Research ArticleTRANSPLANTATION

Donor SIRPα polymorphism modulates the innate immune response to allogeneic grafts

See allHide authors and affiliations

Science Immunology  23 Jun 2017:
Vol. 2, Issue 12, eaam6202
DOI: 10.1126/sciimmunol.aam6202
  • Fig. 1 Magnitude of the host innate alloresponse is influenced by the genetic background of the donor.

    Bone marrow plugs from different donors were transplanted individually under the kidney capsules of separate mice (A) or simultaneously in the contralateral kidneys of the same mouse (B and C). All recipients were BRG unless otherwise stated. Donor strains are shown on the x axis. The number of recipient mono-DC infiltrating the grafts was determined 1 week later as a measure of an innate alloresponse. (A) Responses of BRG recipients to allografts from six common (DBA.2, NOR, C3H, FVB, BALB/c, and NOD) and three wild-derived (Pahari, WSB, and CAST) inbred strains. Statistical significance shown is relative to B6. (B) NOD grafts transplanted to syngeneic NRG mice (NOD to NRG) elicit a much weaker response than NOD grafts transplanted to allogeneic BRG mice (NOD to BRG). (C and D) Innate responses elicited by grafts from distinct donors transplanted under the contralateral kidney capsules of the same BRG recipient (C) or CRG recipient (D). n = 5 to 6 mice per group per experiment. Experiments were performed once or twice. Each dot represents an individual biological replicate. Bars are means. **P < 0.01, ****P < 0.0001; ns, not significant (unpaired two-tailed t test).

  • Fig. 2 Innate alloresponse is determined by a single Mendelian locus in the donor not linked to the Mhc.

    Bone marrow plugs were transplanted individually under the kidney capsules of separate mice. All recipients were BRG except in (D) where they were CRG. Donor strains are shown on the x axis. Recipient mono-DCs in grafts were measured as in Fig. 1. (A) Responses of BRG recipients to grafts from parental NRG and BRG strains or to grafts from (BRG × NRG)F1 and F2 generations. All donors and recipients were on the Rag2−/−γc−/− background. (B) Effect of donor-recipient non-MHC mismatch (BALB.B grafts) or MHC mismatch (B6.C grafts) on the innate alloresponse of BRG recipients. (C) Effects of donor MHC I deficiency (NOD.scid.b2m−/− grafts) on the innate alloresponse of BRG recipients. (D) Effect of MHC II deficiency (B6.MHCII−/− grafts) on the innate alloresponse of CRG recipients. n = 5 to 6 mice per group per experiment, except in F2 experiment (n = 30 mice transplanted in two separate batches). Experiments were performed once or twice. Each dot represents an individual biological replicate. Bars are means. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired two-tailed t test).

  • Fig. 3 Innate alloresponse maps to a small genomic region in the donor containing the Sirpa gene.

    Bone marrow plugs were transplanted individually under the kidney capsules of separate mice (B and D) or simultaneously in the contralateral kidneys of the same mouse (C). All recipients were BRG. Donor strains are shown on the x axis. Recipient mono-DCs in grafts were measured as in Fig. 1. (A) Mouse chromosome 2 region that differs between R7 and R12 congenic strains and contains the Sirpa locus. β2m, β2-microglobulin. (B) Comparison of innate alloresponses elicited by NOR or NOD grafts to those elicited by congenic donors that carry the NOR (R7) or NOD (R12) Sirpa allele. (C) Innate alloresponses elicited by grafts from NOD and R12 congenic mice transplanted into the same BRG recipient. (D) Grafts from BRG mice congenic for the NOD Sirpa allele (BRGS) induce the same alloresponse as those from NRG mice in BRG recipients. n = 6 to 7 mice per group per experiment. Except for the BRGS group, experiments were performed twice. Each dot represents an individual biological replicate. Bars are means. **P < 0.01, ****P < 0.0001 (unpaired two-tailed t test).

  • Fig. 4 Donor SIRPα binding to recipient CD47 is required for triggering the innate alloresponse.

    (A) NOD grafts were transplanted to BRG mice that received either hCD47-Fc, a decoy protein that binds to the NOD SIRPα variant and prevents it from binding to mouse CD47, or isotype control Fc protein (hIgG1 Fc). Recipient mono-DCs in grafts were measured as in Fig. 1. Control untreated recipients (None) from previous experiments are shown for comparison. (B) NOD or BALB/c grafts were transplanted to BRG CD47−/− mice, and the innate alloresponse was compared with that of CD47-sufficient (BRG) recipients from previous experiments. (C and D) Grafts from wild-type (B6 and BALB/c) or mutant (B6 mSIRP-A) mice, which lack the intracellular signaling domain of SIRPα, were transplanted to CRG (C) or BRG (D) recipients to test the effect of removing SIRPα signaling from donor cells on the host response. (E) Proportion of mature (MHCIIhiCD80+) recipient or donor mono-DCs in NRG allografts transplanted to BRG mice. (F) Absolute number of mature host mono-DC in allogeneic (NRG) versus syngeneic (BRG) grafts transplanted to BRG recipients. n = 6 mice per group per experiment. Experiments were performed once or twice. Each dot represents an individual biological replicate. Bars are means. *P < 0.05, **P < 0.01, ****P < 0.0001 (unpaired two-tailed t test).

  • Fig. 5 Mouse SIRPα amino acid polymorphism modulates binding to CD47.

    (A) Amino acid variability in mouse SIRPα protein based on alignment of sequences from 19 mouse strains. Variability was calculated for each position along the sequence and aligned against the denoted SIRPα protein domains. IgV domain (red box) had the highest frequency of amino acid polymorphisms (number of vertical blue lines) and contained polymorphisms with the greatest degree of variability (height of vertical blue lines). (B) Phylogram representation of SIRPα IgV domain amino acid variation among the 19 mouse strains. Blue, common inbred strains; red, wild-derived inbred strains; bold, mouse strains tested in Fig. 1; scale, proportion of amino acid substitutions; circles, mouse strains that share similar or identical CD47 IgV domains. (C) Binding of mCD47-Fc to splenic monocytes (LinCD11b+CD11cF4/80 cells) from NOD and CAST compared with B6, BALB/c, and C3H mice. Top: Serial dilution of mCD47-Fc. (D) SIRPα expression on monocytes from all strains tested. Histograms are representative of two to three biological replicates from two independent experiments (unpaired two-tailed t test).

  • Fig. 6 Donor SIRPα polymorphism modulates monocyte proliferation.

    (A) BRG mice were immunized intraperitoneally with irradiated allogeneic (BALB/c) or syngeneic (B6) splenocytes. Spleen cells were analyzed 1 week later. Mice were pulsed with EdU 1 hour before spleen harvest. Representative flow plots of EdU staining of myeloid cell populations are shown. Arrows indicate EdU+ cell population. cDC, conventional DC. (B) BRG or BRG CD47−/− mice were stimulated as in (A) with splenocytes from mouse strains shown on the x axis, and the proportion of EdU+ cells in Ly6Chi monocyte subset was determined and divided by the proportion of EdU+ cells in mice immunized with syngeneic (B6) splenocytes to determine the proliferation index. n = 3 mice per group per experiment × 2 experiments except for B6 group where total n = 9. Each dot represents an individual biological replicate. Bars are means. *P < 0.05, ***P < 0.001 (unpaired two-tailed t test).

  • Fig. 7 Innate alloactivation is triggered by mismatch between donor and recipient SIRPα.

    (A) Top: Balance between activating signals (+) mediated by CD47 and inhibitory signals (−) mediated by SIRPα in recipient monocytes in the syngeneic transplantation setting. Bottom: Imbalance in the allogeneic setting when donor SIRPα (red) has greater affinity to CD47 than recipient SIRPα (green). The net result of this imbalance is recipient monocyte differentiation to mono-DC. (B) Imbalance is created if the syngeneic graft lacks CD47. Bar graph shows results from B6 CD47−/− and B6 wild-type grafts transplanted to separate BRG recipients. Graft-infiltrating mono-DCs were quantified as in Fig. 1. (C) Reversing direction of allotransplantation, such that donor SIRPα has weaker binding to CD47 than recipient SIRPα, inhibits the innate alloresponse. Experimental data are shown in bar graph. n = 5 to 6 mice per group per experiment. Experiments were performed once or twice. *P < 0.05, ****P < 0.0001 (unpaired two-tailed t test).

Supplementary Materials

  • Supplementary Materials

    Supplementary Material for:

    Donor SIRPα polymorphism modulates the innate immune response to allogeneic grafts

    Hehua Dai, Andrew J. Friday, Khodor I. Abou-Daya, Amanda L. Williams, Steven Mortin-Toth, Matthew L. Nicotra, David M. Rothstein, Warren D. Shlomchik, Takashi Matozaki, Jeffrey S. Isenberg, Martin H. Oberbarnscheidt, Jayne S. Danska,* Fadi G. Lakkis*

    *Corresponding authors. Email: jayne.danska{at}sickkids.ca (J.S.D.); lakkisf{at}upmc.edu (F.G.L.)

    Published 23 June 2017, Sci. Immunol. 2, eaam6202 (2017)
    DOI: 10.1126/sciimmunol.aam6202

    This PDF file includes:

    • Fig. S1. Gating strategy for identifying recipient-derived graft-infiltrating mono-DCs.
    • Fig. S2. Alignment of predicted SIRPα protein sequences from 19 mouse strains.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data file.

    Files in this Data Supplement:

Navigate This Article