Non-self recognition by monocytes initiates allograft rejection

Abstract

Maturation of T cell-activating APCs directly links innate and adaptive immunity and is typically triggered by microbial infection. Transplantation of allografts, which are sterile, generates strong T cell responses; however, it is unclear how grafts induce APC maturation in the absence of microbial-derived signals. A widely accepted hypothesis is that dying cells in the graft release "danger" molecules that induce APC maturation and initiate the adaptive alloimmune response. Here, we demonstrated that danger signals associated with dying cells are not sufficient to initiate alloimmunity, but that recognition of allogeneic non-self by the innate immune system is required. In WT as well as in T cell-, B cell-, and innate lymphoid cell-deficient mice, allogeneic grafts elicited persistent differentiation of monocytes into mature DCs that expressed IL-12 and stimulated T cell proliferation and IFN-γ production. In contrast, syngeneic grafts in the same mice elicited transient and less pronounced differentiation of monocytes into DCs, which neither expressed IL-12 nor stimulated IFN-γ production. In a model in which T cell recognition is restricted to a single foreign antigen on the graft, rejection occurred only if the allogeneic non-self signal was also sensed by the host's innate immune system. These findings underscore the importance of innate recognition of allogeneic non-self by monocytes in initiating graft rejection.

Figure 8. MyD88 and NALP3 pathways are not necessary for host monocyte response to allografts.

Syngeneic heart grafts (B6; black circles) were transplanted into B6 wild-type mice. Allogeneic heart grafts (BALB/c; white circles) were transplanted into B6 wild-type, Myd88^–/–, or Nalp3^–/– mice. Host mature mono-DCs that infiltrated the grafts were enumerated 3 days after transplantation. Recipient strains are indicated on the x axis. P values by Mann-Whitney U test are shown.

Figure 7. Monocyte depletion blunts T cell–mediated rejection.

(A–C) B6 CD11b-DTR chimeras (white circles) and B6 wild-type (WT) control chimeras (black circles) were transplanted with BALB/c hearts and treated with 12.5 ng/g DT on days 0, 2, 4, and 6. Grafts were harvested on day 7 after transplantation. (A) Enumeration of intragraft CD3⁺ cells. Micrographs show graft sections stained with anti-CD3 (red). Original magnification, ×2 (top panels) and ×40 (bottom panels). (B) Flow cytometric enumeration of total intragraft CD4⁺ and CD8⁺ T cells (left panel) and IFN-γ–producing T cells (right panel). (C) Enumeration of neutrophils (N), monocytes (M), macrophages (Mϕ), mono-DCs (mDC), and conventional DCs (cDC) in heart allografts. (D–F) Wild-type B6 mice were transplanted with BALB/c heart grafts and were treated with either 1A8 (neutrophil-depleting anti-mouse Ly6G Ab; white circles) or 2A3 (isotype control antibody; black circles). The same enumeration was performed as in A–C. n = 3 and 5 mice/group. *P < 0.05 compared with control groups.

Figure 6. Innate sensing of allogeneic non-self precipitates graft rejection.

(A) Heart grafts from multiple donor strains (shown on x axis) were transplanted into B6 RAG^–/–γc^–/–CX₃CR1^gfp/+ recipients, and host-derived mature mono-DCs present in the grafts were enumerated by flow cytometry 3 days later. (B and C) Graft recipients, as in A, received 5 × 10⁵ OT-II cells 2 days after transplantation. Grafts and spleens were harvested on the day of rejection or at termination of the experiment (day 42). IFN-γ production by OT-II cells in the spleen is shown in B. Allograft survival, number of graft-infiltrating T cells, and representative graft sections stained with anti-CD3 (red) and hematoxylin (blue) are shown in C (original magnification, ×2). *P < 0.05 compared with B6 and B6-OVA groups.

Figure 5. Graft mono-DCs present antigen to CD4 and CD8 T cells ex vivo.

(A) T cell proliferation (dilution of CTV) and IFN-γ production in response to control splenic CD11b⁺ DCs sorted from naive mice and fed LPS-free OVA in the presence or absence of exogenous LPS. (B) T cell proliferation and IFN-γ production in response to graft mono-DCs fed LPS-free OVA in the absence of exogenous LPS. Mono-DCs were sorted from syngeneic heart grafts on day 3 after transplantation and from heart allografts on either day 3 or day 42. Same donor-recipient strain combination as in Figure 2, A–C. Flow cytometry was performed 5 days after in vitro stimulation of T cells with DCs. DC/T cell ratio = 1:50. Percentages indicate CFSE-diluted cells. *P < 0.05 compared with the syngeneic group.

Figure 4. Analysis of monocyte and mono-DC infiltration of kidney grafts transplanted into lymphoid cell–deficient recipients.

B6 (Syn; black circles) or BALB/c (Allo; white circles) kidneys were transplanted into B6 RAG^–/–γc^–/–CX₃CR1^gfp/+ mice. Kidneys were imaged by intravital 2-photon microscopy either immediately (day 0) or on day 3 after transplantation. Enumeration of total (A), intravascular (i), and extravascular (e) (B), and round and dendrite-shaped GFP⁺ cells (C) in kidney grafts. Each data point in A and B represents 1 image volume. Image volume = 510 × 510 × 25 εm. Representative micrographs show GFP⁺ cells in green and capillaries in red. Scale bars: 50 εm (A) and 10 εm (C). n = 3–4 mice/group (1–5 image volumes/mouse). *P < 0.05 compared with the corresponding syngeneic group.

Figure 3. Enumeration of mature mono-DC infiltrate in bone marrow plug grafts transplanted into lymphoid cell–deficient recipients.

B6 and NOD grafts on the WT or RAG^–/–γc^–/– background were transplanted under the contralateral kidney capsules of B6 RAG^–/–γc^–/– recipients. Mature mono-DCs that infiltrated the grafts were enumerated 1 week later. Each data point represents 1 graft; n = 6 grafts/experiment; each experiment was performed twice. Donor strains are indicated on the x axis. ***P < 0.001.

Figure 2. Analysis of monocyte and mono-DC infiltrate in heart grafts transplanted into lymphoid cell–deficient recipients.

B6 RAG^–/– (syngeneic) or BALB/c RAG^–/– (allogeneic) heart grafts, except where indicated, were transplanted into B6 RAG^–/–γc^–/–CX₃CR1^gfp/+ recipients. Infiltrating cells were analyzed by flow cytometry at multiple time points after transplantation. (A–C) Enumeration of host monocytes (A), total mono-DCs (B), and mature (CD80⁺) mono-DCs (C) in syngeneic (black circles) and allogeneic (white circles) grafts. Percentage of mature mono-DCs and percentage of mature mono-DCs that are IL-12p40⁺ or TNF-α⁺ are shown in C. Data represent the mean ± SD; n = 4 grafts/group/time point for days 1, 5, 10, 21, and 42. n = 6 grafts/group for day 3. ND, none detected. (D) Effect of donor MHC (BALB/c, H-2d vs. BALB.B, H-2b) and donor non-MHC (NOR, H-2g⁷ vs. NOD, H-2g⁷) genotype on the number of mature host mono-DCs in heart allografts 3 days after transplantation into B6 mice (H-2b). (E) Effect of NK depletion in the donor on the number of host-derived mature mono-DCs and their IL-12 production in heart allografts 10 days after transplantation. Same donor-recipient strain combination as in A–C. *P < 0.05.

Figure 1. Analysis of myeloid cell infiltrate in heart grafts transplanted into immunocompetent recipients.

B6 (syngeneic) or BALB/c (allogeneic) CD45.2 heart grafts were transplanted into B6 CX₃CR1^gfp/+ CD45.1 recipients, except where indicated. Infiltrating cells were analyzed by flow cytometry 1 day after transplantation; n = 4 grafts/group. (A) Representative flow plot depicting lineage^– (CD19^–CD90^–NK1.1^–DX5^–) recipient (CD45.1⁺) and donor (CD45.2⁺) myeloid cells present in syngeneic (Syn) and allogeneic (Allo) grafts. (B) Enumeration of recipient myeloid populations in syngeneic (black bars) and allogeneic (white bars) grafts. Cell surface markers used to identify cell populations are shown in the text box and in the gating strategy in Supplemental Figure 1. N, neutrophils; Mono, monocytes; DC, dendritic cells; Mϕ, macrophages. (C) Enumeration and representative flow plot of monocyte subsets (Inf, inflammatory Ly-6C^hiCX3CR1^lo; Pat, patrolling Ly-6C^loCX3CR1^hi) in syngeneic (black circles represent black events) and allogeneic (white circles represent gray events) grafts. (D) Representative flow plot depicting phenotype of recipient-derived DCs in an allogeneic graft. (E) Enumeration of mature (CD80⁺) mono-DCs in syngeneic (black circles) and allogeneic (white circles) grafts. Each data point represents 1 graft. Donor-recipient strain combination is indicated on the x axis. *P < 0.05.