B Lymphocyte Antigen CD19
CD19 was first identified as biomarker of normal and neoplastic B lymphocytes as B4 antigen with the use of anti-B4 monoclonal antibody against CD19 (Anderson et al. 1984). Anti-B4 antibody in turn was encoded from cDNA clones of human tonsillar cDNA library, by selectively hybridizing with RNA from CD19+ cell lines. Both human and mouse CD19 molecules are highly homologous with a conserved cytoplasmic domain with no homology with other known proteins and an immunoglobulin-like extracellular domain, which gave CD19 molecule the status of a member of Ig superfamily (Tedder and Isaacs 1989).
The CD19 molecule is a 95 KDa cell surface protein of B lymphocytes and follicular dendritic cells (FDC). It is considered as a biomarker of B cell because of its continued expression throughout B cell differentiation stages starting from late pro-B cell stage until terminally differentiated plasma cells (Nadler et al. 1983; Schriever et al. 1989). It is a co-receptor of B cell receptor complex having an important role in BCR-mediated signaling for B cell differentiation and activation.
Genomic Organization and Protein Structure
CD19 protein is 556 amino acids long and is classified as class I transmembrane protein. Structurally it is divided into extracellular, transmembrane, and cytoplasmic domains having specific interaction sites for various signaling molecules. Extracellular domain consists of two C2-type Ig-like domains separated by a non-Ig-like, potentially disulfide-linked domain and N-linked carbohydrate addition sites. This domain is 276 amino acids long, and through this domain, CD19 interacts with CD21 and CD81 (Bradbury et al. 1992).
CD19 Expression on B Cell and Its Regulation
CD19 expression on B cell surface starts from as early as late pro-B cell stage (on DH-JH rearranged pro-B cells) and continues until differentiated plasmablast stage. Its continuous presence on B cell surface advocates their role in B cell development and activation. Progression through early pro-/prestage to immature B cell stage requires augmentation of pre-BCR signaling through CD19; in addition, it also helps in positive and negative selection of immature B cells. CD19 role in early B cell development was evident with impaired B cell proliferation in CD19−/− mice due to impairment of pre-BCR signaling (Otero et al. 2003). CD19 also has an extended role in peripheral B cell development, which starts from the egress of B cell from the bone marrow to the periphery. CD19 expression is threefold higher in mature B cell than that of immature B cell and is slightly more on B1 cells compared to B2 cells (Carter et al. 2002). B1 cell differentiation and maturation are dependent on CD19-mediated signaling as CD19−/− mice show diminished population of B1 cells, while overexpression of CD19 leads to extended B1 cell population. In the subpopulation of B2 cells, both marginal zone and follicular B cell compartment require survival signal from CD19 for their maintenance (Otero et al. 2003).
CD19 expression is vital for B cell development and function; hence, its density on B cell surface is tightly regulated; moreover, its expression is lineage specific, and it expresses only on B cell and follicular dendritic cells among all members of the hematopoietic system. CD19 expression level is crucial for correct B cell development as it has been shown through studies on CD19−/− mice and overexpressing CD19 transgenic mice that both low (absence of positive selection) and high (negative selection) expressions of CD19 lead to defect in B cell development and disturbed pro-/pre- to mature B cell ratio (Carter and Fearon 1992; Engel et al. 1995). Transcription factor PAX5 (also known as BSAP: B cell lineage-specific activator protein) expressed at all B cell developmental stages except terminally differentiated plasma cells and considered as master regulator of B cell commitment and differentiation of early lymphoid progenitors correlates with CD19 expression (Fuxa and Busslinger 2007). Cloning and characterization of CD19 gene have reported several PAX5-binding site, and in particular a high-affinity binding site was identified in the promoter region instead of TATA box (Kozmik et al. 1992). Studies have shown that Pax5 is required for normal expression of CD19, starting from late pro-B cell stage until terminal differentiation to plasma cell. Surface expression of CD19 molecules requires the presence of tetraspanin molecule CD81, and in the absence of CD81, expression of CD19 on B cell surface is greatly reduced as CD81 regulates the transport of CD19 to plasma membrane. CD81-deficient mice show almost 50% reduction in CD19 expression compared to controls, and patients deficient in CD81 did not express CD19 on the B cell surface (Shoham et al. 2003).
Role of CD19 in B Cell Signaling
CD19 in Pre-BCR and BCR Signaling
CD19 expresses on the surface of B cell at late pro-B cell stage prior to the expression of BCR and thus functions independently of BCR. It plays a role in late pro-B cell to large pre-B (proliferating) cell stage transition by modulating the proliferative signal emanating through pre-BCR. Another important role of CD19 is during positive and negative selection of immature B lymphocyte. Based on the BCR signaling threshold, a B cell can be either positively selected or eliminated. As CD19 augments the BCR signals, it may have a role in these selection events. CD19−/− mice show reduced number of mature B cell suggesting a defective positive selection of immature B cell (von Muenchow et al. 2014).
Naïve B cell, on their surface, expresses BCR with an antigen-recognizing domain immunoglobulins (Ig), immunoreceptor tyrosine-based activation motifs (ITAMs) containing signaling components Igα (CD79a) and Igβ (CD79b), and signal-modulating co-receptor components CD19-C21 complex. Upon antigen recognition and ligand binding, phosphorylation of ITAMs occurs and recruits tyrosine kinases Lyn (hyperlink) and Syk (hyperlink). Both tyrosine kinases phosphorylate several adaptor proteins and the co-receptor CD19. Phosphorylated Cd19 further recruits several other molecules as Vav, Bruton’s tyrosine kinase (Btk), and PI3 kinase (PI3K) via its cytoplasmic domains and lowers the threshold for B cell activation. CD19 interacts with a variety of proteins through which it involves intracellular signal transduction downstream of BCR.
CD19 and Lyn
CD19 is primarily phosphorylated by Src family protein tyrosine kinase Lyn at tyrosine 513, as this phosphorylation event is absent in Lyn-deficient B cell before or after BCR ligation. However, reports suggest that CD19-deficient primary B cells have greatly compromised Lyn phosphorylation; hence, there exist an interdependence between CD19 and Lyn. In addition, data exist in support of independent Lyn and CD19 activity as well. By regulating Lyn kinase activity, CD19 regulates the B cell signaling threshold, and this interaction of Lyn with co-receptor complex may reduce the threshold of BCR activation by up to 104-fold (Fearon and Carroll 2000).
CD19 and Vav
Vav proteins are highly expressed in peripheral lymphocytes and spleen cells and have been shown to modulate BCR-mediated PI3K signaling. The Vav family of proteins is cytoplasmic guanosine nucleotide exchange factor for Rho family GTPases. CD19 phosphorylation recruits Vav into a signaling complex of CD19 via SH2 interaction, which facilitates its subsequent phosphorylation by activated PTK. Vav binds with PIP3 produced by PI3K and thus its activation is controlled by PI3K. On the other hand, Vav regulates PI3K through Rac1, a member of the Rho family GTPases (Bustelo 2014).
CD19 and PI3K
CD19 and Complement
The complement system is a cascade of serum-soluble protein that constitutes an important part of the innate immune system. Activation of complement is an essential component of early response against infection. Cleavage products of complement rapidly recruit effector cells and facilitate lysis and phagocytosis of microbes. In addition these cleavage products collectively called as C3d (g) bind with CR2 receptor (CD21) on B cell surface and FDC and bridge innate and adaptive arm of the immune system. Upon engagement of CD21 with C3d (g) bound with antigen, recruits CD19-CD21-CD81 complex to the lipid raft and augment B cell signaling through CD19 cytoplasmic chain. However, co-engagement of BCR is required for CD19 recruitment to raft and its signaling (Del Nagro et al. 2005).
CD19 and Toll-Like Receptors
Toll-like receptor (TLR) families play an important role in innate immune response, which serves as a first line of defense and is T cell independent. Upon encounter with pathogenic microorganisms, innate immune system recognizes specific molecular patterns on their cell components such as lipopolysaccharide (LPS), peptidoglycan, and bacterial DNA and RNA. LPS, a component of gram-negative bacterial cell wall, is recognized by TLR, a pattern recognition receptor, expressed by many cell types. In human and mice, there are 10–13 different types of TLRs which mostly signal via adaptor protein myeloid differentiation factor 88 (MyD88) and TIR domain-containing adaptor inducing interferon-beta (TRIF). However, two different types of TLRs are expressed on B cell along with BCR, TLR4, and RP105, out of which RP105 is preferentially expressed on mature B cell. CD19 plays an important role in the regulation of TLR signaling through B cell-specific receptor RP105. Upon RP105 ligation, CD19 recruits Lyn and Vav and regulates intracellular Ca2+ mobilization (Hua and Hou 2013).
CD19 Animal Models
Both CD19-deficient (CD19−/−) mice (Engel et al. 1995) and human CD19 transgenic mice (hCD19TG) (Zhou et al. 1994) that overexpress CD19 show a dramatic decrease in peripheral B lymphocyte numbers. This decrease in number in case of hCD19TG is because of increased cell surface signaling through CD19 and resulted feedback signaling to inhibit the development of bone marrow precursors of B cells, while in CD19−/− mice B cell precursors, development is largely unaffected; instead later stages of B cell growth are affected with a special reduction in marginal zone B cell. Upon BCR cross-linking and LPS stimulation, CD19−/− B cell shows reduced proliferation, while this is increased in hCD19TG mice depending on gene dosage. In both cases B cells were able to proliferate clonally and secrete antibody. Antibody secretion in hCD19TG is skewed toward IgG2b, while there is an overall decrease in all isotypes in CD19−/− mice. T cell-dependent humoral response is reduced in CD19−/− mice with reduced proliferation and reduced germinal center formation and memory cell selection. These phenotypes of both mice suggest that CD19 acts as a regulator of cell surface receptor signaling having crucial role both at early development in the bone marrow and at later stages of clonal expansion after antigen encounter in peripheral B cell pool.
CD19 and Its Association with Human Diseases
Studies have shown that B cell regulates immune response in a variety of ways including antigen presentation, cytokine production, antibody production, and T cell help and influence on the function of other immune cells including dendritic cells and macrophages. On the surface of B cell, CD19 molecule along with its co-receptor complex members CD21, CD81, and CCL25 signals with BCR to lower the threshold of BCR-dependent signaling and regulates the immune response (Carter and Fearon 1992). CD19 is considered as positive response regulator of BCR signaling. Any defect or deficiency of CD19 affects the humoral immune response and leads to malfunction of immune system resulting into disease.
Mutation in cd19 gene in mice leads to hypogammaglobulinemia, low CD5+/B1-B cells, impaired T cell-dependent germinal center formation, and impaired B cell memory. CD19 mutation in humans has been reported: insertion and deletion of base pair in exons 6 and 11, respectively, result in frameshift mutation and insertion of an early stop codon prior to tyrosine residues in the cytoplasmic chain which are critical for CD19-mediated signaling. Depending on the type of mutation, there could be either complete lack of CD19 surface expression or a severely reduced expression. Phenotype of disease includes a normal number of precursor mature B cell but reduced CD5+ B cell and CD27+ memory B lymphocytes along with decreased serum level of IgG antibodies. These patients show hypogammaglobulinemia similar to CD19−/− mice because of poor antigen-specific response by mature B cell, poor response to vaccination (toward rabies vaccine), and increased susceptibility to bacterial infection (van Zelm et al. 2006).
A subset of common variable immunodeficiency (CVID) is linked with mutation of CD19 gene. Reported case of one patient with mutation in splice acceptor site of intron 5 had similar immunological and clinical phenotype as observed before with loss of CD19 surface expression and antibody deficiency (Kanegane et al. 2007).
As we have already seen, CD19 acts as a positive regulator of BCR signaling, while negative regulators for BCR signaling are CD22, CD72, and FcγRIIB that dampen BCR signals. These are collectively called as response regulators, which establish signaling threshold that controls the duration and intensity of B cell activation. Any alteration in CD19/CD22 loop activities contributes to autoimmunity both in mice and human. hCD19Tg mice which express threefold more CD19 are reported to be autoimmune prone, and they show increased proliferation in response to antigen and have anti-dsDNA serum antibodies. An autoimmune disease systemic sclerosis (SSc) is characterized by tissue fibrosis and production of disease-specific autoantibodies. B cells from SSc patients show higher expression level of CD19 by 20% and upregulated CD19 signaling pathway resulting in chronic hyperactivation of B cells, precisely CD27+ memory B cells compared to naïve B cells (Yoshizaki and Sato 2015). Dysregulated (both high and low) CD19 expression has been reported in the case of systemic lupus erythematosus (SLE), another kind of autoimmune disease where autoantibodies and immune complex aid into the pathogenesis (Sato et al. 2000).
CD19 antigen is a specific B cell marker and is expressed by all normal and malignant B cells and is used as a marker to differentiate between B and T cell leukemia. It is expressed in acute lymphoblastic leukemias (ALL), chronic lymphocytic leukemias (CLL), and B cell lymphomas. Expression level of CD19 in different types of malignancy differs significantly and ranges from normal to high levels and is also a useful marker as diagnostic tool. Occasionally CD19 expression also associates with non-B cell malignancies such as acute myeloid leukemias (AML) and multiple myeloma (MM) and is considered as a result of aberrant regulation of CD19 by PAX5. Studies in CD19−/− mice have identified the role of CD19 in the stabilization of c-Myc protein, which is an established proto-oncogene in human cancers. CD19/c-Myc activation loop has been shown to have a role in malignant B cell transformation and lymphoma genesis (Wang et al. 2012).
As CD19 is expressed by majority of B lymphoid malignant cells and autoreactive B cells, antibody-mediated therapy targeting CD19 is a new advancement in the field of antibody therapy. CD19 targeting has evolved through years, and considerable advancement has been made for its precise use. Initially CD19 monoclonal antibodies have been used for lymphoma therapy, and transient reduction of tumor cells was reported; later anti-CD19 monoclonal antibodies were conjugated with immunotoxins such as ricin A and saporin and evaluated successfully against human and murine malignant B cells. Combination therapy of anti-CD19 with chemotherapy also gave a successful result in experimental models. Combination of anti-CD19 with cytokine treatment induces antibody-mediated cellular toxicity mechanisms (Hammer 2012). Advancement and improvement to this antibody therapy have been done with the use of humanized anti-CD19 antibody (Medi-551), which is a fucosylated antibody and has increased affinity to FcγRIIIA. Targeted immunotherapy against CD19 has been developed in the form of bi-specific T cell engager (BiTE) antibody, a monoclonal antibody (blinatumomab) having antibody-binding site specific for both CD19 and CD3. It engages cytotoxic T cell and then guides it to CD19-expressing B cells and subsequently leads to lysis of malignant cells; however, normal B cells get lysed too. Alternative approach toward B cell targeting is chimeric antigen receptor (CAR) approach. CAR T cell immunotherapy involves patient-derived T cells, which are genetically modified to express synthetic antigen receptors having specificity toward CD19. They are composed of three domains, an extracellular CD19 recognition domain derived from a single-chain variable fragment (scFv), which is linked via a flexible hinge region to transmembrane domain, and a cytoplasmic signaling domain that triggers the T cell activation. CARs have undergone a series of modifications; first-generation CARs utilized cytoplasmic CD2ζ region, while second- and third-generation CARs included a variety of co-stimulatory molecules for improved T cell stimulation. Clinical trials of CARs are undergoing and have shown promising results in B cell malignancies (Katz and Herishanu 2014).
Other than B cell malignancy, anti-CD19 antibodies are also considered for the treatment of autoimmune diseases. Previously anti-B cell approaches have been used for targeting B cells in non-Hodgkin lymphoma, rheumatoid arthritis, and other autoimmune diseases as SLE and ANCA-associated vasculitis using rituximab (anti-CD20) and belimumab. However, these approaches are not suitable for targeting neither pre-B cell nor antibody-secreting plasma cell where CD20 is not expressed; hence, CD19 appears to be a promising approach for specific targeting of B cells as it starts expressing before CD20 (B1 antigen) at pro-B cell and continues till plasma cell differentiation (Mei et al. 2012).
CD19 is a biomarker for B cells and function as a co-receptor for BCR on B cell surface. CD19 along with its signaling complex plays an important role in signaling cascade emanating downstream of BCR and is involved in positive regulation of activation threshold of BCR. CD19 plays an important role in B cell development, and any deregulation of CD19 results in impairment of B cell development, which is associated with either B cell deficiency or autoimmunity in patients. Hence, it maintains a balance among humoral response and tolerance induction. CD19-based immunotherapy is emerging as a promising clinical development for the management of B cell malignancies and autoimmunity. CD19 monoclonal antibodies anti-B4-br, BiTE, SAR 3419, MEDI-551, and chimeric antigen receptor toward B cells (anti-CD19-CAR) are under clinical trials in different phases and look promising for the treatment of B cell malignancies.
CSIR-IITR manuscript communication no.: 3431.