Thus, while safety of cells from match effector function represents a well-documented, evolutionarily conserved process with significant implications in human disease (62C64, 69, 70), the ability of match to likewise negatively regulate Ab formation following engagement of RBCs provides a previously unrecognized additional ability of match to differentially regulate adaptive immunity. The ability of Abs in general to control immune function represents a long recognized, yet poorly understood process previously suggested to reflect a key regulatory loop in preventing or reducing Ab formation (71, 72). anti-HOD and anti-KEL IgG subclass distribution following transfusion. B6 mice were transfused with either HOD or KEL RBCs, followed by evaluation of anti-KEL or anti-HOD IgG1 (A), IgG2b (B), IgG2c (C), Calcifediol monohydrate and IgG3 (D) in the serum on day time 21 post-transfusion by circulation cross-match. (ACD) ns?=?not significant. Means??SD shown. Image_3.tif (716K) GUID:?EAB1FA4C-D7F8-46F6-B674-7BAD0119C30C Number S4: Anti-KEL antibodies fail to induce detectable C3b/iC3b deposition about circulating KEL reddish blood cells (RBCs) post-transfusion. C3b/iC3b deposited on circulating DiI-labeled KEL RBCs was measured at 10?min (A), 4?h (B), FLN2 day time 1 (C), and day time 3 (D) post-transfusion into KEL, B6, and C3 KO mice. (ACD) ns?=?not significant. Means??SD shown. Image_4.tif (964K) GUID:?66DE1CF8-361B-4BE3-A892-0F6DB83AA1EA Abstract Red blood cell (RBC) alloimmunization can make it hard to procure compatible RBCs for long term transfusion, directly leading to increased morbidity Calcifediol monohydrate and mortality in transfusion-dependent individuals. However, the factors that regulate RBC alloimmunization remain incompletely recognized. As complement offers been shown to serve as a key adjuvant in the development of antibody (Ab) reactions against microbes, we examined the effect of match on RBC alloimmunization. In contrast to the effect of match component 3 (C3) in the development of an immune response following microbial exposure, transfusion of C3 knockout (C3 KO) recipients with RBCs expressing KEL (KEL RBCs) actually resulted in an enhanced anti-KEL Ab response. The Calcifediol monohydrate effect of C3 appeared to be specific to KEL, as transfusion of RBCs bearing another model antigen, the chimeric HOD antigen (hen egg lysozyme, ovalbumin and Duffy), into C3 KO recipients failed to result in a similar increase in Ab formation. KEL RBCs experienced enhanced C3 deposition and loss of detectable target antigen over time when compared to HOD RBCs, suggesting that C3 may inhibit Ab formation by impacting the convenience of the prospective KEL antigen. Loss of detectable KEL within the RBC surface did not reflect antigen masking by C3, but instead appeared to result from actual removal of the KEL antigen, as western blot analysis shown complete loss of detectable KEL protein. Consistent with this, exposure of wild-type B6 or C3 KO recipients to KEL RBCs with reduced levels of detectable KEL antigen resulted in a significantly reduced anti-KEL Ab response. These results suggest that C3 possesses a unique ability to actually suppress Ab formation following transfusion by reducing the availability of the prospective antigen within the RBC surface. with 1:2 PBS to isolate platelet-rich plasma, as carried out previously (40, 42). WBCs, platelets, or RBCs were then stained with polyclonal anti-KEL Ab or polyclonal anti-HEL Ab diluted 1:100 in FACS buffer. Following washing in FACS buffer, cells were stained with anti-mouse IgG (Jackson Immunoresearch, Western Grove, PA) diluted 1:100 in FACS buffer. After washing in FACS buffer, cells were stained with anti-CD45 (BD) for WBCs, anti-CD41 (BD) for platelets, or anti-Ter119 (BD) for RBCs. Stained RBCs were then washed 3 in FACS buffer and diluted to a final total volume of 100?L in FACS buffer. Circulation Cytometry After staining, 50?L of each set of stained cells in FACS buffer was then added to 400?L of FACS buffer and the level of detectable antigen, Abdominal bound, or match deposition was measured by a FACSCalibur circulation cytometer by gating specifically on RBCs (Number S1 in Supplementary Material). Data acquisition was accomplished by CellQuest Pro and was analyzed using FlowJo software (33, 34, 37). Mean fluorescence intensity (MFI) was used to assess the levels of detectable antigen, Ab bound, or match deposition. For the level of detectable antigen, the MFI of experimental mice was indicated as a percentage Calcifediol monohydrate of the MFI of DiI-KEL RBCs transfused into KEL mice or DiI-HOD RBCs transfused into HOD mice. Seroanalysis To detect anti-KEL or anti-HOD alloantibody development in the serum, a Calcifediol monohydrate circulation cross-match was performed, as previously explained (32, 33, 40, 41, 43). Briefly, 10?L of serum was incubated for 15?min at room temp with 3?L of either KEL or HOD RBCs. RBCs were then washed 3 in FACS buffer, followed by.