Host natural killer T cells induce an interleukin-4-dependent expansion of donor CD4+CD25+Foxp3+ T regulatory cells that protects against graft-versus-host disease - PubMed (original) (raw)

Host natural killer T cells induce an interleukin-4-dependent expansion of donor CD4+CD25+Foxp3+ T regulatory cells that protects against graft-versus-host disease

Asha B Pillai et al. Blood. 2009.

Abstract

Although CD4(+)CD25(+) T cells (T regulatory cells [Tregs]) and natural killer T cells (NKT cells) each protect against graft-versus-host disease (GVHD), interactions between these 2 regulatory cell populations after allogeneic bone marrow transplantation (BMT) have not been studied. We show that host NKT cells can induce an in vivo expansion of donor Tregs that prevents lethal GVHD in mice after conditioning with fractionated lymphoid irradiation (TLI) and anti-T-cell antibodies, a regimen that models human GVHD-protective nonmyeloablative protocols using TLI and antithymocyte globulin (ATG), followed by allogeneic hematopoietic cell transplantation (HCT). GVHD protection was lost in NKT-cell-deficient Jalpha18(-/-) hosts and interleukin-4 (IL-4)(-/-) hosts, or when the donor transplant was Treg depleted. Add-back of donor Tregs or wild-type host NKT cells restored GVHD protection. Donor Treg proliferation was lost in IL-4(-/-) hosts or when IL-4(-/-) mice were used as the source of NKT cells for adoptive transfer, indicating that host NKT cell augmentation of donor Treg proliferation after TLI/antithymocyte serum is IL-4 dependent. Our results demonstrate that host NKT cells and donor Tregs can act synergistically after BMT, and provide a mechanism by which strategies designed to preserve host regulatory cells can augment in vivo donor Treg expansion to regulate GVHD after allogeneic HCT.

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Figures

Figure 1

Figure 1

Protection against GVHD after TLI/ATS conditioning is dependent on CD4+ T cells in the donor transplant. (A) Wild-type (WT) BALB/c host survival after 800 cGy TBI/ATS (n = 10) versus TLI/ATS (n = 8), followed by transplantation of 50 × 106 whole bone marrow cells and 60 × 106 splenocytes from wild-type C57BL/6 donors. Each group represents survival combined from 2 separate experiments. (B) Wild-type BALB/c host survival after TLI/ATS, followed by transplantation of 50 × 106 whole bone marrow and 60 × 106 splenocytes from CD4 T cell–deficient donors (CD4−/−; n = 10), CD8 T cell–deficient donors (CD8−/−; n = 10), invariant NKT cell–deficient donors (Jα18−/−; n = 8), or WT donors (n = 8). Each group represents survival combined from 2 to 3 separate experiments. (C) Mean body weight (± SE) at serial time points after transplantation in wild-type BALB/c hosts receiving either 800 cGy TBI/ATS or TLI/ATS and 50 × 106 whole bone marrow and 60 × 106 splenocytes from wild-type donors, or wild-type TLI/ATS-conditioned hosts given CD4−/− donor bone marrow and splenocyte transplants. + indicates analysis was stopped when 2 hosts remained in the group. (D) Mean (± SE) histopathologic GVHD scores of colon, skin, and liver at day 6 from 3 groups of hosts from panel C. n = 5-8 for all groups. (E) Microscopic examination of hematoxylin/eosin-stained sections of colon at day 6 in 1 representative host from each group shown in panels C and D. WT BALB/c host given TBI/ATS with WT donor cells shows evidence of inflammatory infiltrate (*) and loss of normal crypt architecture in the colon, with separation of crypts by inflammation, lifting off of crypts from the basement membrane, and multiple areas of apoptotic crypt nuclei (→). WT BALB/c host given TLI/ATS conditioning with WT donor cells shows normal appearance. WT host given TLI/ATS and CD4−/− donor cells shows inflammatory infiltrate (*) and loss of normal crypt architecture in the host colon, with separation of crypts by inflammation and lifting off of crypts from the basement membrane. Specimens shown are at 300× final magnification.

Figure 2

Figure 2

Protection against GVHD after TLI/ATS is dependent on both donor CD4+CD25+ Tregs and host invariant NKT cells. (A) Representative FACS patterns of the donor transplant inoculum before (PRE) and after (POST) CD25+ T-cell depletion. Percentage of CD4+CD25+ T cells among gated CD4+TCRαβ+ T cells is shown enclosed in boxes. (B) Survival of wild-type BALB/c hosts after TLI/ATS conditioning, followed by transplantation of either undepleted (UNDEP DONOR, WT HOST; n = 8) or CD25+ T cell–depleted (CD25-DEP DONOR, WT HOST; n = 9) donor grafts consisting of 50 × 106 bone marrow and 60 × 106 splenocytes from wild-type C57BL/6 donors. Also shown is survival of NKT cell–deficient Jα18−/− (n = 10) hosts conditioned with TLI/ATS and injected with 50 × 106 untreated whole bone marrow and 60 × 106 untreated splenocytes from wild-type C57BL/6 donors (UNT DONOR, Jα18−/− HOST). Each group represents survival combined from 2 to 3 separate experiments. (C) Mean body weight (± SE) of TLI/ATS-conditioned wild-type BALB/c hosts receiving either undepleted or CD25+ T cell–depleted donor grafts or TLI/ATS-conditioned NKT cell–deficient Jα18−/− hosts receiving untreated donor grafts shown in panels A and B, at serial time points after transplantation. + indicates analysis was stopped when 2 hosts remained in the group. (D) Mean (± SE) histopathologic GVHD scores of colon, skin, and liver at day 6 from wild-type (WT) hosts given untreated or CD25-depleted transplants or Jα18−/− hosts given untreated transplants. n = 5 for all groups. (E) Microscopic examination of hematoxylin/eosin-stained sections of colon at day 6 in a representative host from each group shown in panel C. Comparison of WT BALB/c host given TLI/ATS and an undepleted transplant and WT BALB/c host given TLI/ATS and a CD25+ T cell–depleted transplant demonstrates severe crypt apoptosis (→), inflammatory infiltrates (*), and crypt atrophy with loss of goblet cells in the latter compared with the former. NKT cell–deficient Jα18−/− host given TLI/ATS and untreated transplant reveals severe crypt apoptosis (→), inflammatory infiltrates (*), and crypt atrophy with loss of goblet cells. All micrographs are at 300× final magnification.

Figure 3

Figure 3

Host NKT cells facilitate the early proliferation of chimeric donor CD4+Foxp3+ Tregs. (A) Representative FACS analyses of CD4 versus intracellular Foxp3 staining at day 6 of gated H-2Kb+CD4+ splenocytes from normal untreated (UNT) C57BL/6 control mice and from the following hosts: 800 cGy TBI/ATS wild-type BALB/c hosts or TLI/ATS wild-type BALB/c hosts given untreated transplants, TLI/ATS wild-type BALB/c hosts given CD25 T-cell–depleted transplants, TLI/ATS Jα18−/− BALB/c hosts given untreated transplants, TLI/ATS Jα18−/− BALB/c hosts given 0.5 × 106 sorted wild-type BALB/c NKT cells with untreated donor cell transplants, and TLI/ATS Jα18−/− BALB/c hosts given 0.5 × 106 sorted BALB/c NKT cells with CD25+ T-cell–depleted donor cell transplants. Percentage of CD4+Foxp3+ cells among gated CD4+ donor cells is shown enclosed in boxes. (B) Mean (± SE; × 105) absolute number of H-2Kb+CD4+CD25+Foxp3+ cells isolated from spleen at day 6 in groups of transplanted BALB/c hosts from panel A. n = 5-10 per group. (C) Representative FACS plots showing CD4 versus intracellular Foxp3 staining in gated H-2Kb+CD4+ cells from MLN and colon of wild-type BALB/c hosts conditioned with either TBI/ATS or TLI/ATS at day 6 after injection of untreated donor cell transplants. Percentage of Foxp3+ cells is shown. (D) Representative histograms showing CFSE-staining intensity versus cell number for gated CD45.1+CD4+TCRαβ+Foxp3+ cells and CD45.1+CD8+TCRαβ+ cells from the pretransplant donor cell inoculum at day 0 (DONOR C57BL/6 [PRE]) and cells isolated from spleen of transplanted TLI/ATS-conditioned hosts at day 6. SYN, Wild-type CD45.2 C57BL/6 TLI/ATS-conditioned hosts given C57BL/6 CD45.1+ splenocytes and CD45.2+ marrow cells; ALLO, wild-type CD45.2 BALB/c TLI/ATS-conditioned hosts given C57BL/6 CD45.1+ splenocytes and CD45.2+ marrow cells. n = 2-3 pooled animals per group. Percentage of cells with 2 or more divisions is shown. (E) Mean (± SE; %) gated CD45.1+CD4+TCRαβ+Foxp3+ cells and CD45.1+CD8+TCRαβ+ CFSElow cells in the groups shown in panel D. Mean of 2 to 3 experiments. n = 2-3 pooled animals per group. Determination of CFSElow cells is detailed in “Methods.”

Figure 4

Figure 4

Host NKT cells and donor CD4+CD25+ T cells inhibit accumulation of donor CD8+ T cells in TLI/ATS-conditioned host tissues. (A) Representative analyses of gated donor H-2Kb+TCRαβ+ cells, showing CD4 versus CD8 in spleen, MLN, and colon at day 6 after bone marrow and splenocyte transplantation in TBI/ATS-conditioned BALB/c hosts given wild-type C57BL/6 transplants and in TLI/ATS-conditioned hosts in the following groups: wild-type BALB/c hosts given untreated C57BL/6 transplants, wild-type BALB/c hosts given CD25+ T cell–depleted C57BL/6 transplants, invariant NKT cell–deficient Jα18−/− BALB/c hosts given untreated C57BL/6 transplants, and Jα18−/− BALB/c hosts receiving 0.5 × 106 purified wild-type BALB/c NKT cells 4 hours before either untreated or CD25 T cell–depleted C57BL/6 transplants. Percentages of CD4 or CD8 T cells are shown enclosed in boxes. (B) Comparison of mean absolute number (± SE; ×106) of H-2Kb+CD8+TCRαβ+ cells isolated at day 6 from the colon of groups shown in panel A. n = 4-8 per group.

Figure 5

Figure 5

Host IL-4 is required for protection against GVHD, NKT-cell regulatory activity, and proliferation of donor Tregs after transplantation. (A) Wild-type (WT) versus IL-4−/− BALB/c host survival after TLI/ATS, followed by transplantation of 50 × 106 bone marrow and 60 × 106 splenocytes from wild-type C57BL/6 donors (IL-4−/−, n = 10; WT, n = 7). Each group represents survival combined from 2 to 3 separate experiments. (B) Mean body weight (± SE) at serial time points after transplantation in hosts shown in panel A. + indicates analysis was stopped when 2 hosts remained in the group. (C) Microscopic examination of hematoxylin/eosin-stained sections of colon at day 6 in one representative host from each group shown in panels A,B. IL-4−/− BALB/c host given TLI/ATS with WT donor cells shows evidence of inflammatory infiltrate (*) in colonic crypts and multiple areas of apoptotic crypt nuclei (→). WT BALB/c host given TLI/ATS conditioning with WT donor cells shows normal appearance. Specimens shown are at ×300 final magnification. (D) Representative FACS analyses of CD4 versus intracellular Foxp3 staining of gated H-2Kb+CD4+ splenocytes (top panels) and CD4 versus CD8 staining of gated H-2Kb+TCRαβ+ splenocytes (bottom panels) 6 days after transplantation of untreated donor cells into TLI/ATS-treated IL-4−/− BALB/c hosts with and without injections of 0.5 × 106 sorted wild-type BALB/c NKT cells. NKT cells were injected 4 hours before donor cell transplantation. (E) Mean (± SE; ×105) absolute number of H-2Kb+CD4+CD25+Foxp3+ cells isolated from spleen at day 6 from groups of transplanted BALB/c hosts from Figure 3D. n = 4-6 per group. Values for additional control wild-type BALB/c hosts given untreated donor cell transplants are shown for comparison (n = 5). (F) Representative histograms from 1 of 2 to 3 similar experiments showing CFSE-staining intensity versus cell number for gated CD45.1+CD4+TCRαβ+Foxp3+ cells and CD45.1+CD8+TCRαβ+ cells from the pretransplant donor cell inoculum (DONOR C57BL/6 (PRE)) and cells isolated from spleen of transplanted TLI/ATS-conditioned hosts at day 6. SYN, Wild-type CD45.2 C57BL/6 TLI/ATS-conditioned hosts given C57BL/6 CD45.1+ splenocytes and CD45.2+ marrow cells; IL-4−/− CD45.2 BALB/c TLI/ATS-conditioned hosts were given no NKT cells (IL-4−/− BALB/c HOST), 0.5 × 106 sorted wild-type BALB/c NKT cells (IL-4−/− BALB/c HOST + WT NKT), or 0.5 × 106 sorted IL-4−/− BALB/c NKT cells (IL-4−/− BALB/c HOST + IL-4−/− NKT) 4 hours before untreated donor cell transplantation. n = 2-3 pooled animals per group. (G) Mean ± SD (%) gated CD45.1+CD4+TCRαβ+Foxp3+ cells and CD45.1+CD8+TCRαβ+ CFSElow cells in the groups shown in panel F. Mean of 2 to 3 experiments. n = 2-3 pooled animals per group.

Figure 6

Figure 6

Donor Tregs added to CD4−/− donor transplants protect against donor CD8+ T-cell infiltration in the colon. (A) Mean absolute number (± SD) H-2Kb+TCRαβ+CD8+ cells recovered from MLN of wild-type BALB/c hosts given 800 cGy TBI/ATS and wld-type C57BL/6 donor bone marrow and splenocytes, or TLI/ATS and transplantation of 50 × 106 bone marrow and 60 × 106 splenocytes from CD4−/− C57BL/6 donors with and without addition of 1.5 × 106 purified wild-type C57BL/6 CD4+CD25+ Tregs (n = 3, TBI/ATS; n = 5, CD4−/− TLI/ATS; n = 4, CD4−/− TLI/ATS + Treg). Each group represents data combined from 2 separate experiments. (B) FACS analyses of gated donor H-2Kb+TCRαβ+ cells, showing CD4 versus CD8 in MLN at day 6 after bone marrow and splenocyte transplantation in 1 representative host from each group shown in panel A. (C) Mean absolute number (± SD) H-2Kb+TCRαβ+CD8+ cells recovered from colons of wild-type BALB/c hosts shown in panel A. (D) FACS analyses of gated donor H-2Kb+TCRαβ+ cells, showing CD4 versus CD8 in colon at day 6 after bone marrow and splenocyte transplantation in 1 representative host from each group shown in panel C.

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