Alloimmunization to transfused platelets requires priming of CD4+ T cells in the splenic microenvironment in a murine model

Abstract

Background: Alloantibodies are a clinically significant sequelae of platelet (PLT) transfusion, potentially rendering patients refractory to ongoing PLT transfusion support. These antibodies are often IgG class switched, suggesting the involvement of CD4+ T-cell help; however, PLT-specific CD4+ T cells have not been visualized in vivo, and specifics of their stimulation are not completely understood.

Study design and methods: A murine model of alloimmunization to transfused PLTs was developed to allow in vivo assessment and characterization of CD4+ T cells specific for PLT major histocompatibility complex (MHC) alloantigen. PLTs were harvested from BALB/c mice, filter leukoreduced, and transfused into C57BL/6 recipients. PLT-specific CD4+ T-cell responses were visualized by using a T-cell receptor transgenic mouse that detects peptide from donor MHC I presented on recipient MHC II. Antibody responses were determined by indirect immunofluorescence using BALB/c donor targets.

Results: C57BL/6 recipients of BALB/c leukoreduced PLT transfusions produced BALB/c antibodies, with proliferation of antigen-specific CD4+ T cells seen in the spleen but not lymph nodes or liver. Depletion of recipient CD4+ cells or splenectomy independently abrogated the alloantibody response.

Conclusion: We report a novel model to study antigen-specific CD4+ T cells during alloimmunization to PLT transfusion. The presented data support a critical role for CD4+ T-cell help in the humoral response to PLT transfusion and establish the spleen as a required microenvironment for effective CD4+ T-cell priming against donor PLT-derived MHC I.

Figure 1. Antigen specific CD4⁺ T cell help is required for the anti-donor antibody response to transfused LR-PLTs

(A) Analysis of CD4⁺ T cell levels following treatment with the CD4-depleting antibody GK1.5. Recipients were injected i.p. at 4 and 2 days prior to each LR-PLT transfusion. CD3⁺CD4⁺ T cells are shown from the spleen of representative animals at days 0 and 21. Numbers of CD4⁺ T cell numbers in treated animals (center panels) dropped to baseline signal seen with isotype control (right panels) throughout the time of LR-PLT transfusion. Flow plots are representative of two independent experiments with two mice per group. (B) C57BL/6 recipients were either untreated or injected i.p. with GK1.5, and then received four weekly BALB/c or syngeneic LR-PLT transfusions. The total Ig anti-donor antibody response was quantified by indirect immunofluorescence against BALB/c splenocyte targets. (left panel: individual animals, right panel: combined data). (C) 3A9 TCR transgenic mice and non-transgenic littermates received four weekly BALB/c LR-PLT transfusions. The total Ig anti-donor antibody response was quantified by indirect immunofluorescence against BALB/c splenocyte targets. (left panel: individual animals, right panel: combined data). In panels B and C, the horizontal dashed line marks two standard deviations above the mean signal for mice receiving syngeneic LR-PLTs, and thus represents the cut-off for a statistically significant response. Combined data from three independent experiments are shown in panels B and C, with three mice per group per experiment (n=9 mice total/group). Error bars indicate standard deviation.

Figure 2. Characterization of in vivo CD4⁺ T cell response to alloantigen on transfused LR-PLTs

(A) 1 × 10⁶ CFSE labeled TCR75 × Thy1.1 splenocytes were adoptively transferred into wild-type C57BL/6 (Thy1.2) recipients and were visualized by staining with anti-CD4 and anti-Thy1.1 (B) C57BL/6 recipients were adoptively transferred with 1 × 10⁶ CFSE labeled TCR75 splenocytes and were then given a single transfusion of LR-PLT 24 hours later from either BALB/c (dark line), BALB.B (dashed line) or syngeneic C57BL/6 (shaded histogram) donors. Representative histograms are from three independent experiments with three mice per group (n=9). (C) Sera were collected from the TCR75 adoptively transferred C57BL/6 recipients 7 and 14 days post-LR-PLT transfusion. The total Ig anti-donor antibody response was quantified by indirect immunofluorescence against BALB/c splenocyte targets (left panel: individual animals, right panel: combined data). The horizontal dashed line marks two standard deviations above the mean signal for mice receiving syngeneic LR-PLTs, and thus represents the cut-off for a statistically significant response. Combined data from three independent experiments with three mice per group (n=9). Error bars indicate standard deviation.

Figure 3. The splenic microenvironment is required for antigen-specific CD4⁺ T cell division and humoral alloimmunization

(A) Sham-operated and (B) splenectomized recipients were adoptively transferred with 1 × 10⁶ CFSE labeled TCR75 splenocytes, followed by a single LR-PLT transfusion 24 hours later. Five days after transfusion, division was assessed in the spleen, liver, and lymph nodes by gating on CD4⁺ Thy1.1⁺ cells and assessing CFSE dilution. Representative histograms are from three independent experiments, with three mice per group (n=9). (C) Intact and splenectomized recipients were adoptively transferred with 1 × 10⁶ CFSE labeled TCR75 splenocytes, followed by a single BALB/c or C57BL/6 LR-PLT transfusion 24 hours later. The total Ig anti-donor antibody response was quantified by indirect immunofluorescence against BALB/c splenocyte targets 7 and 14 days post-LR-PLT transfusion. (left panel: individual animals, right panel: combined data). The horizontal dashed line marks two standard deviations above the mean signal for mice receiving syngeneic LR-PLTs, and thus represents the cut-off for a statistically significant response.

Figure 4. A spleen is required for the anti-donor antibody response to LR-PLTs but not whole blood

Sham-operated and splenectomized C57BL/6 recipients received four weekly BALB/c or C57BL/6 LR-PLT transfusions (A) or a single transfusion of whole blood (B). The total Ig anti-donor antibody response was quantified by indirect immunofluorescence against BALB/c splenocyte targets at the indicated time-points post-transfusion. (left panel: individual animals, right panel: combined data). The horizontal dashed line marks two standard deviations above the mean signal for mice receiving syngeneic transfusions, and thus represents the cut-off for a statistically significant response. Combined data from three independent experiments with at least three mice per group are shown for all panels. Error bars indicate standard deviation.