CD19, from bench to bedside

Accepted Manuscript Title: CD19, from Bench to Bedside Authors: Xinchen Li, Ying Ding, Mengting Zi, Li Sun, Wenjie Zhang, Shun Chen, Yuekang Xu PII: DOI: Reference:

S0165-2478(16)30236-X http://dx.doi.org/doi:10.1016/j.imlet.2017.01.010 IMLET 5974

To appear in:

Immunology Letters

Received date: Revised date: Accepted date:

30-10-2016 19-1-2017 20-1-2017

Please cite this article as: Li Xinchen, Ding Ying, Zi Mengting, Sun Li, Zhang Wenjie, Chen Shun, Xu Yuekang.CD19, from Bench to Bedside.Immunology Letters http://dx.doi.org/10.1016/j.imlet.2017.01.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

CD19, from Bench to Bedside

Xinchen Lia, Ying Dinga, Mengting Zia, Li Suna, Wenjie Zhanga, Shun Chena and Yuekang Xua* a

School of Life Science, Anhui Normal University, Anhui, China.

*Corresponding author: Prof. Yuekang Xu, School of Life Science, Anhui Normal University, Wuhu 241000, China. Tel: 86-553-3883539. Fax: 86-553-3869571. E-mail: [email protected].

Highlights 

CD19 is a double-edged sword, which functions as both positive and negative regulator in BCR signaling.



The biochemical basis of CD19 as BCR regulator is the recruitment of secondary signaling molecules like PI3K via its cytoplasmic tail, which may be inside or outside of lipid rafts, depending on extracellular environments, to either promote or suppress BCR signals from antigens respectively.



Abnormal CD19 expression on B cells is associated with tumor development and autoimmune diseases. Thus CD19 can be as a valid therapeutic target for these diseases.

Abstract As a 95-kDa member of the immunoglobulin super-family expressed exclusively on B lymphocytes, CD19 is a critical co-receptor for B cell antigen receptor (BCR) signal transduction. Co-ligation of CD19 with the BCR synergistically enhances calcium release, mitogen-activated protein kinase activity and cell proliferation. However, CD19 deficient animals also display hyper-responsiveness under certain 1

circumstances, indicating potential negative regulatory functions in BCR signaling. Thus CD19, like many other signaling molecules, is a double-edged sword and its abnormal expression can result in B cell related diseases. Here in this review, we summarize the latest development on the major functions of CD19 as both positive and negative regulator of BCR signaling in different situations and highlight the correlation and mechanisms of disturbed CD19 expression with autoimmune diseases and B cell lymphomas. Hopefully, the knowledge derived could shed an interesting light on the mechanistic insights of this important B cell surface molecule in both physiological and pathological conditions.

Abbreviations: BCR: B cell antigen receptor; SH2: Src-homology domain 2; MAPK: mitogen-activated protein kinase; PTKs: Src-family protein tyrosine kinases; phosphatidylinositol-3 kinase;

NF-κB: nuclear factor of κ-binding;

PI3K:

IP3: inositol

1,4,5-trisphosphate; PIP2: phosphatidylinositol 4, 5-bisphosphate; PIP5K: phosphatidylinositol 4-phosphate 5-kinase; mIg: membrane Ig; BCAP: B cell adaptor for PI3Ks; Erk2: extracellular signal-regulated kinase 2; kinase;

SSc: systemic sclerosis;

SLE: systemic lupus erythematosus;

cell activating factor of the TNF family; encephalomyelitis;

JNK1: c-Jun N-terminal

EAE: Experimental autoimmune

CLL: chronic lymphocytic leukaemia;

lymphoblastic leukemias; monoclonal antibody;

BAFF: B

ALL: acute

MOG: myelin oligodendrocyte glycoprotein;

CAR: chimeric antigen receptor;

mAb：

ADCC:

antibody-dependent cellular cytotoxicity.

Keywords: CD19, BCR, signal transduction, autoimmunity, B cell lymphomas.

2

1. Introduction B cell development and function are regulated by signals transuded through the B cell antigen receptor (BCR) and cell surface regulatory molecules including CD21, CD22, CD72, CD32b and CD19 [1-5]. CD19 is a B cell specific member of the immunoglobulin super-family expressed by early pre-B cells from the time of heavy chain rearrangement until plasma cell differentiation [6,7]. CD19 has a 240-amino acid cytoplasmic domain that contains nine conserved tyrosine residues, which play a critical role in the transduction of CD19-mediated signals [8-10]. Some of the tyrosine residues can be rapidly phosphorylated following BCR ligation to generate functionally active Src-homology domain 2 (SH2) recognition motifs that mediate the recruitment of regulatory molecules to the cell surface. For example, Src-family protein tyrosine kinases (PTKs), Fyn, Lyn, and Lck are present in the immunoprecipitated CD19 complexes following BCR ligation [11,12]. The phosphorylated tyrosines in the cytoplasm domain of CD19 then interact with effector molecules downstream of BCR signaling, such as phosphatidylinositol-3 kinase （PI3K）and the adapter proteins Vav, Cbl, Shc and c-AbI for signaling transduction towards gene transcription inside the B cells [13-20]. Historically, CD19 is considered as a positive regulator of B cell function because it was reported to amplify Src family PTK activation, enhance mitogen-activated protein kinase (MAPK) activity and promote cell proliferation [17,21,22]. Moreover, CD19 and BCR co-ligation greatly augments BCR-induced Ca2+ responses [23] and dramatically lowers the threshold for B cell activation in vitro [21]. While these positive functions of CD19 on BCR signaling are well documented, the roles of CD19 in inhibition of BCR signaling were also reported, in which cross linking of CD19 was found to suppress calcium release and proliferation following BCR ligation [24-27]. In line with the negative regulatory roles of CD19 in BCR signaling, we recently identified a biochemical mechanism to explain the elevated MAPK activity, calcium release and proliferation after BCR stimulation in the absence of CD19 [28]. Therefore, the roles of CD19 in BCR signaling are complex and cannot be attributed to positive regulation alone. 3

The diverse and important signaling roles of CD19 are ultimately manifested by its indispensible physiological functions, and abnormal expression will lead to B cell related diseases. CD19 is critical for normal B cell response, as loss of CD19 will cause immune-deficiency. Gene disruption studies have showed that CD19-deficiency in humans and mice led to an overall impaired humoral response with increased susceptibility to infection [29-31]. In addition, diminished CD19 expression is associated with B cell related lymphoma, including chronic lymphocytic leukaemia (CLL), follicular lymphoma and diffuse large B cell lymphoma [32,33]. On the other hand, elevated CD19 expression correlates with autoimmune diseases, such as systemic sclerosis (SSc) [34,35].

In this review, we make a timely summary on the

major roles of this important B cell co-receptor as both positive and negative regulator in BCR signaling, outline the impact of CD19 expression on autoimmunity and B-cell lymphomas, and overview the rationale for CD19 as a molecular target in these disease settings. 2. BCR signaling The BCR consists of an antigen-binding transmembrane immunoglobulin molecule and a non-covalently associated Igα-Igβ heterodimer as signal transduction module.

Both Igα and Igβ contain a cytoplasmic immunoreceptor tyrosine based

activation motif, which is rapidly phosphorylated on tyrosine following antigen binding to the BCR. Signals transmitted by several branched pathways from the phosphorylated tyrosines with their recruited molecules in the activated BCR lead to proliferation, differentiation, and survival of B cells.

Most prominent are the PI3K

pathway，the MAPK pathway, and PLCγ2/Ca2+ pathway [36]. The PI3K pathway is central to the maintenance and activation of mature B cells [37]. Disrupted PI3K pathway via the loss of p85α/β, p110δ or adaptor proteins BCAP (B cell adaptor for PI3Ks) and TC21, a small GTPase encoded by Rras2, resulted in impaired homeostasis [38,39]. PI3K converts membrane-associated phosphatidylinositol 4, 5-bisphosphate (PIP2) into phosphatidylinositol (3,4,5)-trisphosphate (PIP3), and then to activate the downstream transcription factor FOXO1, which regulates the expression of genes critical for progress through consecutive steps of B-cell 4

differentiation [40]. MAPK pathway also has multiple effects on B cells, including enhanced proliferation and survival. Stimulation of B cells with antigen activates MAP kinase pathways, leading to a strong increase in Ras GTP levels with subsequent activation of the Ras-Raf-Mek-Erk cascade, and regulation of the BCL-6 transcription factor stability in B cells, which is crucial for the control of B-cell differentiation [41-43]. In addition, proliferation in isotype-switched B cells can be enhanced by Grb2 association with the cytoplasmic tail of the heavy chain in the IgG and IgE BCR [44]. Btk is primarily responsible for activating PLCγ2, leading to the production of inositol 1,4,5-trisphosphate (IP3) and activation of Ca2+ signaling pathways as well as the transcription factor, such as nuclear factor of κ-binding（NF-κB）, which plays an important role in B-cell survival and differentiation [45,46]. Collectively, these prominent BCR signaling pathways convert to constitute the biochemical basis for the diverse B cell behaviors.

However, they are all subjected to the tight regulation by a

BCR co-receptor molecule, CD19. 3. The structure of CD19 complex As a 95,000 Mr cell-surface protein of B cells, CD19 belongs to the immunoglobulin super-family [6] and is classified as a type I transmembrane protein, with extracellular N-terminus, a single transmembrane domain, and a cytoplasmic C-terminus. With two C2-type Ig-like domains divided by a smaller potential disulfide linked non-Ig-like domain, as well as N-linked carbohydrate addition sites, the extracellular part of CD19 forms a complex with CD21 (CR2, complement receptor 2), CD81 and Leu-13 on cell surface [47]. The CD21 is composed of 15 or 16 short consensus repeats, a transmembrane region, and a 34 amino acid cytoplasmic domain [48,49]. The short CD21 cytoplasmic domain is devoid of known signaling motifs, but required for CD21 internalization upon ligand binding. Activated C3 forms covalent bonds with foreign Ags to generate C3d(g)-Ag complexes that bind to CD21[50] and regulate B cell function by signaling through the CD19 complex [51]. CD81 is a tetraspanin molecule, belonging to an evolutionarily conserved family of proteins. The partnership of CD81 with CD19 was first demonstrated using co-immunoprecipitation studies [52], and later reduced CD19 cell surface expression 5

was found in three independent lines of CD81 deficient mice [53-55], providing genetic evidence that CD81 is necessary for normal trafficking of CD19 to the cell surface. The function of Leu-13 on the cell surface, however, is not fully understood as yet, although it was envisioned that CD19 and its associated CD81/Leu-13 molecules provide signaling to inform B cells of complement activation in their microenvironment [56]. The single transmembrane domain of CD19 is essential for the complex formation, as replacement of the CD19 transmembrane with those of L-selectin resulted in the loss of CD19 complex formation [8]. The most important part of CD19 structure is its cytoplasmic domain, which consists of 242 amino acids with nine conserved tyrosine residues near the C-terminus [9,30,57]. They are highly conserved among human, mouse and guinea pig [10,58], indicating the importance of this domain in the transduction of CD19-mediated signals. Phosphorylation of CD19 tyrosines provides SH2-recognition motifs that recruit various signaling molecules to the cell surface. For example, CD19 phosphorylated dually on Y513 and Y482 binds with the tandem SH2 domains of p85 subunit of PI3K in vitro, and their mutation in mice reproduces most of the phenotypes found in CD19 deficient mice, such as reduced differentiation of B-1 and marginal zone B cells, and T-dependent and -independent antibody responses [18,59-63]. Phosphorylation of CD19-Y391 is essential for the activation of Vav [19,64], which in turn activates PI3K [65] and phosphatidylinositol 4-phosphate 5-kinase (PIP5K) [15], the key effectors in CD19 functions. 4. The functions of CD19 complex The unique extracelluar domain complexed with the extended cytoplasmic tails containing multiple tyrosine phosphorylated sites constitutes CD19 as an ideal signaling modulator. By modifying the engagement and signal transduction of BCR, CD19 plays a crucial role in mature B cell development as best exemplified by the finding that CD19 deficient mice have severely reduced mature B cell compartments [66,67]. Although B cells from CD19-deficient mice mature normally within the periphery, they are hypo-responsive to most transmembrane signals, including BCR ligation, LPS, and CD40 ligation plus IL-4, which leads to significant deficiencies in 6

proliferation, clonal expansion, and differentiation [6,7,67,68]. Genetic deletion of CD19 (CD19-/-) in mice exhibit hypo-responsiveness to transmembrane signals, severe reduction in serum Ig levels and weak T cell dependent antibody production, leading to an overall impaired humoral immune responses [29,30]. Hence, CD19 is essential for primary B-cell activation by T-cell-dependent antigens as well as the differentiation into memory B cells [66]. On the other hand, not only the mere expression of CD19 is essential for B cell biology, but also the amount of CD19 expression plays a critical role in their developmental process. Nup-98–HoxB4 immortalized hematopoietic stem cells over-expressing CD19 showed upon transplantation an impaired pro/pre B to immature B cell transition in the bone marrow, whereas Nup-98–HoxB4 hematopoietic stem cells expressing a shRNA that down-modulated but not knocked-out CD19 expression displayed a strongly reduced mature B cell compartment post-transplantation [69]. In addition, transgenic mice that over-express CD19 resulted in the development of autoimmune disease [70]. Collectively, the immune-deficiency associated with the loss of CD19 and the autoimmune tendency correlated with too much CD19 indicate indispensible functions of this B cell co-receptor, which mainly acts to regulate BCR signal transduction. Consistent with the mice studies, information derive from patients also suggest critical functions of CD19 in human. For examples, mutations of the CD19 gene were associated with severe antibody deficiency and autoimmune diseases in various clinical case studies. The first reports on CD19 deficiency in human revealed homozygous frame shift mutations in the CD19 gene in four patients from two separate families. Patients with this type of CD19 gene mutations showed normal numbers of precursors and total B cells, but decreased numbers of CD27+ memory B cells and CD5+ B cells. Their B cells exhibit normal levels of CD81, but decreased CD21 and very low to undetectable levels of CD19. The patients developed hypogammaglobulinemia, showing impaired antigen-induced BCR response and poor antibody response to rabies vaccination, as well as increased susceptibility to infection [31].

In another case study, similar observations were reported in conjunction with 7

two additional CD19 mutations in a single patient, whose CD19 deficiency was suspected to relate to his low platelet counts, possibly linking the CD19 mutations to the development of autoimmune disease [71]. Similarly, mutations that cause over-expression of CD19 in human, like those found in systemic sclerosis patients, also disrupted B-cell regulated autoimmunity and resulted in autoimmune disorders [72]. The first case of a genetic CD21 deficiency in human was presented in a patient with hypogammaglobulinemia and a severe reduction in IgD-CD27+ memory B cells [73]. This deficiency was also associated with an immune defect, common variable immunodeficiency (CVID) [73,74]. CD81 in human was mostly studied with respect to viral and parasite infections. Both hepatitis C virus and Plasmodium sporozoites interact with CD81 to infect hepatocytes [75,76], and HIV particle assembly in infected T cells critically depends on CD81 [77]. A mutation in the CD81 gene can also lead to antibody deficiency, which in CD81 deficiency patient was remarkably similar to that of patients with CD19 deficiency, characterized by impaired antibody responses upon vaccination and impaired memory B cell formation [78]. 5. Regulation of BCR signaling by CD19 5.1 Positive regulation Acting as a critical co-receptor for BCR signaling, CD19 is generally considered as a positive B cell signaling regulator [70]. The mechanisms for its positive regulation of BCR signaling by CD19 are illustrated in Figure 1A and can be summarized as following: Promote calcium mobilization: Since intracellular calcium flux is an important index for cell response, the importance of CD19 as a signaling molecule on calcium flux was first examined by cross-linking it alone with monoclonal antibodies and an elevated intracellular calcium was observed [26]. Later on, co-ligation of CD19 with surface Ig was believed to synergistically increase intracellular calcium levels, as the response achieved by simultaneous ligation was greater than the sum of individual ligation of the two receptors [23], further demonstrating the promoting effect of CD19 on calcium flux in B cells. Biochemically, the effect of CD19 on Ca2+ flux is associated with the enhanced 8

IP3 generation，which is responsible for the release of intracellular calcium storage. The IP3 comes from PIP2, synthesized by a PIP5K. Studies have revealed that PIP5K can be modestly activated in primary B cell when CD19 is ligated alone, but strongly activated when CD19 is co-ligated to membrane Ig (mIg) [15]. This suggested that CD19 may contribute to the B cell calcium response by regulating the availability of PIP2. Previous studies proposed the mechanisms for CD19 to regulate calcium flux via phosphorylation of Y484 and Y515 in its cytoplasm tails to activate PI3K and Btk [61,63]. In accordance with this finding, CD19 malfunction results in PI3K activation defects by BCR [79], and the phosphorylated CD19 leads to recruitment and activation of PI3K and generation of PIP3, which then functions to recruit and activates Btk by binding its PH domain, causing CD19 to enhance calcium flux [61]. However, opposite results in terms of calcium pathway activation were also reported, for example, PI3K activity and Akt activation were not compromised in CD19-deficient mice [80]. These discrepant results obtained from different laboratories could be due to the existence of CD19-dependent and -independent regulation [80], with the sum of these signaling pathways having specific quantitative and qualitative effects on Ca2+ responses. Enhance MAP kinase activation: The other biochemical basis to explain the positive role of CD19 in primary B cells is the enhanced activation of three MAP kinases: extracellular signal-regulated kinase 2（Erk2）, c-Jun N-terminal kinase （JNK1）and p38, relative to the effects of BCR ligation alone in primary murine B cells [17]. Similar results were also achieved in human B cell lines in which co-ligation of CD19 with BCR caused synergistic, prolonged enhancement of MAP kinase activity relative to ligation of either receptor complex alone [81]. These data indicate that CD19 has a positive impact on MAP kinases activation. Erk2 is the terminal kinase in the well-defined MAPK cascade that consists of Ras, Raf, MEK1, and Erk2. Activation of Ras and Raf by mIg has been reported previously, most likely through the Shc/SOS/Grb2 complex [82-84]. Co-ligation of CD19 and BCR enhanced the interaction of Shc with SOS/Grb2 in both transformed 9

and normal human B cells [14]. In addition, SOS/Grb2 complexes were also found to be associated with native CD19. Mapping studies with altered constructs demonstrated that Y330 in the cytoplasm tail of CD19 was necessary for binding to Grb2/SOS [85]. Therefore, enhanced tyrosine phosphorylation and membrane translocation of Shc/SOS/Grb2 complex could form an additional mechanism for activation of MAPK by co-stimulation of CD19. The pathway leading to the specific Erk2 activation by CD19 was further dissected in Daudi human B lymphoblastoid cells and compared with that from BCR. Ligation of mIgM alone, but not CD19 alone, resulted in activation of Ras, Raf, and MEK1 [81]. Neither Ras nor Raf activation was enhanced after co-ligation of CD19 and mIgM, relative to mIgM alone [81]. In contrast, MEK1 was activated synergistically by CD19-mIgM co-ligation, indicating a different pathway from CD19 to Erk2 activation. Furthermore, Synergistic activation of Erk2 by CD19-mIgM co-ligation did not require increased intracellular Ca2+ or PKC activity [81]. Collectively, these data demonstrated a positive effect of CD19 on MAP kinase signaling cascade independent of other signaling pathways. Amplify Src PTK activation: Src family PTKs are implicated in the initiation and propagation of BCR signaling, which might be utilized by CD19 in the promotion of BCR signaling. As the predominant Src family member in B cells, Lyn has a important initiating role in BCR signaling [86]. The expression of Lyn was reportedly required for CD19 tyrosine phosphorylation in primary B cells [22]. It was believed that once phosphorylated, CD19 provides functionally active SH2 recognition motifs and serves as an efficient substrate for Src family PTK phosphorylation and binding to regulate the activity of the Src family PTK. The mechanism of CD19 to amplify Lyn activity in this way was demonstrated using in vitro kinase assays with purified Lyn in the presence or absence of recombinant CD19 fusion proteins [22]. CD19-Y513 was the primary Lyn phosphorylation and binding site, which was proposed to result in processive phosphorylation of CD19-Y482 that recruited a second Lyn molecule, allowing for transphosphorylation and amplification of Lyn kinase activity [22]. However, this model fails to explain why the CD19 and Lyn 10

double knockout mice didn’t look like that of CD19-deficient mice as would be expected [87]. On the contrary, we demonstrated that the activation and subsequent regulatory roles of Lyn and CD19 after B cell receptor ligation were independent [88]. Therefore, the Src-family PTK amplification loop initiated and regulated by CD19 as a crucial mechanism to modulate signaling thresholds in B cells requires further investigation. Prolong BCR signaling in lipid rafts: Lipid rafts, which are detergent-resistant, sphingolipid, and cholesterol-rich membrane fractions, have a central feature of segregating membrane proteins, thereby providing a mechanism to physically compartmentalize BCR signaling proteins for regulation [89,90]. The association of the CD19/CD21complex with the translocation of the BCR into lipid rafts was explored first in the CH27 mouse B cell line [91]. After co-ligating CD19 with BCR by complement-tagged antigens, the CD19/CD21 complex was found to translocate together with the BCR into lipid rafts. Moreover, the presence of CD19/CD21 in the lipid raft caused by the co-ligation was demonstrated to significantly sustain BCR residency in lipid rafts, resulting in prolonged BCR signaling [91]. The observation in this study is further explored by a new finding that the enhanced synaptic recruitment of the lipid rafts by CD19 and BCR co-ligation is dependent on PI3K activation [92]. Apart from BCR components, co-ligation of the BCR with the CD19 molecule also results in a marked increase of tyrosine phosphorylation of Shc and rapidly translocation of Shc complex towards the lipid rafts on plasma membrane in normal human B cells [14]. Collectively, these results provide a novel additional mechanism to explain the positive function of CD19, which involves assembling important BCR signaling molecules into lipid rafts to facilitate their interaction and activation. 5.2 Negative regulation Although predominant roles of CD19 were found to be positive regulation of B cell signal transduction, old and recent studies have also revealed negative effects of this BCR co-receptor in certain situations (Figure 1B). The potential negative regulation of BCR signaling by CD19 was identified in following major signaling pathways: 11

Calcium pathway:

It is well established that Ca2+ serves as a universal second

messenger required for signal transduction in B cells, as the increased intracellular Ca2+ induced by BCR leads to the activation of several transcription factors such as CREB (cAMP response element-binding protein), NF-κB and NFAT (nuclear factor of activated T cells), as well as Ca2+-dependent protein kinase C (PKCs) and Ras guanine nucleotide exchange factor（RasGEFs）[46,93,94].

Early studies have

showed delayed but significantly elevated calcium in CD19-deficient B cells after BCR ligation, indicating a possible negative regulation of calcium release by CD19 [7]. Moreover, this possibility was further explored in the wild-type mice, in which the enhanced [Ca2+] response caused by anti-IgM F(ab’)2 antibody fragments (40μg/ml) in combination with anti-CD19 antibody at 10μg/ml

was decreased and

delayed when more anti-CD19 antibody (20μg/ml or 40μg/ml) was added with anti-IgM antibody (40μg/ml) [25]. Thus CD19 engagement can qualitatively regulate BCR signaling through dose-dependent manner, suggesting a potential negative role for CD19 in regulation of BCR induced calcium flux. Consistent with the negative regulation of calcium by CD19, we recently confirmed that when primary B cells were stimulated with F(ab’)2 anti-IgM, they displayed an exaggerated Ca2+ flux in the absence of CD19 compared to their wild-type counterparts [28]. PI3K pathways: The phosphorylated substrates of the PI3K promote specific recruitment of selected signaling proteins to the plasma membrane, where important signaling complexes are formed to mediate the differentiation, selection, survival, and activation of B cells [95]. Such an essential cellular pathway was found to be subjected to CD19 modulation. For example, a study demonstrated that in the absence of CD19 the baseline PI3K activity was up to 50% higher than wild-type B cells, and remained higher at 1 and 5 min following BCR engagement although the fold of increase is low [80]. Furthermore, the activation of PI3K downstream protein kinase Akt was also examined in both CD19 deficient primary B cells and A20 cells following IgM and IgG cross-linking respectively. IgM-induced Akt phosphorylation increased faster in CD19-/- primary B cells and higher constitutive Akt phosphorylation and faster IgG-induced phosphorylation were observed in the 12

A20-CD19neg cells [80]. Overall, these data suggest that CD19 might negatively regulate PI3K pathway in B cells. However, the mechanisms behind this observation were not explored further until recently we found the same hyper-sensitivity in the absence of CD19 and investigated the spatial relationship between CD19, BCR and Src kinase in lipid rafts. We found that in resting B cells, small amounts of PI3K were constitutively bound to CD19 outside the lipid raft, and further increased following BCR engagement. Interestingly, the BCR detected by the anti-IgM blot was also located outside the lipid raft, separated from the Src kinase Lyn inside the raft. Co-ligation with CD19 brought both BCR and CD19 into lipid rafts[28]. Collectively, these data suggest that CD19 could sequester from lipid raft via its cytoplasmic tail a critical level of positive signaling molecules including PI3K, which are activated only when CD19 is co-ligated with the BCR into the lipid raft. In the absence of CD19, however, the CD19 associated PI3Ks are available to bind to other adaptor proteins, such as BCAP and TC21，which interact constitutively with the BCR and display receptor specific activation properties [39,96-98]. These adaptor proteins can also recruit the p85a subunit of PI3K through their YxxM motifs, so more PI3K associated with these adaptor proteins would be translocated into the lipid rafts in response to BCR ligation for signaling amplification. Cell proliferation: Proliferation is one of the important behaviors that cells response to signaling. CD19 was reported by several studies to suppress B cell proliferation following BCR ligation 20 years ago [24,26,27]. Furthermore, in mice that over expressed human CD19 gene, the development of bone marrow immature B cells have been severely impaired and the number of mature B cells in the periphery was dramatically decreased [99]. In concert with these studies, our recently studies also found the inhibitive role of CD19 in B cell proliferation. CD19-deficient B cells proliferated faster and also entered the first division earlier in response to BCR cross-linking. The hyper-proliferative nature of CD19-/- primary B cells is BCR specific in the context of anti-IgM stimulation, as they proliferated normally in response to TLR ligation. Furthermore, we found that the BCR-induced hyper-proliferation in the absence of CD19 were dependent on PI3K as CD19 13

deficient primary B cells demonstrated elevated PI3K activity and proliferation potential in response to BCR ligation, but their hyper-proliferative phenotype was lost once PI3K activity was brought to normal [28]. 6. Disturbed expression of CD19 in autoimmune diseases and B cell lymphomas Given the above-mentioned important roles of CD19 in BCR signaling, and its overall expression pattern from pre-B cells until the terminal differentiation to plasma cells, it is not surprising that abnormal CD19 expression on B cells is associated with autoimmune diseases and B cell lymphomas development (Figure 2). 6.1 Autoimmune diseases Dysregulated CD19 expression has been implicated in several autoimmune diseases [100-102], including systemic sclerosis (SSc), systemic lupus erythematosus (SLE) and experimental autoimmune encephalomyelitis (EAE). SSc: SSc is a connective tissue disease characterized by excessive extracellular matrix deposition in the skin and visceral organs, with an autoimmune background [103]. As observed in the flow cytometric analyses of blood from SSc patients, the surface density of CD19 in SSc B cells was significantly higher by ∼20% than that in healthy individuals [35]. However, whether this small increase of CD19 expression is related to autoimmunity was far from clear. Therefore, the pathogenic significance of the 20% increase in CD19 expression was assessed in animal by generating transgenic mice that overexpressed CD19 to a similar extent as human SSc did [99,104]. Transgenic mice that overexpressed CD19 by 20% had significantly elevated levels of various autoantibodies, including SSc-specific anti-topo I Ab, anti-DNA Ab, anti-histone Ab, and rheumatoid factor [104,105]. These results suggest that the small increase in CD19 expression observed in human SSc may be sufficient to induce autoantibody production. To further examine the role of CD19 in systemic autoimmunity and tissue fibrosis, CD19 deficiency was investigated in the bleomycin-induced SSc mouse models [34]. The absence of CD19 inhibited the development of skin and lung fibrosis, hyper-γ-globulinemia, and autoantibody production in the bleomycin-induced SSc mouse models [34]. Although the exact mechanism remains to be determined, these findings suggest that CD19 is required for 14

the disease development and thus CD19 signaling in B cells could be potential therapeutic targets in the treatment of human SSc. SLE: SLE is a prototypic multisystem autoimmune disease characterized by the production of auto-antibodies and the involvement of most organ systems [106]. B cells play a pivotal role in the pathogenesis of SLE not only by producing pathogenic auto-antibodies but also by modulating immune responses via production of cytokines and chemokines [107]. The involvement of CD19 in SLE development is associated with B cell activating factor of the TNF family (BAFF), a TNF-like cytokine essential for the maturation and survival of peripheral B cells [108-110].

BAFF transgenic

(BAFF-Tg) mice develop abnormal B-cell hyperplasia and a lupus-like phenotype with high titres of anti-double-strand DNA antibody, proteinuria and autoimmune nephritis [111] because their B cell compartment is enlarged, in particular the marginal zone and B1 B cell compartments in which auto-reactive B cells are known to accumulate [110,112,113]. A recent study reported that BAFF-Tg mice simultaneously lacking CD19 are protected from splenomegaly and the development of autoimmune disorders [114]. The loss of CD19 had a dominant effect over BAFF including marginal zone and B1 cell expansion, splenomegaly and the development of autoimmunity, indicating an essential role of CD19 in the autoimmune pathogenesis [114]. Collectively, these data suggest that the expression of CD19 might be related to the pathogenesis of SLE, at least in the BAFF-driven SLE. Therefore, CD19 could serve as both a diagnostic marker and therapeutic target for the autoimmune disease. EAE: EAE is an inflammatory demyelinating disease of the central nerve system primarily mediated by CD4+ T cells, in which cytokines play a key role in the disease development and remission [115].

Fillatreau et al [116] have suggested a

regulatory role of B cells in EAE by demonstrating that IL-10 production from B cells plays a key role in resolving the disease. Interestingly, study using mice deficient for CD19 have demonstrated increased severity of EAE [117]. Consistent with this, a latter study demonstrated that B cells from CD19-/- mice with EAE produced significantly higher levels of interferon-γ in response to myelin oligodendrocyte glycoprotein (MOG) and anti-CD40 antibody than wild-type mice, whereas MOG and 15

anti-CD40 antibody induced less IL-10 production in B cells from CD19-/- mice with EAE than those from wild-type mice with EAE. In addition, CD19 loss resulted in increased severity as well as delayed recovery of the disease, suggesting an inhibitory role of CD19 in the etiology of EAE [118]. Collectively, these findings suggest that diminished expression of CD19 might be associated with the disease progression of EAE. 6.2 B cell lymphomas Since the majority of human leukemias and lymphomas are of B-cell origin [119], and CD19 is expressed in most acute lymphoblastic leukemias (ALL), CLL and other B cell lymphomas [120], it is not surprising that CD19 correlates or influences the development of these blood cancers. ALL: ALL is a heterogeneous group of diseases involving clonal expansion of early lymphoid progenitor cells, invariably leading to death unless treated [121]. CD19 represents the most commonly expressed antigen in pre-B-ALL, as noted from immunephenotyping studies in a series of 451 cases of B-ALL, in which all cases were positive for CD19 [122]. Although its link with ALL development has not been well established, CD19 is the most densely expressed B cell surface antigen in the patient with ALL, and thus a highly attractive target for drug development. For example, the antibody-drug conjugate SAR3419 and chimeric antigen receptors (CARs) T-cells that are engineered to target CD19 are among the growing list of anti-CD19 agents being developed for ALL treatment [123,124]. CLL: CLL is an indolent neoplasia characterized by progressive accumulation of small mature B cells in the peripheral blood, bone marrow, and secondary lymphoid organs [125]. Early studies using quantitative flow cytometry to detect the levels of CD19 expression in B cell malignancies in comparison with normal B cells demonstrated the low levels of CD19 expression in CLL [33]. Although the levels of CD19 expression is low in CLL, whether CD19 expression influence CLL development is unclear. Recently however, a study has showed that CD19 stimulation rapidly induced homotypic aggregation in a subpopulation of CLL cells, designated as CD19-responsive cells [126]. This CD19-responsive subpopulation expressed higher 16

levels of membrane CD19, c-myc mRNA, correlated with clinical disease progression, and reacted differently to anti-CLL therapeutics compared with CD19-nonresponsive cells of the same clone. The finding that CD19 responsiveness subset in CLL cells correlates with disease progression points to the physiological importance of CD19 signaling response in CLL development in spite of the overall low CD19 expression in the patients [126]. Other B cell lymphomas:

Follicular B cell lymphoma has lower CD19 levels

more frequently than any other lymphoma subtypes. Low CD19 expression is also more common in CD10-positive than in CD10-negative diffuse large B cell lymphoma [32]. Although the effects of CD19 expression on malignant B cell transformation or growth in vivo is less reported, a recent study demonstrated that a high order of CD19 clustering induces intense Akt activation, which is followed by up-regulation of c-Myc expression, in a manner independent of other components of the BCR, indicating a possible association of CD19 signaling with the oncogenesis [127]. In keeping with these findings, it was shown that the level of CD19 expression may serve as a poor prognostic factor in human lymphomas. Furthermore, recent studies have constructed one model of lymphomagenesis involving CD19 and the proto-oncogene c-Myc. Constitutive c-Myc overexpression caused CD19 up-regulated and phosphorylated. Up-regulated CD19 expression serves to further promote and stabilize c-Myc signaling, whose downstream effectors include important cell cycle regulators like cyclin D2. Dysregulation in these regulators subsequently enhance lymphomagenesis [29]. These studies have shown that CD19 expression can accelerate lymphomagenesis and is associated with increased disease severity. On the other hand, c-myc transgenic mice with CD19 deficiency, as compared to those expressing CD19, exhibited reduced malignancies and significantly higher (>80%) increase in survival and life spans. Thus CD19 regulatory loop can positively influence B cell transformation and lymphoma progression [29]. 7. CD19 antibody mediated therapies Overall, Due to its close association with the autoimmune diseases and B cell lymphoma, the rationale for CD19 as a molecular target in these disease settings 17

includes the application of both free soluble monoclonal antibody (mAb) and Chimeric Antigen Receptors (CARs) engineered T cells utilizing specificity of CD19 antibody . mAb: CD19 is a B cell-specific differentiation antigen that has gained wide attention in the design of new therapeutic strategies for B cell lymphomas. Several recombinant CD19 mAbs are currently being developed for clinical trials: SAR3419, a humanized IgG1 anti-CD19 antibody, has been reported to delay the progression of four CD19+ B-cell precursor-ALL and three mixed lineage leukemia-ALL xenografts, though not effective against T-lineage ALL xenografts [128]. XmAb5871, another anti-CD19 antibody, was found to effectively suppress multiple functions of B cells from RA patients [129]. Biochemically, it causes phosphorylation of the ITIM of FcγRIIb and inhibits BCR-induced calcium mobilization, proliferation, and costimulatory expression of human B cells from both healthy donors and SLE cases [130]. MEDI-551, the most advanced compound, is an IgG1k anti-CD19 mAb. Its effect on B cell depletion is primarily mediated by antibody-dependent cellular cytotoxicity (ADCC) [131]. MEDI-551 also demonstrated a robust ADCC activity against B-cell leukemia and lymphoma cell lines in vitro, as well as against CLL and ALL samples. The safety, pharmacokinetics, immunogenicity, and clinical activity of MEDI-551 in patients with B-cell malignancies, including diffuse large B cell lymphoma, CLL, follicular lymphoma, or SSc were recently evaluated in a phase 1 study [132]. CARs: Apart from those free soluble monoclonal antibodies, another CD19 antibody mediated immunotherapy approach is the application of CAR-engineered T cells. Although CD19 specific CARs genetically encoded in T cells have experienced various difficulties, satisfactory effects was finally achieved for B cell malignancies after incorporation of co-stimulation domains, and applied in conjunction with conditioning chemotherapy. The most widely studied and successful applications of CD19-targeted CAR therapy have been in treatment of B-cell leukemia and lymphoma [133]. In advanced follicular B-cell Non-Hodgkin’s Lymphoma, anti-CD19 CAR therapy resulted in dramatic regression, with peripheral blood B-cells 18

absent for at least 39 weeks after treatment, but no acute toxicities occurred [134]. In CLL, treatment has resulted in partial or complete remissions in a trial of 3 patients with advanced disease, but no persistent toxicities occurring other than B-cell aplasia [135]. Moreover, clinical trials of anti-CD19 CAR in relapsed/refractory B-cell ALL have also shown remarkable response rates. Two trials of 15 and 30 patients both had complete remission rates of roughly 90%, and lasting remissions up to 24 months were observed in the latter. In the larger of the two studies, cytokine-release syndrome was observed in all patients and responding patients also experienced B-cell aplasia [123,136]. Early human trials evaluating CD19-directed CAR T cells for chronic CLL showed limited responses until these CARs were incorporated co-stimulation domains, and conditioning chemotherapy was given before infusion of the genetically modified T cells (Please see recent review [137] for details). 8. Conclusion and future direction As a hallmark antigen of the B-cell lineage, CD19 is a well-known co-receptor for BCR signaling. CD19 is critically involved in establishing intrinsic B cell signaling thresholds through modulating BCR signaling. The functions of CD19 in BCR signaling regulation, however, have more than one face, dependent on how CD19 is engaged.

On the one hand, co-ligation of CD19 with the BCR

synergistically enhances MAP kinase activity, calcium release and proliferation. On the other hand, ligation of CD19 away from BCR complex suppresses BCR signal transduction. The recent findings of inhibitory roles for CD19 in BCR signaling add diversity to the signaling functions of this surface molecule. Since CD19 is expressed throughout B-cell development and plays a critical role in maintaining the balance between humoral, antigen-induced immune response and tolerance induction, CD19 targeted immunotherapy is emerging as a promising approach for autoimmunity diseases and B cell lymphomas. Detailed signaling mechanisms of CD19 under different stimulation conditions await further study in the future.

Understanding the

exact roles of CD19 signaling in B cell biology as well as pathology will extend its therapeutic potential from merely antibody-based treatment to the design of small molecule mimics or inhibitors for precise manipulation of B cell behaviors in different 19

situation.

Conflict of interests The authors declare no financial or commercial conflict of interest.

Acknowledgement This work was supported by the start-up fund from Anhui Normal University, China (004061439); Anhui International Collaborative Project, China (160521602); and Anhui Natural Science Research Fund, China (160511608) to Y.X.

References [1]

Karnell JL, Dimasi N, Karnell FG, 3rd, Fleming R, Kuta E, Wilson M, et al. CD19 and CD32b differentially regulate human B cell responsiveness. Journal of immunology 2014;192:1480-1490.

[2]

Tedder TF, Poe JC, Haas KM. CD22: a multifunctional receptor that regulates B lymphocyte survival and signal transduction. Advances in immunology 2005;88:1-50.

[3]

Poe JC, Hasegawa M, Tedder TF. CD19, CD21, and CD22: multifaceted response regulators of B lymphocyte signal transduction. International reviews of immunology 2001;20:739-762.

[4]

Adachi T, Wakabayashi C, Nakayama T, Yakura H, Tsubata T. CD72 negatively regulates signaling through the antigen receptor of B cells. Journal of immunology 2000;164:1223-1229.

[5]

Buhl AM, Cambier JC. Co-receptor and accessory regulation of B-cell antigen receptor signal transduction. Immunological reviews 1997;160:127-138.

[6]

Sato S, Ono N, Steeber DA, Pisetsky DS, Tedder TF. CD19 regulates B lymphocyte signaling thresholds critical for the development of B-1 lineage 20

cells and autoimmunity. Journal of immunology 1996;157:4371-4378. [7]

Sato S, Steeber DA, Jansen PJ, Tedder TF. CD19 expression levels regulate B lymphocyte development: human CD19 restores normal function in mice lacking endogenous CD19. Journal of immunology 1997;158:4662-4669.

[8]

Bradbury LE, Goldmacher VS, Tedder TF. The CD19 signal transduction complex of B lymphocytes. Deletion of the CD19 cytoplasmic domain alters signal transduction but not complex formation with TAPA-1 and Leu 13. Journal of immunology 1993;151:2915-2927.

[9]

Tedder TF, Isaacs CM. Isolation of cDNAs encoding the CD19 antigen of human and mouse B lymphocytes. A new member of the immunoglobulin superfamily. Journal of immunology 1989;143:712-717.

[10]

Zhou LJ, Ord DC, Hughes AL, Tedder TF. Structure and domain organization of the CD19 antigen of human, mouse, and guinea pig B lymphocytes. Conservation of the extensive cytoplasmic domain. Journal of immunology 1991;147:1424-1432.

[11]

Fujimoto M, Poe JC, Jansen PJ, Sato S, Tedder TF. CD19 amplifies B lymphocyte signal transduction by regulating Src-family protein tyrosine kinase activation. Journal of immunology 1999;162:7088-7094.

[12]

Chalupny NJ, Kanner SB, Schieven GL, Wee SF, Gilliland LK, Aruffo A, et al. Tyrosine phosphorylation of CD19 in pre-B and mature B cells. The EMBO journal 1993;12:2691-2696.

[13]

Beckwith M, Jorgensen G, Longo DL. The protein product of the proto-oncogene c-cbl forms a complex with phosphatidylinositol 3-kinase p85 and CD19 in anti-IgM-stimulated human B-lymphoma cells. Blood 1996;88:3502-3507.

[14]

Lankester AC, van Schijndel GM, Rood PM, Verhoeven AJ, van Lier RA. B cell antigen receptor cross-linking induces tyrosine phosphorylation and membrane translocation of a multimeric Shc complex that is augmented by CD19 co-ligation. European journal of immunology 1994;24:2818-2825.

[15]

O'Rourke LM, Tooze R, Turner M, Sandoval DM, Carter RH, Tybulewicz VL, 21

et al. CD19 as a membrane-anchored adaptor protein of B lymphocytes: costimulation of lipid and protein kinases by recruitment of Vav. Immunity 1998;8:635-645. [16]

Inaoki M, Sato S, Weintraub BC, Goodnow CC, Tedder TF. CD19-regulated signaling thresholds control peripheral tolerance and autoantibody production in B lymphocytes. The Journal of experimental medicine 1997;186:1923-1931.

[17]

Tooze RM, Doody GM, Fearon DT. Counterregulation by the coreceptors CD19 and CD22 of MAP kinase activation by membrane immunoglobulin. Immunity 1997;7:59-67.

[18]

Tuveson DA, Carter RH, Soltoff SP, Fearon DT. CD19 of B cells as a surrogate kinase insert region to bind phosphatidylinositol 3-kinase. Science 1993;260:986-989.

[19]

Weng WK, Jarvis L, LeBien TW. Signaling through CD19 activates Vav/mitogen-activated protein kinase pathway and induces formation of a CD19/Vav/phosphatidylinositol 3-kinase complex in human B cell precursors. The Journal of biological chemistry 1994;269:32514-32521.

[20]

Zipfel PA, Grove M, Blackburn K, Fujimoto M, Tedder TF, Pendergast AM. The c-Abl tyrosine kinase is regulated downstream of the B cell antigen receptor and interacts with CD19. Journal of immunology 2000;165:6872-6879.

[21]

Carter RH, Fearon DT. CD19: lowering the threshold for antigen receptor stimulation of B lymphocytes. Science 1992;256:105-107.

[22]

Fujimoto M, Fujimoto Y, Poe JC, Jansen PJ, Lowell CA, DeFranco AL, et al. CD19 regulates Src family protein tyrosine kinase activation in B lymphocytes through processive amplification. Immunity 2000;13:47-57.

[23]

Carter RH, Tuveson DA, Park DJ, Rhee SG, Fearon DT. The CD19 complex of B lymphocytes. Activation of phospholipase C by a protein tyrosine kinase-dependent pathway that can be enhanced by the membrane IgM complex. Journal of immunology 1991;147:3663-3671. 22

[24]

Callard RE, Rigley KP, Smith SH, Thurstan S, Shields JG. CD19 regulation of human B cell responses. B cell proliferation and antibody secretion are inhibited or enhanced by ligation of the CD19 surface glycoprotein depending on the stimulating signal used. Journal of immunology 1992;148:2983-2987.

[25]

Fujimoto M, Poe JC, Hasegawa M, Tedder TF. CD19 amplification of B lymphocyte Ca2+ responses: a role for Lyn sequestration in extinguishing negative regulation. The Journal of biological chemistry 2001;276:44820-44827.

[26]

Pezzutto A, Dorken B, Rabinovitch PS, Ledbetter JA, Moldenhauer G, Clark EA. CD19 monoclonal antibody HD37 inhibits anti-immunoglobulin-induced B cell activation and proliferation. Journal of immunology 1987;138:2793-2799.

[27]

Rigley KP, Callard RE. Inhibition of B cell proliferation with anti-CD19 monoclonal antibodies: anti-CD19 antibodies do not interfere with early signaling events triggered by anti-IgM or interleukin 4. European journal of immunology 1991;21:535-540.

[28]

Xu Y, Fairfax K, Light A, Huntington ND, Tarlinton DM. CD19 differentially regulates BCR signalling through the recruitment of PI3K. Autoimmunity 2014;47:430-437.

[29]

Poe JC, Minard-Colin V, Kountikov EI, Haas KM, Tedder TF. A c-Myc and surface CD19 signaling amplification loop promotes B cell lymphoma development and progression in mice. Journal of immunology 2012;189:2318-2325.

[30]

Tedder TF. CD19: a promising B cell target for rheumatoid arthritis. Nature reviews Rheumatology 2009;5:572-577.

[31]

van Zelm MC, Reisli I, van der Burg M, Castano D, van Noesel CJ, van Tol MJ, et al. An antibody-deficiency syndrome due to mutations in the CD19 gene. The New England journal of medicine 2006;354:1901-1912.

[32]

Yang W, Agrawal N, Patel J, Edinger A, Osei E, Thut D, et al. Diminished expression of CD19 in B-cell lymphomas. Cytometry Part B, Clinical 23

cytometry 2005;63:28-35. [33]

Ginaldi L, De Martinis M, Matutes E, Farahat N, Morilla R, Catovsky D. Levels of expression of CD19 and CD20 in chronic B cell leukaemias. Journal of clinical pathology 1998;51:364-369.

[34]

Yoshizaki A, Iwata Y, Komura K, Ogawa F, Hara T, Muroi E, et al. CD19 regulates skin and lung fibrosis via Toll-like receptor signaling in a model of bleomycin-induced scleroderma. The American journal of pathology 2008;172:1650-1663.

[35]

Sato S, Hasegawa M, Fujimoto M, Tedder TF, Takehara K. Quantitative genetic variation in CD19 expression correlates with autoimmunity. Journal of immunology 2000;165:6635-6643.

[36]

Hobeika E, Nielsen PJ, Medgyesi D. Signaling mechanisms regulating B-lymphocyte activation and tolerance. Journal of molecular medicine 2015;93:143-158.

[37]

Srinivasan L, Sasaki Y, Calado DP, Zhang B, Paik JH, DePinho RA, et al. PI3 kinase signals BCR-dependent mature B cell survival. Cell 2009;139:573-586.

[38]

Oak JS, Chen J, Peralta RQ, Deane JA, Fruman DA. The p85beta regulatory subunit of phosphoinositide 3-kinase has unique and redundant functions in B cells. Autoimmunity 2009;42:447-458.

[39]

Delgado P, Cubelos B, Calleja E, Martinez-Martin N, Cipres A, Merida I, et al. Essential function for the GTPase TC21 in homeostatic antigen receptor signaling. Nature immunology 2009;10:880-888.

[40]

Szydlowski M, Jablonska E, Juszczynski P. FOXO1 transcription factor: a critical effector of the PI3K-AKT axis in B-cell development. International reviews of immunology 2014;33:146-157.

[41]

Limnander A, Weiss A. Ca-dependent Ras/Erk signaling mediates negative selection of autoreactive B cells. Small GTPases 2011;2:282-288.

[42]

Genot E, Cantrell DA. Ras regulation and function in lymphocytes. Current opinion in immunology 2000;12:289-294.

[43]

Niu H, Ye BH, Dalla-Favera R. Antigen receptor signaling induces MAP 24

kinase-mediated phosphorylation and degradation of the BCL-6 transcription factor. Genes & development 1998;12:1953-1961. [44]

Engels N, Konig LM, Heemann C, Lutz J, Tsubata T, Griep S, et al. Recruitment of the cytoplasmic adaptor Grb2 to surface IgG and IgE provides antigen receptor-intrinsic costimulation to class-switched B cells. Nature immunology 2009;10:1018-1025.

[45]

Hendriks RW, Yuvaraj S, Kil LP. Targeting Bruton's tyrosine kinase in B cell malignancies. Nature reviews Cancer 2014;14:219-232.

[46]

Oh-hora M, Rao A. Calcium signaling in lymphocytes. Current opinion in immunology 2008;20:250-258.

[47]

Sato S, Miller AS, Howard MC, Tedder TF. Regulation of B lymphocyte development and activation by the CD19/CD21/CD81/Leu 13 complex requires the cytoplasmic domain of CD19. Journal of immunology 1997;159:3278-3287.

[48]

Moore MD, Cooper NR, Tack BF, Nemerow GR. Molecular cloning of the cDNA encoding the Epstein-Barr virus/C3d receptor (complement receptor type 2) of human B lymphocytes. Proceedings of the National Academy of Sciences of the United States of America 1987;84:9194-9198.

[49]

Weis JJ, Fearon DT, Klickstein LB, Wong WW, Richards SA, de Bruyn Kops A, et al. Identification of a partial cDNA clone for the C3d/Epstein-Barr virus receptor of human B lymphocytes: homology with the receptor for fragments C3b and C4b of the third and fourth components of complement. Proceedings of the National Academy of Sciences of the United States of America 1986;83:5639-5643.

[50]

Fearon DT, Carroll MC. Regulation of B lymphocyte responses to foreign and self-antigens by the CD19/CD21 complex. Annual review of immunology 2000;18:393-422.

[51]

Lee Y, Haas KM, Gor DO, Ding X, Karp DR, Greenspan NS, et al. Complement component C3d-antigen complexes can either augment or inhibit B lymphocyte activation and humoral immunity in mice depending on the 25

degree of CD21/CD19 complex engagement. Journal of immunology 2005;175:8011-8023. [52]

Bradbury LE, Kansas GS, Levy S, Evans RL, Tedder TF. The CD19/CD21 signal transducing complex of human B lymphocytes includes the target of antiproliferative antibody-1 and Leu-13 molecules. Journal of immunology 1992;149:2841-2850.

[53]

Tsitsikov EN, Gutierrez-Ramos JC, Geha RS. Impaired CD19 expression and signaling, enhanced antibody response to type II T independent antigen and reduction of B-1 cells in CD81-deficient mice. Proceedings of the National Academy of Sciences of the United States of America 1997;94:10844-10849.

[54]

Miyazaki T, Muller U, Campbell KS. Normal development but differentially altered proliferative responses of lymphocytes in mice lacking CD81. The EMBO journal 1997;16:4217-4225.

[55]

Maecker HT, Levy S. Normal lymphocyte development but delayed humoral immune response in CD81-null mice. The Journal of experimental medicine 1997;185:1505-1510.

[56]

Tedder TF, Sato S, Poe JC, Fujimoto M. CD19 and CD22 regulate a B lymphocyte signal transduction pathway that contributes to autoimmunity. The Keio journal of medicine 2000;49:1-13.

[57]

Haas KM, Tedder TF. Role of the CD19 and CD21/35 receptor complex in innate immunity, host defense and autoimmunity. Advances in experimental medicine and biology 2005;560:125-139.

[58]

Zhou LJ, Ord DC, Omori SA, Tedder TF. Structure of the genes encoding the CD19 antigen of human and mouse B lymphocytes. Immunogenetics 1992;35:102-111.

[59]

Wang Y, Carter RH. CD19 regulates B cell maturation, proliferation, and positive selection in the FDC zone of murine splenic germinal centers. Immunity 2005;22:749-761.

[60]

Wang Y, Brooks SR, Li X, Anzelon AN, Rickert RC, Carter RH. The physiologic role of CD19 cytoplasmic tyrosines. Immunity 2002;17:501-514. 26

[61]

Buhl AM, Cambier JC. Phosphorylation of CD19 Y484 and Y515, and linked activation of phosphatidylinositol 3-kinase, are required for B cell antigen receptor-mediated activation of Bruton's tyrosine kinase. Journal of immunology 1999;162:4438-4446.

[62]

Carter RH, Doody GM, Bolen JB, Fearon DT. Membrane IgM-induced tyrosine phosphorylation of CD19 requires a CD19 domain that mediates association with components of the B cell antigen receptor complex. Journal of immunology 1997;158:3062-3069.

[63]

Buhl AM, Pleiman CM, Rickert RC, Cambier JC. Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization. The Journal of experimental medicine 1997;186:1897-1910.

[64]

Li X, Sandoval D, Freeberg L, Carter RH. Role of CD19 tyrosine 391 in synergistic activation of B lymphocytes by coligation of CD19 and membrane Ig. Journal of immunology 1997;158:5649-5657.

[65]

Vigorito E, Bardi G, Glassford J, Lam EW, Clayton E, Turner M. Vav-dependent and vav-independent phosphatidylinositol 3-kinase activation in murine B cells determined by the nature of the stimulus. Journal of immunology 2004;173:3209-3214.

[66]

Rickert RC, Rajewsky K, Roes J. Impairment of T-cell-dependent B-cell responses and B-1 cell development in CD19-deficient mice. Nature 1995;376:352-355.

[67]

Engel P, Zhou LJ, Ord DC, Sato S, Koller B, Tedder TF. Abnormal B lymphocyte development, activation, and differentiation in mice that lack or overexpress the CD19 signal transduction molecule. Immunity 1995;3:39-50.

[68]

Sato S, Steeber DA, Tedder TF. The CD19 signal transduction molecule is a response regulator of B-lymphocyte differentiation. Proceedings of the National Academy of Sciences of the United States of America 1995;92:11558-11562.

[69]

von Muenchow L, Engdahl C, Karjalainen K, Rolink AG. The selection of 27

mature B cells is critically dependent on the expression level of the co-receptor CD19. Immunology letters 2014;160:113-119. [70]

Tedder TF, Inaoki M, Sato S. The CD19-CD21 complex regulates signal transduction thresholds governing humoral immunity and autoimmunity. Immunity 1997;6:107-118.

[71]

Kanegane H, Agematsu K, Futatani T, Sira MM, Suga K, Sekiguchi T, et al. Novel mutations in a Japanese patient with CD19 deficiency. Genes and immunity 2007;8:663-670.

[72]

Tedder TF, Poe JC, Fujimoto M, Haas KM, Sato S. The CD19-CD21 signal transduction complex of B lymphocytes regulates the balance between health and autoimmune disease: systemic sclerosis as a model system. Current directions in autoimmunity 2005;8:55-90.

[73]

Thiel J, Kimmig L, Salzer U, Grudzien M, Lebrecht D, Hagena T, et al. Genetic CD21 deficiency is associated with hypogammaglobulinemia. The Journal of allergy and clinical immunology 2012;129:801-810 e806.

[74]

Frank MM. CD21 deficiency, complement, and the development of common variable immunodeficiency. The Journal of allergy and clinical immunology 2012;129:811-813.

[75]

Cormier EG, Tsamis F, Kajumo F, Durso RJ, Gardner JP, Dragic T. CD81 is an entry coreceptor for hepatitis C virus. Proceedings of the National Academy of Sciences of the United States of America 2004;101:7270-7274.

[76]

Silvie O, Rubinstein E, Franetich JF, Prenant M, Belnoue E, Renia L, et al. Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity. Nat Med 2003;9:93-96.

[77]

Grigorov B, Attuil-Audenis V, Perugi F, Nedelec M, Watson S, Pique C, et al. A role for CD81 on the late steps of HIV-1 replication in a chronically infected T cell line. Retrovirology 2009;6:28.

[78]

van Zelm MC, Smet J, Adams B, Mascart F, Schandene L, Janssen F, et al. CD81 gene defect in humans disrupts CD19 complex formation and leads to antibody deficiency. The Journal of clinical investigation 28

2010;120:1265-1274. [79]

Keppler SJ, Gasparrini F, Burbage M, Aggarwal S, Frederico B, Geha RS, et al. Wiskott-Aldrich Syndrome Interacting Protein Deficiency Uncovers the Role of the Co-receptor CD19 as a Generic Hub for PI3 Kinase Signaling in B Cells. Immunity 2015;43:660-673.

[80]

Fujimoto M, Poe JC, Satterthwaite AB, Wahl MI, Witte ON, Tedder TF. Complementary roles for CD19 and Bruton's tyrosine kinase in B lymphocyte signal transduction. Journal of immunology 2002;168:5465-5476.

[81]

Li X, Carter RH. Convergence of CD19 and B cell antigen receptor signals at MEK1 in the ERK2 activation cascade. Journal of immunology 1998;161:5901-5908.

[82]

Tordai A, Franklin RA, Patel H, Gardner AM, Johnson GL, Gelfand EW. Cross-linking of surface IgM stimulates the Ras/Raf-1/MEK/MAPK cascade in human B lymphocytes. The Journal of biological chemistry 1994;269:7538-7543.

[83]

Saxton TM, van Oostveen I, Bowtell D, Aebersold R, Gold MR. B cell antigen receptor cross-linking induces phosphorylation of the p21ras oncoprotein activators SHC and mSOS1 as well as assembly of complexes containing SHC, GRB-2, mSOS1, and a 145-kDa tyrosine-phosphorylated protein. Journal of immunology 1994;153:623-636.

[84]

Harwood AE, Cambier JC. B cell antigen receptor cross-linking triggers rapid protein kinase C independent activation of p21ras1. Journal of immunology 1993;151:4513-4522.

[85]

Brooks SR, Li X, Volanakis EJ, Carter RH. Systematic analysis of the role of CD19 cytoplasmic tyrosines in enhancement of activation in Daudi human B cells: clustering of phospholipase C and Vav and of Grb2 and Sos with different CD19 tyrosines. Journal of immunology 2000;164:3123-3131.

[86]

DeFranco AL, Chan VW, Lowell CA. Positive and negative roles of the tyrosine kinase Lyn in B cell function. Seminars in immunology 1998;10:299-307. 29

[87]

Hasegawa M, Fujimoto M, Poe JC, Steeber DA, Lowell CA, Tedder TF. A CD19-dependent signaling pathway regulates autoimmunity in Lyn-deficient mice. Journal of immunology 2001;167:2469-2478.

[88]

Xu Y, Beavitt SJ, Harder KW, Hibbs ML, Tarlinton DM. The activation and subsequent regulatory roles of Lyn and CD19 after B cell receptor ligation are independent. Journal of immunology 2002;169:6910-6918.

[89]

Gupta N, Wollscheid B, Watts JD, Scheer B, Aebersold R, DeFranco AL. Quantitative proteomic analysis of B cell lipid rafts reveals that ezrin regulates antigen receptor-mediated lipid raft dynamics. Nature immunology 2006;7:625-633.

[90]

Gupta N, DeFranco AL. Visualizing lipid raft dynamics and early signaling events during antigen receptor-mediated B-lymphocyte activation. Molecular biology of the cell 2003;14:432-444.

[91]

Cherukuri A, Cheng PC, Sohn HW, Pierce SK. The CD19/CD21 complex functions to prolong B cell antigen receptor signaling from lipid rafts. Immunity 2001;14:169-179.

[92]

Xu L, Auzins A, Sun X, Xu Y, Harnischfeger F, Lu Y, et al. The synaptic recruitment of lipid rafts is dependent on CD19-PI3K module and cytoskeleton remodeling molecules. Journal of leukocyte biology 2015;98:223-234.

[93]

Baba Y, Kurosaki T. Impact of Ca2+ signaling on B cell function. Trends in immunology 2011;32:589-594.

[94]

Lyubchenko T. Ca(2)+ signaling in B cells. TheScientificWorldJournal 2010;10:2254-2264.

[95]

Werner M, Hobeika E, Jumaa H. Role of PI3K in the generation and survival of B cells. Immunological reviews 2010;237:55-71.

[96]

Baracho GV, Miletic AV, Omori SA, Cato MH, Rickert RC. Emergence of the PI3-kinase pathway as a central modulator of normal and aberrant B cell differentiation. Current opinion in immunology 2011;23:178-183.

[97]

Aiba Y, Kameyama M, Yamazaki T, Tedder TF, Kurosaki T. Regulation of 30

B-cell development by BCAP and CD19 through their binding to phosphoinositide 3-kinase. Blood 2008;111:1497-1503. [98]

Okada T, Maeda A, Iwamatsu A, Gotoh K, Kurosaki T. BCAP: the tyrosine kinase substrate that connects B cell receptor to phosphoinositide 3-kinase activation. Immunity 2000;13:817-827.

[99]

Zhou LJ, Smith HM, Waldschmidt TJ, Schwarting R, Daley J, Tedder TF. Tissue-specific expression of the human CD19 gene in transgenic mice inhibits antigen-independent B-lymphocyte development. Molecular and cellular biology 1994;14:3884-3894.

[100] Ziegler AI, Le Page MA, Maxwell MJ, Stolp J, Guo H, Jayasimhan A, et al. The CD19 signalling molecule is elevated in NOD mice and controls type 1 diabetes development. Diabetologia 2013;56:2659-2668. [101] Gabrielli A, Avvedimento EV, Krieg T. Scleroderma. The New England journal of medicine 2009;360:1989-2003. [102] Edwards JC, Cambridge G. B-cell targeting in rheumatoid arthritis and other autoimmune diseases. Nature reviews Immunology 2006;6:394-403. [103] Hachulla E, Launay D. Diagnosis and classification of systemic sclerosis. Clinical reviews in allergy & immunology 2011;40:78-83. [104] Sato S, Fujimoto M, Hasegawa M, Takehara K. Altered blood B lymphocyte homeostasis in systemic sclerosis: expanded naive B cells and diminished but activated memory B cells. Arthritis and rheumatism 2004;50:1918-1927. [105] Asano N, Fujimoto M, Yazawa N, Shirasawa S, Hasegawa M, Okochi H, et al. B Lymphocyte signaling established by the CD19/CD22 loop regulates autoimmunity in the tight-skin mouse. The American journal of pathology 2004;165:641-650. [106] Rahman A, Isenberg DA. Systemic lupus erythematosus. The New England journal of medicine 2008;358:929-939. [107] Sabahi R, Anolik JH. B-cell-targeted therapy for systemic lupus erythematosus. Drugs 2006;66:1933-1948. [108] Vincent FB, Morand EF, Schneider P, Mackay F. The BAFF/APRIL system in 31

SLE pathogenesis. Nature reviews Rheumatology 2014;10:365-373. [109] Fairfax K, Mackay IR, Mackay F. BAFF/BLyS inhibitors: A new prospect for treatment of systemic lupus erythematosus. IUBMB life 2012;64:595-602. [110] Thompson JS, Schneider P, Kalled SL, Wang L, Lefevre EA, Cachero TG, et al. BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population. The Journal of experimental medicine 2000;192:129-135. [111] Mackay F, Woodcock SA, Lawton P, Ambrose C, Baetscher M, Schneider P, et al. Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations. The Journal of experimental medicine 1999;190:1697-1710. [112] Amezcua Vesely MC, Schwartz M, Bermejo DA, Montes CL, Cautivo KM, Kalergis AM, et al. FcgammaRIIb and BAFF differentially regulate peritoneal B1 cell survival. Journal of immunology 2012;188:4792-4800. [113] Batten M, Groom J, Cachero TG, Qian F, Schneider P, Tschopp J, et al. BAFF mediates survival of peripheral immature B lymphocytes. The Journal of experimental medicine 2000;192:1453-1466. [114] Fairfax KA, Tsantikos E, Figgett WA, Vincent FB, Quah PS, LePage M, et al. BAFF-driven autoimmunity requires CD19 expression. Journal of autoimmunity 2015;62:1-10. [115] Constantinescu CS, Farooqi N, O'Brien K, Gran B. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). British journal of pharmacology 2011;164:1079-1106. [116] Fillatreau S, Sweenie CH, McGeachy MJ, Gray D, Anderton SM. B cells regulate autoimmunity by provision of IL-10. Nature immunology 2002;3:944-950. [117] Du C, Sriram S. Increased severity of experimental allergic encephalomyelitis in lyn-/- mice in the absence of elevated proinflammatory cytokine response in the central nervous system. Journal of immunology 2002;168:3105-3112. [118] Matsushita T, Fujimoto M, Hasegawa M, Komura K, Takehara K, Tedder TF, 32

et al. Inhibitory role of CD19 in the progression of experimental autoimmune encephalomyelitis by regulating cytokine response. The American journal of pathology 2006;168:812-821. [119] Kuppers R. Mechanisms of B-cell lymphoma pathogenesis. Nature reviews Cancer 2005;5:251-262. [120] Cooper LJ, Al-Kadhimi Z, DiGiusto D, Kalos M, Colcher D, Raubitschek A, et al. Development and application of CD19-specific T cells for adoptive immunotherapy of B cell malignancies. Blood cells, molecules & diseases 2004;33:83-89. [121] Kaplan JB, Grischenko M, Giles FJ. Blinatumomab for the treatment of acute lymphoblastic leukemia. Invest New Drugs 2015;33:1271-1279. [122] Raponi S, De Propris MS, Intoppa S, Milani ML, Vitale A, Elia L, et al. Flow cytometric study of potential target antigens (CD19, CD20, CD22, CD33) for antibody-based immunotherapy in acute lymphoblastic leukemia: analysis of 552 cases. Leukemia & lymphoma 2011;52:1098-1107. [123] Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. The New England journal of medicine 2014;371:1507-1517. [124] Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. The New England journal of medicine 2013;368:1509-1518. [125] Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia. The New England journal of medicine 2005;352:804-815. [126] Herishanu Y, Kay S, Dezorella N, Baron S, Hazan-Halevy I, Porat Z, et al. Divergence in CD19-mediated signaling unfolds intraclonal diversity in chronic lymphocytic leukemia, which correlates with disease progression. Journal of immunology 2013;190:784-793. [127] Chung EY, Psathas JN, Yu D, Li Y, Weiss MJ, Thomas-Tikhonenko A. CD19 is a major B cell receptor-independent activator of MYC-driven B-lymphomagenesis. The Journal of clinical investigation 33

2012;122:2257-2266. [128] Carol H, Szymanska B, Evans K, Boehm I, Houghton PJ, Smith MA, et al. The anti-CD19 antibody-drug conjugate SAR3419 prevents hematolymphoid relapse postinduction therapy in preclinical models of pediatric acute lymphoblastic leukemia. Clinical cancer research : an official journal of the American Association for Cancer Research 2013;19:1795-1805. [129] Chu SY, Yeter K, Kotha R, Pong E, Miranda Y, Phung S, et al. Suppression of rheumatoid arthritis B cells by XmAb5871, an anti-CD19 antibody that coengages B cell antigen receptor complex and Fcgamma receptor IIb inhibitory receptor. Arthritis & rheumatology 2014;66:1153-1164. [130] Horton HM, Chu SY, Ortiz EC, Pong E, Cemerski S, Leung IW, et al. Antibody-mediated coengagement of FcgammaRIIb and B cell receptor complex suppresses humoral immunity in systemic lupus erythematosus. Journal of immunology 2011;186:4223-4233. [131] Herbst R, Wang Y, Gallagher S, Mittereder N, Kuta E, Damschroder M, et al. B-cell depletion in vitro and in vivo with an afucosylated anti-CD19 antibody. The Journal of pharmacology and experimental therapeutics 2010;335:213-222. [132] Schiopu E, Chatterjee S, Hsu V, Flor A, Cimbora D, Patra K, et al. Safety and tolerability of an anti-CD19 monoclonal antibody, MEDI-551, in subjects with systemic sclerosis: a phase I, randomized, placebo-controlled, escalating single-dose study. Arthritis research & therapy 2016;18:131. [133] Magee MS, Snook AE. Challenges to chimeric antigen receptor (CAR)-T cell therapy for cancer. Discov Med 2014;18:265-271. [134] Kochenderfer JN, Wilson WH, Janik JE, Dudley ME, Stetler-Stevenson M, Feldman SA, et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood 2010;116:4099-4102. [135] Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A, et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish 34

memory in patients with advanced leukemia. Sci Transl Med 2011;3:95ra73. [136] Davila ML, Riviere I, Wang X, Bartido S, Park J, Curran K, et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med 2014;6:224ra225. [137] Oluwole OO, Davila ML. At The Bedside: Clinical review of chimeric antigen receptor (CAR) T cell therapy for B cell malignancies. Journal of leukocyte biology 2016.

35

Figure legends

Legend to Figure 1: Differential Regulation of BCR Signaling by CD19 A. Positive regulation: In the case of pathogen invasion, complement-coated pathogenic antigens co-ligate CD19 with BCR into lipid rafts to amplify immune signals from BCR in terms of calcium mobilization, MAPK activity and Lyn activation. CD19 regulates calcium flux via PI3K and Btk, resulting in enhanced IP3 generation, and the release of intracellular calcium storage. Co-ligation of CD19 and BCR enhanced the interaction of Shc with SOS/Grb2, leading to the activation of MAPK. B. Negative regulation: At the end of immune response, CD19 could be pre-ligated away from BCR and lipid rafts by free circulating complements as CD21 ligand, functioning as a molecular sink to attract PI3K and other signaling molecules to its cytoplasmic tail and sequestering them away from the lipid rafts. Therefore, less signaling components will be available for BCR to attenuate unnecessary signaling event. MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol-3 kinase; IP3, inositol 1,4,5-trisphosphate; PIP2, phosphatidylinositol 4, 5-bisphosphate; BCAP, B cell adaptor for PI3Ks.

36

37

Legend to Figure 2: Putative model for CD19 and B Cell-related Diseases On the one hand, although diminished CD19 expression occurs frequently in B-cell lymphomas, in particular FL and DLBCL, CD19 stimulation rapidly induces homotypic aggregation in a subpopulation of CLL cells, which expresses higher levels of c-myc mRNA, whose downstream effectors include cyclin D2, subsequently enhancing lymphomagenesis，and correlates with CLL progression. On the other hand, CD19 expression levels are higher on B cells from autoimmune diseases like SSc etc. Up-regulated CD19 expression results in elevated levels of various pathogenic mediators like auto-antibodies, BAFF and IL-10 that play key roles in SSc, SLE and EAE respectively. FL, follicular lymphoma; DLBCL, diffuse large B cell lymphoma; SSc, systemic sclerosis; Ab: antibody; BAFF, B cell activating factor of the TNF family; EAE: Experimental autoimmune encephalomyelitis.

38