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. 2013 Sep 19;122(12):2125-34.
doi: 10.1182/blood-2012-11-470252. Epub 2013 Jun 27.

Inhibiting retinoic acid signaling ameliorates graft-versus-host disease by modifying T-cell differentiation and intestinal migration

Kazutoshi Aoyama  1 , Asim Saha, Jakub Tolar, Megan J Riddle, Rachelle G Veenstra, Patricia A Taylor, Rune Blomhoff, Angela Panoskaltsis-Mortari, Christopher A Klebanoff, Gérard Socié, David H Munn, William J Murphy, Jonathan S Serody, Leshara M Fulton, Takanori Teshima, Roshantha A Chandraratna, Ethan Dmitrovsky, Yanxia Guo, Randolph J Noelle, Bruce R Blazar
Affiliations

Inhibiting retinoic acid signaling ameliorates graft-versus-host disease by modifying T-cell differentiation and intestinal migration

Kazutoshi Aoyama et al. Blood. .

Abstract

Graft-versus-host disease (GVHD) is a critical complication after allogeneic bone marrow transplantation. During GVHD, donor T cells are activated by host antigen-presenting cells and differentiate into T-effector cells (Teffs) that migrate to GVHD target organs. However, local environmental factors influencing Teff differentiation and migration are largely unknown. Vitamin A metabolism within the intestine produces retinoic acid, which contributes to intestinal homeostasis and tolerance induction. Here, we show that the expression and function of vitamin A-metabolizing enzymes were increased in the intestine and mesenteric lymph nodes in mice with active GVHD. Moreover, transgenic donor T cells expressing a retinoic acid receptor (RAR) response element luciferase reporter responded to increased vitamin A metabolites in GVHD-affected organs. Increasing RAR signaling accelerated GVHD lethality, whereas donor T cells expressing a dominant-negative RARα (dnRARα) showed markedly diminished lethality. The dnRARα transgenic T cells showed reduced Th1 differentiation and α4β7 and CCR9 expression associated with poor intestinal migration, low GVHD pathology, and reduced intestinal permeability, primarily via CD4(+) T cells. The inhibition of RAR signaling augmented donor-induced Treg generation and expansion in vivo, while preserving graft-versus-leukemia effects VSports手机版. Together, these results suggested that reagents blunting donor T-cell RAR signaling may possess therapeutic anti-GVHD properties. .

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Figure 1
Figure 1
Vitamin A metabolism is upregulated during acute GVHD. (A) Lethally irradiated B10.BR recipients were injected with 107 T-cell–depleted (TCD) BM cells and 1.5 × 107 splenocytes from fully MHC-mismatched B6 mice. On days 7 and 14 after BMT, tissues from lung, liver, small intestine, and colon were harvested and analyzed using a B16-DR5 assay (n = 4/group). The fold change was calculated by dividing the value of the non-GVHD group or GVHD group by the value of naive mouse group. (B) Lethally irradiated B10.BR recipients injected with 107 wt B6 TCD-BM cells and 3 × 106 purified RARE-luc B6 T cells. On days 7 and 21 after BMT, tissues from liver, small intestine, and colon were harvested and analyzed using a LC-(MS)-MS assay (n = 4/group). (C) Lethally irradiated B10.BR recipients were injected with 107 BM cells and 1.5 × 107 B6 splenocytes from wt B6 mice. Recipient mice (n = 4/group) were sacrificed on day 14 along with 4 naive B10.BR control mice, and sections from frozen tissue blocks were analyzed for expression of CD11c+ (green), CD45+ (blue), and either retinaldehyde dehydrogenase 1 (RALDH1) (red) or RALDH2 (red). The boxed area in (C) is indicated at higher magnification in (C′, C″). Arrows points out triple labeling (CD11c/CD45/RALDH1 or CD11c/CD45/RALDH2). Data shown are representative of 4 mice/group. Fluorescence was detected using an Olympus FluoView 1000 BX2 Upright confocal laser scanning microscope. (Original magnification ×400.) (D) Lethally irradiated B6-Ly5.2/Cr or B10.BR recipients were injected with 107 BM cells and 1.5 × 107 splenocytes from fully MHC-mismatched B6 mice. Lamina propria lymphocytes from the small intestine and lymphocytes from the MLNs were isolated on day 14 and donor (CD45.1− or H2kb+) CD11c+ and CD11b+ cells were evaluated for ALDH enzymatic activity by measuring the Aldefluor mean fluorescence intensity (MFI) (n = 4/group). (E) Lethally irradiated B6 or B10.BR recipients were transplanted with 107 BM cells and 3 × 106 purified RARE-luc B6 T cells. RARE-luc T cells were quantified by emitted photons over the total body area and within individual organs at serial time points after BMT (n = 3-4/group). *P > .05; **P > .01; and ***P > .001.
Figure 2
Figure 2
Increased RA levels and RAR signaling exacerbates GVHD. Survival and weight curves of lethally irradiated B10.BR recipients injected with 107 BM cells and 5 × 106 splenocytes from B6 mice are shown (n = 16/group). Subgroups were treated with vehicle (filled circles) or ATRA (open circles); P = .017. Data are combined from 2 experiments with similar results. **P > .01; ***P > .001.
Figure 3
Figure 3
Inhibiting RAR signaling in donor T cells prevents GVHD lethality. (A) Survival and weight curves of lethally irradiated B10.BR recipients of 107 BM cells and 107 splenocytes from B6 mice. Recipients of splenocytes from dnRARα-CD4Cre mice (filled circles) survived significantly longer than recipients of dnRARα splenocytes (open circles; P < .001; n = 16/group). (B) Survival, weight, and clinical GVHD scores curves of lethally irradiated BALB/c recipients of 107 BM cells and 5 × 106 splenocytes from B6 mice. Recipients of splenocytes from dnRARα-CD4Cre mice (filled circles) survived significantly longer than recipients of dnRARα splenocytes (open circles) (P < .001; n = 13-14/group). (C) Tissues (lung, liver, spleen, small intestine, and colon) from B10.BR recipients or BALB/c recipients were harvested on day 21 posttransplant, stained with hematoxylin and eosin, and scored for GVHD (means ± standard error). (D) FITC-dextran was orally administered to BALB/c recipients on day 21. Serum FITC-dextran levels were measured 4 hours later. Mice received wt BM cells unless otherwise indicated (P = .007). Data are combined from 2 experiments with similar results or 1 experiment each with 4 to 5 (C) or 5 to 7 (D) mice per group. (C-D) *P > .05; **P > .01; and ***P > .001.
Figure 4
Figure 4
Blocking RAR signaling in donor T cells impairs integrin and chemokine expression and skews T-cell polarity toward a Th2 phenotype. (A-D) Lethally irradiated BALB/c recipients were transplanted with B6 107 BM cells and 1.5 × 106 wt– (filled triangles), dnRARα− (open circles), or dnRARα-CD4Cre– (filled circles) purified T cells. Splenocytes, MLNs, liver cells, and colon lamina propria lymphocytes (LPLs) were isolated on indicated days and analyzed by fluorescence-activated cell sorter. Cells were gated on H-2Kb-positive events. (A) The frequency of CD4+ cells expressing IFN-γ, IL-4, and IL-17 from spleen and colon LPLs is shown. (B) The frequency of CD4+ cells expressing T-bet, GATA3, and RORγt from spleen is shown. (C) The absolute number of CD4+ cells expressing CXCR3, α4β7, and CCR9 in spleen, MLN, and liver cells is depicted. (D) The absolute number of total, CD4+, and CD8+ cells and the absolute number of CD4+ cells expressing CXCR3, α4β7, and CCR9 in colon LPLs are shown. (A) Data were combined from 2 experiments with similar results (n = 4 to 8/group). (B,D) Data were obtained from 1 experiment each with 4 mice per group. (C) Data were from 1 representative of 3 independent experiments. *P < .05; **P < .01; and ***P < .001.
Figure 5
Figure 5
Inhibition of RAR signaling in donor T cells increases Tregs in vivo. Lethally irradiated BALB/c recipients were transplanted with B6 107 BM cells and 1.5 × 106 dnRARα (open circles) or dnRARα-CD4Cre (filled circles) purified T cells. (A) The frequency of CD4+Foxp3+ cells and the absolute number of CD4+Foxp3+ cells in spleen are shown. (B) Liver cells and colon lamina propria lymphocytes were isolated on day 14 and analyzed using fluorescence-activated cell sorter. The frequency of CD4+Foxp3+ cells is shown. (C) Lethally irradiated BALB/c recipients were transplanted with CD45.2+ TCD B6 107 BM cells and 1 × 106 CD45.1+ B6 T cells along with 1 × 106 CD45.2+ T cells from either dnRARα (CD45.1 with dnRARα) or dnRARαCD4Cre (CD45.1 with dnRARα-CD4Cre) mice or were transplanted with CD45.2+TCD-B6 107 BM cells and 2 × 106 dnRARα-CD4Cre T cells alone. The percentages of chimerism of CD4+ cells and frequency of CD4+Foxp3+ cells gated on CD45.2+ (dnRARα or dnRARαCD4Cre) or CD45.1+ cells in spleens on day 14 after BMT are shown. (D) Lethally irradiated BALB/c recipients were injected with 107 BM cells and 5 × 106 CD25-depleted splenocytes or CD25-replete splenocytes from either dnRARα or dnRARα-CD4Cre donors and monitored for survival. (A-D) Data were obtained from 1 experiment each with 4 (A-B), 4 to 5 (C), or 8 (D) mice per group. *P < .05; **P < .01; and ***P < .001.
Figure 6
Figure 6
Blockade of RAR signaling in donor T cells does not abort the GVL effect. Lethally irradiated BALB/c recipients were transplanted with 107 T-cell–depleted BM cells with or without 3 × 105 A20-lymphoma cells on day 0. Subgroups were transplanted with 5 × 106 splenocytes from dnRARα (open circles) and dnRARα-CD4Cre (filled triangles) mice also on day 0. (A-C) Survival, weight, and clinical GVHD scores of lethally irradiated BALB/c recipients transplanted with BM cells only (filled squares), BM cells + A20 (filled circles), or with A20 + 5 × 106 splenocytes from dnRARα (open circles) or dnRARα-CD4Cre (filled triangles) donor mice. (D) Tumor growth was monitored by luciferase imaging at 1, 2, 3, 4, 6, 8, and 15 weeks after BMT. (E) The frequency of CD8+ cells expressing granzyme B was analyzed in spleen on day 14. Data were obtained from 1 experiment each with 5 to 10 (A-D) or 7 to 8 (E) mice per group. (A) BM cells + A20 vs dnRARα-CD4Cre + A20; P < .001. dnRARα+ A20 vs dnRARα-CD4Cre + A20; P < .001. (B-C) dnRARα+ A20 vs dnRARα-CD4Cre + A20 on day 6 to day 27; P < .001. (D) BM cells + A20 vs dnRARα-CD4Cre + A20 at 1, 2, 3, and 4 weeks; P < .001. dnRARα+ A20 vs dnRARα-CD4Cre + A20; P = not significant (ns).
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