BAFF and CD4+ T cells are major survival factors for long-lived splenic plasma cells in a B-cell-depletion context.

Previous data have suggested that B-cell-depletion therapy may induce the settlement of autoreactive long-lived plasma cells (LLPCs) in the spleen of patients with autoimmune cytopenia. To investigate this process, we used the AID-CreERT2-EYFP mouse model to follow plasma cells (PCs) engaged in an immune response. Multiplex polymerase chain reaction at the single-cell level revealed that only a small fraction of splenic PCs had a long-lived signature, whereas PCs present after anti-CD20 antibody treatment appeared more mature, similar to bone marrow PCs. This observation suggested that, in addition to a process of selection, a maturation induced on B-cell depletion drove PCs toward a long-lived program. We showed that B-cell activating factor (BAFF) and CD4+ T cells play a major role in the PC survival niche, because combining anti-CD20 with anti-BAFF or anti-CD4 antibody greatly reduce the number of splenic PCs. Similar results were obtained in the lupus-prone NZB/W model. These different contributions of soluble and cellular components of the PC niche in the spleen demonstrate that the LLPC expression profile is not cell intrinsic but largely depends on signals provided by the splenic microenvironment, implying that interfering with these components at the time of B-cell depletion might improve the response rate in autoimmune cytopenia.

Emergence of long-lived autoreactive plasma cells in the spleen of primary warm auto-immune hemolytic anemia patients treated with rituximab.

Primary warm autoimmune hemolytic anemia (wAIHA) is a rare autoimmune disease in which red blood cells are eliminated by IgG autoantibodies. We analyzed the antibody-secreting cells in the spleen and the peripheral blood of wAIHA patients in various contexts of treatment. Plasmablasts were observed in peripheral blood of newly diagnosed wAIHA patients and, accordingly, active germinal center reactions were present in the spleen of patients receiving short-term corticosteroid therapy. Long-term corticosteroid regimens markedly reduced this response while splenic plasma cells were able to persist, a fraction of them secreting anti-red blood cell IgG in vitro. In wAIHA patients treated by rituximab and who underwent splenectomy because of treatment failure, plasma cells were still present in the spleen, some of them being autoreactive. By using a set of diagnostic genes that allowed us to assess the plasma cell maturation stage, we observed that these cells displayed a long-lived program, differing from the one of plasma cells from healthy donors or from wAIHA patients with various immunosuppressant treatments, and more similar to the one of normal long-lived bone-marrow plasma cells. Interestingly, an increased level of B-cell activating factor (BAFF) was observed in the supernatant of spleen cell cultures from such rituximab-treated wAIHA patients. These results suggest, in line with our previous report on primary immune thrombocytopenia, that the B-cell depletion induced by rituximab promoted a suitable environment for the maturation and survival of auto-immune long-lived plasma cells in the spleen.

A splenic IgM memory subset with antibacterial specificities is sustained from persistent mucosal responses.

To what extent immune responses against the gut flora are compartmentalized within mucosal tissues in homeostatic conditions remains a much-debated issue. We describe here, based on an inducible AID fate-mapping mouse model, that systemic memory B cell subsets, including mainly IgM+ B cells in spleen, together with IgA+ plasma cells in spleen and bone marrow, are generated in mice in the absence of deliberate immunization. While the IgA component appears dependent on the gut flora, IgM memory B cells are still generated in germ-free mice, albeit to a reduced extent. Clonal relationships and renewal kinetics after anti-CD20 treatment reveal that this long-lasting splenic population is mainly sustained by output of B cell clones persisting in mucosal germinal centers. IgM-secreting hybridomas established from splenic IgM memory B cells showed reactivity against various bacterial isolates and endogenous retroviruses. Ongoing activation of B cells in gut-associated lymphoid tissues thus generates a diversified systemic compartment showing long-lasting clonal persistence and protective capacity against systemic bacterial infections.

Long-term complications of splenectomy in adult immune thrombocytopenia.

The recent large decrease in splenectomy use for chronic immune thrombocytopenia (ITP) is partly due to still-unsolved questions about long-term safety. We performed the first single-center exposed/unexposed cohort study evaluating the long-term incidence of splenectomy complications in patients with primary ITP. Overall, 83 patients who underwent splenectomy more than 10 years ago (exposed) were matched with 83 nonsplenectomized patients (unexposed) on the date of ITP diagnosis ±5 years, age and gender. After a median follow-up of 192 months (range 0.5-528), 43 patients (52%) achieved overall response after splenectomy. Splenectomized patients experienced more venous thromboembolism (VTE) than controls (n = 13 vs n = 2, P = 0.005). On multivariate analysis, splenectomy was an independent risk factor of VTE (hazard ratio = 4.006, P = 0.032 [95% confidence interval: 1.13-14.21]). Splenectomized patients presented more severe infections on long-term follow-up: all required hospitalization, and 5/26 (19%) infections led to severe sepsis or septic shock and to death for 3 cases (none in controls). However, the incidence of malignancy was similar in both groups, as was cardiovascular risk, which appeared to be related more to ITP than splenectomy. Finally, splenectomy did not significantly decrease overall survival. Despite the risk of thrombosis and severe sepsis, splenectomy remains an effective and curative treatment for ITP.

Kikuchi Disease-Like Inflammatory Pattern in Cutaneous Inflammatory Infiltrates Without Lymph Node Involvement: A New Clue for the Diagnosis of Lupus?

Kikuchi-Fujimoto disease (KFD) is a rare and benign disorder that usually occurs in young adults with enlarged lymph nodes containing infiltrate of cytotoxic T cells and nuclear debris. It can be a manifestation of systemic lupus erythematosus (SLE) although the strength of this association has varied among studies. Although specific KFD cutaneous lesions are well described, pure cutaneous lesions have never been reported. We studied a series of patients prospectively entered into a database between 2007 and 2014 with skin biopsies showing diffuse or localized inflammatory infiltrates reminiscent of cutaneous KFD, without lymph-node-related KFD. We called these skin lesions "Kikuchi disease-like inflammatory pattern" (KLIP). Twenty-nine patients, whose median age was 49 years at the time of skin biopsy, were selected and retrospectively analyzed using standardized clinical and histology charts. In skin biopsies, KLIP was localized to restricted areas within the inflammatory infiltrate (17%) or diffuse (83%), and was the only histological finding (45%) or accompanied interface dermatitis with or without dermal mucinosis (55%). Clinical dermatological findings varied widely. A definite diagnosis could be established for 24 patients: 75% had connective tissue diseases or vasculitis, mainly cutaneous lupus erythematosus (CLE) (n = 16, 67%), including 5 SLE with satisfying American College of Rheumatology criteria; 3 of the remaining patients had malignant hemopathies. CLE patients were mostly young females with acute (n = 5), subacute (n = 4), or chronic CLE (n = 6) or lupus tumidus (n = 1). Two were classified as having anti-tumor necrosis factor-alpha-induced lupus. Because two-thirds of these patients were finally diagnosed with CLE, we think that KLIP may represent a new histopathological clue for the diagnosis of lupus based on skin biopsy, requiring clinical-immunological comparison to make the correct diagnosis. KLIP should not be considered a variant of classical KFD, but rather as an elementary pattern of cutaneous inflammation, that might be the expression of the same cytotoxic process within skin infiltrates as that involved in KFD. This lesion might reflect a particular T-cell-mediated autoimmune process directed against mononuclear cells within cutaneous lupus infiltrates.

Are anti-proteinase-3 ANCA a useful marker of granulomatosis with polyangiitis (Wegener's) relapses? Results of a retrospective study on 126 patients.

OBJECTIVES: Predicting granulomatosis with polyangiitis (Wegener's) (GPA) relapses based on ANCA titers remains a source of debate. Our objective was to evaluate the relevance of monitoring PR3-ANCA titers for GPA management. METHODS: This retrospective study included 126 patients fulfilling the 1990 ACR criteria for GPA and PR3-ANCA-positive at the time of diagnosis. Disease activity was assessed with BVAS/WG and Disease Extent Index. For each patient, a median of 12 serum samples was analyzed, i.e., one every 5.5months. RESULTS: Induction therapy obtained remission in 88% of the patients. ANCA became negative by IF for 70/115 (60.9%) patients and by ELISA for 90/115 (78.3%). After median follow-up of 70months, 85/126 (67.5%) patients had 154 clinical relapses associated with cANCA and PR3-ANCA-positivity for 122 (79.2%) and 102 (66.2%) of them, respectively. Relapse-free survival was significantly longer for patients who remained PR3-ANCA-negative (HR 0.60 [95% CI 0.39-0.92], P=0.02). Individual ANCA-profile analysis revealed that, for 60% of GPA patients, clinical outcomes and ANCA-titer changes were closely associated, i.e., ANCA were always positive during relapses and negative during remission. The 35 patients with fluctuating ANCA-positivity during remission were in partial remission or had developed grumbling GPA. CONCLUSION: Although ANCA were positive during most systemic relapses or residual disease, no strict clinical-immunological correspondence was observed for 25% of the patients. Thus, GPA management cannot be based on ANCA levels alone.

The essential role of the mitochondria and reactive oxygen species in Cisplatin-mediated enhancement of fas ligand-induced apoptosis in malignant pleural mesothelioma.

Cytotoxic chemotherapeutic drugs such as cisplatin (CDDP) synergistically interact with soluble Fas ligand (sFasL) to mediate profound induction of apoptosis in cancer cells, particularly those refractory to this death-inducing ligand. The goal of this study was to evaluate the roles of the mitochondria-dependent apoptotic cascade and the CDDP-generated reactive oxygen species (ROS) in mediating the supra-additive enhancement of cytotoxicity and apoptosis in combination-treated malignant pleural mesothelioma (MPM) cells. MPM cells were treated with sequential CDDP/sFasL in vitro. Cell viability and apoptosis were determined by MTT and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays. Stable transfectants expressing high levels of Bcl2 were created by retroviral gene transfer. Specific proteolytic activity of caspases 3, 8, and 9 were measured using fluorescent substrates. Pretreating MPM cells with CDDP increased their susceptibility to sFasL by 2- to more than 20-fold. Overexpression of either Bcl-2, the selective caspase 9 inhibitor z-LEHD-fmk, or the antioxidant N-acetylcysteine significantly abrogated combination-induced cytotoxicity and apoptosis. Moreover, the robust activation of caspase 8 in combination-treated cells was completely suppressed by Bcl-2 overexpression, thus implicating a mitochondria-mediated amplification feedback loop. As an in vivo correlate, sequential intraperitoneal administration of CDDP and sFasL significantly inhibited the growth of intraperitoneal MPM human xenografts in nude mice. Our data indicate that the mitochondria-dependent feedback loop of the caspase activation cascade and the generation of ROS are both essential in mediating profound cytotoxicity and apoptosis of MPM cells treated with CDDP and sFasL. This mechanistic study establishes a the translational framework for the clinical application of sequential CDDP/sFasL in the treatment of MPM.