HLA-Restriction of Human Treg Cells Is Not Required for Therapeutic Efficacy of Low-Dose IL-2 in Humanized Mice.

Regulatory T (Treg) cells are essential to maintain immune homeostasis in the intestine and Treg cell dysfunction is associated with several inflammatory and autoimmune disorders including inflammatory bowel disease (IBD). Efforts using low-dose (LD) interleukin-2 (IL-2) to expand autologous Treg cells show therapeutic efficacy for several inflammatory conditions. Whether LD IL-2 is an effective strategy for treating patients with IBD is unknown. Recently, we demonstrated that LD IL-2 was protective against experimental colitis in immune humanized mice in which human CD4+ T cells were restricted to human leukocyte antigen (HLA). Whether HLA restriction is required for human Treg cells to ameliorate colitis following LD IL-2 therapy has not been demonstrated. Here, we show that treatment with LD IL-2 reduced 2,4,6-trinitrobenzensulfonic acid (TNBS) colitis severity in NOD.PrkdcscidIl2rg-/- (NSG) mice reconstituted with human CD34+ hematopoietic stem cells. These data demonstrate the utility of standard immune humanized NSG mice as a pre-clinical model system to evaluate therapeutics targeting human Treg cells to treat IBD.

Optimization of novel reversible Bruton's tyrosine kinase inhibitors identified using Tethering-fragment-based screens.

Since the approval of ibrutinib for the treatment of B-cell malignancies in 2012, numerous clinical trials have been reported using covalent inhibitors to target Bruton's tyrosine kinase (BTK) for oncology indications. However, a formidable challenge for the pharmaceutical industry has been the identification of reversible, selective, potent molecules for inhibition of BTK. Herein, we report application of Tethering-fragment-based screens to identify low molecular weight fragments which were further optimized to improve on-target potency and ADME properties leading to the discovery of reversible, selective, potent BTK inhibitors suitable for pre-clinical proof-of-concept studies.

BCR-dependent lineage plasticity in mature B cells.

B2 cells engage in classical antibody responses, whereas B1 cells are considered carriers of innate immunity, biased toward recognizing epitopes present on the surfaces of common pathogens and self antigens. To explore the role of B cell antigen receptor (BCR) specificity in driving B1 cell differentiation, we developed a transgenic system allowing us to change BCR specificity in B cells in an inducible and programmed manner. Mature B2 cells differentiated into bona fide B1 cells upon acquisition of a B1 cell-typical self-reactive BCR through a phase of proliferative expansion. Thus, B2 cells have B1 cell differentiation potential in addition to their classical capacity to differentiate into memory and plasma cells, and B1 differentiation can be instructed by BCR-mediated self-reactivity, in the absence of B1-lineage precommitment.

CD62L is not a reliable biomarker for predicting PML risk in natalizumab-treated R-MS patients.

OBJECTIVE: To assess if the percentage of CD3(+)CD4(+)CD62L(+) cells in cryopreserved peripheral blood mononuclear cells (PBMCs) (here termed %CD62L) can predict risk of developing progressive multifocal leukoencephalopathy (PML) and better inform the physician for benefit-risk assessment of natalizumab treatment decisions in a global setting. METHODS: Cryopreserved PBMCs from 21 natalizumab-treated patients who developed PML and 104 matched natalizumab-treated patients with multiple sclerosis (MS) without PML collected as a part of Biogen clinical trials were retrospectively examined for CD3, CD4, CCR7, CD45RA, and CD62L by flow cytometry. RESULTS: In this cohort, %CD62L in natalizumab-treated patients did not predict PML risk. Natalizumab-treated patients with MS without PML showed highly variable %CD62L upon serial sampling. In the STRATA study, the distribution of %CD62L in samples collected more than 6 months before a PML diagnosis, at diagnosis, and in natalizumab-treated patients without PML overlapped. No statistical threshold for risk could be determined. In addition, we demonstrated that lymphocyte viability strongly affects %CD62L, supporting previous reports that %CD62L is inherently unstable following cryopreservation and is sensitive to sample collection. CONCLUSION: Data from this well-controlled cohort of natalizumab-treated patients indicate that %CD62L is not a biomarker of PML risk.

Cytoplasmic Ig alpha serine/threonines fine-tune Ig alpha tyrosine phosphorylation and limit bone marrow plasma cell formation.

Igα serine 191 and 197 and threonine 203, which are located in proximity of the Igα ITAM, dampen Igα ITAM tyrosine phosphorylation. In this study, we show that mice with targeted mutations of Igα S191, 197, and T203 displayed elevated serum IgG2c and IgG2b concentrations and had elevated numbers of IgG2c- and IgG2b-secreting cells in the bone marrow. BCR-induced Igα tyrosine phosphorylation was slightly increased in splenic B cells. Our results suggest that Igα serine/threonines limit formation of IgG2c- and IgG2b-secreting bone marrow plasma cells, possibly by fine-tuning Igα tyrosine-mediated BCR signaling.

BAFF activates Akt and Erk through BAFF-R in an IKK1-dependent manner in primary mouse B cells.

B cell activating factor (BAFF) signals through BAFF-R to promote mature B cell survival. Recent analyses of BAFF-induced signaling revealed direct association between augmented B cell metabolic fitness and activation of Akt, one of the key regulators of cell survival. The strongest and most reproducible induction of Akt occurs with significant delay (24 h) after BAFF treatment, where it precedes activation of anabolism. It was also recently shown that BAFF induces sustained Erk activation and increased turnover of the proapoptotic molecule Bim. Here we show that these BAFF-induced signaling pathways are mediated by BAFF-R and represent previously unknown arms of I kappa B kinase (IKK)1-dependent signaling. In combination with the known role of IKK1 in regulating transcription of prosurvival genes, our data underscore the central role of IKK1 in coordinating multiple BAFF-R-mediated signaling pathways controlling mature B cell homeostasis.

Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells.

Normal intestinal mucosa contains abundant immunoglobulin A (IgA)-secreting cells, which are generated from B cells in gut-associated lymphoid tissues (GALT). We show that dendritic cells (DC) from GALT induce T cell-independent expression of IgA and gut-homing receptors on B cells. GALT-DC-derived retinoic acid (RA) alone conferred gut tropism but could not promote IgA secretion. However, RA potently synergized with GALT-DC-derived interleukin-6 (IL-6) or IL-5 to induce IgA secretion. Consequently, mice deficient in the RA precursor vitamin A lacked IgA-secreting cells in the small intestine. Thus, GALT-DC shape mucosal immunity by modulating B cell migration and effector activity through synergistically acting mediators.

Basal immunoglobulin signaling actively maintains developmental stage in immature B cells.

In developing B lymphocytes, a successful V(D)J heavy chain (HC) immunoglobulin (Ig) rearrangement establishes HC allelic exclusion and signals pro-B cells to advance in development to the pre-B stage. A subsequent functional light chain (LC) rearrangement then results in the surface expression of IgM at the immature B cell stage. Here we show that interruption of basal IgM signaling in immature B cells, either by the inducible deletion of surface Ig via Cre-mediated excision or by incubating cells with the tyrosine kinase inhibitor herbimycin A or the phosphatidylinositol 3-kinase inhibitor wortmannin, led to a striking "back-differentiation" of cells to an earlier stage in B cell development, characterized by the expression of pro-B cell genes. Cells undergoing this reversal in development also showed evidence of new LC gene rearrangements, suggesting an important role for basal Ig signaling in the maintenance of LC allelic exclusion. These studies identify a previously unappreciated level of plasticity in the B cell developmental program, and have important implications for our understanding of central tolerance mechanisms.

Plasma cell differentiation and the unfolded protein response intersect at the transcription factor XBP-1.

The transcription factor X-box binding protein 1 (XBP-1) is essential for the differentiation of plasma cells and the unfolded protein response (UPR). Here we show that UPR-induced splicing of XBP-1 by the transmembrane endonuclease IRE1 is required to restore production of immunoglobulin in XBP-1-/- mouse B cells, providing an integral link between XBP-1, the UPR and plasma cell differentiation. Signals involved in plasma cell differentiation, specifically interleukin-4, control the transcription of XBP-1, whereas its post-transcriptional processing is dependent on synthesis of immunoglobulins during B cell differentiation. We also show that XBP-1 is involved in controlling the production of interleukin-6, a cytokine that is essential for plasma cell survival. Thus, signals upstream and downstream of XBP-1 integrate plasma cell differentiation with the UPR.