B cells require Type 1 interferon to produce alloantibodies to transfused KEL-expressing red blood cells in mice

Fig. S1. IFNAR expression is required for alloimmunization to KEL RBCs. Data show experimental replications of Figure 1. Peripheral blood of KEL-expressing transgenic mice was transfused into recipients. Serum anti-KEL IgG was measured by flow cytometric crossmatch. (A) Anti-KEL IgG in serum of WT or IFNAR1^-/- mice 28 days after transfusion. (B) Serum anti-KEL IgG of transfused WT mice injected i.p. with anti-IFNAR1 blocking antibody (MAR1-5A3), an isotype control IgG1 antibody (MOPC-21), or PBS on Day -1, +2, and +7, relative to transfusion on Day 0. (C) Anti-KEL IgG of transfused WT, IFNAR1^-/-, and bone marrow chimeric mice. Recipients were irradiated and reconstituted with donor bone marrow cells, 8 weeks prior to transfusion. “n/a”; non applicable. *p < 0.05 by (A, C) Mann Whitney U test and (B) Kruskal-Wallis test with a Dunn's posttest.

Fig. S2. IFNAR expression by B cells is required for KEL RBC alloimmunization. Data show experimental replications of Figure 2D (A), 3D (B), and 4D (C). (A) Mixed chimeras were generated by reconstituting irradiated CD45.2+ WT recipients with a mixture of Zbtb46-DTR (CD45.1+, CD45.2+) and either IFNAR1^-/- (CD45.2+) or WT (CD45.1+) bone marrow. Serum anti-KEL IgG produced by indicated chimeras treated with PBS or DT prior to transfusion with KEL RBCs. Z46-DTR = Zbtb46-DTR. n.s., not significant by Kruskal-Wallis test with a Dunn's posttest. (B, C) Anti-KEL IgG in serum of indicated chimeras following transfusion with KEL RBCs. *p < 0.05, **p < 0.01 and n.s., not significant, by Mann Whitney U test. (B) Mixed chimeras were generated by reconstituting irradiated IFNAR1^-/- (CD45.2+) recipients with a mixture of TCRα^-/- (CD45.2+) and either IFNAR1^-/- (CD45.2+) or WT (CD45.1+) bone marrow. (C) Mixed chimeras were generated by reconstituting irradiated WT (CD45.2+) recipients with a mixture of muMT- (CD45.2+) and either IFNAR1^-/- (CD45.2+) or WT (CD45.1+) bone marrow. Chimeras reconstituted with only IFNAR1^-/- bone marrow (left) served as negative controls for alloimmunization.

Fig. S3. IFNAR expression promotes germinal center B cell development. Data show experimental replications of Figure 5. IFNAR1^-/- and WT mice were transfused with KEL RBCs. (A-C) Spleen GC B cells (CD19+IgDloFas+GL7hi) from (A) naïve or transfused mice (B) 8 or (C) 36 days after transfusion were quantified by flow cytometry as percent of CD19+ B cells (left) and cell number (right). (D) Serum anti-KEL IgG measured by flow cytometric cross-match 36 days after transfusion. (C, D) Mice received a second transfusion 28 days following the first transfusion. Data are from one of two independent experiments with 3-5 mice per group. *p < 0.05, n.s., not significant, by Mann Whitney U test. Experiments with 3 mice per group were not tested for statistical significance.

Fig. S4. IFNAR expression promotes plasma cell differentiation. Data show experimental replications of Figure 6B-C. WT and IFNAR1^-/- mice were transfused 28 days following an initial transfusion, and plasma cells were analyzed 14 days later. (A) Bone marrow plasma cells (CD19+IgDloB220loCD138+) were quantified by flow cytometry as (B) percent of CD19+ B cells and (C) cell number. Data show one of two independent experiments with 4 mice per group. *p < 0.05, n.s. not significant, by Mann Whitney U test.