Liver and Spleen Work Together to Clear Allogeneic Platelets from the Bloodstream during Transfusion Refractoriness

Background: Transfusing platelets into patients already alloimmunized against the HLA class I antigen can be challenging. Such alloantibodies can result in rapid elimination of the transfused platelets from the bloodstream, leading to transfusion failure known as platelet transfusion refractoriness (PTR). The questions of how and where transfused platelets are eliminated during PTR remains unanswered. Nevertheless, the liver and spleen may serve as potential sites of sequestration, thereby contributing to the rapid clearance of allogeneic platelets. Liver macrophages excel at internalization of desialylated platelets, while the splenic counterpart are highly effective at phagocytosis of opsonized platelets.

Aim: To unravel the mechanisms of clearance of transfused platelets during PTR.

Methods: A previously described murine model of platelet alloimmunization was used to mimick PTR (Couvidou et al. 2023). Fluorescent allogeneic platelets (H2b) were tracked by flow cytometry following transfusion into naïve or alloimmune mice (H2d). 30 min after transfusion, liver and spleen were harvested and processed for histology, and stained for transfused platelets (eGFP⁺), macrophages (F4/80⁺), and endothelium (CD31⁺). Identification of the cells positive for allogeneic platelets was performed 30 min post transfusion on (CD45+ F4/80+ CD11b low) liver/spleen cells by flow cytometry. The relative importance of spleen was studied in splenectomized/sham mice. The role of the liver was assessed by administering a low dose of clodronate liposomes (6.5 mg/kg i.v.) 24 h before transfusion. Asialoglycoprotein receptors were blocked with asialofetuin (8 mg i.v.) just before transfusion. IVIg/human albumin (1.0 g/kg i.p.) 24 h before transfusion or a mAb directed against FcγRIV which blocks FcγRIIIa (clone 9E9) (50 µg/mouse i.p.) 4h before transfusion were injected to study the role of FcRγ. To assess transfusion kinetics, we performed a nonlinear regression fit of the percentage of transfused platelets (n=3 for all experiments).

Results:

Alloimmune mice were confirmed to be in a refractory state 30 min after transfusion, whereas naïve mice showed a different kinetic (p=0.0082) in which platelets were still circulating. Confocal images showed allogeneic platelets present within both splenic and hepatic macrophages only in refractory mice. Flow cytometry analysis identified the Kupffer cells (F4/80+ CD11b low) as the major hepatic cells responsible for platelet uptake during PTR: 54.6% positive for allogeneic platelets versus 2.1% in naïve mice (p=0.05). In the spleen, red pulp macrophages (F4/80+ CD11b low) were identified as the primary population taking up platelets during PTR (19.1% positive for allogeneic platelets compared to 1.4% in naïve mice (p=0.05)). To assess the importance of FcγR in platelet clearance, mice were treated either with IVIg or with mAb 9E9. No difference in the elimination kinetic was shown between treated mice or control (p=0.1978 and p=0.5531, respectively). In line with these observations, splenectomized mice did not show a difference in the elimination of allogeneic platelets upon transfusion as compared to alloimmune sham controls (p=0.7067). While clodronate-treated mice showed different elimination kinetics compared to PBS-liposome-treated individuals (p=0.0021), at least half of the transfused platelets were still eliminated. Platelet elimination upon blockade of asialoglycoprotein receptors was not different compared to mice treated with sialylated equivalent proteins (p=0.4264). Splenectomy in combination with clodronate liposomes did alter the elimination kinetics of allogeneic platelets in alloimmune mice compared to sham controls treated with clodronate (p<0.0001), indicating a role of both the spleen and the liver.

Conclusions:

This work indicates allogeneic platelets end up in both the liver and the spleen during PTR, mainly in Kupffer cells and red pulp macrophages. Although the mechanisms by which these platelets are eliminated remain unclear, they do not seem to depend on asialoglycoprotein receptors, nor FcγR. This suggests there is an alternative or a compensatory mechanism, which sets apart this platelet elimination from other immune platelet pathologies.