Single-cell analyses of immune thrombocytopenic patients reveal multiorgan dissemination of high-affinity autoreactive plasma cells

Abstract

The major therapeutic goal for immune thrombocytopenia (ITP) is to restore normal platelet counts using drugs to promote platelet production or by interfering with mechanisms responsible for platelet destruction. 80% of patients possess anti-integrin αIIbβ3 (GPIIbIIIa) IgG autoantibodies causing platelet opsonization and phagocytosis. The spleen is considered the primary site of autoantibody production by autoreactive B cells and platelet destruction. The immediate failure in ~50% of patients to recover a normal platelet count after anti-CD20 Rituximab-mediated B cell depletion and splenectomy suggest that autoreactive, rituximab-resistant, IgG-secreting B cells (IgG-SC) reside in other anatomical compartments. We analyzed >3,300 single IgG-SC from spleen, bone marrow and/or blood of 27 patients with ITP revealing high inter-individual variability in affinity for GPIIbIIIa with variations over 3 logs. IgG-SC dissemination and range of affinities were however similar per patient. Longitudinal analysis of autoreactive IgG-SC upon treatment with anti-CD38 mAb daratumumab demonstrated variable outcomes, from complete remission to failure with persistence of high-affinity anti-GPIIbIIIa IgG-SC in the bone marrow. This study demonstrates the existence and dissemination of high-affinity autoreactive plasma cells in multiple anatomical compartments of patients with ITP that may cause the failure of current therapies.

Regulation of CD8 T cell by B-cells: A narrative review

“…Response to B-cell depletion with Rituximab is frequent but inconsistent (~60%). One current explanation is that Rituximab refractoriness is characterized by the persistence of long-lived autoreactive plasma cells in the spleen and in other sites, the emergence of which may even be promoted by B-cell depletion itself (12,13). However, alternative and nonexclusive mechanisms may be involved.…”

Rituximab resistance in ITP and beyond

“…While the cause of ITP was initially identified as a humoral factor by observing that the infusion of citrated whole blood or plasma from ITP patients into healthy recipients would result in a decrease in platelet count (6), more recently, the mechanism of ITP has been better understood. Circulating B cells and autoreactive plasma cells are responsible for the overproduction of autoantibodies (7,8). Autoantibody binding with glycoproteins (GPs) IIb/IIIa, Ib, IV and Ia/IIa (9,10) on the platelet surface is the crucial element in the pathogenesis of ITP.…”

“…The results suggest that the long-lived plasma cells in the spleen could explain rituximab resistance. These pathogenic plasma cells have been demonstrated throughout the spleen, peripheral blood, and bone marrow, potentially leading to treatment failure (8). Higher concentration of B-cellactivating factor (BAFF), a member of the tumor necrosis factor family which promotes B-cell survival was detected in the supernatant of spleen cell cultures of rituximab refractory patients (50).…”

Single-Cell Technologies for the Study of Antibody-Secreting Cells

“…An alternative approach to study ASC in droplets is to collect droplets in a horizontal plane and measure changes in droplet fluorescence over multiple timepoints, termed a stationary droplet-assay (14) or DropMap (73,74). DropMap measures fluorescence relocation to a central line of antibody-capturing beads aligned using a magnetic field within each droplet.…”

“…The ability to measure relocation over time within DropMap allows for the determination of both antibody affinity and secretion rate, in addition to determining the proportion of antigen-specific and total ASC within a sample as with other microfluidic techniques. DropMap has been applied to investigate the physiology of ASC in viral infection (75), autoimmunity (74), and fundamental ASC biology (73,76). In our view, DropMap offers a major advancement in current capabilities to functionally characterize ASC, particularly for defining the specificity and affinity repertoire of ASC.…”