Today we have this review on B cells, I have abridged this to focus it on human biology and MS. It will be heavy reading but it gives an alterate view to the ramblings of MD
Lee, D.S.W., Rojas, O.L. & Gommerman, J.L. B cell depletion therapies in autoimmune disease: advances and mechanistic insights. Nat Rev Drug Discov (2020). https://doi.org/10.1038/s41573-020-00092-2
B cells, along with T cells, form the core of the adaptive arm of the immune system. They are generated continually and throughout the life of the organism in the bone marrow, a primary lymphoid tissue, from haematopoietic stem cell progenitors that progress through sequential developmental steps. Each developing B cell expresses a unique B cell receptor (BCR), which is composed of two identical heavy chain proteins and two identical light chain proteins. In both humans and rodents, once a developing B cell expresses a correctly assembled BCR on its cell surface, and that BCR has been confirmed to not be autoreactive, the IgM+ immature B cell exits the bone marrow, enters the blood and migrates to the spleen.
Within the spleen, the architecture of which is somewhat different in mice versus humans, immature B cells undergo additional developmental steps ( The immature B cell is then considered a naive B cell (NBC), which has the capacity to be primed against proteins derived from foreign pathogens (that is, antigens). With a half-life of approximately 6 weeks, the NBCs circulate in the blood and enter lymph node organs through high endothelial venules. Unless they are activated by an antigen, B cells will spend approximately 24 hours in follicles, which are structures found in the lymph node cortex. They are drawn to these structures because NBCs express CXC-chemokine receptor 5 (CXCR5), which binds to its ligand, CXC-chemokine ligand 13 (CXCL13); CXCL13 is expressed in the follicle. Following this brief residence in the follicle, NBCs follow the gradient of a different molecule, sphingosine 1-phosphate, to the medullary cords and out through the efferent lymphatics. After transitioning through the thoracic duct, NBCs re-enter the blood and repeat this journey.
Because of the remarkable diversity of the BCR repertoire, B cells can respond to a seemingly infinite number of antigens. Immune responses to antigens occur within secondary lymphoid organs (lymph nodes, tonsils and spleen): the spleen supports immune responses to blood-borne antigens, whereas lymph nodes sample tissue-derived antigens. Binding of an antigen to the BCR results in intracellular events such as calcium mobilization and protein phosphorylation.
Depending on the molecular structure of the antigen, NBCs may or may not need additional help from T cells to fully respond to the antigen. To induce this help, B cells that have been activated by particular protein antigens form cognate interactions with T cells that are specific for a linear peptide fragment of the same antigen. These cognate B cell–T cell interactions occur in the germinal centres (GCs) in secondary lymphoid organs. Within the GCs, B cells with a range of affinities for the antigen compete for help from these cognate T cells, which express key costimulatory proteins, such as CD40 ligand (CD40L), and also make cytokines that direct the B cell response.
To increase their affinity for antigens, B cells also undergo secondary BCR diversification. The BCR-encoding genes accumulate mutations in their antigen-binding regions through the activity of activation-induced cytidine deaminase (AID). This process results in multiple variants of the original germ line BCR, and the B cells that emerge from this mutagenesis programme with the highest affinity for the antigen will ‘win’ the GC reaction competition and become dominant clones in the immune response. Such B cells can also undergo class switch recombination to generate B cells that produce other classes of antibodies. A proportion of these B cells (switched and unswitched) become memory B cells (MBCs) or plasma cells (PCs), which secrete copious amounts of antibodies. MBCs and long-lived PCs (LLPCs) provide protection against reinfection by the same pathogen.
Activation of B cells
The GC is an important microenvironment that supports the interaction of antigen-specific B and T cells. GCs are readily observed in histological sections of secondary lymphoid organs (tonsils, spleen and lymph nodes) in mice and humans. Somatic hypermutation mediated by AID may increase the affinity of the BCR, but may also reduce its affinity, or even generate an affinity for self-antigen. Due to its anatomical organization , the GC environment imposes a framework of competition among GC-resident B cells such that those B cells with the highest affinity for an antigen persist and become MBCs and PCs. Advances in our understanding of this GC reaction may inform the design of specific therapies that interfere with this specialized microenvironment. For example, in the past 10 years, our understanding of how T follicular helper cells (TFH cells) and regulatory TFH cells engage with B cells has exploded. In mouse models, entanglement of GC-resident TFH cells with GC B cells promotes sustained interactions that select for B cells with the highest affinity for an antigen through a process of feedforward loops involving CD40L, inducible T cell costimulator (ICOS) and BAFF (also known as TNFSF13B). This entanglement is reminiscent of the types of CD40–CD40L-driven B cell–T cell interactions that occur in human tonsils
The GC is not the only environment in which B cells can participate in the immune response. Extrafollicular B cell responses provide an early source of antibodies during infection. Moreover, the extrafollicular response can foster somatic hypermutation. Extrafollicular class switching, driven by the cytokines BAFF and APRIL (also known as TNFSF13), has been documented in human tonsils and gut. Extrafollicular B cell responses have also been observed in the synovium and salivary glands of patients with RA or Sjögren syndrome. The extrafollicular pathway of B cell activation is also an early and potent source of PCs because BLIMP1 (also known as PRDM1) is rapidly upregulated in extrafollicular B cells. When expressed in B cells, BLIMP1 represses the expression of genes involved in BCR signalling and GC B cell function, so cells adopt a PC phenotype.
Extrafollicular responses have implications for autoimmune diseases. Although extrafollicular responses clearly produce class-switched B cells, in contrast, and in keeping with older findings in the literature, GCs are dominated by non-class-switched IgM+ B cells. Since class-switched antibodies are critical in the pathogenesis of diseases, this observation calls into question the utility of targeting the GC itself as a therapeutic strategy.
In humans, it is difficult to study the extrafollicular response because tissue is not readily available. However, some inferences may be made from clinical observations. B cells associated with the tonsillar epithelium can undergo class switch to IgG and IgA and undergo somatic hypermutation in response to locally produced cytokines, again suggesting an extrafollicular response. For the most part, however, extrafollicular responses in humans are inferred because some circulating MBCs are CXCR5− and have relatively low levels of somatic hypermutation of the BCR, suggesting that they matured outside the GC1. From these findings taken together, although we traditionally think of GCs as a hotbed for autoimmune B cell genesis, targeting extrafollicular responses may also have significant utility in treating B cell-mediated autoimmune diseases.
T-bet+ B cells are commonly referred to as double-negative (DN) B cells, as they express neither CD27 (also known as TNFRSF7) nor IgD. Although DN B cells are CD27−, and CD27 usually marks MBCs, DN B cells are considered to be antigen-experienced.
B cells that express high levels of T-bet (T-bethi B cells) can be directly generated from NBC precursors in response to IFNγ (which is locally produced by T cells), and consequently become ‘activated naive’. In addition, some T-bethi B cells have the capacity to quickly differentiate into PCs in an IL-21-dependent manner. These cells also lack CXCR5, and thus extrafollicular responses may have contributed to their generation.
Antibody-independent B cell functions
People who positively respond to BCDT do not necessarily show a corresponding decrease in autoantibody levels. In such cases, BCDT must therefore alter autoimmune diseases in a manner that is independent of antibody production.
One antibody-independent function of B cells is to initiate and/or sustain tertiary lymphoid tissues (TLTs) in inflamed tissues. TLTs have been observed in a number of autoimmune disease settings and in some cases can be driven by the membrane-bound expression of lymphotoxin-αβ (LTαβ) by B cells, although other cell types and other cytokines (IL-17 and IL-22) can also provide this signal.
Beyond their function in TLT formation, B cells produce numerous soluble cytokines. PCs are also important cytokine-producing B cells. Alternative B cell functions beyond antibody production, including cytokine production, provide a potential explanation for the surprising efficacy of BCDT in autoimmune disease.
Regulatory B cells
B cells also have the capacity to quiet inflammation, and IL-10, which is produced by subsets of myeloid cells, B cells and T cells, clearly mediates much (but likely not all) of this anti-inflammatory effect. IL-10-producing regulatory B cells have also been described in humans. Depending on the sequence and/or nature of the activation signal, human B cells can produce IL-10 or, alternatively, secrete proinflammatory cytokines such as soluble LTα3, TNF and IL-6. TLR signalling on B cells may also elicit different cytokine responses, for example producing either the anti-inflammatory IL-10 or the proinflammatory IL-13, depending on the type of TLR and the maturation stage of the B cell (NBC vs MBC). Importantly, the ability to produce IL-10 by regulatory B cells in response to CD40 stimulation can be lost in regulatory B cells.
Anti-inflammatory cytokines can also be produced by PCs. Importantly, in humans, IL10-producing PCs can potentially be localized in the diseased tissue itself.
Our main BCDT tool is anti-CD20 antibodies — this therapy has been extensively tested in numerous autoimmune disease settings.However anti-CD19 and anti-BAFF antibodies have also been tested in autoimmune disease indications
B cells in MS
MS is a chronic demyelinating disease of the CNS with no cure or known cause. Genetic and environmental factors play roles. In MS, a complex interplay of neurodegenerative and immunological processes damages the myelin sheaths that surround neurons. There are three clinical variations of MS: relapsing–remitting MS (RRMS), primary progressive MS (PPMS) and secondary progressive MS (SPMS). RRMS is typified by episodes of clinical relapses — during which immune cells infiltrate the CNS and form focal lesions that can be detected by MRI — followed by clinical improvement and remyelination of lesion areas. Progressive forms of the disease (PPMS and SPMS) are considered to be more neurodegenerative in nature and have lower levels of inflammation and immune activation. Without treatment, most patients with RRMS convert to SPMS, and reductions in conversion rates are often measured in clinical trials as a parameter for success. Not surprisingly, the efficacy of therapeutics differs between MS subtypes.
Historically, MS has been thought of as a T cell-mediated disease. This was fuelled by genetic observations (the MS risk is associated with the HLA-DRB1*15:01 allele), histopathology studies (T cells are much more abundant than B cells in MS lesions) and the key observation that adoptive transfer of activated myelin-specific T cells into naive mice is sufficient to cause EAE. However, in other species, such as the marmoset, B cells are necessary for disease. Ironically, the importance of B cells in MS has been appreciated for many years. One of the earliest diagnostics for MS was the presence of IgG oligoclonal bands in the CSF, implying the presence of intrathecal antibody-producing cells. Evidence of B cell accumulation in CNS lesions and ectopic B cell follicles in the meninges of patients with SPMS (and, to a lesser extent, those with PPMS) further implicated B cells in the aetiopathology of the disease. Deposition of complexes containing antibodies and complement components in MS plaques also suggests that B cells are important in MS pathogenesis, although in a separate study these complexes were not found to be a specific feature of lesions, nor a hallmark of MS in particular. To date, no specific autoantibody has been demonstrated to be pathogenic and/or pathognomonic.
Studies have begun to characterize B cells in the blood of individuals with MS, and have examined the localization of B cells in the inflamed CNS. Specifically, NBCs in the peripheral blood of patients with MS have altered cytokine profiles: typically a decreased ability to produce IL-10, and increased production of IL-6, GM-CSF and LTα3 . Interestingly, these aberrant cytokine profiles are normalized in B cells in the blood of patients with MS who have reconstituted their B cell compartment after BCDT. Therefore, these studies suggest that the proinflammatory cytokine profiles of B cells in the blood may alter the disease in individuals with MS and can be modulated by BCDT
In terms of CNS-resident B cells, patients with active disease can accumulate B cells in the CSF, including antigen-experienced B cell subsets such as MBCs and PCs. Sequencing of the immunglobulin heavy chain variable region repertoire to ascertain clonal relationships between peripheral B cells and CNS-derived B cells revealed clonal relationships between B cells in the CNS and B cells in the cervical lymph nodes. Moreover, by building lineage trees based on the relative amount of affinity maturation compared with a germ line sequence, it was determined that ancestral B cell clones are enriched in the cervical lymph nodes compared with the CNS. This elegant study suggested that, although antigen-experienced B cells are clearly present in the CNS, most B cell maturation and activation occurs in the periphery
B cell infiltration is not equally distributed throughout the CNS. CD20+ B cells tend to accumulate in perivascular spaces and within the subarachnoid space of the leptomeninges rather than in the tissue parenchyma. B cell-enriched TLTs within the subarachnoid space are particularly evident in patients with SPMS, and these B cell-rich and PC-rich structures are typically associated with grey matter pathology in layer I of the underlying cortex (subpial lesions). However, it is difficult to know whether TLT structures are impacted by BCDT as their small size makes them extremely difficult to capture by MRI.
From these findings taken together, B cells show significant alterations in accumulation, localization and activation in patients with RRMS versus healthy controls, and thus BCDT was posited as a treatment option.
Most MS treatments focus on depleting or altering the trafficking of peripheral immune cells. This is very effective for treating RRMS, in which waves of proinflammatory leukocytes from the periphery breach the blood–brain barrier and subsequently promote focal demyelinating lesions. Traditionally, clinical trials in patients with MS use clinical disability and/or radiological evidence of lesions (by MRI) as end points to measure success. Newer trials use a combination of clinical relapse rate, MRI-based evidence of lesions and progression of disability (which together form ‘no evidence of disease activity’, (NEDA-3)) as an end point that is measured 1, 2 or even 5 years after treatment initiation. Disease-modifying therapies, which abrogate specific immune processes involved in RRMS pathogenesis, are very good treatment options for patients with MS. Three BCDTs that target CD20 have shown efficacy in MS.Due to the success of anti-CD20 therapies, other approaches for targeting B cells have been tested in the clinic.
Overall, some BCDTs are excellent treatment options for patients with MS (particularly RRMS, in which active inflammation is a prominent feature). However, the failure of another BCDT, atacicept, highlights our incomplete understanding of the nuances of B cells in MS. Data from these BCDT trials suggest that B cell subsets are heterogeneous in their capacity to regulate the immune response in MS. Moreover, the efficacy of anti-CD20 in MS is not related to a reduction in CSF antibody levels.
Potential mechanism of action of BCDT
The successes of anti-CD20 treatment in autoimmune diseases are some of the most encouraging examples of positive clinical outcomes for disease-modifying therapies in autoimmune diseases that have been traditionally difficult to treat. However, not all B cell-driven diseases respond to BCDT (SLE, for example, does not). The reason for such disparity is still unclear.
On the basis of the success of BCDT in some indications, second-generation anti-CD20 mAbs have rapidly emerged. Different anti-CD20 mAbs evoke distinct cytotoxic mechanisms: complement-dependent cellular cytotoxicity (CDC), FcγR-mediated depletion through cellular effector mechanisms including ADCC and antibody-dependent cellular phagocytosis, and directly inducing cell death. Anti-CD20 mAbs can be divided into two types (type I and type II) on the basis of their ability to redistribute CD20 into plasma membrane lipid rafts, and their potency in assays measuring CDC, homotypic adhesion and programmed cell death induction. The first generation of genetically engineered chimeric anti-CD20 mAbs, such as rituximab and ocrelizumab, are type I mAbs.
The main mechanism through which type I anti-CD20 mAbs deplete B cells is by redistribution of CD20 into lipid rafts: mAb-driven CD20 clustering results in CDC, although the process of cell death is independent of raft formation. However, internalization of type I anti-CD20 mAbs from the surface via FcγRIIb results in resistance to therapy and decreased clinical efficacy. Type II mAbs, binds to a different epitope, and does not induce efficient clustering of CD20 or antibody internalization, thus increasing efficacy and decreasing possible treatment resistance
Tissue and B cell lineage specificity
BCDT has a range of outcomes depending on the disease. One relatively straightforward reason for this variability is that pathogenic B cells in some scenarios are difficult to access (for example, because B cells localize into TLTs in the leptomeninges). The depth of B cell depletion may therefore depend on the target tissue. In MS, B cell depletion is effective in the blood and CSF (selective reading here) , but variable in the CNS. Inflammation in MS can be CNS compartmentalized into TLTs (for example, in the leptomeninges), so B cells may persist in these locations, particularly because survival factors such as BAFF are locally produced by astrocytes. Furthermore, a substantial reservoir of T-bet+ MBCs that express CD11c resides in the spleen and does not circulate. T-bet+ MBCs can be precursors to antibody-producing cells that could play a pathogenic role in autoimmune diseases, It is unclear whether anti-CD20 treatment effectively depletes these tissue-resident cells.
BCDT may also have variable outcomes in different autoimmune diseases if the pathogenic B cell lineage does not express CD20 and turnover is slow. For example, CD20 is largely absent on subsets of LLPCs in healthy human bone marrow and gut. For this reason, anti-CD20 therapy does not substantially deplete LLPCs. When anti-CD20 therapy has robust efficacy in diseases that are known to be mediated by autoantibodies, this efficacy may be due, at least in part, to a loss of short lived PBs or PCs that are not replaced because precursor B cells are also depleted.
On the other end of the efficiency spectrum, anti-CD20 therapy has low efficacy in LupusSLE) . We posit that this may be because pathogenic LLPCs that are CD20− and thus resistant to rituximab have a critical role in SLE. Not only does this potentially explain why anti-CD20 therapy failed in SLE, in which LLPCs are an important source of anti-DNA antibodies, it may also explain why, after BCDT, autoantibodies persist and oligoclonal bands remain in MS and protective antibodies elicited by previous vaccinations can still be found.
Impact on cytokine-producing cells
The efficacy of BCDT in the absence of altered autoantibody levels suggests that antibody-independent B cell functions, such as cytokine production, could be important. Cytokines produced by B cells may alter the function of other immune cells such as T cells and myeloid cells. For example, in MS, B cells produce very high quantities of cytokines such as TNF, IL-6 and GM-CSF. IL-6 produced by B cells has been clearly linked with the generation of a T helper 17 cell inflammatory immune response in EAE, and a similar observation has been made in humans: circulating B cells from patients with MS overproduce IL-6 in vitro, and anti-CD20 treatment normalizes IL-6 production. Indeed, in patients with MS, B cells that were reconstituted following BCDT had totally different cytokine profiles compared with B cells from untreated patients. Reconstituted B cells were characterized by limited proinflammatory cytokine production and higher production of anti-inflammatory IL-10. Thus, BCDT may produce a clean slate for peripheral B cells, providing a much-needed cytokine reset for the patient.
Antigen presentation and MBCs
In addition to producing antibodies and cytokines, B cells can internalize and process antigens, and then present them to CD8+ and CD4+ T cells via MHC class I and MHC class II, respectively. Although B cells are considered to be less efficient antigen-presenting cells than dendritic cells, MBCs can play an important role in antigen presentation, particularly if B cells present the same antigen as the responding T cell.
B cells also mediate the proliferation of brain-homing autoreactive myelin-specific CD4+ T cells independently of any external T cell stimuli, and this function is lost after rituximab treatment. These myelin-specific CD4+ T cells increase in number predominantly in patients with MS who express the HLA-DR15 haplotype and decrease in number after anti-CD20 treatment, providing further evidence that interrupting B cell–T cell interactions by BCDT may be relevant to MS aetiopathology. In summary, it is possible that BCDT is efficacious in part because it abrogates proinflammatory antigen presentation by B cells.
Other hypotheses on mechanism
The effects on non-B cells may explain the success of anti-CD20 therapy in MS, anti-CD20 therapy may be effective partially because it removes CD20+ T cells.In addition to this potential off-target-cell effect, removing B lymphocytes, an entire branch of the immune system, will change the availability of growth and survival factors that were previously consumed by these cells.Thus, one potential mechanism for the efficacy of anti-CD20 therapy in MS is that BAFF levels are increased after treatment and consequently promote the survival of immunoregulatory PCs, particularly gut-derived IgA+ PCs.
New therapeutic directions
On the basis of what we have learned about B cells in the past 10 years, we might now be able to design therapies that provide superior specificity and sensitivity to thwart inflammatory B cells and foster the function and accumulation of immunoregulatory B cells
The field of BCDT began with a rather crude instrument (antibodies that deplete all CD20+ B cells). The clinical experiments performed with anti-CD20 therapy, however, have been surprising and revealing, spurring on research into how B cells impact disease. Our rudimentary idea that PCs and antibodies are ‘bad’ has matured considerably into a more nuanced view of B cells in health and disease. Moving forward, it will be important to not merely ascribe increasingly specific subset designations on the basis of emerging RNA sequencing data in the absence of a holistic view of the environment in which B cells differentiate and exist. For example, the microbiome is a key environmental factor that influences B cell phenotype. Taking cues from our co-evolution with pathogens, new perspectives on the roles of B cells in autoimmune disease will undoubtedly emerge. These perspectives, combined with advances in antibody technologies, should increase the efficacy and specificity of BCDTs in autoimmune diseases.
However after reading this I still shout “its behind you” as there this is a ramble through every thing and means you can research this forever more. Sometimes you have to focus and you may get it wrong if you focus in the wrong place but without focus you spend too much time saying “Wouldn’t it be fun to research this bit or that bit” as MS continues to ravage peoples brains.If I had added all the mousey bits it would be even more confusing. See the message falls on deaf ears:-(