Sensitive B-cell receptor repertoire analysis shows repopulation correlates with clinical response to rituximab in rheumatoid arthritis.

BACKGROUND: Although B-cell depleting therapy in rheumatoid arthritis (RA) is clearly effective, response is variable and does not correlate with B cell depletion itself. METHODS: The B-cell receptor (BCR) repertoire was prospectively analyzed in peripheral blood samples of twenty-eight RA patients undergoing rituximab therapy. Timepoints of achieved BCR-depletion and -repopulation were defined based on the percentage of unmutated BCRs in the repertoire. The predictive value of early BCR-depletion (within one-month post-treatment) and early BCR-repopulation (within 6 months post-treatment) on clinical response was assessed. RESULTS: We observed changes in the peripheral blood BCR repertoire after rituximab treatment, i.e., increased clonal expansion, decreased clonal diversification and increased mutation load which persisted up to 12 months after treatment, but started to revert at month 6. Early BCR depletion was not associated with early clinical response but late depleters did show early response. Patients with early repopulation with unmutated BCRs showed a significant decrease in disease activity in the interval 6 to 12 months. Development of anti-drug antibodies non-significantly correlated with more BCR repopulation. CONCLUSION: Our findings indicate that rather than BCR-depletion it is repopulation with unmutated BCRs, possibly from naïve B cells, which induces remission. This suggests that (pre-existing) differences in B-cell turnover between patients explain the interindividual differences in early clinical effect.

Chronic Lymphocytic Leukemia: Disease Biology.

BACKGROUND: B-cell receptor (BCR) signaling is crucial for normal B-cell development and adaptive immunity. In chronic lymphocytic leukemia (CLL), the malignant B cells display many features of normal mature B lymphocytes, including the expression of functional B-cell receptors (BCRs). Cross talk between CLL cells and the microenvironment in secondary lymphatic organs results in BCR signaling and BCR-driven proliferation of the CLL cells. This critical pathomechanism can be targeted by blocking BCR-related kinases (BTK, PI3K, spleen tyrosine kinase) using small-molecule inhibitors. Among these targets, Bruton tyrosine kinase (BTK) inhibitors have the highest therapeutic efficacy; they effectively block leukemia cell proliferation and generally induce durable remissions in CLL patients, even in patients with high-risk disease. By disrupting tissue homing receptor (i.e., chemokine receptor and adhesion molecule) signaling, these kinase inhibitors also mobilize CLL cells from the lymphatic tissues into the peripheral blood (PB), causing a transient redistribution lymphocytosis, thereby depriving CLL cells from nurturing factors within the tissue niches. SUMMARY: The clinical success of the BTK inhibitors in CLL underscores the central importance of the BCR in CLL pathogenesis. Here, we review CLL pathogenesis with a focus on the role of the BCR and other microenvironment cues. KEY MESSAGES: (i) CLL cells rely on signals from their microenvironment for proliferation and survival. (ii) These signals are mediated by the BCR as well as chemokine and integrin receptors and their respective ligands. (iii) Targeting the CLL/microenvironment interaction with small-molecule inhibitors provides a highly effective treatment strategy, even in high-risk patients.

B-cell receptor profiling before and after IVIG monotherapy in newly diagnosed idiopathic inflammatory myopathies.

OBJECTIVE: To unravel B-cell receptor (BcR) characteristics in muscle tissues and peripheral blood and gain more insight into BcR repertoire changes in peripheral blood in idiopathic inflammatory myopathies (IIMs), and study how this correlates to the clinical response to IVIG. METHODS: Nineteen treatment-naive patients with newly diagnosed IIM were prospectively treated with IVIG monotherapy. RNA-based BcR repertoire sequencing was performed in muscle biopsies collected before, and in peripheral blood (PB) collected before and nine weeks after IVIG treatment. Results were correlated to patients' clinical improvement based on the total improvement score (TIS). RESULTS: Prior to IVIG treatment, BcR clones found in muscle tissue could be retrieved in peripheral blood. Nine weeks after IVIG treatment, new patient-specific dominant BcR clones appeared in peripheral blood while pre-treatment dominant BcR clones disappeared. The cumulative frequency of all dominant BcR clones before treatment was significantly higher in individuals who responded to IVIG compared with those who did not respond to IVIG, and correlated with a higher CK. During follow-up, a decrease in the cumulative frequency of all dominant clones correlated with a higher TIS. CONCLUSION: In treatment-naive patients with newly diagnosed IIM, muscle tissue and peripheral blood share expanded BcR clones. In our study a higher cumulative frequency of dominant BcR clones in blood before treatment was associated with a higher CK and better treatment response, suggesting that response to IVIG may depend on the composition of the pre-treatment BcR repertoire.

Reemergence of pathogenic, autoantibody-producing B cell clones in myasthenia gravis following B cell depletion therapy.

Myasthenia gravis (MG) is an autoantibody-mediated autoimmune disorder of the neuromuscular junction. A small subset of patients (<10%) with MG, have autoantibodies targeting muscle-specific tyrosine kinase (MuSK). MuSK MG patients respond well to CD20-mediated B cell depletion therapy (BCDT); most achieve complete stable remission. However, relapse often occurs. To further understand the immunomechanisms underlying relapse, we studied autoantibody-producing B cells over the course of BCDT. We developed a fluorescently labeled antigen to enrich for MuSK-specific B cells, which was validated with a novel Nalm6 cell line engineered to express a human MuSK-specific B cell receptor. B cells (≅ 2.6 million) from 12 different samples collected from nine MuSK MG patients were screened for MuSK specificity. We successfully isolated two MuSK-specific IgG4 subclass-expressing plasmablasts from two of these patients, who were experiencing a relapse after a BCDT-induced remission. Human recombinant MuSK mAbs were then generated to validate binding specificity and characterize their molecular properties. Both mAbs were strong MuSK binders, they recognized the Ig1-like domain of MuSK, and showed pathogenic capacity when tested in an acetylcholine receptor (AChR) clustering assay. The presence of persistent clonal relatives of these MuSK-specific B cell clones was investigated through B cell receptor repertoire tracing of 63,977 unique clones derived from longitudinal samples collected from these two patients. Clonal variants were detected at multiple timepoints spanning more than five years and reemerged after BCDT-mediated remission, predating disease relapse by several months. These findings demonstrate that a reservoir of rare pathogenic MuSK autoantibody-expressing B cell clones survive BCDT and reemerge into circulation prior to manifestation of clinical relapse. Overall, this study provides both a mechanistic understanding of MuSK MG relapse and a valuable candidate biomarker for relapse prediction.

B-cell receptor signaling induces proteasomal degradation of PDCD4 via MEK1/2 and mTORC1 in malignant B cells.

B-cell receptor (BCR) signaling plays a major role in the pathogenesis of B-cell malignancies and is an established target for therapy, including in chronic lymphocytic leukemia cells (CLL), the most common B-cell malignancy. We previously demonstrated that activation of BCR signaling in primary CLL cells downregulated expression of PDCD4, an inhibitor of the translational initiation factor eIF4A and a potential tumor suppressor in lymphoma. Regulation of the PDCD4/eIF4A axis appeared to be important for expression of the MYC oncoprotein as MYC mRNA translation was increased following BCR stimulation and MYC protein induction was repressed by pharmacological inhibition of eIF4A. Here we show that MYC expression is also associated with PDCD4 down-regulation in CLL cells in vivo and characterize the signaling pathways that mediate BCR-induced PDCD4 down-regulation in CLL and lymphoma cells. PDCD4 downregulation was mediated by proteasomal degradation as it was inhibited by proteasome inhibitors in both primary CLL cells and B-lymphoma cell lines. In lymphoma cells, PDCD4 degradation was predominantly dependent on signaling via the AKT pathway. By contrast, in CLL cells, both ERK and AKT pathways contributed to PDCD4 down-regulation and dual inhibition using ibrutinib with either MEK1/2 or mTORC1 inhibition was required to fully reverse PDCD4 down-regulation. Consistent with this, dual inhibition of BTK with MEK1/2 or mTORC1 resulted in the strongest inhibition of BCR-induced MYC expression. This study provides important new insight into the regulation of mRNA translation in B-cell malignancies and a rationale for combinations of kinase inhibitors to target translation control and MYC expression.

Proapoptotic and immunomodulatory effects of SYK inhibitor entospletinib in combination with obinutuzumab in patients with chronic lymphocytic leukaemia.

Spleen tyrosine kinase (SYK) is indispensable in B-cell receptor signalling. SYK inhibitor entospletinib demonstrated clinical efficacy in patients with chronic lymphocytic leukaemia (CLL). However, pharmacodynamic effects of SYK inhibition in CLL cells and immunomodulatory effects of B-cell receptor-signalling inhibitors in patients with CLL are poorly understood. We conducted a phase 2 trial of entospletinib in combination with obinutuzumab, an anti-CD20 antibody, in 17 patients with relapsed/refractory CLL. Pharmacodynamic analysis demonstrated that treatment with entospletinib led to rapid downmodulation of pSTAT3 and the anti-apoptotic protein MCL1 in CLL cells. Meanwhile, 6 months of combination therapy was accompanied by a reduction in interferon-γ secretion in CD4+ T-cells and a reversal of exhausted phenotype, as evidenced by downregulation of PD-1. Thus, SYK inhibition downmodulates MCL-1 and partially restores T-cell immunity in CLL. Trial registration number NCT03010358.

Anti-ICOSL New Antigen Receptor Domains Inhibit T Cell Proliferation and Reduce the Development of Inflammation in the Collagen-Induced Mouse Model of Rheumatoid Arthritis.

Lymphocyte costimulation plays a central role in immunology, inflammation, and immunotherapy. The inducible T cell costimulator (ICOS) is expressed on T cells following peptide: MHC engagement with CD28 costimulation. The interaction of ICOS with its sole ligand, the inducible T cell costimulatory ligand (ICOSL; also known as B7-related protein-1), triggers a number of key activities of T cells including differentiation and cytokine production. Suppression of T cell activation can be achieved by blocking this interaction and has been shown to be an effective means of ameliorating disease in models of autoimmunity. In this study, we isolated specific anti-ICOSL new antigen receptor domains from a synthetic phage display library and demonstrated their ability to block the ICOS/ICOSL interaction and inhibit T cell proliferation. Anti-mouse ICOSL domains, considered here as surrogates for the use of anti-human ICOSL domains in patient therapy, were tested for efficacy in a collagen-induced mouse model of rheumatoid arthritis where they significantly decreased the inflammation of joints and delayed and reduced overall disease progression and severity.

CD19-CAR trials.

CD19 is a B-lineage-specific transmembrane glycoprotein, the expression of which is maintained on more than 95% B-cell malignancies. This strict lineage restriction makes CD19 an ideal target for immune therapies using chimeric antigen receptors (CARs). Here, we review published phase 1 trials of T cells expressing CARs targeting CD19 and describe briefly the biological questions that they addressed. All patients treated in these trials had relapsed B-cell malignancies, which in many cases were chemorefractory. Nonetheless, major responses have been observed, especially in patients with chronic lymphocytic leukemia and acute lymphoblastic leukemia. Many of these responses were accompanied by a systemic inflammatory reaction syndrome that could be life threatening but was almost always reversible with adequate medical management. Given their remarkable activity, CD19-CAR T cells are likely to be quickly incorporated into the management of B-cell neoplasms; these cells have become the paradigm for similar strategies targeting other cancers.

B cell activation through CD40 and IL4R ligation modulates the response of chronic lymphocytic leukaemia cells to BAFF and APRIL.

The two tumour necrosis factor family proteins BAFF (TNFSF13B) and APRIL (TNFSF13) and their receptors [BAFF-R (TNFRSF13C), TACI (TNFRSF13B), BCMA (TNFRSF17)] play a critical role in the survival of normal B cells. The sensitivity of normal B cells to BAFF and APRIL can be modulated by signals regulated by their receptors. This modulation, however, has not been extensively investigated in chronic lymphocytic leukaemia (CLL) cells. We evaluated the expression, regulation and signalling of BAFF and APRIL receptors in normal and in CLL cells upon stimulation through CD40+IL4R and BCR. We further analysed the prognostic value of BAFF and APRIL receptors expression in patients with CLL. BCMA expression was significantly higher on CLL cells than on normal B cells. BCR and CD40+IL4R stimulation promoted an increase in TACI and BCMA expression, cell viability and activation in normal B cells. A similar effect was observed in CLL cells after CD40+IL4R but not BCR stimulation. BCMA expression correlated with unmutated IGHV genes, poor-risk cytogenetics, and short progression-free survival. These findings further characterize the link between CD40+IL4R regulatory signals, BAFF, APRIL and their receptors and the survival of leukaemic cells and clinical features of CLL.

Human polyclonal anti-hepatitis B surface antigen immunoglobulin reduces the frequency of acute rejection after liver transplantation for chronic hepatitis B.

BACKGROUND: Use of polyclonal anti-hepatitis B surface antigen immunoglobulin (HBIg) has been shown to reduce hepatitis B virus (HBV) recurrence after liver transplantation (LT) and to decrease the frequency of acute cellular rejection (ACR). However, the protective role of HBIg against ACR remains controversial, since HBV infection has been also associated with a lower incidence of ACR. AIM: To assess the relationship between HBIg immunoprophylaxis and the incidence of rejection after LT. METHODS: 260 patients (158 males, 43 +/- 14 years old) submitted to LT were retrospectively evaluated and divided into three groups, according to the presence of HBsAg and the use of HBIg. Group I was comprised of HBsAg-positive patients (n = 12) that received HBIg for more than 6 months. Group II was comprised of HBsAg-positive patients that historically have not received HBIg or have been treated irregularly for less than 3 months (n = 10). Group III was composed of 238 HBsAg-negative subjects that have not received HBIg. RESULTS: HBIg-treated patients (group I) had significantly less ACR episodes, when compared to group II and III. No differences between groups II and III were observed. CONCLUSIONS: Long-term HBIg administration contributes independently to reduce the number of ACR episodes after LT.