CD19 Phosphorylation Research Articles

Deciphering prognostic value of CD22 and its contribution to suppression of proinflammatory cytokines production in patients with IgA nephropathy

BackgroundCD22, mainly expressed in mature B cells, could negatively regulate the function of B cells by binding to sialic acid-positive IgG (SA-IgG). Soluble CD22 (sCD22) is generated by the cleavage of the extracellular domain of CD22 on the membrane surface. However, the role of CD22 in IgA nephropathy (IgAN) remains unknown. MethodsA total of 170 IgAN patients with a mean follow-up of 18 months were included in this study. The sCD22, TGF-β, IL-6 and TNF-α were detected using commercial ELISA kits. SA-IgG were purified to stimulate peripheral blood mononuclear cells (PBMCs) from IgAN patients. ResultsThe plasma levels of sCD22 were lower in IgAN patients in comparison with healthy control. Furthermore, CD22 mRNA levels in PBMCs from patients with IgAN were significantly lower than those of healthy controls. The plasma levels of sCD22 were positively correlated to the mRNA levels of CD22. We found that patients with higher sCD22 levels had a lower level of serum creatinine and a higher level of eGFR on the time of renal biopsy and a higher remission rate of proteinuria and a lower risk of kidney events at the end of follow-up. The logistic regression analysis showed sCD22 was associated with an increased odd of proteinuria remission after being adjusted for eGFR, proteinuria, and SBP. After adjusting for confounding variables, sCD22 was a borderline significant predictor of less kidney composite endpoint. In addition, the sCD22 levels were positively associated with SA-IgG in plasma. The experimental results in vitro showed that addition of SA-IgG enhanced the release of sCD22 in cell supernatant and the phosphorylation of CD22 in PBMCs, further inhibiting the production of IL-6, TNF-α, and TGF-β in cell supernatant in a dose-dependent manner. Pretreatment with CD22-antibody significantly increased the expression of cytokines in PBMCs. ConclusionsThis is the first study to demonstrate that lower plasma soluble CD22 in IgAN patients and high soluble CD22 levels are associated with an increased odd of proteinuria remission and a decreased odd of kidney endpoint. The interaction between CD22 and SA-IgG can inhibit proliferation and inflammation release in PBMCs from IgAN patients.

The Protein Tyrosine Phosphatase SHP-1 (PTPN6) but Not CD45 (PTPRC) Is Essential for the Ligand-Mediated Regulation of CD22 in BCR-Ligated B Cells.

CD22 is an inhibitory B cell coreceptor that regulates B cell development and activation by downregulating BCR signaling through activation of SH2-containing protein tyrosine phosphatase-1 (SHP-1). CD22 recognizes α2,6 sialic acid as a specific ligand and interacts with α2,6 sialic acid-containing membrane molecules, such as CD45, IgM, and CD22, expressed on the same cell. Functional regulation of CD22 by these endogenous ligands enhances BCR ligation-induced signaling and is essential for normal B cell responses to Ags. In this study, we demonstrate that CD45 plays a crucial role in CD22-mediated inhibition of BCR ligation-induced signaling. However, disruption of ligand binding of CD22 enhances CD22 phosphorylation, a process required for CD22-mediated signal inhibition, upon BCR ligation in CD45-/- as well as wild-type mouse B cells but not in mouse B cells expressing a loss-of-function mutant of SHP-1. This result indicates that SHP-1 but not CD45 is required for ligand-mediated regulation of CD22. We further demonstrate that CD22 is a substrate of SHP-1, suggesting that SHP-1 recruited to CD22 dephosphorylates nearby CD22 as well as other substrates. CD22 dephosphorylation by SHP-1 appears to be augmented by homotypic CD22 clustering mediated by recognition of CD22 as a ligand of CD22 because CD22 clustering increases the number of nearby CD22. Our results suggest that CD22 but not CD45 is an endogenous ligand of CD22 that enhances BCR ligation-induced signaling through SHP-1-mediated dephosphorylation of CD22 in CD22 clusters.

CD19 regulates innate immunity by the toll-like receptor RP105 signaling in B lymphocytes

AbstractLipopolysaccharide (LPS) is a major gramnegative bacterial component that stimulates innate immune response and also induces B-lymphocyte activation. Recent studies have revealed that common molecular patterns of microorganisms such as LPS are recognized by toll-like receptors (TLRs). B cells have 2 known TLRs that mediate LPS signaling, TLR4 and RP105 (CD180). While TLR4 is expressed on immune cells of various types, RP105 is preferentially expressed on mature B cells. Here we demonstrate that CD19 plays a major role in regulating signal transduction through RP105. Anti-RP105 ligation induced normal proliferation of B cells from mice deficient for MyD88, an adaptor protein that mediates most TLR pathways. By contrast, the loss of CD19 resulted in modest B-cell proliferation against anti-RP105 stimulation as well as LPS stimulation. LPS induced tyrosine phosphorylation of CD19, which was RP105-dependent but TLR4-independent. CD19 formed a complex with Lyn and Vav following RP105 ligation, and CD19 expression was required for optimal Lyn activation and Vav phosphorylation. Consistently, B cells deficient for CD19 exhibited specific defect in the activation of c-Jun N-terminal kinases following RP105 ligation and LPS stimulation. In contrast, CD19 and phosphatidylinositol 3-kinase independently regulated intracellular calcium mobilization induced by anti-RP105 stimulation. Thus, signaling through the B-cell–specific LPS receptor RP105 is uniquely regulated by the B-cell–specific signaling component, Lyn/CD19/Vav complex.

Astilbin combined with lipopolysaccharide induces IL-10-producing regulatory B cells via the STAT3 signalling pathway

ObjectivesAstilbin exerts immunoregulatory activities and plays anti-inflammatory effects in inflammation-associated diseases. IL-10-producing B cells are the major subset of regulatory B cells (Bregs) and inhibit inflammation and autoimmune diseases. This study aimed to analyse the inducing effect of astilbin on Bregs and investigate the involved molecular mechanisms. MethodsThe frequencies and activities of IL-10-producing Bregs were observed using the co-treatment of astilbin and lipopolysaccharide (LPS) ex vivo. The protective effect of astilbin/LPS-induced Bregs on dextran sulphate sodium (DSS)-induced colitis was confirmed in vivo. The molecular signalling events of Breg induction were checked via Western blot. CD40−/− and toll-like receptor (TLR) 4−/− B cells were treated with astilbin/LPS to determine the modulatory role of CD40 or TLR4 on astilbin/LPS-induced Bregs. ResultsAlthough astilbin alone could not affect Bregs, the co-treatment of astilbin and LPS remarkably induced CD19+ CD1dhi and CD19+ TIM-1+ cells which produced IL-10 ex vivo. Colonic CD19+ CD1dhi and CD19+ TIM-1+ cells were also increased in astilbin-treated mice with DSS-induced colitis. The adoptive transfer of CD19+ TIM-1+ cells pre-induced by astilbin/LPS directly suppressed the progression of DSS-induced colitis. Combined astilbin and LPS stimulated the STAT3 activation of CD19+ TIM-1+ cells but had no effects on SOCS3, AKT, NF-κB, Erk, JNK nor P38. Inhibiting the STAT3 phosphorylation of CD19+ TIM-1+ cells abolished Breg induction by astilbin/LPS. Furthermore, Breg induction was weakened in CD40−/− B cells with the decrease in STAT3 activation, but had disappeared in TLR4−/− B cells with no STAT3 activation, thereby confirming the indispensable role of TLR4 signalling in the induction of IL-10-producing Bregs. ConclusionsThis study reports the new immunoregulatory role of astilbin for promoting IL-10-producing B cells and suggests the possible use of astilbin in the therapy of inflammatory diseases.

AKT2 deficiency impairs formation of the BCR signalosome

BackgroundAKT2 is one of the key molecules that involves in the insulin-induced signaling and the development of cancer. In B cells, the function of AKT2 is unclear.MethodsIn this study, we used AKT2 knockout mice model to study the role of AKT2 in BCR signaling and B cell differentiation.ResultsAKT2 promotes the early activation of B cells by enhancing the BCR signaling and actin remodeling. B cells from AKT2 KO mice exhibited defective spreading and BCR clustering upon stimulation in vitro. Disruption of Btk-mediated signaling caused the impaired differentiation of germinal center B cells, and the serum levels of both sepecific IgM and IgG were decreased in the immunized AKT2 KO mice. In addition, the actin remodeling was affected due to the decreased level of the activation of WASP, the actin polymerization regulator, in AKT2 KO mice as well. As a crucial regulator of both BCR signaling and actin remodeling during early activation of B cells, the phosphorylation of CD19 was decreased in the AKT2 absent B cells, while the transcription level was normal.ConclusionsAKT2 involves in the humoral responses, and promotes the BCR signaling and actin remodeling to enhance the activation of B cells via regulating CD19 phosphorylation.E2Qk5NqwnEMPV-C1CqpUy1Video

N-Linked Glycosylation Regulates CD22 Organization and Function.

The organization and clustering of cell surface proteins plays a critical role in controlling receptor signaling; however, the biophysical mechanisms regulating these parameters are not well understood. Elucidating these mechanisms is highly significant to our understanding of immune function in health and disease, given the importance of B cell receptor (BCR) signaling in directing B cells to produce antibodies for the clearance of pathogens, and the potential deleterious effects of dysregulated BCR signaling, such as in B cell malignancies or autoimmune disease. One of main inhibitory co-receptors on B cells is CD22, a sialic-acid binding protein, which interacts homotypically with other sialylated CD22 molecules, as well as heterotypically with IgM and CD45. Although the importance of CD22 in attenuating BCR signaling is well established, we still do not fully understand what mediates CD22 organization and association to BCRs. CD22 is highly glycosylated, containing 12 N-linked glycosylation sites on its extracellular domain, the function of which remain to be resolved. We were interested in how these glycosylation sites mediate homotypic vs. heterotypic interactions. To this end, we mutated five out of the six N-linked glycosylation residues on CD22 localized closest to the sialic acid binding site. Glycan site N101 was not mutated as this resulted in lack of CD22 expression. We used dual-color super-resolution imaging to investigate the impact of altered glycosylation of CD22 on the nanoscale organization of CD22 and its association with BCR. We show that mutation of these five glycosylation sites increased the clustering tendency of CD22 and resulted in higher density CD22 nanoclusters. Consistent with these findings of altered CD22 organization, we found that mutation of N-glycan sites attenuated CD22 phosphorylation upon BCR stimulation, and consequently, increased BCR signaling. Importantly, we identified that these sites may be ligands for the soluble secreted lectin, galectin-9, and are necessary for galectin-9 mediated inhibition of BCR signaling. Taken together, these findings implicate N-linked glycosylation in the organization and function of CD22, likely through regulating heterotypic interactions between CD22 and its binding partners.

Decreased CD22 expression and intracellular signaling aberrations in B cells of patients with systemic sclerosis.

The objective of the study was to explorethe phenotype and intracellular signaling events of B cells in patients with systemic sclerosis (SSc). Peripheral blood B cell surface markers CD19 and CD22 were evaluated by flow cytometry in 23 patients with SSc and seven healthy individuals. Levels of intracellular kinases Lyn, Syk and P-Y 348 Syk along with phosphatase SHP-1 were examined with Western immunoblotting in selected patients. P-Y 822 CD22 was subsequently evaluated flow cytometrically in antigen receptor stimulated B cells. A statistically significant decrease in CD22 B cell surface expression was found in the diffuse subset of patients (median CD22 MFI ± SD was 5.90 ± 2.35 vs 10.20 ± 1.88 for patients vs healthy controls respectively; p = 0.021), while no statistically significant change was found regarding CD19. CD22 underexpression was more pronounced when interstitial lung disease (ILD) was present (median CD22 MFI ± SD was 5.90 ± 2.25 vs 10.20 ± 1.88 for patients with ILD vs healthy controls respectively; p = 0.011). CD22 phosphorylation following B cell receptor (BCR) stimulation was also found to be impaired in patients with diffuse SSc (median change in MFI ± SD was 0.28 ± 0.09 vs 0.38 ± 0.08 for patients vs healthy controls respectively; p = 0.034). Low CD22 expression was arithmetically correlated with kinase Lyn underexpression (Pearson coefficient 0.926; p = ns) in B cells from a small sample of patients. These results suggest that CD22 underexpression and impaired phosphorylation along with implications for Lyn kinase aberrations could contribute to the activated B cell phenotype in SSc.

Igβ ubiquitination activates PI3K signals required for endosomal sorting.

A wealth of in vitro data has demonstrated a central role for receptor ubiquitination in endocytic sorting. However, how receptor ubiquitination functions in vivo is poorly understood. Herein, we report that ablation of B cell antigen receptor ubiquitination in vivo uncouples the receptor from CD19 phosphorylation and phosphatidylinositol 3-kinase (PI3K) signals. These signals are necessary and sufficient for accumulating phosphatidylinositol (3,4,5)-trisphosphate (PIP3) on B cell receptor-containing early endosomes and proper sorting into the MHC class II antigen-presenting compartment (MIIC). Surprisingly, MIIC targeting is dispensable for T cell-dependent immunity. Rather, it is critical for activating endosomal toll-like receptors and antiviral humoral immunity. These findings demonstrate a novel mechanism of receptor endosomal signaling required for specific peripheral immune responses.

Engagement of CD22 on B cells with the monoclonal antibody epratuzumab stimulates the phosphorylation of upstream inhibitory signals of the B cell receptor.

The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr(822) on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal phosphorylation of other key ITIMs on CD22, in primary human B cells epratuzumab also enhanced phosphorylation of Tyr(807), a residue involved in the recruitment of Grb2. Such initiation events could explain the effects of epratuzumab on downstream signalling in B cells. Finally, we were able to demonstrate that epratuzumab stimulated the phosphorylation of Tyr(292) on the low affinity inhibitory Fc receptor CD32B which would further attenuate BCR-induced signalling. Together, these data demonstrate that engagement of CD22 with epratuzumab leads to the direct phosphorylation of key upstream inhibitory receptors of BCR signalling and may help to explain how this antibody modulates B cell function.

IFITM3 (CD225) Links the B Cell Antigen CD19 to PI3K-AKT Signaling in Human ALL Cells

Background & Hypothesis: IFITM3 (Interferon-induced transmembrane protein 3) also known as CD225/Leu-13 was identified as interferon-inducible molecule in the context of viral infection. IFITM3 encodes a surface receptor, basally expressed on the plasma membrane, that is associated with known B cell co-receptors including CD19, CD81 and CD21. Recent immunotherapy approaches based on CD19-specific engineered chimeric antigen receptors (CART19) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (Grupp et al., 2013). However, in some cases, CART19 treatment was followed by ALL relapse developing from clones that lacked CD19 surface expression. Therefore, we studied factors that regulate CD19 surface expression on human pre-B ALL cells.Results: Studying IFITM3 mRNA levels in ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, patients with higher than median IFITM3 mRNA levels at the time of diagnosis were significantly more likely to experience ALL relapse and had a higher risk of a positive MRD status at the end of induction chemotherapy.To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1. Strikingly, deletion of IFITM3 resulted in loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K-AKT signaling in both B cell progenitors and pre-B ALL cells. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and activation of checkpoint molecules p53 and p21, reduction of BCL2 and BCLXL levels and increased propensity to apoptosis. Reconstitution of IFITM3 reversed these changes.Conversely, forced expression of IFITM3 in patient-derived pre-B ALL cells increased phosphorylation of CD19-Y513 together with downstream SRC, SYK, PI3K signaling. Although human IFITM1 has known as a component of B cell receptor complex, co-immunoprecipitation experiments revealed that the cytoplasmic tail of IFITM3 interacts with CD19, LYN, SYK, PI3K p110δ and AKT. In addition, agonistic antibodies against IFITM3/CD225 trigger CD19/PI3K-AKT signaling, which caused massively increased proliferation of pre-B ALL cells. Interestingly, agonistic antibodies against CD19 had no effect on the proliferation of pre-B ALL cells despite their ability to activate CD19/PI3K-AKT signaling. These findings indicate a specific role of IFITM3 in regulating CD19/PI3K-AKT signaling in malignant pre-B ALL cells compared to their normal pre-B cell counterparts.Conclusion: These findings identify novel role of the IFITM3 (CD255) surface receptor in regulating CD19 phosphorylation and CD19-Y513 mediated PI3K-AKT signaling in pre-B ALL cells. IFITM3 is a central mediator of CD19 surface trafficking and mediates the proliferation and survival signaling via PI3K-AKT in normal pre-B cells and various subtypes of human pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.

CD19 controls Toll-like receptor 9 responses in human B cells

CD19 is a B cell-specific molecule that serves as a major costimulatory molecule for amplifying B-cell receptor (BCR) responses. Biallelic CD19 gene mutations cause common variable immunodeficiency in human subjects. BCR- and Toll-like receptor (TLR) 9-induced B-cell responses are impaired in most patients with common variable immunodeficiency. We sought to analyze whether CD19 is required for TLR9 function in human B cells. Expression of surface activation markers was assessed after anti-IgM or CpG stimulation by using flow cytometry on B cells from patients with 1 or 2 defective CD19 alleles, which decrease or abrogate CD19 expression, respectively. The phosphorylation or interaction of signaling molecules was analyzed by using phospho flow cytometry, immunoblotting, or co-immunoprecipitation in CD19-deficient or control B cells and in a B-cell line in which CD19 has been knocked down with lentivirus-transduced short hairpin RNA. B cells from subjects with 1 or 2 defective CD19 alleles showed defective upregulation in vitro of CD86, transmembrane activator and CAML interactor (TACI), and CD23 activation markers after TLR9 stimulation. TLR9 ligands normally induce phosphorylation of CD19 through myeloid differentiation primary response gene-88 (MYD88)/proline-rich tyrosine kinase 2 (PYK2)/LYN complexes, which allows recruitment of phosphoinositide 3-kinase (PI3K) and phosphorylation of Bruton tyrosine kinase (BTK) and AKT in human B cells with a different kinetic than that of BCRs. In addition, inhibition of PI3K, AKT, or BTK, as well as BTK deficiency, also resulted in TLR9 activation defects in B cells similar to those in patients with CD19 deficiency. CD19 is required for TLR9-induced B-cell activation. Hence CD19/PI3K/AKT/BTK is an essential axis integrating BCRs and TLR9 signaling in human B cells.

Recent Advances of Researches on Expression, Function and Regulation of CD22

CD22 is a type I transmembrane protein expressed on most mature B lymphocyte, and plays a significant role in signal transduction pathways. CD22 acts as a co-receptor of the B-cell receptor (BCR) that inhibits the BCR signaling by antigen-receptor interaction. The phosphorylation of CD22 can be triggered by cross-linking of CD22 with the BCR through antigen, then predominantly triggers the dephosphorylation and inactivation of downstream proteins and inhibit the BCR signaling. Autoimmune disease could be caused by the abnormal expression or dysfunction of CD22 which interrupts BCR signaling and then influences the quantity and function of B cells. The further study of the function and regulation of CD22 would help us understanding the pathogenesis of autoimmune disease and setting theoretical basis for its targeting treatment. In this article, the structure and expression of CD22, the ligands of CD22, the regulation of BCR and transmenbrane signaling, the effect of CD22 on B cells, and CD22 and autoimmune diseases were reviewed.

Immunoglobulin Heavy Chain (IgH) Knockout Inhibits Proliferation of Pre-BCR+ B-Cell Acute Lymphoblastic Leukemia (B-ALL) Via a FOXO1 and MYC Dependent Mechanism

A pivotal step during B-cell development is the expression of the precursor B-cell receptor (pre-BCR) by pre-B lymphocytes (cyto-Igµ+, surface-IgM-). The pre-BCR represents an immature form of the BCR and consists of two immunoglobulin heavy chains (IgH), two surrogate light chains (SLC) and the signal transducing adapter proteins Igα and Igβ. A functional pre-BCR drives proliferation of pre-B-cells, ensuring their further differentiation into mature B-cells. By immunophenotype, ~20% of B-cell acute lymphoblastic leukemia (B-ALL) cases originate from the pre-B-cell stage (pre-B-ALL) of lymphocyte development and might therefore also express the pre-BCR. In view of the importance of pre-BCR signaling for normal pre-B-cell development, we hypothesize that it is exploited by pre-B-ALL for malignant growth and proliferation.A hallmark of active pre-BCR signaling is the continuous internalization of pre-BCRs, resulting in low pre-BCR surface expression. Using this phenotype of active pre-BCR signaling (low pre-BCR expression and high phosphorylation of the pre-BCR associated kinases LYN and SYK), we identified pre-BCR+ ALL cell lines (RCH-ACV, SMS-SB and Nalm6) and xenograft expanded patient samples. To study the role of the pre-BCR in these cells, we rendered RCH-ACV and SMS-SB pre-BCR null by using CRISPR/CAS9 gene editing with guide RNAs specific for the hypervariable region (recombined V, D, and J segments) of their expressed IgH allele. As identified by flow cytometry for the pre-BCR, deficient RCH-ACV and SMS-SB cells exhibited reduced viability and impaired proliferation when compared to their pre-BCR+ controls (Figure 1). Pre-BCR- cells showed reduced baseline phosphorylation of CD19, VAV1 and AKT. Interestingly, BTK and ERK phosphorylation were not affected. These results provide evidence for the dependency of pre-BCR+ ALL on pre-BCR signaling and suggest selective involvement of the PI3K-AKT pathway.We also investigated the effects of pharmacological pre-BCR inhibition by treating pre-BCR+ and pre-BCR- ALL cell lines and xenograft expanded primary patient samples with PRT318, a small-molecule inhibitor of spleen tyrosine kinase (SYK). In pre-BCR+ ALL PRT318 blocked cell proliferation and selectively inhibited AKT phosphorylation, thus mimicking the effects of IgH knockout. Pre-BCR- ALL cells were resistant to PRT318.Key effectors of the pre-BCR during normal B-cell development are FOXO transcription factors. In line with this, we found reduced FOXO1 phosphorylation and increased FOXO1 total protein levels after IgH knockout as well as after treatment with PRT318. This was accompanied by an increase in the FOXO1 transcriptional targets p27 and BLNK, suggesting increased FOXO1 transcriptional activity in response to the inhibition of pre-BCR signaling. To study the contribution of FOXO1 to the effects of IgH knockout and SYK inhibition more thoroughly, we expressed constitutively active FOXO1 (FOXO1-3A) in the pre-BCR+ ALL cell line RCH-ACV and consequently assessed its effects on cell proliferation and protein expression. Similar to IgH knockout and PRT318, FOXO1-3A reduced cell proliferation and increased p27 and BLNK protein levels, confirming FOXO1 as an important downstream target of pre-BCR signaling in B-ALL.To identify additional effectors of the pre-BCR in B-ALL we performed gene expression profiling (GEP) to compare pre-BCR+ and pre-BCR- cells of RCH-ACV and SMS-SB. Gene set enrichment analysis (GSEA) showed that IgH knockout resulted in significant enrichment for gene sets associated with down-modulation of MYC activity. This was confirmed by Western blot analysis of MYC total protein levels, and consistent with the finding of reduced MYC protein in PRT318-treated and FOXO1-3A-expressing pre-BCR+ cells, all indicating that pre-BCR signaling modulates MYC activity through a mechanism involving SYK and FOXO1.In conclusion, we provide evidence for the dependence of certain B-ALL subgroups on pre-BCR signaling. According to our data this is mainly due to pre-BCR-induced inactivation of FOXO1 and the subsequent deregulation of MYC. Importantly, pharmacological inhibition of pre-BCR signaling with the SYK inhibitor PRT318 completely reversed these effects, therefore providing a rationale for the use of SYK inhibitors in pre-BCR+ subgroups of B-ALL.Figure 1: [Display omitted] DisclosuresCoffey:Portola Pharmaceuticals: Employment, Equity Ownership.

Infant B-Cell Signaling after ABO-Incompatible Heart Transplantation (ABOi HTx): Defining the Role of the Inhibitory Molecule CD22

Purpose Immune immaturity allows ABOi HTx to be performed safely in infants and results in B-cell tolerance to donor ABO antigens (Ags). In experimental settings, T-independent (TI) B-cell activation has been shown to be inhibited by binding of the B-cell co-receptor CD22 to CD22 ligands leading to B-cell tolerance. Since immunity to TI Ags, including ABO Ags, are generally reduced in early childhood, we hypothesize a role for CD22, possibly contributing to ABOi HTx tolerance. Previously, we showed that CD22 expression is higher on CD27+IgM+ B-cells in infants when compared to older individuals. In this study we performed functional assays with CD27+IgM+ B-cells to assess the presence of ABO Ag-specific IgM antibody (Ab)-secreting cells (ASC). In addition, we established a phospho-flow assay (PPF) to assess B-cell signaling. Methods and Materials CD27+IgM+ and CD27-IgM+ B-cells were isolated from human splenocytes (n=3 infant, n=6 adult) by cell sorting and stimulated with CpG plus IL-2, IL-10, and IL-15. After 1 week the frequency of ABO Ag-specific ASC was detected by ELISPOT. The PPF was optimized using a Ramos B-cell line and intracellular protein phosphorylation of CD22(pY822) and PLCγ2(pY759) were examined after stimulation with anti-IgM Abs. Results The total number of ASC was lower in infant samples compared to older individuals and ELISPOT analysis revealed that the majority of ABO Ag-specific ASC were derived from CD27+IgM+ B-cells in both groups. Preliminary results by PPF (n=8) showed pY759 signaling beginning at 0.5min and peaking at 4min, with a 7-fold increase in median fluorescence intensity (MFI) of pY759. pY822 signaling began at 2min with a modest peak at 12min, with a 1.6-fold increase in MFl of pY822. Conclusions The overall increased expression of CD22 on infant CD27+IgM+ B-cells, which include ABO Ag-specific ASC precursors, may increase their susceptibility to decreased signaling, leading to inactivation. CD22 may therefore play an inhibitory role in infant immune responses to ABO Ags after ABOi HTx.

B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia

Abstract SCI-25The B-cell receptor (BCR) is a flexible and variable environmental sensor with no fixed ligand. The central component is surface Ig (sIg), which appears to function at several levels, mediating a “tonic” signal essential for survival and also binding to antigens via its variable (V) regions. Expression of sIg and reliance on BCR signals generally persist in neoplastic B cells. Indolent tumors, including chronic lymphocytic leukemia (CLL), allow insight into the pathogenetic role of the BCR prior to therapy as well as revealing key proteins within these pathways for drug targeting. CLL is heterogeneous, arising at two points of differentiation, generating two major subsets, one with unmutated Ig V genes (U-CLL), another with mutated Ig V genes (M-CLL). U-CLL appears to develop from naïve B cells of the natural antibody repertoire aimed against common pathogens. The clinical behavior of the two subsets differs, with U-CLL being of poorer prognosis. Evidence for antigen drive on both subsets comes from detecting “endocytosis in vivo,” whereby sIgM expression and signal capacity in blood cells are variably downmodulated, but can recover in vitro. Mysteriously, sIgD of the same presumed antigen specificity shows no evidence for endocytic downmodulation in vivo. CLL cells apparently engage antigens via sIgM in tissue sites, leading to proliferation and downmodulation, with reexpression gradually occurring during transit through the blood. Expression of CXCR4 closely follows that of sIgM, and clonal analysis reveals subpopulations of potentially dangerous cells with high sIgM/CXCR4 primed for tissue-based proliferative stimulation. In contrast to normal B cells, this is an iterative process exposing the proliferating CLL cells to further genetic changes. Overall higher sIgM levels and increased signal capacity in U-CLL likely account for more aggressive clinical behavior. BCR-induced membrane-proximal events include LYN-mediated phosphorylation of Iga/b followed by recruitment of the tyrosine kinase Syk. Signal propagation then involves Btk and PLCg2. LYN-dependent phosphorylation of CD19 also recruits the p85 subunit of PI3K, a known survival mechanism in CLL. Downstream events include upregulation of MYC proto-oncoprotein expression and induction of MYC-regulated target genes such as cyclin D2, with both proteins detected in proliferation centers. Pathways to increased cell survival include induction of the antiapoptotic MCL1 protein and inactivation of the proapoptotic activity of BIM(EL/L) via enhanced phosphorylation. The ability to phosphorylate BIM(EL) was highly correlated with mutational status and with requirement for treatment. While these events delineate BCR-activated pathways, they provide only the skeleton. sIgM signaling is highly dependent on the polymeric nature of the antigen, with responses to solid-phase stimulus producing a higher and more prolonged signal than the soluble form. Clearly, CLL cells have to integrate BCR signals with those from other receptors for the multitude of microenvironmental factors. This is a two-way process, since BCR signals operate “inside-out” by modulating the expression of molecules involved in migration and adhesion. The fact that the glycan composition of sIgM is also modulated to a mannosylated form, potentially able to bind to mannose-binding lectins, could contribute to the latter. Clinical effects of Syk, Btk and PI3Kd inhibitors, known to affect BCR signaling and potentially other pathways, are both explicable and exciting. Disclosures:No relevant conflicts of interest to declare.

CD22 and Autoimmune Disease

CD22 is a 140-kDa member of the Siglec family of cell surface proteins that is expressed by most mature B-cell lineages. As a co-receptor of the B-cell receptor (BCR), it is known to contribute to the sensitive control of the B-cell response to antigen. Cross-linking of CD22 and the BCR by antigen triggers the phosphorylation of CD22, which leads to activation of signaling molecules such as phosphatases. Signal transduction pathways involving CD22 have been explored in a number of mouse models, some of which have provided evidence that in the absence of functional CD22, B cells have a “hyperactivated” phenotype, and suggest that loss of CD22 function could contribute to the pathogenesis of autoimmune diseases. Modulating CD22 activity has therefore been suggested as a possible therapeutic approach to such diseases. For example, the novel CD22-targeting monoclonal antibody epratuzumab is currently under investigation as a treatment for the connective tissue disorder systemic lupus erythematosus (SLE).

Abstract 2523: The in vitro effects of immobilized epratuzumab, a non-blocking anti-CD22 humanized monoclonal antibody, on malignant B cells

Abstract Epratuzumab (hLL2), a humanized anti-CD22 monoclonal antibody, is currently in advanced trials in non-Hodgkin lymphoma (NHL) and systemic lupus erythematosus (SLE) patients. As a single agent, hLL2 is well tolerated and depletes 30 to 50% of circulating B cells in patients with NHL and SLE. The mechanisms by which hLL2 depletes B cells in vivo is still largely unknown and continues to be a subject of current research interest. In vitro, hLL2 shows moderate antibody-dependent cell-mediated cytotoxicity, but no detectable complement-dependent toxicity, and displays cytotoxicity to CD22-expressing cells, for example, Ramos and Daudi human Burkitt lymphomas, only when immobilized onto plastic plates or in combination with a suboptimal amount (1 μg/mL) of anti-IgM plus a crosslinking secondary antibody. We have previously reported that hLL2, despite a lack of direct cytotoxicity, is effective in inducing the phosphorylation of CD22, translocation of CD22 to lipid rafts, and internalization of CD22. In this study, we evaluated the various effects of immobilized hLL2 on D(1-1), a subclone of Daudi selected for a higher expression of membrane IgM (mIgM). Our findings demonstrate for the first time that many of the intracellular events induced by binding CD22 to immobilized hLL2 were similar to those induced by binding mIgM with a sufficient high concentration (10 μg/mL) of anti-IgM; for example, both were found to induce phosphorylation of CD22, CD79a and CD79b, and the translocation of all three into the lipid rafts, which is critical for the observed cytotoxicity. Immunofluorescence analysis revealed CD22 colocalized with BCR and formed caps before being internalized. Like anti-IgM, immobilized hLL2 induced substantial apoptosis (25 to 40%) in D(1-1), which was caspase-dependent, accompanied by a pronounced increase in both pERKs and pJNKs with a concurrent decrease in the constitutively-activated p38, and preventable by inhibitors to JNKs or caspases. The involvement of mitochondria in the apoptotic pathway was evidenced by changes in mitochondrial membrane potential and generation of reactive oxygen species, as well as upregulation of Bax (pro-apoptotic) and downregulation of Bcl-xl, Mcl-1 and Bcl-2 (anti-apoptotic). Further studies are underway to elucidate how hLL2 interferes with B-cell survival in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2523. doi:1538-7445.AM2012-2523

Targeting Human B-Precursor Acute Lymphoblastic Leukemia Cells with Recombinant Human CD19 Ligand

AbstractAbstract 599CD19 is a 95-kDa B-lineage restricted receptor molecule that functions as a key regulator of transmembrane signals in both B-cells and B-cell precursors. Here we report the cloning and characterization of a novel high-mobility group (HMG)-box protein as the membrane-associated natural ligand of human CD19 receptor (CD19-L) on human immature as well as mature lymphoid cells. We cloned the gene encoding CD19-L from a human thymus cDNA library by yeast two-hybrid screening using the cDNA encoding human CD19 extracellular domain (AA 1 to 273) (CD19ECD) fused to the GAL4 DNA binding domain as the bait plasmid. The cDNA for the surface membrane-associated CD19-L protein is 2290-bp in length encoding a 487-aa protein with a predicted molecular mass of 54-kDa. The comparison of the amino acid sequence of CD19-L protein with reported sequences in Genebank Database revealed that CD19-L is a new member of the HMG-box protein family. CD19-L contains two leucine-rich hydrophobic nuclear export signal (NES) motifs associated with unconventional ER- and Golgi-independent transport of nuclear/cytoplasmic secretory proteins to the surface membrane. Expression of the CD19-L gene expression is limited to the lymphocyte compartment within the human lymphohematopoietic system and particularly abundant in T-lineage cells. CD19-L displays abundant expression on immature double-positive (DP) thymocytes as well as leukemic T-cell precursors from T-lineage ALL patients corresponding to immature double-negative (DN) pro-thymocyte and DP cortico-thymocyte stages of human T-cell ontogeny. CD19-L is also expressed on B-lineage lymphoid cells at all stages of human B-cell ontogeny, including fetal liver derived biphenotypic CD2+CD19+ pro-B/T cells, pro-B cells, pre-B cells and mature B-cells. Soluble recombinant human CD19-L protein produced in a baculovirus expression system exhibited exquisite specificity for the extracellular domain of CD19 and had profound effects on apoptosis-related signaling and gene expression in CD19+ human leukemia cells. Engagement of CD19 co-receptor on B-lineage ALL cells with soluble CD19-L protein perturbed CD19-associated signaling network and triggered tyrosine phosphorylation of CD19 in a time-dependent fashion with peak phosphorylation occurring within 1–5 min. CD19-phosphorylation was associated with rapid and transient activation of SYK tyrosine kinase. Treatment of B-lineage ALL cells with 100 ng/mL CD19-L for 24 h corrupted the regulation of gene expression and altered the expression levels of 13 genes directly involved in regulation of apoptosis. We next examined the ability of CD19-L to induce apoptosis in leukemic B-cell precursors from chemotherapy-resistant CD19-positive human B-lineage ALL cell lines NALM-6 (pre-B ALL), RS4;11 (MLL-AF4+ Pro-B ALL), and ALL-1 (BCR-ABL+ Pre-pre-B ALL). CD19-L (but not control proteins CD19ECD or CD19ICD) caused apoptosis in each of these 3 ALL cell lines. As CD19-L specifically targets CD19ECD, excess CD19ECD protein was able to compete with surface CD19 receptor for CD19-L binding and thereby prevent CD19-L induced apoptosis. Excess CD19 intracellular domain protein (CD19ICD) that was included as a negative control did not affect CD19-L induced apoptosis. CD19-L was also capable of causing apoptosis in chemotherapy-resistant primary leukemic cells from relapsed CD19+ B-lineage ALL patients. This collection of experimental data provides compelling evidence that CD19-L is a potent biotherapeutic new agent candidate against CD19+ lymphoid malignancies. The identification of human CD19-L may lead to therapeutic innovation for B-lineage ALL as well as other B-lineage lymphoid malignancies by providing an effective alternative to CD19-directed monoclonal antibody-based biotherapeutic agents that have encountered several limitations in clinical settings. Disclosures:No relevant conflicts of interest to declare.

Differential phosphorylation of functional tyrosines in CD19 modulates B‐lymphocyte activation

CD19 is a B-cell transmembrane molecule that is critical for B-cell activation. CD19 serves as a scaffold protein for key signal transduction molecules including Lyn, PI3K, and Vav, by providing docking sites for these molecules via phosphorylation of CD19-Y(513), CD19-Y(482), and CD19-Y(391). We investigated the process of CD19 tyrosine phophorylation during B-cell activation using Ab specific for each of these phosphorylated tyrosines. BCR engagement induced differential tyrosine phosphorylation, as CD19-Y(513) phophorylation occurred first, and CD19-Y(482) phosphorylation was delayed and transient. Different BCR isotypes exhibited distinct patterns of CD19 phosphorylation: IgG-BCR ligation resulted in faster phosphorylation of CD19-Y(513) and more intense phosphorylation of CD19-Y(391) than IgM-BCR ligation. This affected CD19-mediated downstream pathways involving Vav, PI3K, and Akt. Additionally, the phosphorylation profile of CD19 differed distinctly according to its plasma membrane location. CD19 phosphorylated at Y(513) was almost exclusively located within lipid rafts, whereas phosphorylated Y(482) and Y(391) were found both inside and outside of the rafts. Furthermore, the phosphorylation of all three tyrosines was remarkably enhanced and prolonged following the simultaneous stimulation of BCR and CD40. Thus, variations in phosphorylation patterns may contribute to the complexity of CD19-regulated signal transduction.

Autoantibody-mediated regulation of B cell responses by functional anti-CD22 autoantibodies in patients with systemic sclerosis.

Studies have demonstrated that B cells play important roles in systemic sclerosis (SSc), especially through the CD19/CD22 autoimmune loop. CD22 is a B cell-specific inhibitory receptor that dampens B cell antigen receptor (BCR) signalling via tyrosine phosphorylation-dependent mechanism. In this study, we examined the presence and functional property of circulating autoantibodies reacting with CD22 in systemic sclerosis. Serum samples from 10 tight skin (TSK/+) mice and 50 SSc patients were assessed for anti-CD22 autoantibodies by enzyme-linked immunosorbent assays using recombinant mouse or human CD22. The association between anti-CD22 antibodies and clinical features was also investigated in SSc patients. Furthermore, the influence of SSc serum including anti-CD22 autoantibodies for CD22 tyrosine phosphorylation was examined by Western blotting using phosphotyrosine-specific antibodies reacting with four major tyrosine motifs of CD22 cytoplasmic domain. Anti-CD22 autoantibodies were positive in 80% of TSK/+ mice and in 22% of SSc patients. Patients positive for anti-CD22 antibodies showed significantly higher modified Rodnan skin thickness score compared with patients negative for anti-CD22 antibodies. Furthermore, anti-CD22 antibodies from patients' sera were capable of reducing phosphorylation of all four CD22 tyrosine motifs, while sera negative for anti-CD22 antibodies did not affect CD22 phosphorylation. Thus, a subset of SSc patients possessed autoantibodies reacting with a major inhibitory B cell response regulator, CD22. Because these antibodies can interfere CD22-mediated suppression onto B cell activation in vitro, SSc B cells produce functional autoantibodies that can enhance their own activation. This unique regulation may contribute to the autoimmune aspect of SSc.