Requirement for interactions of natural killer T cells and myeloid-derived suppressor cells for transplantation tolerance - PubMed (original) (raw)

. 2014 Nov;14(11):2467-77.

doi: 10.1111/ajt.12914. Epub 2014 Oct 13.

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Requirement for interactions of natural killer T cells and myeloid-derived suppressor cells for transplantation tolerance

D Hongo et al. Am J Transplant. 2014 Nov.

Abstract

The goal of the study was to elucidate the cellular and molecular mechanisms by which a clinically applicable immune tolerance regimen of combined bone marrow and heart transplants in mice results in mixed chimerism and graft acceptance. The conditioning regimen of lymphoid irradiation and anti-T cell antibodies changed the balance of cells in the lymphoid tissues to create a tolerogenic microenvironment favoring the increase of natural killer T (NKT) cells, CD4+ CD25+ regulatory T cells and Gr-1+ CD11b+ myeloid-derived suppressor cells (MDSCs), over conventional T cells (Tcons). The depletion of MDSCs abrogated chimerism and tolerance, and add back of these purified cells was restorative. The conditioning regimen activated the MDSCs as judged by the increased expression of arginase-1, IL-4Rα and programmed death ligand 1, and the activated cells gained the capacity to suppress the proliferation of Tcons to alloantigens in the mixed leukocyte reaction. MDSC activation was dependent on the presence of host invariant NKT cells. The conditioning regimen polarized the host invariant NKT cells toward IL-4 secretion, and MDSC activation was dependent on IL-4. In conclusion, there was a requirement for MDSCs for chimerism and tolerance, and their suppressive function was dependent on their interactions with NKT cells and IL-4.

Keywords: Basic (laboratory) research/science; bone marrow/hematopoietic stem cell transplantation; immunosuppression/immune modulation; immunosuppressive regimens; induction; macrophage/monocyte biology: activation; tolerance: chimerism; tolerance: mechanisms.

© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

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Conflict of interest statement

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

Figures

Figure 1

Figure 1. An imbalance favoring NKT cells, Tregs and MDSCs over Tcon cells and upregulation of negative costimulatory receptors creates a tolerogenic microenvironment after TLI/ATS conditioning

(A) Experimental scheme: BALB/c hosts were given donor C57BL/6 neonatal heart transplants (HTX) on day 0, and ATS was injected i.p. on days 0, 2, 6, 8, and 10. Hosts were conditioned over 14 days with 10 doses of 240cGy each. Some hosts were depleted of CD11b+Gr-1+ cells (MDSCs) with a single dose of anti- Gr-1 mAb (280 μg i.p. on day13). On day 15, 50 × 106 C57BL/6 donor bone marrow cells were injected intravenously (BMT), and chimerism and heart graft survival were monitored thereafter. (B) Representative FACS patterns of showing changes in the phenotypes of CD4+ and CD8+ T cell subsets, CD4+CD25+Tregs, CD4+CD1dtetramer+NKT cells, and Gr-1+CD11b+ MDSCs in the spleen of untreated normal wild type mice, hosts conditioned with TLI and ATS without transplantation or with combined bone marrow and heart allografts (TX, SPLEEN) on day 5 after the completion of TLI. Boxes show percentages of each cell type. Arrows shows gating of cells from a box. (C) Mean (± SEM) percentages of CD11b+Gr-1+ cells, TCR αβ +, CD4+CD1dtetramer+NKT, and CD4+CD25+ cells in the spleen of untreated (UNT) controls, of TLI and ATS conditioned BALB/c (TLI/ATS), and of TLI and ATS conditioned and transplanted (TX) hosts (day 5 after injection of donor bone marrow cells) (Upper panels and lower panels respectively) (UNT, N= 8; TX, N= 10; TLI/ATS, N= 8).

Figure 2

Figure 2. Depletion of MDSCs abrogates tolerance to combined bone marrow and heart transplants

(A) Representative spleen FACS patterns of Gr-1 versus CD11b and F4/80 staining are shown in untreated wild type mice, transplanted wild type hosts without anti- Gr-1 mAb treatment (WT TX), and transplanted wild type hosts with anti- Gr-1 mAb treatment. Patterns are also shown for peripheral blood mononuclear cells (PBMCs). Two days prior to end of TLI conditioning (day 13), BALB/c hosts were given a single injection of anti- Gr-1 mAb antibody (280 μg/ml/mouse) intraperitoneally, and spleen cells were obtained for analysis 24 hours after the injection. (B) Heart allograft survival in untreated (Non depl, WT TX, N= 10), or treated with anti- Gr-1 mAb (day 13) (Anti- Gr-1 depl + No add back, N= 8) or treated with anti- Gr-1 mAb and given an add back of sorted (1 × 106) MDSCs (CD11b+Gr-1+) cells (Depl+MDSCs add back, N= 8) from TLI/ATS conditioned wild type mice at the time of donor bone marrow infusion. (C) Shows the mean percentage of donor type cells (H-2Kb+) among peripheral blood mononuclear cells (PBMC) at day 28 after injection of donor bone marrow cells in non depleted, anti- Gr-1 depleted alone, or anti- Gr-1 depleted hosts given an add back of sorted (1 × 106) MDSCs cells. Data are from 3 independent experiments. * p< 0.05, ** p< 0.01, *** p< 0.001

Figure 3

Figure 3. NKT cells regulate Ly6G expression on MDSCs after transplantation

(A) Representative FACs staining of Ly6G versus Ly6C among CD11b+Gr-1+ cell subsets (Gr-1 hi vs Gr-1lo) in the spleen of untreated hosts (UNT WT, N= 12), or in wild type (WT TX, N= 10) or CD1d−/− hosts (CD1d−/− TX, N= 12) after conditioning and transplant. Four subgroups of Gr-1hi or Gr-1lo CD11b+ cells were enclosed in boxes labeled 1, 2, 3, and 4 with percentages shown; Ly6GhiLy6Chi (1), Ly6GhiLy6Clo (2), Ly6GloLy6Clo (3), and Ly6GloLy6Chi (4). (B) Bar graphs showing mean (± SEM) percentages of Ly6G+ Ly6C+ cell subgroups in boxes 1–4 among Gr-1hi cells. (C) Bar graphs show among Gr-1lo cells.

Figure 4

Figure 4. Upregulation of PDL1, Arginase-1, and IL-4Rα on MDSCs after transplantation is dependent on NKT cells

(A–C) Representative histogram plots showing the expression of PDL1, arg-1 (arginase-1), and IL-4Rα (interleukin- 4 receptor alpha) on gated CD11b+Gr-1+ cells from untreated wild type mice (UNT WT, N= 8), or transplanted wild type mice (WT TX, N= 10), or transplanted CD1d−/− mice (CD1d−/− TX, N= 12) 12 days after bone marrow infusion. (D –F) Bar graphs showing means (± SEM) of mean fluorescence intensity (MFI) measurements of PDL-1, arg-1, and IL-4Rα on CD11b+Gr-1+ cells in spleen.

Figure 5

Figure 5. The increase in MDSCs after transplantation is transient

(A–B) The mean (± SEM) percentages of CD11b+Gr-1hi and CD11b+Gr-1lo in the spleen (A) and among PBMCs (B) of TLI/ATS conditioned and transplanted wild type hosts at different time points (CD11b+Gr-1hi, N= 15; CD11b+Gr-1lo, N= 15). Data represents 5 independent experiments.

Figure 6

Figure 6. MDSC suppression of alloreactivity in vitro is dependent on NKT cells and IL-4

(A) Representative FACS patterns showing the CD11b+Gr-1+ enrichment and sorting for assay of in vitro suppression in the mixed leukocyte reactions (MLR). (B) Representative CFSE histogram plots showing CD4+ and CD8+ T cell proliferation in the presence or absence of sorted BALB/c MDSCs (2 × 105) from untreated wild type mice, N= 8 (WT T/A), TLI and ATS conditioned wild type mice N=8 (UNT WT), or TLI and ATS conditioned CD1d−/− mice, N= 10 (CD1d−/− T/A), or TLI and ATS conditioned Jα18−/− mice, N= 8 (Jα18−/− T/A), or TLI and ATS conditioned IL-4−/− mice, N= 5 (IL-4−/− T/A) or TLI and ATS conditioned IL-4−/− mice given an intravenous injection of 0.5 × 106 wild type NKT cells on day 15 after completion of TLI, N= 5 (IL-4−/− T/A + NKT) in day 5 cultures. There were 2×105 C57BL/6 responders (R) cells and 4×105 BALB/c stimulator (S) cells in MLR cultures. The percentages of dull CFSE cells are shown. (C) Mean (± SEM) percentages of CFSE+ dull cells among gated CD4+ and CD8+ T cells after in vitro culture.

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