PIRs mediate innate myeloid cell memory to nonself MHC molecules

Abstract

Immunological memory specific to previously encountered antigens is a cardinal feature of adaptive lymphoid cells. However, it is unknown whether innate myeloid cells retain memory of prior antigenic stimulation and respond to it more vigorously on subsequent encounters. In this work, we show that murine monocytes and macrophages acquire memory specific to major histocompatibility complex I (MHC-I) antigens, and we identify A-type paired immunoglobulin-like receptors (PIR-As) as the MHC-I receptors necessary for the memory response. We demonstrate that deleting PIR-A in the recipient or blocking PIR-A binding to donor MHC-I molecules blocks memory and attenuates kidney and heart allograft rejection. Thus, innate myeloid cells acquire alloantigen-specific memory that can be targeted to improve transplant outcomes.

Fig. 1.. Monocyte and macrophage memory specific to allogeneic MHC molecules.

A-D, Monocyte responses in immunized mice measured as monocyte-derived dendritic cells (Mo-DCs) in the graft (n = 5 to 12/N = 1 to 2). E, Monocytes from immunized mice transfer memory to naive recipients (n = 2 to 4/N = 1 to 3). F, Allospecific killing of CFSE-labeled cells in immunized mice (n = 6/N = 2). G-H, Nonself MHC recognition is necessary for eliciting monocyte (n = 6/N = 1) (G) and macrophage (n = 6/N = 2) (H) memory. Killing of target cells lacking nonself MHC (BALB.B) or MHC-I (B2m^–/–) is significantly diminished (H). Assays were performed 7 days after immunizing B6-Rag^−/−Il2rg^−/− mice. I, Allogeneic MHC-I tetramer binding to macrophages from BALB/c-immunized B6- Rag^−/−Il2rg^−/− hosts (representative of 3 experiments). Mean and individual biological replicates (A-H); one-way ANOVA (A-C, F, H) or two-tailed Student’s t-test (D, E, G).

Fig. 2.. Memory to allogeneic MHC-I is mediated by PIR-A molecules on monocytes and macrophages.

A-B, PIR-A and -B expression on Ly6C^hi monocytes (A) and macrophages (B) before (d.0) and after alloimmunization (d.14 & d.28). Colored histograms = biological replicates (n = 3). C-E, Blocking PIR-A/B inhibits monocyte (n = 12/N = 2) (C-D) and macrophage (n = 6/N = 2) (E) memory. B6-Rag^−/−Il2rg^−/− hosts were immunized with BALB/c splenocytes and rechallenged with BALB/c allografts 7 days later. F-H, H-2Dd MHC-I tetramers bind preferentially to PIR-A3-transfected BWZ.36 (n = 5/N = 1) (F) and 3T3 cells (G). PIR-A3/Fc binds preferentially to H-2Dd⁺ (BALB/c) cells (H). I-J, Specific inhibition of macrophage (n = 5–6/N = 2) (I) and monocyte (n = 6/N = 1 to 2) (J) memory to BALB/c allografts by PIR-A3/Fc. K, Absent memory 7 and 28 days after immunization, but normal primary monocyte alloresponse in Pira^−/− hosts (n = 6/N = 1 to 2). Representative of three experiments (B, G-H); mean and individual biological replicates (C-D, F, I-K); one-way ANOVA (C-E, I-J) or two-tailed Student’s t-test (K).

Fig. 3.. Mechanisms of monocyte memory.

A, Tetramer-positive splenic monocytes 7 days after immunizing B6-Rag^−/−Il2rg^−/− mice (mean and individual biological replicates; n = 3/N = 1 to 3). B-E, Splenic monocyte scRNA-seq analysis 1 and 4 weeks post-immunization (n = 3/N = 1): Pseudotime plots, S = Starting state, E1–3 = Expanding states (B); heatmaps and graphs depicting proportion of S and E states in each immunization group (C); differential expression of Pir genes in E states vs. S state (D); and proportion of monocytes expressing Pira2, Pira3, or both in each state (E). one-way ANOVA (A).

Fig. 4.. Genetic deletion or blocking PIR-A attenuates kidney and heart allograft rejection.

A-D, Survival (A), serum creatinine (B), histology (C), and chronic rejection scores (D) of BALB/c kidneys transplanted to wildtype (wt), Pira^–/– or Pirb^–/– B6 recipients. Arrows point to infiltrates and fibrosis. E-F, Survival (E) and histology (F) of BALB/c hearts transplanted to B6 mice treated with PIR-A3/Fc (± CTLA4Ig) or control mouse IgG1 (Ctrl IgG). Arrows point to chronic rejection vasculopathy. Mean and individual biological replicates (B, D); log-rank (A, E) and one-way ANOVA (B, D).