Proportion versus absolute counts of B cell populations in the assessment of immunological profiles of kidney transplant recipients.

Immunophenotyping lymphocytes in kidney transplant recipients often raises questions as to whether proportions or absolute counts should be considered, especially for longitudinal assessment. Several studies conclude the pathophysiology of rejection based on proportions of naive and memory B cells. We compared the two analytical methods for B cells sampled from 82 clinically stable, adult kidney transplant recipients. Time post-transplant was analyzed both as a continuous variable and as tertiles (<1.5 years, 1.5-8 years, and > 8 years). B cells were stained for CD38 and IgD and were classified according to mature B cells (Bm) classification. The proportion of cells in the naive Bm2 compartment decreased by more than half in the late versus the early tertile, whereas the percentages of memory early Bm5 tripled and that of memory Bm5 cells doubled. In contrast, we observed a substantial reduction in naive B cell counts, but very stable memory B cell counts. Linear regressions showed that the absolute reduction in the Bm2 cell compartment was independent of age, sex, graft function, immunosuppression scheme, and rejection occurrence. In conclusion, the physiological reservoir of naive cells decreases over time post-transplant in kidney recipients, whereas that of memory B cells remains stable. Peripheral B subset percentages should be interpreted cautiously when analyzing pathophysiological processes.

Tacrolimus prevents von Willebrand factor secretion by allostimulated human glomerular endothelium.

Little is known about the endothelial injury caused directly by circulating donor-specific antibodies (DSAs) during antibody-mediated rejection. von Willebrand factor (vWF) is a highly thrombotic glycoprotein stored in Weibel-Palade bodies in endothelial cells. It has been shown that its secretion is triggered by allostimulation. Calcineurin-like phosphatases regulate pathways involved in vWF secretion. Therefore, we hypothesized that tacrolimus would prevent alloantibody-induced glomerular lesions, in part via inhibition of vWF secretion from endothelial cells. Here, we used a human in vitro model of glomerular endothelium expressing HLA class I and II antigens and demonstrated that anti-HLA class II antibodies elicit a higher endothelial release of vWF than do anti-HLA class I antibodies in cell supernatants. We observed that tacrolimus treatment decreased vWF secretion after stimulation with both classes of anti-HLA antibodies and decreased platelet adhesion on allostimulated endothelial cells in a microfluidic chamber. In kidney recipients, tacrolimus trough levels were negatively associated with vWF blood levels. These results indicate that direct disruption of hemostasis via vWF secretion is a potential mechanism of antibody-mediated injury in patients with DSAs. Our results further suggest that the targeting of microcirculation hemostasis may be beneficial to prevent the development of microangiopathic lesions in antibody-mediated rejection.

Effects of alloantibodies to human leukocyte antigen on endothelial expression and serum levels of thrombomodulin.

Essentials The effect of alloantibodies on the endothelial expression of thrombomodulin is unknown. Thrombomodulin was quantified in stimulated endothelial cells and measured in serum samples. Anti-human leukocyte antigen (HLA) I vs. II antibodies have different effects on thrombomodulin. Anti-HLA II antibodies may promote a prothrombotic state and contribute to microangiopathy. SUMMARY: Rationale Thrombomodulin (TBM) is an anticoagulant and anti-inflammatory transmembrane protein expressed on endothelial cells. Donor-specific alloantibodies, particularly those against human leukocyte antigen (HLA) class II, are associated with microvascular endothelial damage in solid allografts. Objective Our aim was to characterize the effects of anti-HLA antibodies on endothelial expression of TBM, and in particular, the differential effects of anti-HLA class I compared with those of anti-HLA class II. Methods We used human glomerular microvascular endothelial cells to examine TBM expression on anti-HLA-treated cells, and we tested sera from transplant recipients for soluble TBM. Results We found that whereas membrane TBM expression increased in a dose-dependent manner in the presence of anti-HLA class I antibodies, treatment with anti-HLA class II led to minimal TBM expression on the endothelial surface but to a cytosolic accumulation. Platelet adhesion studies confirmed the functional impact of anti-HLA class II. Quantitative densitometry of the membrane lysates further suggested that anti-HLA class II impairs TBM glycosylation. Furthermore, we found a significant association between the presence of circulating anti-HLA class II antibodies in transplant recipients and low serum levels of TBM. Conclusion These results indicate that ligation of anti-HLA class I and II antibodies produces different effects on the endothelial expression of TBM and on serum levels of TBM in transplant recipients. Anti-HLA class II antibodies may be associated with a prothrombotic state, which could explain the higher occurrence of microangiopathic lesions in the allograft and the poor outcomes observed in patients with these alloantibodies.