Complement dysregulation is key in the pathogenesis of atypical Haemolytic Uraemic Syndrome (aHUS), but no clear role for complement has been identified in Thrombotic Thrombocytopenic Purpura (TTP). We aimed to assess complement activation and cytokine response in acute antibody-mediated TTP. Complement C3a and C5a and cytokines (interleukin (IL)-2, IL-4, IL-6, IL-10, tumour necrosis factor, interferon-γ and IL-17a) were measured in 20 acute TTP patients and 49 remission cases. Anti-ADAMTS13 immunoglobulin G (IgG) subtypes were measured in acute patients in order to study the association with complement activation. In acute TTP, median C3a and C5a were significantly elevated compared to remission, C3a 63·9 ng/ml vs. 38·2 ng/ml (P < 0·001) and C5a 16·4 ng/ml vs. 9·29 ng/ml (P < 0·001), respectively. Median IL-6 and IL-10 levels were significantly higher in the acute vs. remission groups, IL-6: 8 pg/ml vs. 2 pg/ml (P = 0·003), IL-10: 6 pg/ml vs. 2 pg/ml (P < 0·001). C3a levels correlated with both anti-ADAMTS13 IgG (rs = 0·604, P = 0·017) and IL-10 (rs = 0·692, P = 0·006). No anti-ADAMTS13 IgG subtype was associated with higher complement activation, but patients with the highest C3a levels had 3 or 4 IgG subtypes present. These results suggest complement anaphylatoxin levels are higher in acute TTP cases than in remission, and the complement response seen acutely may relate to anti-ADAMTS13 IgG antibody and IL-10 levels.

SummaryAutoantibodies inhibiting the activity of the metalloproteinase, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), underlie the pathogenesis of thrombotic thrombocytopenic purpura (TTP). Rituximab (RTX) combined with plasma-exchange (PEX) is an effective treatment in TTP. Patients can remain in remission for extended periods following PEX/RTX, and this is associated with continuing reduction in antibodies to ADAMTS13. Factors controlling B cell differentiation to autoantibody production, including stimulation through the B cell receptor and interactions with the B cell-activating factor (BAFF), may thus impact length of remission. In this cross-sectional study, we measured naive and memory B cell phenotypes [using CD19/immunoglobulin (Ig)D/CD27] following PEX/RTX treatment in TTP patients at B cell return (n = 6) and in 12 patients in remission 10-68 months post-RTX. We also investigated relationships among serum BAFF, soluble CD23 (sCD23 -a surrogate measure of acquiring B memory (CD27 + ) phenotype) and BAFF receptor (BAFF-R) expression. At B cell return after PEX/RTX, naive B cells predominated and BAFF-R expression was reduced compared to healthy controls (P < 0·001). In the remission group, despite numbers of CD19 + B cells within normal limits in most patients, the percentage and absolute numbers of pre-switch and memory B cells remained low, with sCD23 levels at the lower end of the normal range. BAFF levels were correlated inversely with BAFF-R expression and time after therapy. In conclusion, the long-term effects of RTX therapy in patients with TTP included slow regeneration of memory B cell subsets and persistently reduced BAFF-R expression across all B cell subpopulations. This may reflect the delay in selection and differentiation of potentially autoreactive (ADAMTS13-specific) B cells, resulting in relatively long periods of low disease activity after therapy.

These data indicate that the modifications increase uptake, alter the antigenicity of HIV- 1 p24 Vacc-4x peptides and increase the breadth of the response to the Vacc-4x peptides. The in vitro cell culture system is a suitable model for the antigenic assessment of peptide antigens.

Background and Aims Thrombotic Thrombocytopenic Purpura (TTP) is due to deficiency or dysfunction of the metalloproteinase ADAMTS13, which cleaves von Willebrand factor. This results in increased platelet adhesion and thrombus formation in small blood vessels. Most cases are associated with autoantibodies (IgM/IgG/IgA) to ADAMTS13, which are somatically mutated (sub-classes being predominantly IgG4 subclass, followed by IgG1). Rituximab (RTX) is an effective treatment for patients with TTP and also for patients with Rheumatoid Arthritis (RA). In both conditions, remission can last for months or even years after RTX but in RA, relapse closer to B-cell return is more common. Factors controlling B-cell maturation, differentiation and autoantibody production probably impact remission. Survival and maturation of B-cells largely depends on interactions with the B-cell cytokine B-cell activating factor (BAFF). In order to explore the possible role of the BAFF/BAFF-receptor (BAFF-R or BR3) system in maintaining long remissions following RTX in TTP patients, we investigated B-cell phenotypes, serum BAFF levels and BAFF-R expression. The relationship between time after treatment, B-cell phenotype and BAFF-R expression with laboratory parameters in TTP patients was also explored. Materials and Methods We compared B-cell phenotypes using FACS analysis (based on IgD/CD27 on CD19+ B-cells) between both diseases at B-cell return following RTX, RA (n = 5), TTP (n = 6) and in the TTP cohort of patients, also in patients in long-term remission (n = 13;10–68 months post-RTX). In this cross-sectional study, we also investigated the temporal relationship between serum BAFF and expression of BAFF-R on B-cell sub-populations. Comparisons between B-cell sub-populations, BAFF-R expression on B-cell sub-populations and between diseases were with non-parametric statistics and relationships with time and serum BAFF levels using Spearman’s rank correlation coefficients. Results B-cell return after RTX followed similar patterns in TTP and RA, with naïve B-cells predominant and BAFF-R expression low in all B-cell subpopulations in both diseases. In TTP patients in long-term remission, naive B-cells remained high (p < 0.01) and post-switch memory B-cells (IgD-CD27+) low (p < 0.05) and BAFF-R expression reduced (p < 0.001 for all B-cell sub-populations compared with healthy controls. BAFF levels were most strongly inversely correlated with BAFF-R expression (MFI) on naïve (IgD+) B-cells (r2 > 0.45; p = 0.01). Conclusions As extended periods of remission after RTX were associated with persistence of naïve B-cell phenotype in TTP and this may reflect the inability of potentially autoreactive B-cells to be selected into the memory compartment or into autoantibody-secreting cells. Reduced BAFF-R expression after RTX may decrease pro-survival B-cell signalling, perhaps reducing opportunities for ADAMTS13 specific B-cells to proliferate and/or undergo class-switch recombination.

Background B cell depletion therapy based on rituximab (RTX) is an effective therapy for Rheumatoid Arthritis (RA) and other autoimmune diseases such as Thrombotic Thrombocytopenic Purpura (TTP). Serum BAFF levels are within the normal range in patients with RA prior to RTX therapy, but are raised in TTP patients1. BAFF levels rise after RTX in both diseases and remain elevated for many months in the majority of patients. We have previously described lower percentages of naive and memory B cells expressing BAFFR at relapse following RTX2. Here, we have explored whether BAFFR expression after B cell repopulation was related to RTX treatment per se by comparing BAFFR expression in patients repopulated but remaining in remission in patients with RA or with TTP after RTX. Objectives To compare expression of BAFFR in naive and memory B cells in TTP and RA patients in remission after RTX. Methods Phenotype analysis of BAFFR expression on naive (CD27-) and memory (CD27+) B cells was performed using combinations of CD19 and CD27 in TTP patients (n:14) in remission after B cell repopulation following RTX and compared with patients with RA (n: 9) also in remission after RTX therapy. Mann Whitney U test was used to compare results. Results The time of sampling following RTX ranged from 8 to 38 months in RA and 10 to 68 months in TTP. Phenotypic analysis showed similar proportions of naïve (CD27-) and memory (CD27+) B cells in both conditions. For naïve B cells, median was 93.5% (range 86.9-98.9%) for TTP and median 96.2% (range 66.0-98.8%) for RA. Similarly for memory B cells: median 6.1% (range 1.1-13.1%) for TTP and median 3.8% (range 1.2-34%) for RA. BAFFR expression on naive B cells from TTP patients was significantly higher with median 94.7% (range 29.4-99.7%) than RA median 52% (range 18.3-88%) (p=0. 01). For memory B cells, BAFFR% median levels were also higher for TTP patients: median 88.3% (range 40.8-97.8%) compared to RA patients median 42.2% (range 22.0-61.8%) (p<0.01). There was no correlation between the percentage of cells expressing BAFFR and months after RTX in either group. Conclusions Repopulation of B cells after RTX follows similar patterns in patients with TTP and RA, mirroring ontogeny with naïve B cells predominating. However, BAFFR expression was significantly reduced following RTX in RA patients compared with patients with TTP. This was not related to the time elapsed following RTX. Downregulation of BAFFR expression on naïve B cell can occur following signalling through B cell receptor and toll-like receptor or in response to raised BAFF levels. The finding that BAFFR expression was lower in B cells in RA but not TTP patients after RTX suggests a disease specific dysregulation, consistent with an autoimmune phenotype already present in repopulating B cells. References B-cell activating factor is elevated in acute idiopathic thrombotic thrombocytopenic purpura. Thomas MR et al. Br J Haematol. 2011;155:620-2 B-cell-activating factor receptor expression on naive and memory B cells: relationship ...