CTLA4-Ig prevents alloantibody production and BMT rejection in response to platelet transfusions in mice

Abstract

Background: Platelet (PLT) transfusions can induce humoral and cellular alloimmunity. HLA antibodies can render patients refractory to subsequent transfusion, and both alloantibodies and cellular alloimmunity can contribute to subsequent bone marrow transplant (BMT) rejection. Currently, there are no approved therapeutic interventions to prevent alloimmunization to PLT transfusions other than leukoreduction. Targeted blockade of T-cell costimulation has shown great promise in inhibiting alloimmunity in the setting of transplantation, but has not been explored in the context of PLT transfusion.

Study design and methods: We tested the hypothesis that the costimulatory blockade reagent CTLA4-Ig would prevent alloreactivity against major and minor alloantigens on transfused PLTs. BALB/c (H-2(d)) mice and C57BL/6 (H-2(b)) mice were used as PLT donors and transfusion recipients, respectively. Alloantibodies were measured by indirect immunofluorescence using BALB/c PLTs and splenocytes as targets. BMTs were carried out under reduced-intensity conditioning using BALB.B (H-2(b) ) donors and C57BL/6 (H-2(b)) recipients to model HLA-identical transplants. Experimental groups were given CTLA4-Ig (before or after PLT transfusion) with control groups receiving isotype-matched antibody.

Results: CTLA4-Ig abrogated both humoral alloimmunization (H-2(d) antibodies) and transfusion-induced BMT rejection. Whereas a single dose of CTLA4-Ig at time of transfusion prevented alloimmunization to subsequent PLT transfusions, administration of CTLA4-Ig after initial PLT transfusion was ineffective. Delaying treatment until after PLT transfusion failed to prevent BMT rejection.

Conclusions: These findings demonstrate a novel strategy using an FDA-approved drug that has the potential to prevent the clinical sequelae of alloimmunization to PLT transfusions.

Figure 1. CTLA4-Ig prevents humoral alloimmunization to LR-PLT

(A) C57BL/6 recipients were administered a 500 µg dose of CTLA4-Ig i.p. two hours prior to each weekly transfusion of BALB/c LR-PLT concentrates. Sera was collected one week after each transfusion and tested for anti-donor antibodies using BALB/c splenocyte targets by indirect immunofluorescence staining. The middle panel shows individual animals whereas the right panel demonstrates combined data. (B) On day 0, C57BL/6 recipients were administered a single 500 µg dose of CTLA4-Ig i.p. two hours prior to a BALB/c LR-PLT transfusion. Recipients received four subsequent transfusions a week apart, on days 21, 28, 35, and 42. Sera was collected one week after each transfusion and tested for anti-BALB antibodies by indirect immunofluorescence staining using BALB/c splenocyte targets. Responders in (A) and (B) were defined as having an MFI two standard deviations above the mean MFI of the background sera from naïve C57BL/6 mice (dotted line). Error bars represent the mean ± SD. The combined data from three independent experiments are shown.

Figure 2. CTLA4-Ig treatment inhibits donor specific CD4⁺ T cell responses to platelet transfusions

(A) Representative dot plots illustrate the ability to readily discriminate adoptively transferred TCR75×Thy1.1 splenocytes from C57BL/6×Thy1.2 recipients using the Thy T cell congenic marker. (B) CFSE labeled TCR75 splenocytes were adoptively transferred into C57BL/6 recipients. Twenty-four hours later, recipients received a LR-PLT transfusion from either BALB/c (dark line) or syngeneic C57BL/6 (shaded histogram) donors. Some mice were treated with 500 µg of CTLA4-Ig i.p two hours prior to transfusion. Five days post-transfusion, splenocytes were harvested and stained with anti-CD4 and anti-Thy1.1; TCR75 cells were visualized by gating on CD4+Thy1.1+ events and division was measured by dilution. The illustrated histograms are representative of the trend observed in three independent experiments. (C) TCR75 splenocytes were adoptively transferred into C57BL/6 recipients. Twenty-four hours later, recipients received a LR-PLT transfusion from either BALB/c or syngeneic C57BL/6 mice. Some animals received 500 µg of CTLA4-Ig i.p two hours prior to transfusion. Sera were collected at seven and fourteen days post transfusion and tested for anti-donor antibodies by indirect immunofluorescence staining using BALB/c splenocyte targets. Error bars represent the mean ± SD. The combined data from three independent experiments are shown.

Figure 3. CTLA4-Ig LR-PLT transfusion induced BMT rejection

(A) Experimental model testing the ability of CTLA4-Ig to prevent platelet induced rejection of an MHC-matched BMT. BALB/c platelet donors were MHC- and mHA-mismatched, whereas BALB.B bone marrow donors were MHC-matched but mHA-mismatched with respect to the C57BL/6 recipients. Recipients received a single 500 µg dose of CTLA4-Ig or human IgG1 i.p. two hours prior to the first transfusion. Recipients received four LR-PLT transfusions, a week apart. After the fourth transfusion, recipients received a BALB.B BMT under reduced intensity conditions. in vivo survival of BALB.B targets was performed after BMT (see Figure 4). (B) The CD229⁺ congenic markers. Representative dot plots illustrate the ability to detect C57BL/6 recipient CD229⁺ (top left panel) and BALB.B donor CD229.1⁺ (right panel) cells by flow cytometry. Engraftment is demonstrated as the presence of a double positive CD229.1⁺ CD229⁺ population (bottom left panel) and rejection as the absence of this double positive population (bottom right panel). Chimerism is indicated by the presence of both the double positive CD229.1⁺ CD229⁺ and the single positive CD229⁺ populations. (C) BALB.B BMT engraftment results. Engraftment is represented as percent CD229.1⁺ cells in the peripheral blood; the horizontal lines denote the mean of each group. Rejection was measured as having a percent CD229.1⁺ cells engraftment two standard deviations above the mean of the positive control group known to reject, recipients treated with isotype control antibody human IgG1. Statistics were generated using column statistics and a one-way ANOVA with Dunnett’s post-test. The combined data from three independent experiments are shown.

Figure 4. Effects of CTLA4-Ig on alloimmunization in BMT recipients transfused with LR-PLT products

BALB specific immunity was assessed by in vivo survival of BALB.B splenocyte targets in BMT recipients. The horizontal line denotes the mean of each group. Statistics were generated using column statistics and a one-way ANOVA with Dunnett’s post-test. The data shown in both panels is the combined data from three separate experiments.

Figure 5. Delayed CTLA4-Ig treatment is ineffective at preventing LR-PLT induced BMT rejection

(A) Experimental model testing the ability of CTLA4-Ig to prevent platelet induced rejection of an MHC-matched BMT when administered at the time of BMT. Recipients received four LR-PLT transfusions, a week apart. After the fourth transfusion, recipients received a BALB.B BMT under reduced intensity conditions with or without a single 500 µg dose of CTLA4-Ig or human IgG1 i.p. two hours prior to transplantation. (B) Engraftment is represented as percent CD229.1⁺ cells in the peripheral blood; the horizontal lines denote the mean of each group. Rejection was measured as having a percent CD229.1⁺ cells engraftment two standard deviations above the mean of the positive control group known to reject, recipients treated with isotype control antibody human IgG1. Statistics were generated using column statistics and a one-way ANOVA with Dunnett’s post-test. The combined data from three independent experiments are shown.