Andrew Getahun

Monophosphorylation of CD79a and CD79b ITAM Motifs Initiates a SHIP-1 Phosphatase-Mediated Inhibitory Signaling Cascade Required for B Cell Anergy

Anergic B cells are characterized by impaired signaling and activation after aggregation of their antigen receptors (BCR). The molecular basis of this impairment is not understood. In studies reported here, Src homology-2 (SH2)-containing inositol 5-phosphatase SHIP-1 and its adaptor Dok-1 were found to be constitutively phosphorylated in anergic B cells, and activation of this inhibitory circuit was dependent on Src-family kinase activity and consequent to biased BCR immunoreceptor tyrosine-based activation motif (ITAM) monophosphorylation. B cell-targeted deletion of SHIP-1 caused severe lupus-like disease. Moreover, absence of SHIP-1 in B cells led to loss of anergy as indicated by restoration of BCR signaling, loss of anergic surface phenotype, and production of autoantibodies. Thus, chronic BCR signals maintain anergy in part via ITAM monophosphorylation-directed activation of an inhibitory signaling circuit involving SHIP-1 and Dok-1.

Molecular underpinning of B cell Anergy

Summary:  A byproduct of the largely stochastic generation of a diverse B‐cell specificity repertoire is production of cells that recognize autoantigens. Indeed, recent studies indicate that more than half of the primary repertoire consists of autoreactive B cells that must be silenced to prevent autoimmunity. While this silencing can occur by multiple mechanisms, it appears that most autoreactive B cells are silenced by anergy, wherein they populate peripheral lymphoid organs and continue to express unoccupied antigen receptors yet are unresponsive to antigen stimulation. Here we review molecular mechanisms that appear operative in maintaining the antigen unresponsiveness of anergic B cells. In addition, we present new data indicating that the failure of anergic B cells to mobilize calcium in response to antigen stimulation is not mediated by inactivation of stromal interacting molecule 1, a critical intermediary in intracellular store depletion‐induced calcium influx.

Of ITIMs, ITAMs and ITAMis, revisiting Immunoglobulin Fc Receptor signaling

Receptors for immunoglobulin Fc regions play multiple critical roles in the immune system, mediating functions as diverse as phagocytosis, triggering degranulation of basophils and mast cells, promoting immunoglobulin class switching, and preventing excessive activation. Transmembrane signaling associated with these functions is mediated primarily by two amino acid sequence motifs, ITAMs (immunoreceptor tyrosine‐based activation motifs) and ITIMs (immunoreceptor tyrosine‐based inhibition motifs) that act as the receptors’ interface with activating and inhibitory signaling pathways, respectively. While ITAMs mobilize activating tyrosine kinases and their consorts, ITIMs mobilize opposing tyrosine and inositol‐lipid phosphatases. In this review, we will discuss our current understanding of signaling by these receptors/motifs and their sometimes blurred lines of function.

Requirement for complement in antibody responses is not explained by the classic pathway activator IgM

Animals lacking complement factors C1q, C2, C3, or C4 have severely impaired Ab responses, suggesting a major role for the classic pathway. The classic pathway is primarily initiated by antigen–Ab complexes. Therefore, its role for primary Ab responses seems paradoxical because only low amounts of specific Abs are present in naive animals. A possible explanation could be that the classic pathway is initiated by IgM from naive mice, binding with sufficient avidity to the antigen. To test this hypothesis, a knock-in mouse strain, Cμ13, with a point mutation in the gene encoding the third constant domain of the μ-heavy chain was constructed. These mice produce IgM in which proline in position 436 is substituted with serine, a mutation previously shown to abrogate the ability of mouse IgM to activate complement. Unexpectedly, the Ab response to sheep erythrocytes and keyhole limpet hemocyanin in Cμ13 mice was similar to that in WT mice. Thus, although secreted IgM and the classic pathway activation are both required for the normal primary Ab response, this does not require that IgM activate C. This led us to test Ab responses in animals lacking one of three other endogenous activators of the classic pathway: specific intracellular adhesion molecule-grabbing nonintegrin R1, serum amyloid P component, and C-reactive protein. Ab responses were also normal in these animals.

Complement Receptors 1 and 2 in Murine Antibody Responses to IgM-Complexed and Uncomplexed Sheep Erythrocytes

Early complement components are important for normal antibody responses. In this process, complement receptors 1 and 2 (CR1/2), expressed on B cells and follicular dendritic cells (FDCs) in mice, play a central role. Complement-activating IgM administered with the antigen it is specific for, enhances the antibody response to this antigen. Here, bone marrow chimeras between Cr2−/− and wildtype mice were used to analyze whether FDCs or B cells must express CR1/2 for antibody responses to sheep erythrocytes (SRBC), either administered alone or together with specific IgM. For robust IgG anti-SRBC responses, CR1/2 must be expressed on FDCs. Occasionally, weak antibody responses were seen when only B cells expressed CR1/2, probably reflecting extrafollicular antibody production enabled by co-crosslinking of CR2/CD19/CD81 and the BCR. When SRBC alone was administered to mice with CR1/2+ FDCs, B cells from wildtype and Cr2−/− mice produced equal amounts of antibodies. Most likely antigen is then deposited on FDCs in a way that optimizes engagement of the B cell receptor, making CR2-facilitated signaling to the B cell superfluous. SRBC bound to IgM will have more C3 fragments, the ligands for CR1/2, on their surface than SRBC administered alone. Specific IgM, forming a complex with SRBC, enhances antibody responses in two ways when FDCs express CR1/2. One is dependent on CR1/2+ B cells and probably acts via increased transport of IgM-SRBC-complement complexes bound to CR1/2 on marginal zone B cells. The other is independent on CR1/2+ B cells and the likely mechanism is that IgM-SRBC-complement complexes bind better to FDCs than SRBC administered alone. These observations suggest that the immune system uses three different CR1/2-mediated effector functions to generate optimal antibody responses: capture by FDCs (playing a dominant role), transport by marginal zone B cells and enhanced B cell signaling.

Impaired antibody responses but normal proliferation of specific CD4+ T cells in mice lacking complement receptors 1 and 2.

Severely impaired Ab responses are seen in animals lacking C (complement) factors C2, C3 or C4 as well as CR1/2 (C receptors 1 and 2). The molecular mechanism behind this phenomenon is not understood. One possibility is that C‐containing immune complexes are endocytosed via CR2 on B cells and presented to specific CD4+ T cells, which would then proliferate and provide efficient help to specific B cells. In vitro, B cells can endocytose immune complexes via CR1/2 and present the Ag to T cells. Whether absence of this Ag presenting function in Cr2−/− mice (mice lacking CR1/2) explains their low Ab response is unclear. To address this question, Cr2−/− and wild type mice were transferred with OVA‐specific T cells, obtained from the DO11.10 strain which has a transgenic TCR recognizing an OVA peptide. The animals were subsequently immunized with sheep red blood cells (SRBC) conjugated to OVA. Interestingly, proliferation of the OVA‐specific T cells was normal in Cr2−/− mice, although their Ab response to both SRBC and OVA was severely impaired. These observations suggest that the impaired Ab response in Cr2−/− mice cannot be explained by a lack of appropriate induction of T cell help.

STING/MPYS Mediates Host Defense against Listeria monocytogenes Infection by Regulating Ly6C hi Monocyte Migration

MPYS (also known as STING, MITA, and TMEM173) is a type I IFN stimulator that is essential for host defense against DNA virus infection and appears important in defense against certain bacteria. The in vivo significance and mechanisms by which MPYS mediates host defense against nonviral pathogens are unknown. Using an MPYS-deficient mouse (Tmem173), we determined that, distinct from the IFNAR−/− mice, MPYS deficiency leads to increased bacterial burden in the liver upon Listeria monocytogenes infection. The increase was correlated with the diminished MCP-1 and MCP-3 chemokine production and decreased blood and liver Ly6Chi monocyte frequency. We further demonstrate that MPYS-deficient Ly6Chi monocytes are intrinsically defective in migration to the liver. Lastly, adoptive transfer of wild-type Ly6Chi monocyte into MPYS-deficient mice decreases their liver bacterial burden. Our findings reveal a novel in vivo function of MPYS that is distinct from its role in activating type I IFN production.

A Balance between B Cell Receptor and Inhibitory Receptor Signaling Controls Plasma Cell Differentiation by Maintaining Optimal Ets1 Levels

Signaling through the BCR can drive B cell activation and contribute to B cell differentiation into Ab-secreting plasma cells. The positive BCR signal is counterbalanced by a number of membrane-localized inhibitory receptors that limit B cell activation and plasma cell differentiation. Deficiencies in these negative signaling pathways may cause autoantibody generation and autoimmune disease in both animal models and human patients. We have previously shown that the transcription factor Ets1 can restrain B cell differentiation into plasma cells. In this study, we tested the roles of the BCR and inhibitory receptors in controlling the expression of Ets1 in mouse B cells. We found that Ets1 is downregulated in B cells by BCR or TLR signaling through a pathway dependent on PI3K, Btk, IKK2, and JNK. Deficiencies in inhibitory pathways, such as a loss of the tyrosine kinase Lyn, the phosphatase Src homology region 2 domain–containing phosphatase 1 (SHP1) or membrane receptors CD22 and/or Siglec-G, result in enhanced BCR signaling and decreased Ets1 expression. Restoring Ets1 expression in Lyn- or SHP1-deficient B cells inhibits their enhanced plasma cell differentiation. Our findings indicate that downregulation of Ets1 occurs in response to B cell activation via either BCR or TLR signaling, thereby allowing B cell differentiation and that the maintenance of Ets1 expression is an important function of the inhibitory Lyn → CD22/SiglecG → SHP1 pathway in B cells.

Targeting B cells in treatment of autoimmunity.

B cells have emerged as effective targets for therapeutic intervention in autoimmunities in which the ultimate effectors are antibodies, as well as those in which T cells are primary drivers of inflammation. Proof of this principle has come primarily from studies of the efficacy of Rituximab, an anti-CD20 mAb that depletes B cells, in various autoimmune settings. These successes have inspired efforts to develop more effective anti-CD20s tailored for specific needs, as well as biologicals and small molecules that suppress B cell function without the risks inherent in B cell depletion. Here we review the current status of B cell-targeted therapies for autoimmunity.

Phosphatase regulation of immunoreceptor signaling in T cells, B cells and mast cells

Recent progress has begun to reveal the often complex and changing roles of phosphotyrosine and phosphoinositide phosphatases in regulation of immunoreceptor signaling. The resultant confusion has been further increased by discoveries of new players. Here we provide a review of recent progress in defining the roles of these enzymes in immunoreceptor-dependent mast cell, T cell and B cell activation.