B-cell subpopulations in humans and their differential susceptibility to depletion with anti-CD20 monoclonal antibodies

In humans, different B-cell subpopulations can be distinguished in peripheral blood and other tissues on the basis of differential expression of various surface markers. These different subsets correspond to different stages of maturation, activation and differentiation. B-cell depletion therapy based on rituximab, an anti-CD20 mAb, is widely used in the treatment of various malignant and autoimmune diseases. Rituximab induces a very significant depletion of B-cell subpopulations in the peripheral blood usually for a period of 6 to 9 months after one cycle of therapy. Cells detected circulating during depletion are mainly CD20 negative plasmablasts. Data on depletion of CD20-expressing B cells in solid tissues are limited but show that depletion is significant but not complete, with bone marrow and spleen being more easily depleted than lymph nodes. Factors influencing depletion are thought to include not only the total drug dose administered and distribution into various tissues, but also B-cell intrinsic and microenvironment factors influencing recruitment of effector mechanisms and antigen and effector modulation. Available studies show that the degree of depletion varies between individuals, even if treated with the same dose, but that it tends to be consistent in the same individual. This suggests that individual factors are important in determining the final extent of depletion.


Introduction to B-cell subpopulations
In humans from birth all new B cells originate from common precursors in the bone marrow. In the bone marrow, peripheral blood and secondary lymphoid tissues, diff erent B-cell subpopulations can be distinguished corresponding to diff erent stages of maturation, activation and diff erentiation. B-cell subpopulations are characterised mainly by the diff erential expression of diff erent cell surface markers that include various cluster of diff erentiation (CD) molecules and diff erent surface immuno globulin isotypes (B-cell antigen receptor). B-cell develop ment can be separated into an earlier antigenindependent phase, which takes place in the bone marrow, and a later antigen-dependent phase that takes place mainly in secondary lymphoid tissues. In a simplifi ed way, the diff erent B-cell lineage subsets include pro-B cells, pre-B cells, immature and transitional B cells, mature naïve B cells, memory B cells, plasmablasts and plasma cells (Figure 1). Plasmablasts are recently diff erentiated antibody-producing cells that are usually shortlived but can recirculate and home to tissues such as the mucosa or the bone marrow, where they can diff erentiate into fully mature plasma cells. In addition, centroblasts and centro cytes are B cells participating in germinal centre reactions.
B-cell precursor subpopulations are found in the bone marrow. In the peripheral blood, transitional, naïve mature and memory B cells and plasmablasts, and more rarely plasma cells, can be identifi ed. Plasma cells are more frequently seen in the bone marrow and peripheral lymphoid tissues. Centrocytes and centroblasts are found in secondary lymphoid tissues where germinal centre reactions take place, and are not found circulating in peripheral blood. Marginal zone B cells can be found in the marginal zone of the spleen and similar populations are described in particular locations in other secondary lymphoid tissues [1]. Marginal zone B cells in human adults are mainly memory B cells. Th ere is still controversy on what drives formation of human marginal zone B cells, to what extent they are similar to mice marginal zone B cells and what is their relationship with circulating IgM + memory B-cell subsets [1,2].
Immunophenotyping of B cells with multiparameter fl ow cytometry has allowed identifi cation of an increasing number of diff erent subpopulations, increas ing our Abstract In humans, diff erent B-cell subpopulations can be distinguished in peripheral blood and other tissues on the basis of diff erential expression of various surface markers. These diff erent subsets correspond to diff erent stages of maturation, activation and diff erentiation. B-cell depletion therapy based on rituximab, an anti-CD20 mAb, is widely used in the treatment of various malignant and autoimmune diseases. Rituximab induces a very signifi cant depletion of B-cell subpopulations in the peripheral blood usually for a period of 6 to 9 months after one cycle of therapy. Cells detected circulating during depletion are mainly CD20 negative plasmablasts. Data on depletion of CD20-expressing B cells in solid tissues are limited but show that depletion is signifi cant but not complete, with bone marrow and spleen being more easily depleted than lymph nodes. Factors infl uencing depletion are thought to include not only the total drug dose administered and distribution into various tissues, but also B-cell intrinsic and microenvironment factors infl uencing recruitment of eff ector mechanisms and antigen and eff ector modulation. Available studies show that the degree of depletion varies between individuals, even if treated with the same dose, but that it tends to be consistent in the same individual. This suggests that individual factors are important in determining the fi nal extent of depletion.
know ledge of normal B-cell biology and, in particular, changes associated with diff erent disease states. For example, diff erent memory B-cell subsets have now been described in peripheral blood including sub sets that do not express CD27, a marker previously thought to be present on all memory B cells [3,4]. Memory B-cell subpopulations include pre-switch IgD + IgM + CD27 + memory B cells, IgD -IgM + CD27 + memory B cells (IgMonly memory B cells), post-switch IgA + CD27 + and IgG + CD27 + memory B cells and also IgA + CD27and IgG + CD27memory B cells [5]. Th ese memory subpopulations show diff erent frequen cies of somatic mutation and diff erent replication histories that are thought to refl ect their formation on primary or secondary germinal centres or outside germinal centre reactions [5]. A potential new marker for human memory B-cell subpopulations has been identifi ed recently [6]. A proposal has been made that immunophenotyping of peripheral blood B cells should include the markers CD19, CD20, CD24, CD27, CD38 and IgD to be able to distinguish the major subpopulations [7]. More detailed information including separation into further subsets and subtle diff erences in activation status that may be impor tant when looking at disease states may require use of other markers such as diff erent immunoglobulin iso topes, activation markers or chemo kine receptors [6,[8][9][10][11][12][13][14].

Anti-CD20 monoclonal antibodies -rituximab
Anti-CD20 mAbs were developed in the late 1980s and in the 1990s for the treatment of non-Hodgkin's lymphoma of B-cell origin. Rituximab (MabTh era®, Rituxan®; Roche, Basel, Switzerland) was licensed for the treatment of follicular lymphoma in 1997/98 and later for diff use large non-Hodgkin's lymphoma and chronic lymphocytic leukaemia. In 2006 rituximab was licensed for the treatment of rheumatoid arthritis (RA). Rituximab is also used off -license for the treatment of other B-cell malignant diseases, in transplantation and for the treat ment of a variety of other autoimmune diseases, pre domi nantly diseases associated with the presence of auto antibodies. Various other therapeutic anti-CD20 mAbs are either avail able on the market (Ofatumumab -Arzerra®; GlaxoSmithKlein, UK -licensed for the treatment of chronic lymphocytic leukaemia), undergoing clinical trials or under development [15].
Th e CD20 antigen is expressed by the majority of cells in the B-lymphocyte lineage, but not by haematopoietic stem cells, the earliest B-cell precursors (pro-B cells) or terminally diff erentiated plasmablasts and plasma cells ( Figure 1). Th e CD20 molecule is a transmembrane protein thought to function as a calcium channel and to be involved in B-cell activation and proliferation. A recent case report of a patient with CD20 defi ciency suggested a role in T-cell-independent antibody responses [16].
Because haematopoietic stem cells are not directly depleted by anti-CD20 antibodies, one course of treatment with rituximab is followed by B-cell repopulation of the peripheral blood starting usually within 6 to 9 months -but it can take several months or even years for total B-cell numbers in the peripheral blood to recover to pretreatment levels. Repopulation occurs mainly with naïve B cells, with increased frequency and numbers of transitional B cells similar to that seen after bone marrow transplantation [14,17]. Th e time at which B-cell repopulation of the peripheral blood starts is probably determined by the extent of earlier depletion, drug clearance and the capacity of the bone marrow to regenerate. Variability in time to repopulation in primate animal models did not seem to be dose dependent [18]. Factors infl uencing B-cell precursor formation in humans are poorly understood, as are factors that determine to what extent a fully functional B-cell repertoire is regenerated and how long it takes. Whether age or other individual characteristics infl uence repopulation is not known [19,20].
Th e fact that plasma cells are also not directly depleted by anti-CD20 antibodies explains why, in the majority of patients, serum total immunoglobulin levels remain within the normal range after treatment with one course of rituximab. Several studies have shown that serum levels of several autoantibodies decrease after treatment with rituximab (although they do not usually become undetectable) and do so proportionally more than total immunoglobulin levels or anti-microbial antibodies [21][22][23]. Th is observation suggests that these autoantibodies are produced by proportionally more shortlived plasma cells and therefore are more dependent on the formation of new plasma cells, which is interrupted by B-cell depletion [23].
Treatment with rituximab is associated with major depletion of normal B cells in vivo. Depletion in the peripheral blood is frequently higher than 99% but depletion in other tissues has been less well studied, with several studies documenting that depletion in solid tissues with rituximab is frequently not complete and can show considerable variation between individuals. In vitro, rituximab depletes malignant B cells by antibody-dependent cellular cytotoxicity, complement-mediated cyto toxicity and induction of apoptosis. In vivo, rituxi mab is thought to act mainly by inducing antibody-dependent cellular cytotoxicity with activation of complement also contribut ing [24]. One of the consistent fi ndings in several of the animal and earlier human studies is the variability of depletion seen with anti-CD20 mAbs in diff erent individuals even when treated with the same dose [18,25,26]. Interestingly, depletion in the same individual tends to be consistent in diff erent tissues, suggesting that individual characteristics are important.

Resistance to depletion with anti-CD20 monoclonal antibodies
Because depletion is achieved by binding of the mAbs to the cell surface CD20 molecules, the fi nal extent of depletion will necessarily depend on the relationship between total number of B cells and total dose of rituximab administered, on accessibility of the drug and eff ector immune cells to the tissues where B cells are located, on intrinsic or extrinsic factors that may infl uence B-cell survival and on the effi cacy of recruited host immune mechanisms responsible for depletion.
Former small dose-ranging studies in lymphoma and in animal models have shown that B cells in the peripheral blood are readily killed by anti-CD20 antibodies but that higher doses and higher serum levels are needed for depletion in extravascular sites [18,24,25].
Factors infl uencing antigen and eff ector modulation are thought to be important in determining the fi nal extent of depletion achieved (Table 1) [18,27,28]. Antigen modulation refers to antigen endocytosis/modulation after binding to the antibody. Contrary to what was originally thought, this can be seen with the CD20 molecule after binding with certain anti-CD20 antibodies including rituximab [29]. Th is can lead to less recruitment of Fcγ receptors on eff ector immune cells and to decreased serum drug levels. Eff ector modulation refers to genetic and acquired mechanisms that can enhance or diminish eff ector immune cell function and therefore infl uence the extent of depletion. For example, a Fcγ receptor IIIa polymorphism that can infl uence affi nity for IgG has been associated with clinical response in lymphoma [28]. Profound complement depletion as seen during treatment of chronic lymphocytic leukaemia with rituximab can be a limiting factor for further depletion [28].
Intrinsic B-cell factors that may infl uence depletion include high expression of complement regulatory proteins as seen in chronic lymphocytic leukaemia [28]. In cynomolgus monkeys, diff erent sensitivities to rituximab were associated with, but not fully explained by, diff erent levels of expression of CD20 [30]. Binding of rituximab to CD20 leads to translocation of the CD20 molecule to lipid rafts. Altera tions in lipid raft composition and treatment with statins have been associated with less good responses to rituximab [28]. To what extent external B-cell survival factors, in particular the cytokine B-cell activating factor (BAFF), infl uence deple tion is not known, although it has been suggested that local high levels of BAFF may contribute to resis tance to depletion by rituximab [31].
In animal models, certain subpopulations have been shown to be more resistant to depletion with anti-CD20 antibodies but this varies with the mice strain used and whether they were studies using human CD20 transgenic mice treated with anti-human CD20 mAbs or nontransgenic mice treated with anti-mouse CD20 mAbs [32,33]. Populations that were found to be more resistant to depletion were peritoneal B1-type B cells, germinal centre B cells and marginal zone B cells [32,33]. Insufficient depletion of peritoneal B1 cells is thought to be due to the lack of eff ector cells in the peritoneal space [33]. Diff erential sensitivity of germinal centre and marginal zone B cells to anti-CD20 antibodies has also been described in cyno mo logous monkeys, with diff er ences appearing more prominent in the lymph nodes than in the spleen [30]. Th e relative resistance of some populations is thought to be related to B-cell and microenvironment diff erences responsible for antigen or eff ector modulation or related to direct resistance of the B cells involved. In an autoimmune mouse model of lupus, B cells were more resistant to depletion when com pared with nonauto immune mice and more frequent administration of larger doses increased effi cacy of depletion [34]. Less good depletion has also been associated with acquired defects in antibody-dependent cellular cytotoxicity in the same autoimmune mouse model of lupus [35].
To what extent the diff erential susceptibility of various B-cell subsets demonstrated in some of the animal models refl ects what happens in humans in vivo is not known. Diff erent B-cell malignancies deriving from B cells at diff erent stages of diff erentiation and diff erent tumour locations are also associated with diff erential responses to treatment with anti-CD20 mAbs but susceptibility of the correspondent normal human B-cell subpopulations is expected to be substantially diff erent. Whether there are any diff erences in susceptibility to depletion of autoreactive human B-cell clones when compared with nonautoreactive ones, as suggested by mouse models [34], and whether there are any signifi cant diff erences in susceptibility to depletion of disease-associated B-cell clones between diff erent autoimmune diseases are also not known.
In addition, administration of chimaeric anti-CD20 mAbs such as rituximab can be associated with formation of human anti-chimaeric antibodies that can infl uence drug action and clearance. Although most large studies show no association between the presence of human anti-chimaeric antibodies and clinical response or depletion, this association has been described, for example, in small studies in systemic lupus erythematosus patients [36,37].
With evidence showing that not all B cells that bind rituximab are depleted there is an interest in knowing what exactly happens to these cells in vivo during the period of depletion. Are they eventually depleted later on, particularly if they recirculate in peripheral blood? Are they functionally impaired? Are they able to expand in an environment with less competition and raised BAFF levels? Kamburova and colleagues tried to address some of these issues by studying the in vitro eff ects of incuba tion with rituximab on proliferation, activation and diff erentiation of nondepleted human normal peripheral blood B cells [38]. Th ey reported that incubation with rituxi mab (for 30 minutes at 5 μl/ml) inhibited the pro liferation of stimulated CD27naïve B cells but not of CD27 + memory B cells and this was associated with a relative increase of B cells with an activated naïve pheno type. B cells stimulated in the presence of rituximab induced stronger T-cell proliferation and the T-cell popu lation showed a more Th 2-like phenotype. Th ese results suggest that B cells which are exposed to rituximab but are not depleted may have altered function and that naïve and memory B cell populations may be diff erentially aff ected. Whether any of these phenomena occur in vivo and what their implications would be are unclear. Interestingly, and similar to what happens after bone marrow transplantation, the residual B cells are not able to expand and repopulate the peripheral blood, even in the presence of abundant BAFF.

B-cell depletion in peripheral blood
Administration of rituximab is usually associated with a rapid and profound depletion of circulating B cells in the peripheral blood [18]. Major depletion eff ector cells are probably macrophages from the reticulo-endothelial system [24]. Studies in autoimmune diseases -in particular, RA and systemic lupus erythematosus -have documented variable degrees and durations of B-cell depletion in peripheral blood in diff erent individuals following treatment with rituximab with standard doses [17,36,37,[39][40][41]. Incomplete B-cell depletion in the peripheral blood, as defi ned by B-cell counts >5 cells/μl after treatment with rituximab, has been well documented in cases of patients with autoimmune diseases, more frequently in systemic lupus erythematosus than in RA [17,36,37]. Persistent presence of circulating B cells has also been documented with high-sensitivity fl ow cytometry and has been associated with no or less good response to treatment [39,40]. Insuffi cient depletion can be seen on retreatment with documented very rapid clearance of rituximab in association with a marked human antichimaeric antibody response [42]. Other mechanisms under lying incomplete depletion in the peripheral blood have not been well studied but are probably a consequence of more rapid clearance of the drug and/or antigen and eff ector modulation phenomena [17,24,36,37].
Th e very small numbers of circulating B cells that can be detected during periods of depletion usually show a phenotype of plasmablasts but cells with memory or even naïve B cells have also been reported [17,40,41,43]. Th e CD20 antigen cannot usually be detected in these memory B cells, suggesting that it is masked by binding to rituximab because the drug can be detected in the circulation for several months [26]. Mei and colleagues described that, similarly to their controls, the majority of circulating plasmablasts/plasma cells detected during depletion were positive for IgA and a reasonable proportion expressed markers suggesting they had been formed in mucosal tissue and were circulating back to mucosal areas [44]. Th ese results suggest that depletion in mucosal-associated lymphoid tissue may be particularly less pronounced.
Repopulation of the peripheral blood after treatment with a standard dose of rituximab usually starts 6 to 9 months after treatment with predominantly transitional and naïve B cells as previously mentioned. Frequently, repopulation with larger numbers of memory B cells and/ or plasmablasts has been associated with earlier relapse [17,40,45]. At repopulation, the decrease from baseline in the frequency of pre-switch memory B cells (CD27 + IgD + ) was larger than the decrease in the switched memory B-cell population (CD27 + IgD -) [46]. However, to what extent circulating memory B cells at repopulation are old memory B cells that have not been depleted by rituximab or recently diff erentiated memory B cells is not known. We therefore do not know whether relative frequencies of the diff erent B-cell subpopulations at repopulation can tell us anything about the subpopulations of cells that may have resisted depletion.
In RA, nonresponse has been associated with higher numbers of plasmablasts before treatment and early relapse has been associated with higher numbers of CD27 + memory B cells before treatment [39,45]. Again, to what extent this may indicate less susceptibility and insuffi cient depletion of memory B-cell subsets in association with no response or with a shorter response is not known.

B-cell depletion in bone marrow and secondary lymphoid tissues
Unfortunately, there are limited data on the degree of depletion of normal B cells in secondary lymphoid organs and other solid tissues in human individuals treated with rituximab, and hardly any data on diff erential susceptibility to depletion of diff erent subpopulations in diff erent tissues except for the expected resistance of CD20plasmablasts and plasma cells to depletion [47]. Animal studies in primates showed that increasingly higher doses are needed to deplete bone marrow, spleen and lymph nodes in this order [18,48,49]. Th ese studies also showed that B-cell depletion in solid tissues was frequently signifi cant, but not complete, and that it varied from site to site and from individual to individual even when the same doses were used. Interestingly, consistency regard ing the degree of depletion achieved in diff erent lymph nodes in the same individual was described [18,20,48,49]. As previously mentioned, mice studies suggested that B cells resident in tissues other than peripheral blood may be partly resistant to depletion by anti-CD20 antibodies either because of local defective eff ector mechanisms or because the B cells have a particular phenotype that renders them resistant to depletion in association with their specifi c state of maturation, activation or diff erentiation.
In bone marrow samples of RA patients treated with rituximab a relatively high number of B-cell precursors subpopulations can be seen [50][51][52]. Th is has been documented at 1 month or 3 to 4 months after treatment, at a time when peripheral blood repopulation had not yet started [50,51]. Persistence of CD20plasma cells has been observed as expected [50,51]. In the two studies where phenotyping was more detailed, the cells found were mainly B-cell precursors and recirculating memory B cells [50,52]. Once again, variability between individuals was observed [50,52].
Th e presence of cells of B-cell lineage that presumably should be expressing CD20 has therefore been well documented and rituximab is probably still present and binds to the CD20 molecule, preventing its detection in fl ow cytometry as discussed above [50,51]. Alternatively, antigen endocytosis/modu lation could occur. Whether the developing B cells are eventually depleted by anti-CD20 recruited mecha nisms or whether their full maturation is prevented by binding of rituximab to CD20 is not known.
In a study of autopsy samples of lymph node and spleen of patients with lymphoma treated with rituximab monotherapy or with rituximab and chemotherapy, a substantial reduction of B-cell populations was documented -with only three out of eight patients showing any reactivity for markers of cells of B-cell lineage in the lymph nodes and only one out of eight in the spleen by immunohistochemistry [53]. Similarly, a study in patients with idiopathic thrombocytopenic purpura showed major and prolonged depletion of B cells in the spleen of 10 patients treated with rituximab [54]. Th e number of residual B cells correlated with time from rituximab treatment but was <5% of spleen lymphocytes in eight out of nine patients studied up to 10 months after rituximab treatment. Plasma cells were detected at increased frequencies when compared with patients with idiopathic thrombocyto penic purpura not treated with rituximab. In a patient with idiopathic thrombocytopenic purpura, analysis of spleen and bone marrow samples by fl ow cytometry revealed complete depletion of B cells 3 months after treatment with rituximab [55]. In another patient with idiopathic thrombocytopenic purpura, B cells in the spleen 3 months after rituximab treatment were only present in very low numbers (around 0.1%) [56]. Interest ingly, in this later study persistence of memory B cells against vaccinia virus in the spleen of patients previously treated with rituximab was documented [56]. In kidney transplant patients that had a splenectomy 3 to 12 days after treatment with rituximab, naïve B cells were reduced but not memory B cells or plasma cells [57].
Vaccination studies in patients treated with rituximab can provide indirect data on B-cell subpopulations that may be resistant to depletion with anti-CD20 mAbs. However, published data are diffi cult to interpret because of the small number of patients, eff ects of concomitant therapy and the background disease itself on the humoral response to vaccines and, in particular, because studies included patients at various stages of B-cell depletion or repopulation at the time of vaccination. Most studies have looked at responses to infl uenza vaccines and showed absent or decreased humoral responses to vaccination in patients previously treated with rituximab when compared with normal controls or patients not treated with rituximab [58][59][60][61][62][63][64]. Some studies described a positive relationship between the antibody responses to vaccination and number of circulating B cells at the time of vaccination [64] or the time from last rituximab treatment [60,62]. Interestingly, when circulating infl uenzaspecifi c B cells were studied 6 days after vaccination, specifi c IgM-B cells were decreased in patients treated with rituximab 6 months previously when compared with controls but IgA B cells and IgG B cells were similar [61]. In a study in lymphoma patients, responses to recall antigens in the infl uenza vaccine were also seen but not to the new antigen [65]. Th ese studies suggest that memory B cells are more resistant to depletion than naïve B cells and can survive treatment with rituximab and be recruited in a secondary immune response.

B-cell depletion in other solid tissues
In patients with RA, several studies have documented signifi cant but variable depletion of B cells in samples of synovial tissue of involved joints and persistence of CD20plasma cells [66][67][68]. Variability in depletion between individuals was not explained by diff erences in rituximab serum levels [69]. In a study in patients with Sjogren's syndrome, repeated salivary gland biopsies 3 months after treatment with rituximab showed in com plete depletion of B cells [70]. A previous study had shown complete depletion at 4 months [71]. In a study of renal explanted grafts in two patients treated with one dose (4 months earlier) or two doses (10 months earlier) of rituximab, despite depletion of peripheral blood, tertiary lymphoid structures containing B cells were seen [72].

Conclusion
In summary, although there are several studies looking at the degree and duration of B-cell depletion induced by rituximab in the peripheral blood, there is very little information on the exact degree of depletion in solid tissues -and, in particular, few defi nite data on whether diff erent subtypes of CD20-expressing B cells are more or less susceptible to depletion by anti-CD20 antibodies. Th e data available suggest that there is variability between individuals on the extent and duration of depletion induced and that this may have clinical correlations with response and duration of response in autoimmune diseases. Understanding what underlies this variabilityand, in particular, whether drug clearance and antigen and eff ector modulation phenomena are involved -has the potential to lead to more eff ective B-cell depleting strategies and to increasing our understanding of the role that diff erent B-cell subtypes play in the pathogenesis of the diff erent autoimmune diseases.

Competing interests
The author has received consultancy fees and funding to attend international medical meetings from Roche Pharmaceuticals and consultancy fees and research funding from GlaxoSmithKlein.

Declarations
This article has been published as part of Arthritis Research & Therapy Volume 15 Supplement 1, 2013: B cells in autoimmune diseases: Part 2. The supplement was proposed by the journal and content was developed in consultation with the Editors-in-Chief. Articles have been independently prepared by the authors and have undergone the journal's standard peer review process. Publication of the supplement was supported by Medimmune.