CD20 Deficiency, Lessons Related to Therapeutic Biologicals and Primary Immunodeficiency
CD20 was one of the first B-cell-specific differentiation antigens identified (Stashenko et al. 1980). Nowadays, anti-CD20 immunotherapy using chimeric monoclonal antibodies (MoAbs) is used for the treatment of B-cell neoplasia, EBV-associated immunopathology, and a growing list of diseases with presumed autoimmune origin (Franks et al. 2016; Miano 2016; Claes et al. 2015; Froissart et al. 2015).
CD20, encoded by membrane-spanning 4 domains, subfamily a, member 1 (MS4A1; OMIM*112210), belongs to the MS4A family of molecules with multiple membrane-spanning domains (Liang et al. 2001; Liang and Tedder 2001) and is expressed on pre-B and mature B cells but is downregulated upon differentiation into plasma cells (Tedder and Engel 1994). CD20 is unlikely to have a natural ligand, but in vitro studies with CD20, MoAbs have demonstrated its involvement in the regulation of B-cell activation and proliferation (Tedder et al. 1985, 1986).
The CD20 gene structure and expression pattern are strongly conserved between mouse and human (Tedder et al. 1988, 1989; Uchida et al. 2004a), and CD20-knockout mice have been independently generated by two groups (Uchida et al. 2004a; O’Keefe et al. 1998). CD20–/– B cells develop and function normally, but spleen B cells exhibit demonstrable alterations in BCR- and CD19-induced Ca2+ responses (Uchida et al. 2004a). The importance of CD20 for the generation and function of human B cells has yet to be clarified.
B-Cell Development and Mechanistic Role for CD20
Peripheral B-cell survival relies on signals from the BCR and the BAFFR (O’Keefe et al. 1998). The BCR is a heterotrimeric complex consisting of Ag-binding Ig and the signaling Igα/Igβ heterodimers.
Crosslinking the BCR on mature B cells with Ag or anti-IgM Abs initiates multiple intracellular signaling cascades, which eventually lead to the activation of ERK, NF-kB, and NFAT pathways. Among these, NF-kB appears to play a prominently protective role in the survival of Ag-stimulated B cells by inducing the expression of several antiapoptotic proteins such as Bcl-2, Bcl-xL, and Bfl-1 (Mackay et al. 2010; Grumont et al. 1998, 1999). BCR signaling activates the canonical NF-kB pathway, which is characterized by the phosphorylation and ubiquitin-mediated degradation of IkB inhibitory proteins, in particular IkB-alpha. This leads to the translocation of NF-kB1 into the nucleus to activate target gene transcription. BAFFR is a member of the TNFR family.
Deficiency of BAFF or BAFFR results in an almost complete loss of follicular and marginal zone (MZ) B cells (Saijo et al. 2002; Schiemann et al. 2001; Thompson et al. 2001), demonstrating a critical role for BAFFR-mediated signaling in B-cell survival. In contrast to BCR, BAFFR activates the noncanonical NF-kB pathway, which depends on the proteolytic processing of p100 to p52 to generate p52/RelB (NF-kB2) nuclear complexes (Tedder et al. 1986, 1988, 1989; Uchida et al. 2004a; Claudio et al. 2002). Both BCR and BAFFR are required for the maintenance of peripheral B-cell homeostasis. It has been shown that signals from the BCR and BAFFR cooperate to allow B-cell survival at multiple stages of peripheral B-cell differentiation and during immune responses. BCR promotes BAFFR-mediated signals through at least two mechanisms by upregulating the expression of BAFFR and by supplying the noncanonical NF-kB pathway substrate p100 (Cancro 2009).
CD20 is a component of a multimeric cell surface complex that regulates Ca2+ transport across the plasma membrane (Bubien et al. 1993; Kanzaki et al. 1997). B-cell depletion using rituximab does not affect the CD19+CD20− pro-B cell and CD20−CD138+ plasma cell populations, and within 6–8 months following treatment, the CD20+ B-cell compartment begins to replenish.
Although CD20 MoAb immunotherapy depletes normal and malignant B cells in vivo by antibody-dependent activation of the innate monocytic network (Uchida et al. 2004b), crosslinking of CD20 alters Ca2+ homeostasis, which influences cell cycle progression and can lead to apoptosis of normal and leukemic B cells in vitro (Bubien et al. 1993; van der Kolk et al. 2002). Yet the precise functions of CD20 and the therapeutic MoAbs are still controversial issues (Okroj et al. 2013).
Clinical Presentation, Differential Diagnosis, and Treatment
To date, only a single patient has been reported that completely lacked surface CD20 expression on B cells as a result of a homozygous mutation in the CD20 gene (Kuijpers et al. 2010). We found that the patient had a persistent hypogammaglobulinemia, normal B-cell numbers, and a strong reduction in circulating memory B cells. A decreased frequency of somatic hypermutations in IgG heavy chain genes was found. After repeated vaccinations the patient mounted proper responses to recall antigens but displayed a strongly reduced ability to respond to pneumococcal polysaccharides. In agreement with a conserved role of CD20 in the generation of T-cell-independent (TI) antibody responses, we found that CD20-deficient mice had a reduced ability to respond to TI antigens. We concluded that CD20 has a nonredundant role in the generation of optimal B-cell responses.
Human CD20 deficiency results in decreased IgG antibody levels and relatively increased IgM levels with weak responses against polysaccharides after vaccination. Importantly, we found that CD20 deficiency in mice also results in an impaired ability to make TI antibody responses. Thus, although the phenotypes of CD20-deficient humans and mice are subtly different, the current data would imply that CD20 signals are essential to enable B cells to optimally respond to antigenic stimuli.
The severe reduction of class-switched sIgD–CD27+ memory B cells might reflect suboptimal germinal center formation and is likely to cause the profound hypogammaglobulinemia. Although IgG antibodies and circulating memory B cells are not defective in the CD20-knockout mice (Uchida et al. 2004a; O’Keefe et al. 1998), the selection of naive B cells into the non-switched (sIgD+CD27+) MZ B-cell compartment seems disturbed in the patient. In healthy donors, the transition of naive B cells into MZ B cells is accompanied by a reduction of the mean IgVH-CDR3 lengths and acquisition of IgV mutations (Tsuiji et al. 2006; Weller et al. 2004). Concomitantly, a counterselection against poly- and self-reactive antibodies and enrichment for antibodies reactive with specific bacterial polysaccharides occur (Tsuiji et al. 2006). This selection process is potentially driven by tonic BCR signals that may be assumed to be different between BCRs with longer and shorter IgVH-CDR3 lengths. Alternatively, particular self- or commensal-related antigens, recognized with low avidity, may provide positive BCR signals, favoring the selection of B cells that have short IgVH-CDR3 lengths in the MZ B cell compartment. In the MZ B cells of the patient, no counterselection for long IgVH-CDR3 lengths was observed, which is in accordance with a diminished response against polysaccharide vaccination. The notion of a disturbed counterselection against poly- and self-reactive antibodies is strengthened by the observation that, in the absence of any clinical signs of autoimmunity, antinuclear antibodies in the serum of the patient were found repeatedly (Kuijpers et al. 2010).
New Avenues to Approach CD20
Apart from cell-death-inducing capacity, murine anti-CD20 MoAbs were shown to also strongly inhibit IgM and IgG secretion by B cells in vitro (Golay et al. 1985). The first therapeutic anti-CD20 MoAb rituximab and new generation anti-CD20 MoAbs are, however, widely used now to treat autoimmune diseases and organ transplant rejection. Also in this case, the mechanism of action of rituximab is still unclear, as response does not simply correlate with B-cell depletion (Townsend et al. 2010). Thus, the data together suggest that in autoimmune diseases, anti-CD20 MoAbs may be therapeutically active through both killing the autoimmune B cells and inhibition of differentiation of the residual B cells into plasma cells secreting autoimmune antibodies. Experiments are required to verify this hypothesis and to determine whether all stimuli and all B-cell subsets may respond to such inhibition rather than cell death only.
Alternatively, low percentages of CD20-expressing T cells in human blood may be present with a pro-inflammatory role in autoimmunity as well. This minor T-cell subset was first described in 1993 (Hultin et al. 1993) and related to disease (Wilk et al. 2009), but the existence of this rather rare T-cell subset has also been disputed as an artifact (Henry et al. 2010). Others have found that CD20-expressing T cells can exhibit pro-inflammatory capacity. In rheumatoid arthritis (RA), CD20+ T cells make up a larger percentage of Th17 cells when compared with healthy individuals (Eggleton et al. 2011), although the overall percentage of CD20+ T cells among all T cells did not differ between RA patients and healthy individuals. Clearly, the pathological relevance, if any, of CD20+ T cells in autoimmune diseases remains unknown, but these findings give new impetus for the important avenue of investigation to understand the therapeutic effects of CD20 MoAbs – in particular in disease considered to be largely caused by T cells.
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