The Role of B and T Lymphocytes in Recurrent Acquired Thrombotic Thrombocytopenic Purpura.
Authors: Mariani Mariagabriella , Cairo Andrea , Palla Roberta , Lotta Luca Andrea , Rovati Andrea , Trisolini Silvia Maria , Peyvandi Flora 
Published: October 2019

Abstract
Abstract 3521
Poster Board III-458
Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening disease characterized by thrombocytopenia, microangiopathic haemolytic anemia and widespread microvascular thrombosis, resulting in multiorgan ischemia. Acquired TTP, which accounts for approximately 95% of cases, can be either associated to anti ADAMTS13 autoantibodies or secondary to a number of associated conditions (tumors, organ transplantation, use of drugs, pregnancy). There are several key questions that remain unanswered, including the importance of cellular immunity in immunomediated TTP, and the search for laboratory markers that predict disease relapse, an event that occurs in 20% to 50% of patients who survive the acute initial episode. Since alterations of peripheral B and T cell subsets in patients with autoimmune diseases (i.e. rheumatoid arthritis and systemic lupus erythematous) are well established, the aim of this study was to analyze the role of B and T cells in acquired TTP and during its recurrence.

Methods
36 healthy controls and 36 consecutive patients affected by acquired TTP during remission (defined as the maintenance of normalization of clinical and laboratory data for at least 30 days after the last plasma therapy following the resolution of the last acute episode) were characterized by flow cytometry for the quantification of:
- different peripheral B cell subsets, using labeled surface markers anti-CD19-PerCP, anti-IgD-PE, anti-IgM-FITC, anti-CD27-APC, anti-CD38-FITC;
- different peripheral T cell subsets, using labeled surface markers anti-CD3-FITC, anti-CD4-PE, anti-CD8-APC, anti-CD25-FITC. For Treg cell quantification (only 17 patients were analyzed), anti-CD3-PerCP, anti-CD4-FITC, anti-CD25-PE and the intracellular marker FoxP3 were used.
Patients were classified in two subgroups: those who developed at least two episodes of TTP (n=19, with recurrence) and those who experienced a single episode only and no relapse during at least one year of retrospective observational time (n=17).
ADAMTS13 activity was measured by residual collagen binding assay (Gerritsen et al, Thromb Haemost 1999). The presence of anti-ADAMTS13 IgG was evaluated by Western blotting and ELISA assays, using recombinant ADAMTS13 protein as antigen and patients' plasma as a source of antibody. The presence of anti-ADAMTS13 IgA, IgM, IgG subclasses (IgG1, 2, 3, 4) were evaluated by ELISA assays.
For continuous variables, differences between controls and patients and between patients with or without recurrence were evaluated by the t-test; for discrete variables, by the chi square test. P values smaller than 0.05 were considered statistically significant. Analyses were performed using the SPSS package version 17.0.

Results
1) TTP patients had an increased number of CD19+ B cells (mean ± SD 13% ± 5) compared with the control group (10% ± 3, p=0.001). No difference was observed in T cells subsets.
2) The results of the characterization of the two groups of patients (with and without recurrence) are reported in the table.
Patients with and without recurrence did not differ either in the amount of Treg FoxP3 or in the presence of IgA, IgM and IgG subclasses.

Discussion
The increased B cell numbers in acquired TTP indicates an enhanced activation of cellular immunity. Analysis of B cell subsets, particularly of memory B cells, and of T cells CD24+CD25+ during remission might provide information on the likelihood of recurrence in TTP.
In conclusion, in recurrent TTP patients the higher amount of B cells might result in persistent autoantibodies production whilst the decreased level of T cells CD4+CD25+ may lead to a decreased inhibition of autoreactive T cells. These findings may explain the higher level of recurrence in these patients.

Disclosures:
Peyvandi: Archemix Corporation: Consultancy.