Page 39 - Haematologica - Vol. 105 n. 6 - June 2020

CD20: functions and regulation
dimeric and homo-tetrameric oligomers associated with other cell-surface and cytoplasmic proteins contributing to the signal transduction.17,19,20 Tetraspanin proteins tend to associate with multiple other proteins in membrane microdomains (Figure 2).21 Energy transfer experiments indicate that CD20 is in close proximity to other tetraspan molecules, such as CD53, CD81, and CD82, forming supramolecular complexes (Figure 2).22 CD20 is also known to be physically coupled to major histocompatibil- ity complex class II (MHCII), CD40 molecule, BCR, and the C-terminal src kinase-binding protein (CBP) that inter- acts with Src kinases such as LYN, FYN, and LCK (Figure 2).20,23,24 Besides the transmembrane form of CD20, circu- lating CD20 was reported in CLL patients’ plasma;25 how- ever, this is likely to be part of a larger protein complex or a cell membrane fragment originating from cell break- down.
CD20 is a general B-cell marker expressed by the major- ity of B cells starting from late pre-B lymphocytes (it is not expressed by pro-B lymphocytes), and its expression is lost in terminally differentiated plasmablasts and plasma cells. Recently, a subset of CD20+ T cells with immune- regulatory and pro-inflammatory activity has been described; however, the clinical relevance of this remains to be determined.26 In B-cell malignancies, the level of CD20 expression is extremely variable depending on the specific neoplasm, with the lowest CD20 expression usu- ally being observed in patients with CLL and the highest CD20 cell-surface expression on DLBCL and hairy cell leukemia cells.27,28 Within CLL, it was noted that CD20 expression was also relatively higher in a disease subtype with a mutated variable region of immunoglobulin gene (IGHV) than in the subtype with unmutated IGHV.29 Some studies described that higher CD20 expression levels cor- relate with longer overall survival in patients with B-cell lymphomas treated with rituximab,30,31 although this remains controversial.32,33 Notably, CD20 levels are hetero- geneous not only among patients with the same malig- nancy, but also within the intraclonal cell subpopulations in an individual patient.34
CD20 function: a link to B-cell receptor signaling and microenvironmental interactions
The biological function of CD20 in B cells and its phys- iological ligand, if any, remain unclear. Some light on CD20 function has been shed by a case report of a patient with a common variable immunodeficiency and CD20 loss caused by a homozygous mutation in an exon 5 splicing site of MS4A1. The mutation led to alternative splicing with complete deletion of exon 5 and insertion of intron sequences and thus a truncated form of MS4A1 mRNA.35 Due to this homozygous mutation, the patient completely lacked cell-surface CD20. This did not disturb precursor B-cell differentiation in the bone marrow, as the patient had normal serum IgM levels and normal B-cell numbers. However, CD20 deficiency resulted in a reduced number of circulating memory B cells, reduced isotype switching of Ig, and decreased IgG antibody lev- els. In agreement with this observation, challenging the patient’s primary B cells in vitro using T-dependent and T- independent antigens led to the normal proliferation and secretion of IgM but reduced production of IgG. Given these data it is surprising that after repeated vaccinations the patient displayed a reduced ability to respond to T- independent antigens (pneumococcal polysaccharide vac-
cine), but a normal reaction to T-dependent antigens (anti-tetanus toxoid IgG).
Cases of a homozygous mutation in the MS4A1 gene in humans are extremely rare, which prompted the genera- tion of mouse models. This is a reasonable approach, since human and mice CD20 proteins share most struc- tural features and a conserved amino acid sequence (~75% homology) with only a few structural modifica- tions in the transmembrane and N- and C-terminal cyto- plasmic domains.36 CD20 in both humans and mice is B- cell specific, being first expressed by late pre-B cells in the bone marrow, predominantly after Ig heavy chain rearrangement. Uchida et al. created a mouse model with a homozygous mutation in the MS4A1 gene.36 These CD20-less mice had normal B-cell differentiation, isotype switching, maturation, mitogen-induced proliferation, and tissue localization. Similarly, CD20 deletion was not observed to have any effect on proliferation and differen- tiation in mice with MS4A1 disruption, generated by Neuberger’s group.37 CD20-/- mice immunized with T- dependent antigens showed impaired humoral immunity and primary and secondary immune responses connected with reduced numbers of germinal center B cells.38 Altogether, these studies in human and murine CD20- deficient B cells suggest that CD20 is required for both optimal T-independent humoral immunity, and also for a response to T-dependent antigens. However, it should be taken into consideration that the T-dependent immune response might be impaired due to the loss of CD20 in a small CD20+ population of T cells whose specific role in the immune system remains unclear.26 Overall, the rela- tively mild phenotype resulting from CD20 loss in humans and mice is somewhat surprising since CD20 was reported to be physically and functionally coupled to MHCII and CD40 (Figure 2),23 which are both critical for B- and T-cell interactions.
The development of humoral immunity requires a functional BCR signaling pathway, and CD20 was report- ed to be co-localized in lipid rafts39 and to interact directly physically with BCR.20 Additionally, it has been observed that CD20 becomes heavily phosphorylated after mito- gen stimulation, and it has been proposed that it might function as a calcium channel and be involved in B-cell activation.17,19 This is in line with in vitro data showing that BCR-activated calcium flux was reduced after siRNA- mediated CD20 down-modulation in human B-cell lines.34,40 Moreover, direct CD20 crosslinking induces acute signaling similar to BCR crosslinking, including cal- cium flux, and overlapping transcription patterns in human lymphoma cell lines.41,42 Kheirallah et al. also demonstrated that pretreatment of lymphoma cell lines with rituximab interferes with BCR signaling cascade stimulation, suggesting that both cell-surface proteins might share the same signaling pathway components and activate negative feedback regulatory mechanisms, including BCR downmodulation.43 We and others have shown that levels of cell-surface CD20 on primary CLL cells are correlated (and possibly co-regulated) with cell- surface BCR expression.34 Additionally, we observed in vivo that CLL cells that have recently exited the lymph node microenvironment to the peripheral blood are char- acterized by a marked upregulation of CD20 levels.44 This stems from the activation of CXCR4 by SDF1 chemokine, which leads to transcriptional activation of CD20 expression. Moreover, CD20 cell-surface levels are
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