Therapeutic tactics for targeting B lymphocytes in autoimmunity and cancer.

B lymphocytes have become a very popular therapeutic target in a number of autoimmune indications due to their newly appreciated roles, and approachability, in these diseases. Many of the therapies now applied in autoimmunity were initially developed to deplete malignant B cells. These strategies have also been found to benefit patients suffering from such autoimmune diseases as multiple sclerosis, type I diabetes, systemic lupus erythematosus, and rheumatoid arthritis, to name a few. These observations have supported the expansion of research addressing the mechanistic contributions of B cells in these diseases, as well as blossoming of therapeutics that target them. This review seeks to summarize cutting-edge modalities for targeting B cells, including monoclonal antibodies, bispecific antibodies, antibody-drug conjugates, chimeric antigen receptor-T cells, and small molecule inhibitors. Efforts to refine B-cell targeted therapy to eliminate only pathogenic autoreactive cells will be addressed as well as the potential for future B-cell-based cellular therapeutics. Finally, we also address approaches that seek to silence B-cell function without depletion.

A partial human LCK defect causes a T cell immunodeficiency with intestinal inflammation.

Lymphocyte-specific protein tyrosine kinase (LCK) is essential for T cell antigen receptor (TCR)-mediated signal transduction. Here, we report two siblings homozygous for a novel LCK variant (c.1318C>T; P440S) characterized by T cell lymphopenia with skewed memory phenotype, infant-onset recurrent infections, failure to thrive, and protracted diarrhea. The patients' T cells show residual TCR signal transduction and proliferation following anti-CD3/CD28 and phytohemagglutinin (PHA) stimulation. We demonstrate in mouse models that complete (Lck-/-) versus partial (LckP440S/P440S) loss-of-function LCK causes disease with differing phenotypes. While both Lck-/- and LckP440S/P440S mice exhibit arrested thymic T cell development and profound T cell lymphopenia, only LckP440S/P440S mice show residual T cell proliferation, cytokine production, and intestinal inflammation. Furthermore, the intestinal disease in the LckP440S/P440S mice is prevented by CD4+ T cell depletion or regulatory T cell transfer. These findings demonstrate that P440S LCK spares sufficient T cell function to allow the maturation of some conventional T cells but not regulatory T cells-leading to intestinal inflammation.

Preclinical Analysis of Candidate Anti-Human CD79 Therapeutic Antibodies Using a Humanized CD79 Mouse Model.

The BCR comprises a membrane-bound Ig that is noncovalently associated with a heterodimer of CD79A and CD79B. While the BCR Ig component functions to sense extracellular Ag, CD79 subunits contain cytoplasmic ITAMs that mediate intracellular propagation of BCR signals critical for B cell development, survival, and Ag-induced activation. CD79 is therefore an attractive target for Ab and chimeric Ag receptor T cell therapies for autoimmunity and B cell neoplasia. Although the mouse is an attractive model for preclinical testing, due to its well-defined immune system, an obstacle is the lack of cross-reactivity of candidate therapeutic anti-human mAbs with mouse CD79. To overcome this problem, we generated knockin mice in which the extracellular Ig-like domains of CD79A and CD79B were replaced with human equivalents. In this study, we describe the generation and characterization of mice expressing chimeric CD79 and report studies that demonstrate their utility in preclinical analysis of anti-human CD79 therapy. We demonstrate that human and mouse CD79 extracellular domains are functionally interchangeable, and that anti-human CD79 lacking Fc region effector function does not cause significant B cell depletion, but induces 1) decreased expression of plasma membrane-associated IgM and IgD, 2) uncoupling of BCR-induced tyrosine phosphorylation and calcium mobilization, and 3) increased expression of PTEN, consistent with the levels observed in anergic B cells. Finally, anti-human CD79 treatment prevents disease development in two mouse models of autoimmunity. We also present evidence that anti-human CD79 treatment may inhibit Ab secretion by terminally differentiated plasmablasts and plasma cells in vitro.

Inhibitory Receptor Trap: A Platform for Discovery of Inhibitory Receptors That Utilize Inositol Lipid and Phosphotyrosine Phosphatase Effectors.

Among the areas of most impactful recent progress in immunology is the discovery of inhibitory receptors and the subsequent translation of this knowledge to the clinic. Although the original and canonical member of this family is FcγRIIB, more recent studies defined PD1 as an inhibitory receptor that constrains T cell immunity to tumors. These studies led to development of "checkpoint blockade" immunotherapies (CBT) for cancers in which PD1 interactions with its ligand are blocked. Unfortunately, although very effective in some patients, only a small proportion respond to this therapy. This suggests that additional as yet undescribed inhibitory receptors exist, which could be exploited. Here, we describe a new platform, termed inhibitory receptor trap (IRT), for discovery of members of this family. The approach takes advantage of the fact that many of the known inhibitory receptors mediate signaling by phospho-immunoreceptor tyrosine-based inhibition motif (ITIM) mediated recruitment of Src Homology 2 (SH2) domain-containing phosphatases including the SH2 domain-containing inositol phosphatase SHIP1 encoded by the INPP5D gene and the SH2 domain-containing phosphotyrosine phosphatases SHP1 and SHP2 encoded by the PTPN6 and PTPN11 genes respectively. Here, we describe the IRT discovery platform in which the SH2 domains of inhibitory phosphatases are used for affinity-based isolation and subsequent identification of candidate effectors via immunoblotting and high sensitivity liquid chromatography-mass spectrometry. These receptors may represent alternative targets that can be exploited for improved CBT. Salient observations from these studies include the following: SH2 domains derived from the respective phosphatases bind distinct sets of candidates from different cell types. Thus, cells of different identity and different activation states express partially distinct repertoires of up and downstream phosphatase effectors. Phosphorylated PD1 binds not only SHP2 but also SHIP1, thus the latter may be important in its inhibitory function. B cell antigen receptor signaling leads predominantly to CD79 mono-phosphorylation as indicated by much greater binding to LynSH2 than Syk(SH2)2. This balance of ITAM mono- versus bi-phosphorylation likely tunes signaling by varying activation of inhibitory (Lyn) and stimulatory (Syk) pathways.

Selective Loss of Responsiveness to Exogenous but Not Endogenous Cyclic-Dinucleotides in Mice Expressing STING-R231H.

Stimulator of interferon genes (STING) plays a central role in innate immune responses to viral and intracellular bacterial infections, and cellular damage. STING is a cytosolic sensor of cyclic dinucleotides (CDNs) including those produced by pathogenic bacteria and those arising endogenously as products of the DNA sensor cGAS (e.g., 2'3' cGAMP). The two most common alternative allelic variants of STING in humans are STING-R71H-G230A-R293Q (STING-HAQ) and STING-R232H that are found in 20.4% and 13.7-17.6% of the population, respectively. To determine the biologic consequences of these genotypic variations, we generated knock-in mice containing the murine equivalents of each variant and studied their responsiveness to CDNs. Homozygous STING-HAQ (R71H-I229A-R292Q) and STING-R231H mice were found to be unresponsive to all exogenous CDNs tested (ci-di-GMP, ci-di-AMP, 3'3' cGAMP and Rp,Rp-CDA). Responses of homozygous STING-HAQ mice to endogenous 2'3' cGAMP was also greatly impaired. However, homozygous STING-R231H mice are fully responsive to 2'3' cGAMP. Analysis of heterozygous mice revealed reduced responsiveness to exogenous and endogenous CDNs in mice carrying a single copy of STING-HAQ, while STING-R231H heterozygous mice exhibit reduced responsiveness to exogenous but not endogenous CDNs. These findings confirm and extend previous reports by demonstrating differing impact of allelic variation of STING on the ability to sense and respond to exogenous vs. endogenous CDNs. Finally, the STING-R231H variant mouse represents a useful tool with which to examine the relative contributions of STING sensing of exogenous and endogenous CDNs in the context of bacterial infections and CDN-based cancer immunotherapeutics.

A Precision B Cell-Targeted Therapeutic Approach to Autoimmunity Caused by Phosphatidylinositol 3-Kinase Pathway Dysregulation.

The inositol lipid phosphatases PTEN and SHIP-1 play a crucial role in maintaining B cell anergy and are reduced in expression in B cells from systemic lupus erythematosus and type 1 diabetes patients, consequent to aberrant regulation by miRNA-7 and 155. With an eye toward eventual use in precision medicine therapeutic approaches in autoimmunity, we explored the ability of p110δ inhibition to compensate for PI3K pathway dysregulation in mouse models of autoimmunity. Low dosages of the p110δ inhibitor idelalisib, which spare the ability to mount an immune response to exogenous immunogens, are able to block the development of autoimmunity driven by compromised PI3K pathway regulation resultant from acutely induced B cell-targeted haploinsufficiency of PTEN and SHIP-1. These conditions do not block autoimmunity driven by B cell loss of the regulatory tyrosine phosphatase SHP-1. Finally, we show that B cells in NOD mice express reduced PTEN, and low-dosage p110δ inhibitor therapy blocks disease progression in this model of type 1 diabetes. These studies may aid in the development of precision treatments that act by enforcing PI3K pathway regulation in patients carrying specific risk alleles.

Protective role of B cells in sterile particulate-induced lung injury.

Susceptibility to chronic beryllium (Be) disease is linked to HLA-DP molecules possessing a glutamic acid at the 69th position of the ß-chain (ßGlu69), with the most prevalent ßGlu69-containing molecule being HLA-DP2. We have previously shown that HLA-DP2 transgenic (Tg) mice exposed to Be oxide (BeO) develop mononuclear infiltrates in a peribronchovascular distribution and a beryllium-specific, HLA-DP2-restricted CD4+ T cell response. In addition to T cells, B cells constituted a major portion of infiltrated leukocytes in the lung of BeO-exposed HLA-DP2 Tg mice and sequester BeO particles within ectopic lymphoid aggregates and granulomas. B cell depletion was associated with a loss of lymphoid aggregates and granulomas as well as a significant increase in lung injury in BeO-exposed mice. The protective role of B cells was innate in origin, and BeO-induced B cell recruitment to the lung was dependent on MyD88 signaling. Similar to BeO-exposed HLA-DP2 mice, B cells also accumulate in the lungs of CBD subjects, located at the periphery and surrounding the granuloma. Overall, our data suggest a novel modulatory role for B cells in the protection of the lung against sterile particulate exposure, with B cell recruitment to the inflamed lung occurring in an antigen-independent and MyD88-dependent manner.

Non-Antibody-Secreting Functions of B Cells and Their Contribution to Autoimmune Disease.

B cells play multiple important roles in the pathophysiology of autoimmune disease. Beyond producing pathogenic autoantibodies, B cells can act as antigen-presenting cells and producers of cytokines, including both proinflammatory and anti-inflammatory cytokines. Here we review our current understanding of the non-antibody-secreting roles that B cells may play during development of autoimmunity, as learned primarily from reductionist preclinical models. Attention is also given to concepts emerging from clinical studies using B cell depletion therapy, which shed light on the roles of these mechanisms in human autoimmune disease.

Targeting DDR2 enhances tumor response to anti-PD-1 immunotherapy.

While a fraction of cancer patients treated with anti-PD-1 show durable therapeutic responses, most remain unresponsive, highlighting the need to better understand and improve these therapies. Using an in vivo screening approach with a customized shRNA pooled library, we identified DDR2 as a leading target for the enhancement of response to anti-PD-1 immunotherapy. Using isogenic in vivo murine models across five different tumor histologies-bladder, breast, colon, sarcoma, and melanoma-we show that DDR2 depletion increases sensitivity to anti-PD-1 treatment compared to monotherapy. Combination treatment of tumor-bearing mice with anti-PD-1 and dasatinib, a tyrosine kinase inhibitor of DDR2, led to tumor load reduction. RNA-seq and CyTOF analysis revealed higher CD8+ T cell populations in tumors with DDR2 depletion and those treated with dasatinib when either was combined with anti-PD-1 treatment. Our work provides strong scientific rationale for targeting DDR2 in combination with PD-1 inhibitors.

The c-Myc/miR17-92/PTEN Axis Tunes PI3K Activity to Control Expression of Recombination Activating Genes in Early B Cell Development.

Appropriate PI3K signals generated by the antigen receptor are essential to promote B cell development. Regulation of recombination activating gene (RAG)-1 and RAG-2 expression is one key process that is mediated by PI3K to ensure developmental progression and selection. When PI3K signals are too high or too low, expression of RAGs does not turn off and B cell development is impaired or blocked. Yet, the mechanism which tunes PI3K activity to control RAG expression during B cell development in the bone marrow is unknown. Recently we showed that a c-Myc/miR17-92/PTEN axis regulates PI3K activity for positive and negative selection of immature B cells. Here, we show that the c-Myc/miR17-92/PTEN axis tunes PI3K activity to control the expression of RAGs in proB cells. Using different genetically engineered mouse models we show that impaired function of the c-Myc/miR17-92/PTEN axis alters the PI3K/Akt/Foxo1 pathway to result in dis-regulated expression of RAG and a block in B cell development. Studies using 38c-13 B lymphoma cells, where RAGs are constitutively expressed, suggest that this regulatory effect is mediated post-translationally through Foxo1.

Activation of thyroid antigen-reactive B cells in recent onset autoimmune thyroid disease patients.

Autoimmune thyroid disease (AITD), including Hashimoto's thyroiditis (HT) and Graves' disease (GD), is the most common autoimmune disorder in the United States, affecting over 20 million people. At the time of diagnosis, both HD and GD are characterized by the accumulation of B and T lymphocytes in the thyroid gland and production of autoantibodies targeting the thyroid, indicating that a breach in tolerance of autoreactive lymphocytes has occurred. However, few studies have sought to understand the underlying pathogenesis of AITD that ultimately leads to production of autoantibodies and loss of thyroid function. In this study, we analyzed the phenotype of thyroid antigen-reactive B cells in the peripheral blood of recent onset and long standing AITD patients. We found that in recent onset patients thyroid antigen-reactive B cells in blood no longer appear anergic, rather they express CD86, a marker of activation. This likely reflects activation of these cells leading to their production of autoantibodies. Hence, this study reports the early loss of anergy in thyroid antigen-reactive B cells, an event that contributes to development of AITD.

Impaired B cell function during viral infections due to PTEN-mediated inhibition of the PI3K pathway.

Transient suppression of B cell function often accompanies acute viral infection. However, the molecular signaling circuitry that enforces this hyporesponsiveness is undefined. In this study, experiments identify up-regulation of the inositol phosphatase PTEN (phosphatase and tensin homolog) as primarily responsible for defects in B lymphocyte migration and antibody responses that accompany acute viral infection. B cells from mice acutely infected with gammaherpesvirus 68 are defective in BCR- and CXCR4-mediated activation of the PI3K pathway, and this, we show, is associated with increased PTEN expression. This viral infection-induced PTEN overexpression appears responsible for the suppression of antibody responses observed in infected mice because PTEN deficiency or expression of a constitutively active PI3K rescued function of B cells in infected mice. Conversely, induced overexpression of PTEN in B cells in uninfected mice led to suppression of antibody responses. Finally, we demonstrate that PTEN up-regulation is a common mechanism by which infection induces suppression of antibody responses. Collectively, these findings identify a novel role for PTEN during infection and identify regulation of the PI3K pathway, a mechanism previously shown to silence autoreactive B cells, as a key physiological target to control antibody responses.

Continuous inhibitory signaling by both SHP-1 and SHIP-1 pathways is required to maintain unresponsiveness of anergic B cells.

Many autoreactive B cells persist in the periphery in a state of unresponsiveness called anergy. This unresponsiveness is rapidly reversible, requiring continuous BCR interaction with self-antigen and resultant regulatory signaling for its maintenance. Using adoptive transfer of anergic B cells with subsequent acute induction of gene deletion or expression, we demonstrate that the continuous activities of independent inhibitory signaling pathways involving the tyrosine phosphatase SHP-1 and the inositol phosphatase SHIP-1 are required to maintain anergy. Acute breach of anergy by compromise of either of these pathways leads to rapid cell activation, proliferation, and generation of short-lived plasma cells that reside in extrafollicular foci. Results are consistent with predicted/observed reduction in the Lyn-SHIP-1-PTEN-SHP-1 axis function in B cells from systemic lupus erythematosus patients.

γδ T Cells Shape Preimmune Peripheral B Cell Populations.

We previously reported that selective ablation of certain γδ T cell subsets, rather than removal of all γδ T cells, strongly affects serum Ab levels in nonimmunized mice. This type of manipulation also changed T cells, including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4(+) and Vγ6(+) γδ T cells (B6.TCR-Vγ4(-/-)/6(-/-)), we observed expanded Vγ1(+) cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αß T cells as well as spontaneous germinal center formation in the spleen, and elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4(-/-)/6(-/-) mice and other peripheral B cell populations were diminished, most of all splenic marginal zone (MZ) B cells. However, relative frequencies and absolute numbers of Ab-producing cells, as well as serum levels of Abs, IL-4, and BAFF, were increased. Cell transfers confirmed that these changes are directly dependent on the altered γδ T cells in this strain and on their enhanced potential of producing IL-4. Further evidence suggests the possibility of direct interactions between γδ T cells and B cells in the splenic MZ. Taken together, these data demonstrate the capability of γδ T cells of modulating size and productivity of preimmune peripheral B cell populations.

Imbalanced PTEN and PI3K Signaling Impairs Class Switch Recombination.

Class switch recombination (CSR) generates isotype-switched Abs with distinct effector functions. B cells express phosphatase and tensin homolog (PTEN) and multiple isoforms of class IA PI3K catalytic subunits, including p110α and p110δ, whose roles in CSR remain unknown or controversial. In this article, we demonstrate a direct effect of PTEN on CSR signaling by acute deletion of Pten specifically in mature B cells, thereby excluding the developmental impact of Pten deletion. We show that mature B cell-specific PTEN overexpression enhances CSR. More importantly, we establish a critical role for p110α in CSR. Furthermore, we identify a cooperative role for p110α and p110δ in suppressing CSR. Mechanistically, dysregulation of p110α or PTEN inversely affects activation-induced deaminase expression via modulating AKT activity. Thus, our study reveals that a signaling balance between PTEN and PI3K isoforms is essential to maintain normal CSR.

B cell expression of the SH2-containing inositol 5-phosphatase (SHIP-1) is required to establish anergy to high affinity, proteinacious autoantigens.

Many self-reactive B cells exist in the periphery in a rapidly reversible state of unresponsiveness referred to as anergy. Reversibility of anergy indicates that chronically occupied BCR must transduce non-durable regulatory signals that maintain unresponsiveness. Consistent with such a mechanism, studies of immunoglobulin transgenic, as well as naturally occurring polyclonal autoreactive B cells demonstrate activation of the inositol 5-phosphatase SHIP-1 in anergic cells, and low affinity chromatin autoantigen-reactive B cells have been shown to require expression of this phosphatase to maintain anergy. However, it has been reported that anergy of B cells recognizing high affinity soluble antigen may not require SHIP-1, and is instead mediated by upregulation of the inositol 3-phosphatase PTEN. To further explore this apparent difference in mechanism we analyzed the effect of B cell-targeted SHIP-1 deletion on immune tolerance of high affinity anti-HEL B cells in mice expressing soluble HEL (MD4.ML-5). We report that SHIP-1 functions to dampen responses of naïve and low-dose antigen-primed B cells in vitro, and is required for induction of B cell tolerance. Thus, while anergy of B cells reactive with low affinity and likely polyvalent chromatin antigens is maintained by activation of inhibitory signaling circuitry involving SHIP-1, anergy of B cells recognizing soluble self antigen with high affinity also requires increased activity of SHIP-1.

Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production.

Activated caspases are a hallmark of apoptosis induced by the intrinsic pathway, but they are dispensable for cell death and the apoptotic clearance of cells in vivo. This has led to the suggestion that caspases are activated not just to kill but to prevent dying cells from triggering a host immune response. Here, we show that the caspase cascade suppresses type I interferon (IFN) production by cells undergoing Bak/Bax-mediated apoptosis. Bak and Bax trigger the release of mitochondrial DNA. This is recognized by the cGAS/STING-dependent DNA sensing pathway, which initiates IFN production. Activated caspases attenuate this response. Pharmacological caspase inhibition or genetic deletion of caspase-9, Apaf-1, or caspase-3/7 causes dying cells to secrete IFN-ß. In vivo, this precipitates an elevation in IFN-ß levels and consequent hematopoietic stem cell dysfunction, which is corrected by loss of Bak and Bax. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent.

Cyclic-di-GMP and cyclic-di-AMP activate the NLRP3 inflammasome.

The cyclic dinucleotides 3'-5'diadenylate (c-diAMP) and 3'-5' diguanylate (c-diGMP) are important bacterial second messengers that have recently been shown to stimulate the secretion of type I Interferons (IFN-Is) through the c-diGMP-binding protein MPYS/STING. Here, we show that physiologically relevant levels of cyclic dinucleotides also stimulate a robust secretion of IL-1ß through the NLRP3 inflammasome. Intriguingly, this response is independent of MPYS/STING. Consistent with most NLRP3 inflammasome activators, the response to c-diGMP is dependent on the mobilization of potassium and calcium ions. However, in contrast to other NLRP3 inflammasome activators, this response is not associated with significant changes in mitochondrial potential or the generation of mitochondrial reactive oxygen species. Thus, cyclic dinucleotides activate the NLRP3 inflammasome through a unique pathway that could have evolved to detect pervasive bacterial pathogen-associated molecular patterns associated with intracellular infections.

Retention of anergy and inhibition of antibody responses during acute γ herpesvirus 68 infection.

The majority of the human population becomes infected early in life by the gammaherpesvirus EBV. Some findings suggest that there is an association between EBV infection and the appearance of pathogenic Abs found in lupus. Gammaherpesvirus 68 infection of adult mice (an EBV model) was shown to induce polyclonal B cell activation and hypergammaglobulinemia, as well as increased production of autoantibodies. In this study, we explored the possibility that this breach of tolerance reflects loss of B cell anergy. Our findings show that, although anergic B cells transiently acquire an activated phenotype early during infection, they do not become responsive to autoantigen, as measured by the ability to mobilize Ca2+ following AgR cross-linking or mount Ab responses following immunization. Indeed, naive B cells also acquire an activated phenotype during acute infection but are unable to mount Ab responses to either T cell-dependent or T cell-independent Ags. In acutely infected animals, Ag stimulation leads to upregulation of costimulatory molecules and relocalization of Ag-specific B cells to the B-T cell border; however, these cells do not proliferate or differentiate into Ab-secreting cells. Adoptive-transfer experiments show that the suppressed state is reversible and is dictated by the environment in the infected host. Finally, B cells in infected mice deficient of CD4+ T cells are not suppressed, suggesting a role for CD4+ T cells in enforcing unresponsiveness. Thus, rather than promoting loss of tolerance, gammaherpesvirus 68 infection induces an immunosuppressed state, reminiscent of compensatory anti-inflammatory response syndrome.

B cell receptor signal transduction in the GC is short-circuited by high phosphatase activity.

Germinal centers (GCs) generate memory B and plasma cells, which are essential for long-lived humoral immunity. GC B cells with high-affinity B cell receptors (BCRs) are selectively expanded. To enable this selection, BCRs of such cells are thought to signal differently from those with lower affinity. We show that, surprisingly, most proliferating GC B cells did not demonstrate active BCR signaling. Rather, spontaneous and induced signaling was limited by increased phosphatase activity. Accordingly, both SH2 domain-containing phosphatase-1 (SHP-1) and SH2 domain-containing inositol 5 phosphatase were hyperphosphorylated in GC cells and remained colocalized with BCRs after ligation. Furthermore, SHP-1 was required for GC maintenance. Intriguingly, GC B cells in the cell-cycle G(2) period regained responsiveness to BCR stimulation. These data have implications for how higher-affinity B cells are selected in the GC.