Donor SIRPα polymorphism modulates the innate immune response to allogeneic grafts

Abstract

Mice devoid of T, B, and natural killer (NK) cells distinguish between self and allogeneic nonself despite the absence of an adaptive immune system. When challenged with an allograft, they mount an innate response characterized by accumulation of mature, monocyte-derived dendritic cells (DCs) that produce interleukin-12 and present antigen to T cells. However, the molecular mechanisms by which the innate immune system detects allogeneic nonself to generate these DCs are not known. To address this question, we studied the innate response of Rag2^-/- γc^-/- mice, which lack T, B, and NK cells, to grafts from allogeneic donors. By positional cloning, we identified that donor polymorphism in the gene encoding signal regulatory protein α (SIRPα) is a key modulator of the recipient's innate allorecognition response. Donors that differed from the recipient in one or both Sirpa alleles elicited an innate alloresponse. The response was mediated by binding of donor SIRPα to recipient CD47 and was modulated by the strength of the SIRPα-CD47 interaction. Therefore, sensing SIRPα polymorphism by CD47 provides a molecular mechanism by which the innate immune system distinguishes between self and allogeneic nonself independently of T, B, and NK cells.

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, C). All recipients were B6.Rag2^−/−γc^−/− (BRG) unless otherwise stated. Donor strains are shown on the x-axis. Number of recipient monocyte-derived DCs (Mono-DC) infiltrating the grafts was determined 1 wk later as a measure of an innate alloresponse. (A) Responses of BRG recipients to allografts from 6 common (DBA.2, NOR, C3H, FVB, BALB/c, & NOD) and 3 wild-derived (Pahari, WSB, and CAST) inbred strains. Statistical significance shown is relative to B6. (B) NOD grafts transplanted to syngeneic NOD.Rag2^−/−γc^−/− mice (NOD to NRG) elicit a much weaker response than NOD grafts transplanted to allogeneic BRG mice (NOD to BRG). (C, D) Innate responses elicited by grafts from distinct donors transplanted under the contralateral kidney capsules of the same BRG recipient (C) or BALB/c.Rag2^−/−γc^−/−(CRG) recipient (D). n = 5–6 mice/group/experiment. Experiments were performed once or twice. Each dot represents an individual biological replicate. Bars are means. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns = not significant (unpaired, two-tailed t-test). BRG = B6.Rag2^−/−γc^−/−; NRG = NOD.Rag2^−/−γc^−/−; CRG = BALB/c.Rag2^−/−γc^−/−.

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 B6.Rag2^−/−γc^−/− (BRG) except in D where they were BALB/c.Rag2^−/−γc^−/− (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 (BRGxNRG)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 MHCI-deficiency (NOD.scid.b2m−/− grafts) on the innate alloresponse of BRG recipients. (D) Effect of MHCII-deficiency (B6.MHCII−/− grafts) on the innate alloresponse of CRG recipients. n = 5–6 mice/group/experiment, except in F2 experiment (n = 30 mice transplanted in 2 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; ns = not significant (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, D), or simultaneously in the contralateral kidneys of the same mouse (C). All recipients were B6.Rag2^−/−γc^−/−(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 NOD.NOR-R7 (R7) and NOD.NOR-R12 (R12) congenic strains and contains the Sirpa locus. (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 grafts from NOD.Rag2^−/−γc^−/− (NRG) mice in BRG recipients. n = 6–7 mice/group/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; ns = not significant (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 B6.Rag2^−/−γc^−/− (BRG) mice that received either hCD47-Fc, a decoy protein that binds to the NOD SIRPα variant and prevents it 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 prior experiments are shown for comparison. (B) NOD or BALB/c grafts were transplanted to B6.Rag2^−/−γc^−/−CD47^−/− mice (BRG CD47−/−) and the innate alloresponse was compared to that of CD47-sufficient (BRG) recipients from prior experiments. (C, D) Grafts from wildtype (B6 and BALB/c) or mutant (B6 mSIRP-A) mice, which lack the intracellular signaling domain of SIRPα, were transplanted to BALB/c.Rag2^−/−γc^−/− (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 (MHCII^hiCD80⁺) recipient or donor mono-DCs in NOD.Rag2^−/−γc^−/− (NRG) allografts transplanted to BRG mice. (F) Absolute number of mature host mono-DC in allogeneic (NRG) vs syngeneic (BRG) grafts transplanted to BRG recipients. n = 6 mice/group/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; ns = not significant (unpaired, two-tailed t-test).

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

(A) Amino acid (AA) 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 (shaded in red) had the highest frequency of AA 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 AA variation among the 19 mouse strains. Blue font = common inbred strains; red font = wild-derived inbred strains; bold font = mouse strains tested in Fig. 1; scale = proportion of AA substitutions; circles = mouse strains that share similar or identical CD47 IgV domains. (C) Binding of mCD47-Fc to splenic monocytes (Lin⁻CD11b⁺CD11c⁻F4/80⁻ cells) from NOD and CAST compared to B6, BALB/c, and C3H mice. Serial dilution of mCD47-Fc shown on top. (D) SIRPα expression on monocytes from all strains tested. Histograms are representative of 2–3 biological replicates from 2 independent experiments (unpaired, two-tailed t-test).

Fig. 6. Donor SIRPα polymorphism modulates monocyte proliferation

(A) B6.Rag2^−/−γc^−/− (BRG) mice were immunized i.p. with irradiated allogeneic (BALB/c) or syngeneic (B6) splenocytes. Spleen cells were analyzed 1 wk later. Mice were pulsed with EdU 1 hr prior to spleen harvest. Representative flow plots of EdU staining of myeloid cell populations are shown. Arrows indicate EdU+ cell population. (B) BRG or BRG CD47−/− mice were stimulated as in A with splenocytes from mouse strains shown on x-axis and the proportion of EdU+ cells in Ly6C^hi monocyte subset was determined and divided by proportion of EdU+ cells in mice immunized with syngeneic (B6) splenocytes to determine the Proliferation Index. n = 3 mice/group/experiment x 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; ns = not significant (unpaired, two-tailed t-test).

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

(A) Upper panel depicts balance between activating signals (+) mediated by CD47 and inhibitory signals (−) mediated by SIRPα in recipient monocytes in the syngeneic transplantation setting. Lower panel depicts the imbalance in the allogeneic setting when donor SIRPα (red) has greater affinity to CD47 than recipient SIRPα (green). 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 wildtype grafts transplanted to separate B6.Rag2^−/−γc^−/− (BRG) recipients. Graft-infiltrating mono-DC 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. NRG = NOD.Rag2^−/−γc^−/−. Experimental data are shown in bar graph. n = 5–6 mice/group/experiment. Experiments were performed once or twice. ****p<0.0001; *p<0.05 (unpaired, two-tailed t-test).