Tolerance induction or sensitization in mice exposed to noninherited maternal antigens (NIMA)

Abstract

Developmental exposure to noninherited maternal antigens (NIMA) exerts a tolerizing or sensitizing influence on clinical transplantation in humans and experimental animals. The aim of this study was to determine if strain and gender differences influence the NIMA effect. Six different mouse strain backcross matings of F(1) females with homozygous males ('NIMA backcross') and corresponding control breedings of F1 males with homozygous females were performed. H-2 homozygous offspring underwent heterotopic heart transplantation from fully allogeneic donors expressing noninherited H-2 antigens. A NIMA tolerizing effect on heart allograft outcome was found in three of six breeding models. In all three cases, the tolerizing antigens were from an H-2(d+) strain. The tolerogenic effect was greatest in male as compared with female recipients. Offspring from the three breeding models in which no tolerance was seen, appeared to be sensitized based on poorer graft survival, or enhanced T- or B-cell responses to the noninherited H-2(b or k) antigens. Significantly higher percentages of maternal antigen(+) cells were found in the peripheral blood of tolerant versus nontolerant strains of backcross mice prior to transplant. Our findings imply that transplants are predisposed to tolerance or rejection due to recipient developmental history and immunogenetic background.

Figure 1. Heterotopic heart allograft survival of all strain combinations tested

A) D1 model in which H-2^d non-exposed controls and NIMA H-2^d exposed offspring are transplanted with a DBA/2 donor heart. n=61 for NIMA-exposed and n=12 for non-exposed controls. B) D2 model in which H-2^k non-exposed controls and NIMA H-2^d exposed offspring are transplanted with a DBA/2 donor heart. n=12 for NIMA-exposed and n=6 for non-exposed controls. C) D3 model in which H-2^b non-exposed controls and NIMA H-2^d exposed offspring are transplanted with a BALB/c donor heart. n=8 for NIMA-exposed and n=5 for non-exposed controls. D) B1 model in which H-2^k non-exposed controls and NIMA H-2^b exposed offspring are transplanted with a B6 donor heart. n=11 for NIMA-exposed and n=6 for non-exposed controls. E) B2 model in which H-2^d non-exposed controls and NIMA H-2^b exposed offspring are transplanted with a B6 donor heart. n=8 for NIMA-exposed and n=7 for non-exposed controls. F) K model in which H-2^b non-exposed controls and NIMA H-2^k exposed offspring are transplanted with a C3H donor heart. n=9 for NIMA-exposed and n=6 for non-exposed controls. P values in graft represent comparison between NIMA-exposed and non-exposed controls.

Figure 2. Heterotopic heart allograft survival of D1 model separated by gender

A) Female (n=22 for NIMA-exposed and n=6 for non-exposed controls) and B) male (n=39 for NIMA-exposed and n=6 for non-exposed controls). P values in graft represent comparison between NIMA-exposed and non-exposed controls.

Figure 3. Post transplant serum analysis for allo-antibody against the non-inherited H-2 shows that NIMA-exposed mice from the D1 model produce significantly less compliment fixing antibody compared to NIMA-exposed from the B1 model, who appear sensitized

Serum samples were obtained from mice post transplant at various time points and analyzed for the production of complement fixing anti-NIMA IgG₂ antibodies and anti-NIMA IgG₁ in mice from the A) D1 model and B) B1 model. n=5 for each group of mice from D1 model, and n=3 for NIMA-exposed and n=5 for non-exposed controls from the B1 model. *p= <.05 for NIMA-exposed versus non-exposed controls.

Figure 4. Pre-transplant analysis for alloreactive IFN-γproducing T cells indicates that NIMA-exposed mice from the K model are sensitized to maternal antigens compared to NIMA-exposed mice from the D1 and B1 models

Splenocytes were harvested from NIMA-exposed and non-exposed control mice and stimulated with irradiated B6D2F₁ (D1 model) or B6C3F₁ (K and B1 models) splenocytes for ELISpot analysis of IFN-γproduction. Background spots resulting from stimulation with irradiated homozygous splenocytes (B6 for D1 and K models, C3H for B1 model) was subtracted from the number of spots obtained with semi-allo stimulation. Results shown as mean ± SD spot/million cells over background. n=5–9 mice for all groups tested.

Figure 5. Flow cytometric analysis comparing three stains shows that NIMA-exposed mice from the D1 model have significantly more NIMA⁺ cells compared their non-exposed controls, and to NIMA-exposed mice from the K and B1 models

PBMC obtained from mice ages 4–8 weeks of age was collected and analyzed by flow cytometry for non-inherited antigen⁺ cells. A) Representative flow cytometric analysis for non-inherited antigen⁺ cells (H-2K^d+ for the D1 model, H-2K^k+ for the K model, and H-2K^bD^b for the B1 model) in PBMC from NIMA-exposed and non-exposed mice from the D1, K, and B1 models. B) NIMA-exposed mice from the D1 model had significantly more H-2K^d+ cells compared to non-exposed controls. No differences were found between NIMA-exposed and non-exposed controls in the other two models tested. Results shown as mean ± SD. n= 6–13 mice for all groups tested.