Objective-Our objective was to determine a mechanism for the thrombocytopenia of murine Wiskott-Aldrich syndrome (WAS).Materials and Methods-Consumption rates of WAS protein (WASP)( −) and wild-type (WT) platelets were measured by injection of 5-chloromethylfluorescein diacetate (CMFDA)-labeled platelets into WT or WASP(−) recipients, and by in vivo biotinylation. Platelet and reticulated platelet counts were performed using quantitative flow cytometry. Bone marrow megakaryocyte number and ploidy was assessed by flow cytometry. Phagocytosis of CMFDA-labeled, opsonized platelets was assessed using bone marrow-derived macrophages. Serum antiplatelet antibodies were assayed via their binding to WT platelets.Results-CMFDA-labeled WASP(−) platelets are consumed more rapidly than WT platelets in either WT or WASP(−) recipients. In vivo biotinylation studies corroborate these findings and show a normal consumption rate for WASP(−) reticulated platelets. The number of reticulated platelets is reduced in WASP(−) mice, but a significant number of the mice show an increased proportion of reticulated platelets and more severe thrombocytopenia. Sera from some of the latter group contain antiplatelet antibodies. Compared to WT platelets, WASP(−) platelets opsonized with anti-CD61 or 6A6 antibody are taken up more rapidly by bone marrow-derived macrophages. In vivo consumption rates of WASP(−) platelets are more accelerated by opsonization than are those of WT platelets.Conclusion-Both rapid clearance and impaired production contribute to the thrombocytopenia of murine WAS. Increased susceptibility of opsonized WASP(−) platelets to phagocytosis leads to increased in vivo clearance. This correlates with a higher incidence of individuals with an elevated fraction of reticulated platelets, a more severe thrombocytopenia, and antiplatelet antibodies. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe Wiskott-Aldrich syndrome (WAS) is an X-linked recessive condition of variable penetrance, classically described as a triad of immunodeficiency, eczema, and thrombocytopenia [1,2]. It is caused by mutations that reduce the level of the WAS protein (WASP). WASP is a 54-kDa polypeptide that is expressed primarily, but not exclusively [3,4], in hematopoietic cells. WASP transmits and integrates signals arising at the cell membrane that result in actin polymerization. This can, in turn, have multiple effects, including but not limited to changes in cell shape and motility. While its function in T cells and macrophages has been studied in some detail [5,6], its biological role in platelets is not clear.WAS patients can have severe thrombocytopenia, with platelet counts ranging from 10,000 to 50,000 per uL in one series [7]. In some cases, the thrombocytopenia is the predominant clinical abnormality. These cases, formerly termed X-linked thrombocytopenia, are frequently due to missense mutations in the first four exons of the gene [8], and can show a fluctuating course resembling immune t...

BackgroundThe linkage between periodontal disease and rheumatoid arthritis is well established. Commonalities among the two are that both are chronic inflammatory diseases characterized by bone loss, an association with the shared epitope susceptibility allele, and anti-citrullinated protein antibodies.MethodsTo explore immune mechanisms that may connect the two seemingly disparate disorders, we measured host immune responses including T-cell phenotype and anti-citrullinated protein antibody production in human leukocyte antigen (HLA)-DR1 humanized C57BL/6 mice following exposure to the Gram-negative anaerobic periodontal disease pathogen Porphyromonas gingivalis. We measured autoimmune arthritis disease expression in mice exposed to P. gingivalis, and also in arthritis-resistant mice by flow cytometry and multiplex cytokine-linked and enzyme-linked immunosorbent assays. We also measured femoral bone density by microcomputed tomography and systemic cytokine production.ResultsExposure of the gingiva of DR1 mice to P. gingivalis results in a transient increase in the percentage of Th17 cells, both in peripheral blood and cervical lymph nodes, a burst of systemic cytokine activity, a loss in femoral bone density, and the generation of anti-citrullinated protein antibodies. Importantly, these antibodies are not produced in response to P. gingivalis treatment of wild-type C57BL/6 mice, and P. gingivalis exposure triggered expression of arthritis in arthritis-resistant mice.ConclusionsExposure of gingival tissues to P. gingivalis has systemic effects that can result in disease pathology in tissues that are spatially removed from the initial site of infection, providing evidence for systemic effects of this periodontal pathogen. The elicitation of anti-citrullinated protein antibodies in an HLA-DR1-restricted fashion by mice exposed to P. gingivalis provides support for the role of the shared epitope in both periodontal disease and rheumatoid arthritis. The ability of P. gingivalis to induce disease expression in arthritis-resistant mice provides support for the idea that periodontal infection may be able to trigger autoimmunity if other disease-eliciting factors are already present.

Objective: To study the role of anti-platelet antibodies in the thrombocytopenia of murine WAS. Methods: A flow cytometric method was developed for detection of serum anti-platelet antibodies via their binding to intact target platelets lacking surface antibodies. Platelets were labeled with CMFDA in order to track their clearance from the circulation. WASP(−)μMT(−/−) mice were generated by standard breeding methods. Results: Serum anti-platelet antibodies were detected in approximately 40% of WASP(−) males. The mean level of reticulated platelets is significantly increased in these antibody(+) males. While WASP(−) males show an approximately 50% reduction in platelet counts, 5-10% of them show a more severe thrombocytopenia associated with increased reticulated platelets, suggesting the presence of clearance-inducing antiplatelet antibodies (CIAA). In support of that inference, 90% of the latter mice show detectable serum antiplatelet antibodies. The antibodies are primarily IgG, and are also detected in over 30% of CD47(−/−) males. WASP(−)μMT(−/−) males, which demonstrate no serum or platelet associated antibodies, show a degree of thrombocytopenia similar to that of WASP(−) males. Their platelet clearance rates remain accelerated – more so in WASP(−)μMT(−/−) than WASP(+)μMT(−/−) recipients. Conclusions: These findings suggest that platelet WASP deficiency results in an increase in platelet clearance rates by two mechanisms: an antibody independent mechanism which largely requires WASP deficiency in trans, and an antibody dependent mechanism which does not. Both an increased incidence of antiplatelet antibodies and an increased susceptibility to their effects contribute to antibody dependent clearance of WASP(−) platelets.

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While antibodies to citrullinated proteins have become a diagnostic hallmark in rheumatoid arthritis (RA), we still do not understand how the autoimmune T cell response is influenced by these citrullinated proteins. To investigate the role of citrullinated antigens in HLA-DR1- and DR4-restricted T cell responses, we utilized mouse models that express these MHC-II alleles to determine the relationship between citrullinated peptide affinity for these DR molecules and the ability of these peptides to induce a T cell response. Using a set of peptides from proteins thought to be targeted by the autoimmune T cell responses in RA, aggrecan, vimentin, fibrinogen, and type II collagen, we found that while citrullination can enhance the binding affinity for these DR alleles, it does not always do so, even when in the critical P4 position. Moreover, if peptide citrullination does enhance HLA-DR binding affinity, it does not necessarily predict the generation of a T cell response. Conversely, citrullinated peptides can stimulate T cells without changing the peptide binding affinity for HLA-DR1 or DR4. Furthermore, citrullination of an autoantigen, type II collagen, which enhances binding affinity to HLA-DR1 did not enhance the severity of autoimmune arthritis in HLA-DR1 transgenic mice. Additional analysis of clonal T cell populations stimulated by these peptides indicated cross recognition of citrullinated and wild type peptides can occur in some instances, while in others cases the citrullination generates a novel T cell epitope. Finally, cytokine profiles of the wild type and citrullinated peptide stimulated T cells unveiled a significant disconnect between proliferation and cytokine production. Altogether, these data demonstrate the lack of support for a simplified model with universal correlation between affinity for HLA-DR alleles, immunogenicity and arthritogenicity of citrullinated peptides. Additionally they highlight the complexity of both T cell receptor recognition of citrulline as well as its potential conformational effects on the peptide:HLA-DR complex as recognized by a self-reactive cell receptor.

The study of ex vivo phagocytosis via flow cytometry requires that one distinguish experimentally between uptake and adsorption of fluorescently labeled targets by phagocytes. Removal of the latter quantity from the analysis is the most common means of analyzing such data. Because the probability of phagocytosis is a function of the probability of adsorption, and because partially quenched fluorescence after uptake often overlaps with that of negative controls, this approach is suboptimal at best. Here, we describe a numerical analysis model which overcomes these limitations. We posit that the random adsorption of targets to macrophages, and subsequent phagocytosis, is a function of three parameters: the ratio of targets to macrophages (m), the mean fluorescence intensity imparted to the phagocyte by the internalized target (alpha), and the probability of phagocytosis per adsorbed target (p). The potential values of these parameters define a parameter space and their values at any point in parameter space can be used to predict the fraction of adsorption(+) and [adsorption(−), phagocytosis(+)] cells that might be observed experimentally. By systematically evaluating the points in parameter space for the latter two values and comparing them to experimental data, the model arrives at sets of parameter values that optimally predict such data. Using activated THP-1 cells as macrophages and platelets as targets, we validate the model by demonstrating that it can distinguish between the effects of experimental changes in m, alpha, and p. Finally, we use the model to demonstrate that platelets from a congenitally thrombocytopenic WAS patient show an increased probability of ex vivo phagocytosis. This finding correlates with other evidence that rapid in vivo platelet consumption contributes significantly to the thrombocytopenia of WAS. Our numerical analysis method represents a useful and innovative approach to multivariate analysis.

Ag-specific immunotherapy is a long-term goal for the treatment of autoimmune diseases; however developing a means of therapeutically targeting autoimmune T cells in an Ag-specific manner has been difficult. Through the engineering of an HLA-DR1 chimeric Ag receptor (CAR), we have produced CD8+ CAR T cells that target CD4+ T cells in an Ag-specific manner and tested their ability to inhibit the development of autoimmune arthritis in a mouse model. The DR1 CAR molecule was engineered to contain CD3ζ activation and CD28 signaling domains and a covalently linked autoantigenic peptide from type II collagen (CII; DR1-CII) to provide specificity for targeting the autoimmune T cells. Stimulation of the DR1-CII CAR T cells by an anti-DR Ab induced cytokine production, indicating that the DR1-CAR functions as a chimeric molecule. In vitro CTL assays using cloned CD4+ T cells as target cells demonstrated that the DR1-CII CAR T cells efficiently recognize and kill CD4+ T cells that are specific for the CII autoantigen. The CTL function was highly specific, as no killing was observed using DR1-restricted CD4+ T cells that recognize other Ags. When B6.DR1 mice, in which autoimmune arthritis had been induced, were treated with the DR1-CII CAR T cells, the CII-specific autoimmune CD4+ T cell response was significantly decreased, autoantibody production was suppressed, and the incidence and severity of the autoimmune arthritis was diminished. These data demonstrate that HLA-DR CAR T cells have the potential to provide a highly specific therapeutic approach for the treatment of autoimmune disease.

Thrombocytopenia due to rapid platelet consumption contributes to the severe thrombocytopenia of the Wiskott Aldrich Syndrome (WAS), and to the milder thrombocytopenia seen in murine WAS. Here we show that rapid clearance of 111In labeled murine WASP(−) platelets correlates with enhanced splenic uptake. Using platelets labeled with a pH sensitive fluorescent marker (pHrodo), we quantify normal platelet uptake by red pulp macrophages (RPM), and demonstrate its enhancement after in vivo opsonization of platelets. The spleens of WASP(−) mice contain an increased number of RPM, and rapid clearance of WASP(−) platelets in WASP(−) mice in turn generates an increased number of pHrodo(+) splenic RPM. To separately assess the platelet intrinsic and recipient dependent functions involved in the clearance and splenic phagocyte uptake of WASP(−) platelets, we performed “crossed” pHrodo(+) platelet injection studies (WT to WASP(−), WASP(−) to WT). We show that an extrinsic effect of recipient WASP deficiency on the clearance of WASP(−)platelets correlates with increased platelet uptake by RPM. An intrinsic effect of platelet WASP deficiency on platelet clearance does not, however, correlate with increased total uptake by WT or WASP(−) RPM. In contrast to other published findings, we find no evidence of a baseline or antibody-induced increase in phosphatidyl serine exposure on WASP(−) platelets. Our findings suggest that an increased number of RPM in WASP(−) mice contributes significantly to the increased platelet consumption rate in WASP(−) mice. This may explain the consistent efficacy of splenectomy in murine and clinical WAS.