Frequency: Gut aGVHD vs. No-event, p < 0.001). (B) The comparison of the number of MAIT cells among gut aGVHD group, infection or fever group, and no-event group (Number: Gut aGVHD vs. No-event, p = 0.025). (C) The comparison of T-bet+ MAIT (MAIT1) and Rorγt+ MAIT (MAIT17) among the three groups. (D) The comparison of the transcription factors among the three groups (Rorγt: Gut aGVHD vs. No-event, p = 0.037; T-bet: Gut aGVHD vs. No-event, p = 0.018; Gut aGVHD vs. Infection or fever, p = 0.023). (E) The comparison of the chemokine factors and markers among the three groups (Gut aGVHD vs. No-event, p = 0.02; Gut aGVHD vs. Infection or fever, p = 0.011). (F) Cluster heat map of species abundance at the genus level. (G) LDA score distribution histogram and cladogram. Levels of significances are given as p-values with ***≤0.01 and **≤0.5.
Figure 7
Figure 7
The in vitro inhibition experiment of mucosa-associated invariant T (MAIT) cells on CD4+ T cells and immunofluorescence experiment of intestinal tissue. (A) MAIT cells and CD4+ T cells were isolated from the peripheral blood of four healthy donors. Here, 5,6-carboxy-fluorescein diacetate succinimidyl ester (CFSE)-labeled CD4+ T cells with CD3/CD28 bead stimulation and MAIT cells or CD4+ T cells were mixed and cultured at a ratio of 2:1, 1:2, 1:4, and 1:8 for 4 days. Representative flow cytometric analysis of CFSE dilutions in CD4+ T cells. (B) Immunofluorescence of intestinal tissues of healthy people, gut aGVHD lesion sites and peri-gut aGVHD sites of gut aGVHD patients. (C) Comparison of CD161 staining intensity, CD161-positive cell ratio, number, and density between healthy person intestinal tissue (n = 2) and intestinal lesion tissue of gut aGVHD patients (n = 5). Since only one case of gut aGVHD patient in the experiment received the intestinal tissue of peri-gut aGVHD site, there is no peri-gut aGVHD group in the comparison. (D) Comparison of CD8+CD161+ cell ratio, number, and density between healthy person intestinal tissue (n = 2) and intestinal lesion tissue of gut aGVHD patients (n = 5).
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