Platelets are currently stored at room temperature before transfusion to maximize circulation time. This approach has numerous downsides, including limited storage duration, bacterial growth risk, and increased costs. Cold storage could alleviate these problems. However, the functional consequences of cold exposure for platelets are poorly understood. In the present study, we compared the function of cold-stored platelets (CSP) and room temperature-stored platelets (RSP) in vitro, in vivo, and post-transfusion. CSP formed larger aggregates under in vitro shear while generating similar contractile forces compared to RSP. We found significantly reduced GPVI levels after cold exposure of 5-7 days. After transfusion in humans, CSP were mostly equivalent to RSP yet aggregated significantly less to the GPVI agonist collagen. In a mouse model of platelet transfusion, we found a significantly lower response to the GPVI-dependent agonist convulxin and significantly lower GPVI levels on the surface of transfused platelets after cold storage. In summary, our data support an immediate but short-lived benefit of CSP and highlight the need for thorough investigations of this product. (NCT03787927)

Red blood cells expressing alloantigens are well known to be capable of inducing robust humoral alloantibody responses both in transfusion and pregnancy. However, the majority of transfusion recipients and pregnant women never make alloantibodies, even after repeat exposure to foreign RBCs. More recently, RBCs have been used as a cellular therapeutic—very much like transfusion, engineered RBCs are highly immunogenic in some cases but not others. In animal models of both transfusion and RBC based therapeutics, RBCs that do not induce an immune response also cause tolerance. Despite a robust phenomenology, the mechanisms of what regulates immunity vs. tolerance to RBCs remains unclear. However, it has been reported that copy number of alloantigens on the RBCs is a critical factor, with a very low copy number causing non-responsiveness (in both humans and mice) and also leading to tolerance in mice. Recently, we reported that an IgG2c specific for an RBC antigen can substantially enhance the humoral immune response upon transfusion of RBCs expressing that antigen. Herein, we report that an IgG2c converts RBCs with low antigen copy number from a tolerogenic to an immunogenic stimulus. These findings report the first known stimulus that induces humoral alloimmunization to a low copy number RBC alloantigen and identify a previously undescribed molecular switch that has the ability to affect responder vs. non-responder phenotypes of transfusion recipients.

Background: Failure of humoral tolerance to red blood cell (RBC) antigens may lead to autoimmune hemolytic anemia (AIHA), a severe and sometimes fatal disease. Previous studies have shown that although tolerance is robust in HOD mice, autoantibodies are generated upon adoptive transfer of OTII CD4 + T cells, which are specific for an epitope contained within the HOD antigen. These data imply that antigen-presenting cells (APCs) are presenting RBCderived autoantigen(s) and are capable of driving T-cell activation. Given that multiple APCs participate in erythrophagocytosis, we used a transgenic approach to determine which cellular subsets were required for autoantigen presentation and subsequent autoreactive T-cell activation. Study Design and Methods: HOD mice, which express an RBC-specific antigen consisting of hen egg lysozyme, ovalbumin, and human blood group molecule Duffy, were bred with IAb fl/fl and Cre-expressing transgenic animals to generate mice that lack I-A b expression on particular cell subsets. OTII CD4 + T cell proliferation was assessed in vivo in HOD + I-Ab fl/fl xCre + mice and in vitro upon coculture with sorted APCs. Results: Analysis of HOD + I-Ab fl/fl xCre + mice demonstrated that splenic conventional dendritic cells (DCs), but not macrophages or monocytes, were required for autoantigen presentation to OTII CD4 + T cells. Subsequent in vitro coculture experiments revealed that both CD8 + and CD8 − DC subsets participate in erythrophagocytosis, present RBC-derived autoantigen and stimulate autoreactive T-cell proliferation. Conclusion: These data suggest that if erythrocyte T-cell tolerance fails, DCs are capable of initiating autoimmune responses. As such, targeting DCs may be a fruitful strategy for AIHA therapies.

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Background The current approach to manufacture cold‐stored platelets (CSP) replicates that of room temperature–stored platelets (RSP). However, this production method is associated with aggregate formation in CSP, a major pitfall that leads to significant wastage. We hypothesized that isolating platelets from whole blood as platelet‐rich plasma (PRP) and storing them at a lower concentration reduces aggregates and that conventional bedside transfusion filtration removes CSP aggregates. Methods We collected platelets from healthy humans by apheresis (AP) and by phlebotomy, from which we generated platelet‐rich plasma (PRP). We split each AP and PRP platelets into two equal aliquots, storing one at 22°C (RT‐PRP and RT‐AP) and the other at 4°C (4C‐PRP and 4C‐AP). We evaluated platelets on day 0 and day 7 of storage. After storage, we measured platelet counts, aggregates, and other key characteristics before and after filtration by a bedside filter. Results After storage, the 4C‐AP platelet counts decreased significantly. 4C‐PRP preserved glucose better and prevented a significant increase in lactate contrary to 4C‐AP. Filtration led to significantly lower platelet counts in both 4C‐PRP and 4C‐AP but not in their RT counterparts. Post filtration, we observed 50% fewer aggregates only in 4C‐AP, whereas 4C‐PRP showed an unexpected but significant increase in aggregates. Testing confirmed activation during storage but filtration did not further activate platelets. Conclusion We provide evidence that 4C‐PRP is an alternative to 4C‐AP and that bedside filters reduce aggregates from 4C‐AP. Further studies are needed to evaluate the hemostatic potential of 4C‐PRP and the management of aggregates.