Allogeneic major histocompatibility complex antigens are necessary and sufficient for partial tolerance induced by transfusion of pathogen reduced platelets in mice

Abstract

Background and objectives: Alloimmunization is common following transfusion with platelet-rich plasma (PRP) and can cause complications such as platelet refractoriness or transplant rejection. It has previously been shown that pathogen reduction of PRP with riboflavin and UV light (UV+R) can protect against alloimmunization in mice and induce partial tolerance to subsequent transfusions.

Materials and methods: Using B6 H2^d congenic mice, this study evaluated the relative contributions of major histocompatibility complex (MHC) antigens and minor antigens to both the alloresponse to PRP transfusion and the partial tolerance induced by UV+R treatment.

Results: Both total and MHC-specific alloantibody responses were highest when both MHC and minor antigens were mismatched, with lower alloantibody responses observed with MHC mismatch alone, demonstrating that allogeneic minor antigens can enhance the response to allogeneic MHC. There was a weak, but significant alloantibody response to minor antigens only. UV+R treatment protected against both major and minor antigen alloimmunization. Both allogeneic MHC and minor antigens primed an enhanced cytokine response ex vivo, though this was weaker with minor antigens, and both responses were blocked with UV+R treatment.

Conclusion: Allogeneic MHC is both necessary and sufficient to induce the partial tolerance associated with UV+R treatment.

Keywords: alloimmunization; major histocompatibility complex; minor antigens; pathogen reduction; tolerance.

Figure 1.. Allogeneic minor antigens enhance the antibody response to allogeneic MHC.

(A) Schematic of genetic differences between donor and recipient. (B) Schematic of experimental groups: B6 recipient mice received either no transfusion (No Tx) or were given a single transfusion with untreated PRP from syngeneic B6 donors, untreated PRP from fully allogeneic BALB/c donors (where both MHC and minor antigens are allogeneic), or PRP from congenic B6 H2^d donors (BALB/c MHC on B6 background, only MHC alloantigens present) with or without pathogen reduction with UV+R. The final group was given a first transfusion of UV+R PRP from B6 H2^d donors followed 2 weeks later by second transfusion with untreated B6 H2^d PRP. Two weeks after last transfusion, serum from recipients was screened for antibodies against (C) BALB/c target cells (antibodies against both MHC and minor antigen) and (D) B6 H2^d target cells (antibodies against MHC only) by flow cytometry. *p<0.05, **p<0.01, ****p<0.0001.

Figure 2.. Allogeneic MHC is sufficient for PRT induced tolerance.

Splenocytes from mice described in Figure 1 were collected 2 weeks after final transfusion and cultured with mitomycin C treated splenocytes from BALB/c (open circle) or B6 H2^d (grey filled circle) mice for 48 hours. Culture supernatants were collected and screened for cytokines. (A) IFN-γ, GM-CSF, IL-10, (B) IL-4, IL-5, and IL-13 are plotted as examples. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3.. Transfusion of allogeneic minor antigens induces a small alloantibody response that is blocked with PRT.

(A) Schematic of genetic differences between donor and recipient with different donor types. (B) Schematic of experimental groups: BALB/c recipient mice received either no transfusion (No Tx) or were given a single transfusion with untreated PRP from fully allogeneic B6 donors (where both MHC and minor antigens are allogeneic), or PRP from congenic B6 H2^d donors (BALB/c MHC on B6 background, only minor alloantigens present) with or without pathogen reduction with UV+R. The final group was given a first transfusion of UV+R PRP from B6 H2^d donors followed 2 weeks later by second transfusion with untreated B6 H2^d PRP. Two weeks after last transfusion, serum from recipients was screened for antibodies against (B) B6 target cells (antibodies against both MHC and minor antigen) and (C) B6 H2^d target cells (antibodies against allogeneic minor antigens only) by flow cytometry. *p<0.05, **p<0.01, ****p<0.0001.

Figure 4.. Allogeneic MHC is necessary for PRT induced tolerance.

Splenocytes from mice described in Figure 3 were collected 2 weeks after final transfusion and cultured with mitomycin C treated splenocytes from B6 (black filled circle) or B6 H2^d (grey filled circle) mice for 48 hours. Culture supernatants were collected and screened for cytokines. (A) IFN-γ, GM-CSF, IL-10, (B) IL-4, IL-5, and IL-13 are plotted as examples. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5.. Global changes by group and stimulation type.

For each antibody or cytokine measurement, values for each animal were divided by the mean value for non-transfused controls, and the means of these fold changes values are plotted as heat maps. Values exceeding the upper limit of the map range labeled with fold-change values. (A) The experiments using B6 recipients are plotted for BALB/c target/stimulatory cells (allogeneic MHC and minor antigens) in the upper panel and B6 H2^d target/stimulatory cells (allogeneic MHC only) in the lower panel. (B) The experiments using BALB/c recipients are plotted for B6 target/stimulatory cells (allogeneic MHC and minor antigens) in the upper panel and B6 H2^d target/stimulatory cells (allogeneic minor antigens only) in the lower panel.