Invariant natural killer T cell-natural killer cell interactions dictate transplantation outcome after alpha-galactosylceramide administration - PubMed (original) (raw)

. 2009 Jun 4;113(23):5999-6010.

doi: 10.1182/blood-2008-10-183335. Epub 2009 Apr 15.

Edward S Morris, Kelli P A Macdonald, Kate A Markey, Helen M Morris, Neil C Raffelt, Tatjana Banovic, Alistair L J Don, Vanessa Rowe, Angela C Burman, Andrew D Clouston, Camile Farah, Gurdyal S Besra, Petr A Illarionov, Mark J Smyth, Steven A Porcelli, Geoffrey R Hill

Affiliations

Invariant natural killer T cell-natural killer cell interactions dictate transplantation outcome after alpha-galactosylceramide administration

Rachel D Kuns et al. Blood. 2009.

Abstract

Invariant natural killer T cells (iNKT cells) have pivotal roles in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects. iNKT cells are activated through their T-cell receptors by glycolipid moieties (typically the alpha-galactosylceramide [alpha-GalCer] derivative KRN7000) presented within CD1d. We investigated the ability of modified alpha-GalCer molecules to differentially modulate alloreactivity and GVL. KRN7000 and the N-acyl variant, C20:2, were administered in multiple well-established murine models of allogeneic stem cell transplantation. The highly potent and specific activation of all type I NKT cells with C20:2 failed to exacerbate and in most settings inhibited GVHD late after transplantation, whereas effects on GVL were variable. In contrast, the administration of KRN7000 induced hyperacute GVHD and early mortality in all models tested. Administration of KRN7000, but not C20:2, was found to result in downstream interleukin (IL)-12 and dendritic cell (DC)-dependent natural killer (NK)- and conventional T-cell activation. Specific depletion of host DCs, IL-12, or donor NK cells prevented this pathogenic response and the induction of hyperacute GVHD. These data demonstrate the ability of profound iNKT activation to modulate both the innate and adaptive immune response via the DC-NK-cell interaction and raise concern for the use of alpha-GalCer therapeutically to modulate GVHD and GVL effects.

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Figures

Figure 1

Figure 1

Differential effects of KRN7000 and C20:2 on GVHD and GVL. (A) Irradiated B6D2F1 mice received whole spleen from G-CSF–mobilized syngeneic donors, followed by control diluent, C20:2, or KRN7000 IP on days +1 and +4. Cohorts of mice received leukemic challenge with P815Luc+ or no challenge (control + P815Luc+, n = 8; C20:2 + P815Luc+, n = 5; KRN7000 + P815Luc+, n = 5; no leukemic challenge, n = 4-6). Overall survival by Kaplan-Meier analysis. Images are representative biphotonic bioluminescence images captured using the Xenogen system. Color scale represents signal intensity code (photons/s/cm2/sr). (B) Irradiated B6D2F1 mice received whole spleen from G-CSF–mobilized B6 allogeneic donors plus P815Luc+, followed by control diluent, C20:2, or KRN7000 on days +1 and +4 (control and C20:2, n = 24 per group; KRN7000, n = 16). Overall survival by Kaplan-Meier analysis; combined data from 3 identical experiments. *P < .001 KRN7000 vs control or C20:2. **P = .02 control vs C20:2. (C) GVHD mortality by Kaplan-Meier analysis for the experiments in panel B. (D) Leukemic mortality by Kaplan-Meier analysis for the experiments in panel B. ***P = .0379 (control vs C20:2).

Figure 2

Figure 2

KRN7000 induces hyperacute GVHD in multiple models. (A) Irradiated B6D2F1 received whole spleen from G-CSF–mobilized syngeneic B6D2F1 or allogeneic B6 donors, followed by control diluent, C20:2 at 2 μg or 5 μg per mouse, or KRN7000 at 0.5 μg or 2 μg per mouse IP on days +1 and +4. Overall survival by Kaplan-Meier analysis of combined data from 2 experiments (control allo, n = 14; C20:2 2 μg allo, n = 12; C20:2 5 μg allo, n = 16; KRN7000 2 μg allo, n = 10; KRN7000 0.5 μg allo, n = 16; KRN7000 syn, n = 8). **P < .001 for KRN7000 2 μg allo vs C20:2 2 μg and 5 μg allo. *P < .04 for KRN7000 0.5 μg allo vs C20:2 2 μg and 5 μg allo. (B) Irradiated B6 mice received whole or TCD spleen from G-CSF–mobilized BALB/c donors, followed by control diluent, C20:2, or KRN7000 IP on days +1 and +4. Overall survival by Kaplan-Meier analysis and mean ± SE of clinical scores in surviving mice at the end of each experiment (allo groups, n = 20 each; TCD group, n = 12). Combined data from 3 identical experiments. **P < .02 for C20:2 allo vs control and KRN7000 allo. *P < .05 for control allo clinical scores vs C20:2 and KRN7000 allo. (C) Leukemic relapse in irradiated B6 mice receiving whole or TCD spleen from G-CSF–mobilized BALB/c donors with the addition of host-type luciferase expressing EL4 leukemia, followed by control diluent or C20:2 IP on days +1 and +4. Relapse by Kaplan-Meier analysis (allo groups, n = 24 each; TCD group, n = 10). Combined data from 3 identical experiments. *P = .019 for C20:2 allo vs control allo. (D) Representative bioluminescence images of recipients shown in panel C at day 14 after transplant from 2 separate experiments. (E) Irradiated BALB/c mice received whole or TCD spleen from G-CSF–mobilized B10.D2 donors, followed by control diluent, C20:2, or KRN7000 IP on days +1 and +4. Overall survival by Kaplan-Meier analysis and mean (± SE) of clinical scores in surviving mice at the end of each experiment (allo groups, n = 21 each; TCD group, n = 13). Combined data from 3 identical experiments. **P < .02 for KRN7000 allo vs control and C20:2 allo. *P < .05 for C20:2 allo clinical scores vs control allo. (F) Donor T-cell engraftment (Ly9.1neg) at day 50 in transplant recipients from panel E. n = 4-5 in allo groups; n = 3 in the TCD group.

Figure 3

Figure 3

KRN7000 promotes proinflammatory cytokine secretion after allogeneic SCT. (A) Irradiated B6D2F1 mice received whole spleen from G-CSF–mobilized B6 donors, followed by control diluent, C20:2, or KRN7000 IP on days +1 and +4. IFN-γ, TNF, IL-5, IL-4, and IL-10 were determined in the sera at various times thereafter by cytometric bead array (n = 8-12 per group and time point, combined from a minimum of 2 experiments, except for IL-5 and IL-4 at 48 hours and IL-10, where n = 3-4). *P < .05 vs control diluent; #P < .03 vs C20:2 and control diluent. (B) Irradiated B6 mice received whole spleen from G-CSF–mobilized BALB/c donors, followed by control diluent, C20:2, or KRN7000 IP on days +1 and +4. IFN-γ, TNF, IL-5, IL-4, and IL-10 were determined in the sera at various times thereafter by cytometric bead array (n = 8-12 per group and time point combined from a minimum of 2 experiments, except IL-5 and IL-4 at 48 hours and IL-10, where n = 4). *P < .05 vs control diluent; #P < .03 vs C20:2 and control diluent; ##P < .001 vs C20:2 and control diluent.

Figure 4

Figure 4

KRN7000 promotes NK- and T-cell activation after SCT. (A) Irradiated B6 or B6D2F1 mice received whole spleen from G-CSF–mobilized BALB/c or B6 allogeneic donors, respectively, followed by control diluent, C20:2, or KRN7000 IP on days +1 and +4. Hepatic lymphocytes were pooled from 4 to 5 mice per group at days 2 and 5 after transplant, gradient-purified, and IFN-γ secretion by cell subsets determined by cytokine secretion assay. Representative plots from 3 experiments in each strain. (B) Irradiated B6D2F1 mice received whole spleen from G-CSF–mobilized B6 allogeneic donors, followed by control diluent, C20:2, or KRN7000 IP on day +1 and hepatic NK (NK1.1+CD3neg)- and T-cell (CD3posNK1.1neg) subsets enumerated 24 and 48 hours after glycolipid injection. Mean ± SE from 5 to 7 mice per group. *P < .05 vs control diluent; **P < .05 vs control diluent and C20:2. (C) Irradiated B6D2F1 mice received whole CFSE-labeled spleen from G-CSF–mobilized B6 allogeneic donors, followed by control diluent, C20:2, or KRN7000 IP on day +1. Proliferation of liver NK cells was determined by CFSE dilution on day +3. Proliferation indices of individual mice were determined by Modfit analysis and are shown as mean ± SE (n = 5 per group). Representative CFSE fluorescence-activated cell sorter (FACS) plots are also shown. *P < .02 vs control diluent. (D) Irradiated CD45.2+ B6D2F1 mice received whole spleen from CD45.1+ G-CSF–mobilized B6 allogeneic donors, followed by control diluent, C20:2, or KRN7000 IP on days +1 and +4. Donor engraftment (CD45.1+CD45.2neg) in NK- and T-cell subsets (gated as above) was determined at day +5 in hepatic lymphocytes pooled from 4 to 5 mice per group.

Figure 5

Figure 5

GVHD induction by KRN7000 is NK cell–dependent. (A) B6 donors received isotype control or anti-asialo GM1, as described in “Cellular depletion,” and specific NK-cell depletion of the graft confirmed before transplantation by FACS. (B) Irradiated B6D2F1 mice received G-CSF–mobilized spleen from B6 donors pretreated with isotype control or anti-asialo GM1 (corrected to transfer equivalent numbers of T cells to each group) or T-cell–depleted spleen, followed by control diluent or KRN7000 IP on days +1 and +4. (TCD, n = 5; all other groups, n = 21). Combined data from 3 experiments shown. Overall survival by Kaplan-Meier analysis. *P < .05 vs all other groups. Differences between all other T-cell–replete groups are not significant. (C) Irradiated B6 mice received whole spleen from G-CSF–mobilized BALB/c allogeneic donors, followed by control diluent, C20:2, or KRN7000 IP on day +1, and IL-12 levels were measured in sera 6 hours later (n = 8-12 per group combined from 2 experiments). Data expressed as mean ± SE; *P = .002 vs control; #P = .02 vs C20:2. (D) Irradiated B6.Jα18−/− mice received whole spleen from G-CSF–mobilized BALB/c.Jα18−/− donors, followed by control diluent, C20:2, or KRN7000 IP on day +1. IL-12 levels were measured in sera 6 hours later (n = 5 per group). Data are expressed as mean ± SE. (E) Irradiated B6.WT or B6.IL-12−/− mice received whole spleen from G-CSF–mobilized BALB/c allogeneic donors, followed by control diluent, C20:2, or KRN7000 IP on day +1, and IFN-γ levels were measured in sera 24 hours later (n = 8-14 per group, combined from 3 experiments). Data are expressed as mean ± SE; **P = .001, KRN7000 B6.WT vs B6.IL-12−/− recipients. (F) IFN-γ secretion assay on hepatic NK- and T-cell populations 24 hours after glycolipid administration in transplants, as described in panel E, one of 3 representative experiments shown.

Figure 6

Figure 6

Host DCs, IL-12, and IFN-γ are critical for the hyperacute GVHD induced by KRN7000. (A) Irradiated B6.WT or B6.IL-12−/− mice received whole spleen from G-CSF–mobilized syngeneic B6 or allogeneic BALB/c donors, followed by control diluent or KRN7000 IP on days +1 and +4. Overall survival by Kaplan-Meier analysis, combined data from 2 experiments (n = 4, syn KRN7000; n = 16, allo B6.WT groups; n = 13, allo KRN7000 B6.IL-12−/− group). *P < .005 allo KRN7000 B6.WT vs allo KRN7000 B6.IL-12−/− and allo control B6.WT. (B) Irradiated B6 mice received whole spleen from G-CSF–mobilized BALB/c donors, followed by control diluent, C20:2, or KRN7000 IP on day +1. DCs were sort-purified from spleens pooled from 4 mice per group 6 hours after glycolipid injection, and their IL12a expression relative to β_2m_ was determined by real-time PCR. (C) Irradiated B6.WT or B6.IFN-γR−/− recipients were transplanted with G-CSF–mobilized BALB/c grafts and KRN7000 or control diluent administered at day +1 (n = 6 per group). Numbers of host (H-2Dd−) DCs remaining in the spleen were elucidated 3 days after transplant. **P < .01 vs other groups. Combined data from 2 replicate experiments. (D) B6.IFN-γR−/−→B6.WT chimeras were transplanted with whole spleen from G-CSF–mobilized syngeneic B6 or allogeneic BALB/c donors, followed by control diluent or KRN7000 on days +1 and +4. Overall survival by Kaplan-Meier analysis (n = 4, syn group; n = 8, allo groups). P = .33 between allo groups. (E) Irradiated B6.WT or B6.CD11c.DTR mice were injected with diphtheria toxin IP, transplanted with whole spleen from G-CSF–mobilized BALB/c donors 1 hour later, and given KRN7000 IP on day +1 (24 hours later). Effective depletion of splenic DCs was confirmed 24 hours after KRN7000 administration. Representative FACS plots are shown, with the mean ± SE of residual DC percentages given alongside (n = 9-10). (F) Serum IL-12 was determined at 6 hours, and serum IFN-γ at 24 hours, after KRN7000 injection, in transplant recipients from panel E. Data show mean ± SE of combined data from 2 identical experiments (n = 8-10). **P < .001. (G) Hepatic lymphocytes from transplants described in panel E were pooled from 5 mice per group at 24 hours after glycolipid injection and gradient-purified, and IFN-γ secretion by cell subsets was determined by cytokine secretion assay. One of 2 representative experiments is shown.

References

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