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2 Activation of B-Cells Through CD4 T-Cell Interactions

2 Activation of B-Cells Through CD4 T-Cell Interactions

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U.M. Selvaraj et al.

proliferate for several days [11]. This is effectively the first phase of primary

humoral immune response, and has previously been considered to only occur in the

CNS during rare pathology [12]. Recent work by Louveau and colleagues [13],

however, identified lymphatic vessels within both murine and human brains, which

challenges the dogma that the CNS is predominantly immune-privileged and lacks

the lymphatic system found in the periphery. Thus, future studies will need to determine if germinal centers develop, either within the parenchyma or adjacent cortical

lymphatics, under pathological states that involve antigen presentation and immune

activation, as occurs following stroke [14].


B-Cell Differentiation and Relevant Subsets

Activated B-cells in germinal centers, which are continually refined during the

entire germinal center response, differentiate into antibody-secreting plasma cells

or memory B-cells [15]. B-cells differentiate into plasmablasts secreting highaffinity antibody at rapid rates, but lose MHC II expression and thus the ability to

activate CD4 T-cells [16, 17]. Plasma and memory cells exhibit a wide range of

lifespan, with some longer-lived plasmablasts accounting for a persistent, longlasting source of high-affinity antibody [18]. As reviewed below, these long-lasting

B-cell populations may have prolonged effects in clinical stroke recovery [19, 20].


Innate-Like B-Cells

Apart from the B-cells described above which stem from the bone marrow (i.e., B-2

cells), there are heterogeneous populations of unconventional B-cells (e.g., B-1

cells, marginal zone (MZ) B-cells) which play major roles in innate sensing and

rapid immune response [21, 22]. B-1 cells produce more mature naive cells in

peripheral lymphoid tissues, in contrast to conventional B-2 cells, which only divide

upon antigen exposure. B-1 cells secrete antibodies which are polyreactive, lowaffinity antibodies at steady state with broad reactivity [22]. When they get activated, they can rapidly acquire immune regulatory activities through the secretion

of natural immunoglobulin (Ig)M and interleukin (IL)-10. Thus, these innate-like

B-cells constitute an important source of IL-10-producing regulatory B-cells

(Bregs), which have been shown to play critical roles in autoimmunity, inflammation, and infection following stroke, as described below.


B-Regulatory Cells and IL-10

Multiple murine disease models for both autoimmune [23–25] and inflammatory

diseases [26] confirm the potential for B-cells to downregulate pro-inflammatory

immune responses. In particular, Bregs exert immunosuppressive activity through

B-Cells in Stroke and Preconditioning-Induced Protection Against Stroke


both direct and indirect mechanisms, including production of cytokines (e.g., IL-10,

IL-35, and transforming growth factor β (TGF-β) [27, 28], and expression of inhibitory molecules to downregulate the expansion of pathogenic lymphocytes in a cell

contact-dependent manner [28–30]. Regulatory T-cells are classically identified by

the expression of Foxp3 marker [31, 32]. However, there is no known master transcription factor for Bregs, which are instead defined based on their functional specificities [31, 32]. Similar to regulatory T-cells, Bregs need to be activated before

exerting their functions, with different inflammatory environments inducing different types of IL-10-producing regulatory B-cells [31, 33, 34]. Breg populations are

highly heterogeneous, though it has been proposed to classify Bregs as “innate

type” and “adaptive type” [29].

“Innate” Bregs are derived from innate-like B-cells, and are characterized by the

capacity to rapidly produce high amounts of IL-10 and IgM antibodies upon activation [35, 36]. These “innate” Bregs are activated following toll-like receptor (TLR)

or microbial stimulation. Some TLR agonists, like lipopolysaccharide (LPS) from

gram-negative bacteria, induce IL-10-producing Bregs and reduce disease severity

in a murine model of autoimmune disease [37]. Interestingly, systemic LPS preconditioning can induce tolerance to stroke injury [38, 39], though a link between LPSinduced “innate” Bregs and neuroprotection has yet to be established. On the other

hand, “adaptive” Bregs demonstrate antigen specificity and are generated via BCR

and CD40 responses, and possibly TLR signaling. In the murine autoimmune disease experimental autoimmune encephalomyelitis (EAE) model (described below),

B-cells re-stimulated with auto-antigen produced IL-10 through CD40, and loss of

Breg induction exacerbated disease [23].


Understanding the Contribution of B-Cells to Other

Disease States

B-cells are mainly considered to function through antibody production, with

effects limited to downstream events including antibodies coating antigen, activation of the complement pathway, and antibody-dependent cellular cytotoxicity [40].

Though antibodies do play a major role in defense against foreign pathogens, antibodies directed to self-antigen (i.e., autoreactive) can directly induce inflammatory

conditions resulting in tissue damage. For example, B-cells are present in airway

mucosa, and IgE antibodies are involved in allergic respiratory diseases such as

asthma [41, 42]. But apart from antibody production, B-cells can also function as

antigen-presenting cells, produce inflammatory and regulatory cytokines, as well

as anti-inflammatory cytokines. These latter functions, in particular, may contribute to B-cell-mediated protection in the injured CNS. Unfortunately, there is a

paucity of B-cell research compared to investigations of post-stroke monocyte,

neutrophil, and T-cell populations. As such, it is first necessary to review the role

of B-cells established through studies in other inflammatory disease states.



U.M. Selvaraj et al.

The Detrimental Role of B-Cells in Autoimmune Disease

The primary function of immune system is to differentiate between self and foreign

antigens, and produce effector responses toward the foreign pathogens, whilst

avoiding destructive self-targeting [43]. Autoimmune diseases generally involve

abnormal recognition of self-antigens, resulting in severe inflammatory conditions

and subsequent tissue damage. This occurs due to a combination of both environmental and genetic factors, which leads to a loss of tolerance toward self-antigens.

B-cells are usually considered to be detrimental with respect to autoimmune diseases because of the secretion of autoantibodies, which damage tissue and bind

receptors, acting as agonists to either activate or inactivate function [44, 45].

Furthermore, B-cells activate the complement system, leading to the release of anaphylatoxins which increase the secretion of pro-inflammatory cytokines and attract

other effector cells to the site of injury [46, 47]. Antibody-independent pathogenic

functions include providing accessory signals to T-cells to activate an autoreactive

T-cell response [48–50]. B-cells secrete pro-inflammatory cytokines like tumor

necrosis factor (TNF)α and interferon (IFN)γ [51], and areas of chronic inflammation form ectopic germinal centers [52]. These germinal centers are similar to the

germinal centers of the secondary lymphoid organs described above, though the

plasma cells in these structures secrete autoantibodies [53]. While at first these diseases may seem irrelevant to stroke, there is a call for the medical community to

re-evaluate the role of chronic inflammatory diseases in the development of atherosclerosis, a predominant risk factor for stroke [54].


Multiple Sclerosis and EAE

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the human

CNS. Most patients with MS exhibit lesions comprised of immune cell infiltrates,

demyelination plaques, and axonal damage, with major heterogeneity of the pathological features in patients [55, 56]. EAE is an induced autoimmune disease in mice

to model MS, though it is predominantly considered T-cell-mediated, as the adoptive transfer of myelin-specific autoreactive T-cells are sufficient for disease induction [57, 58]. CD4 T-cells specific for CNS antigens, such as myelin oligodendrocyte

glycoprotein (MOG), induce inflammation and result in demyelination of the CNS,

though IL-10-producing T-cells ameliorate disease [59–61]. The importance of

the role of B-cells in MS was first stressed by the reports of a presence of high levels

of immunoglobulins in the cerebrospinal fluid (CSF) MS patients [62]. While EAE

induction does not require autoantibody production, they can exacerbate the existing inflammatory environment [63, 64]. Studies involving B-cell depletion using

anti-IgM antibodies found that B-cell depleted mice were more resistant to the

induction of EAE via recombinant MOG protein [64–66]. On the contrary, B-cell

depletion during the later parts of the disease resulted in reducing the disease symptoms [57], possibly as B-cell antigen presentation and activation affects the entry of

autoreactive T-cells into the CNS [67, 68].

B-Cells in Stroke and Preconditioning-Induced Protection Against Stroke



Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disorder characterized by a significant increase in autoantibodies against a variety of predominantly

nuclear self-antigens, (e.g., DNA, ribonucleoproteins, and phospholipids) [69]. SLE

patients exhibit diverse symptoms because of the heterogeneous impact of the disease on different tissues and organs, including kidney [70–73] and brain [74], resulting in a challenge to diagnosis the disease. B-cell depletion in SLE mouse models

resulted in the absence of nephritis and vasculitis [75], and decreased activation of

T-cells [76]. Considering the significance of B-cells in the initiation and progression

of SLE, treatments targeting B-cell survival and differentiation for B-cell depletion

therapy are being evaluated for clinical applications [77].


Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune disease which results in inflamed

synovial tissue due to infiltration of inflammatory cells into the affected joints [78].

The synovial membrane contains numerous activated CD4 T-cells, which are important for the pathogenesis of RA. B-cells function as APCs to prime autoreactive

T-cells in experimental RA [79]. B-cells surround the T-cell aggregate, with activation dependent on the presence of B-cells [78]. Experiments where inflamed tissue

samples from RA patients were transplanted into mice lacking lymphocytes confirmed the requirement of B-cells for both disease initiation and direct sustainment

of chronic inflammation [80, 81]. B-cells also produce rheumatoid factor (RF) antibody production [82], with the presence of RF antibody correlated with increased

mortality and morbidity in RA patients [83].


Current FDA-Approved B-Cell Therapeutics

CD20 is a B-cell surface marker expressed in different stages of B-cell development. Patients with autoimmune diseases, including MS, SLE, and RA, positively

respond to B-cell depletion treatments using an anti-CD20 antibody, commercially

known as Rituximab [77, 84–86]. In contrast, B-cell depletion exacerbates disease

severity in patients with certain T-cell-mediated autoimmune conditions, including

ulcerative colitis and psoriasis [87, 88]. Determination of efficacy of B-cell depletion is limited to changes in B-cell populations in the peripheral circulation.

Therefore, the full effect on B-cells population in secondary lymphoid tissues outside of bone marrow, including the CNS, remains unknown [89].

Other B-cell-specific therapies are also under development, including the targeting of B-cell receptors involved in proliferation. A double-blind phase-II study with


U.M. Selvaraj et al.

Belimumab, a monoclonal antibody affecting the function of the B-cell receptor

through the B-cell-activating factor (BAFF) [90]. BAFF is essential for B-cell survival and maturation [91, 92], with Belimumab significantly lowering B-cell counts

for active RA patients [90]. Atacicept (i.e., antibodies against transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), another

BAFF receptor required for B-cell maintenance and survival) also decreased B-cells

in preclinical studies [93], with a Phase-Ib study with patients having moderate to

severe RA, demonstrating efficacy [93, 94]. Finally, BR3-Fc, a recombinant protein

that blocks BAFF signaling, is under evaluation in clinical trials [95].

Currently, most B-cell therapeutics are focused on B-cell depletion and antibody efficiency [96]. Induction of Bregs, however, also offers a potential therapeutic strategy, though further studies are required to establish the functional stability

of Bregs [44]. However, there are some clinical trial results which support this

avenue of intervention. Anti-IL-6R (Tocilizumab) treatment for RA increased

TGF-β expression in CD25high B-cells, and could potentially function by regulating

Bregs [97]. Also Laquinimod, a drug under development for MS and in phase-III

clinical trials for SLE, increases the percentage of CD25high B-cells and their IL-10

expression, thereby expanding subpopulations of Bregs [98, 99]. Finally, Copaxone

could prove efficacious in upregulating Bregs after stoke, as its effects include

promoting the conversion of inflammatory CD4 T-cells to Tregs [100], the upregulation of B-cell-derived IL10 [101, 102], and the neuronal production of growth

factors [103], though efficacy in experimental stroke models has been mixed (see

Sect. 3.2).


B-Cells During Stroke Injury and Recovery

Stroke occurs when there is a loss of blood supply to the brain, either due to a vessel

blockage (ischemic stroke) or vessel rupture (hemorrhagic stroke), and is a major

cause of death and disability in the global adult population [104–106]. Despite significant improvements in the understanding and identification of factors contributing to post-stroke CNS tissue damage, therapeutic options for stroke are highly

limited. Apart from the primary tissue damage due to lack of blood supply, there is

a secondary ischemia-reperfusion injury due to the inflammatory cascade. Following

the occurrence of either ischemic or hemorrhagic stroke, there is disruption of the

blood–brain barrier (BBB), allowing endogenous CNS antigens access to activated

lymphocytes [107]. Shortly after experimental stroke onset, endothelial cells within

the BBB are activated to recruit peripherally circulating leukocytes to infiltrate into

the brain parenchyma. This early CNS sterile inflammation induces an immediate

and strong antigen-nonspecific innate immune response against the exposed CNS

molecules, resulting in brain tissue damage and functional impairment, which later

results in an antigen-specific adaptive immune response [108]. However, not all of

the post-stroke immune responses are inherently detrimental to functional recovery.

This section will review post-stroke inflammation, with a focus on B-cell-mediated

mechanisms of both injury and repair in the CNS.

B-Cells in Stroke and Preconditioning-Induced Protection Against Stroke



Post-Stroke Immune Responses Within the CNS

After stroke, there is a loss of BBB integrity, while reactive oxygen species (ROS)

and other inflammatory mediators activate endothelial cells and leukocytes to

increase expression of adhesion molecules [109, 110]. This results in the migration

and recruitment of peripherally circulating leukocytes to the ischemic cortex, allowing the immune cells to access novel CNS antigens. While this is the first step of the

inflammatory cascade, the acute inflammatory response consists of both infiltrating

leukocytes and tissue-resident immune cells, including microglia (Chap. 8) [111].

Resident microglia are activated immediately after stroke and secrete proinflammatory mediators (e.g., IL-1β, TNF-α). Neutrophils are one of the first leukocytes to infiltrate the ischemic tissue, entering within a few hours after experimental

stroke [112, 113]. Neutrophils secrete granules containing pro-inflammatory factors, elastases, myeloperoxidases, and multiple matrix metalloproteinases. The

macrophages and mast cells residing in the perivascular space also release multiple

pro-inflammatory factors, like TNF-α, IL-8, and IL-1β, as well as vasoactive mediators like histamine and proteases [114–117]. This highly enhances the infiltration of

peripheral immune cells into the infarct region in a feed-forward mechanism that

collectively constitutes the acute phase of the post-stroke innate immune response.

Lymphocytes, including T-cells and B-cells, enter the brain parenchyma early

after onset as well, though T-cells function in the acute phase without any antigen

specificity through secretion of cytokines and ROS generation [112, 113]. The dead

and damaged cells in the infarct release an array of molecular signals called dangerassociated molecular pattern molecules (DAMPs) [118]. These bind to and activate

TLRs and scavenger receptors present on APCs to prime them for antigen presentation. This results in the CNS antigens being presented to B-cells and T-cells [14],

and the subsequent development of cellular and humoral immunity against the CNS

antigens. Data concerning antigen-dependent post-stroke adaptive immune

responses are contradictory, as both beneficial and harmful effects to the brain have

been shown in experimental stroke models [119–121].

Ortega and colleagues recently characterized the time course of autoreactivity to

myelin-derived antigens (e.g., MOG, myelin basic protein (MBP)) and neuronalderived antigens (e.g., microtubule-associated protein 2 (MAP2), N-methyl-Daspartate (NMDA) receptor subunit, NR2A) [14]. Experimental stroke induced a

high autoreactivity to CNS antigen as early as 4 days after onset for both B- and

T-cell populations isolated from the spleen and cervical lymph node (CLN). The

CLNs are draining lymph nodes for brain, but the rapid response may reflect a

previously unstudied APC activation of B- and T-cells occurring in newly identified

lymphatic vessels within the CNS [13]. High autoreactive responses to MAP2 and

myelin occurred in the mice with the smallest infarct volumes, suggesting autoimmune responses after stroke may not be solely detrimental to recovery. This corroborates a clinical study in stroke patients, wherein Planas and colleagues

demonstrated that increased APC presentation of MAP2 and NR2A correlated with

smaller infarct volumes and better long-term outcome [122]. This study also found

that higher reactivity for MBP correlated with larger infarctions and greater stroke


U.M. Selvaraj et al.

severity [122], though T-regulatory cells specific for MBP [103], and MBP antigen

presentation before stroke [123] improved long-term recovery in animals [14].

Thus, further studies are required to completely elucidate this complex and ambiguous adaptive immune response to understand the mechanisms related to CNS recovery that could be harnessed as potential therapeutic targets.


B-Cell Potential to Limit Detrimental Acute Post-Stroke


As mentioned previously, regulatory T- and B-cells produce anti-inflammatory

cytokines that suppress the function of infiltrating pro-inflammatory cells. In fact,

IL-10 expression is elevated during major CNS diseases [124], and regulatory lymphocytes play a protective role, limiting the CNS damage due to inflammation in

autoimmune diseases such as MS and SLE [44, 61, 125]. Therefore, it is hardly

surprising that in the absence of B-cells, there is increased infiltration of multiple

leukocyte subsets into the ischemic parenchyma, including neutrophils, monocytes,

macrophages, and T-cells, resulting in larger infarct volumes [124]. Confirmatory

studies used transgenic mice that have a nonsense mutation introduced into the

transmembrane exon of the IgM heavy chain (μMT−/−), which results in the total

deletion of B-cells [126]. These mice developed larger infarct volumes, more severe

functional deficits, and had a higher mortality rate following middle cerebral artery

occlusion (MCAo), a common experimental model of stroke [127]. The complete

absence of B-cells also resulted in higher numbers of activated T-cells, macrophages, microglial cells, and neutrophils in the ischemic brain of μMT−/− mice following MCAo [127].

This phenotype was rescued after the adoptive transfer of highly enriched populations of wild-type B-cells to B-cell-deficient μMT−/− recipient mice before MCAo

[127]. The neuroprotective, anti-inflammatory effect was not observed, however,

when IL-10-deficient B-cells were adoptively transferred prior to stroke. Mice

receiving B-cells as a neurotherapeutic exhibited higher numbers of regulatory cells

in the periphery concomitant with lower numbers of activated inflammatory T-cells

[128]. After LPS stimulation, purified B-cells produced significant amounts of

IL-10. Adoptive transfer of these cells prior to stroke decreased infarct volumes,

confirming the beneficial role of IL-10-producing B-cell subsets on stroke outcome

in the acute phase of recovery [124]. These B-cell subsets also increased the regulatory T-cell numbers, co-inhibitory receptor PD-1 expression, and significantly

reduced peripheral pro-inflammatory immune cells [124].

As mentioned previously, Copaxone as a therapeutic can induce IL-10 from

Bregs [102]. Copaxone treatment (200 μg plus an adjuvant to boost the immune

response) decreased infarct volumes in rats 7 days post-MCAo [129]. More recent

studies, however, found that Copaxone (70 μg, no adjuvant) administered 30 mins

prior to stroke did not reduce infarct volumes at 1 day after stroke [130], nor did

Copaxone administration (400 μg, no adjuvant) immediately after MCAo reduce

B-Cells in Stroke and Preconditioning-Induced Protection Against Stroke


infarct volumes at 3 and 7 days post-stroke [131]. But the latter study also did not

find an upregulation of IL-10, which suggests that the lack of protection in these

studies may be confounded by the lack of adjuvant, which increases inflammation,

and thus Copaxone uptake, at the site of injection. This may also explain the shortened timeframe given for Copaxone to generate a neuroprotective effect, particularly with regard to the 1-day post-stroke study [130]. Therefore future studies

should consider Copaxone as a pharmacologic preconditioning agent (similar to

LPS, 4.4.1) when determining if it can induce neuroprotection via Breg upregulation prior to stroke onset.

4.3 Post-Stroke Immune Responses Occurring

in the Periphery

Stroke induces systemic immune changes, which affect clinical outcomes for weeks

and months following stroke onset [132, 133]. As mentioned above, the initial

response to tissue injury following stroke is carried out primarily by the innate

immune system, as innate cells are recruited to the ischemic parenchyma and contribute to infarct progression [112]. These inflammatory responses are not confined

to the brain, however, but are also concurrent with immunological changes in blood,

spleen, bone marrow, and secondary lymphoid organs [134, 135]. Gene expression

signatures from the peripheral blood after stroke distinguish different causes of

stroke, and this implies a characteristic immune response to CNS injury [136]. Stroke

induces systemic immunodepression within days of onset, especially in the cases of

large infarct volumes [137]. In fact, most patients with larger strokes have higher

levels of circulating IL-6 and IL-10, lower lymphocyte counts, and increased susceptibility to infections (e.g., respiratory, urinary tract) that impede long-term recovery

[138, 139]. Liesz et al. used different ischemia models to demonstrate the effect of

stroke on systemic immunological and microbiological parameters [140]. They

found that post-stroke changes associated with immune system, as well as infectious

complications, depended on infarct volume. Only huge infarct volumes induced leucopenia (i.e., reduced numbers of lymphocyte counts) in spleen, lymph nodes, and

thymus both during acute (after 24 h) and long-term (after 7 days) recovery.

A majority of these systemic immune changes can be attributed to downstream

signaling of noradrenaline (NA) receptors, present on almost all immune cells,

including 30–50 % of B-cells [141]. NA receptors are activated by epinephrine

released from post-ganglionic sympathetic nerves within hours of experimental

stroke onset [142]. Thus, the sympathetic nervous system dramatically alters poststroke immune responses to increase plasma cortisol, catecholamines, and

monocyte-derived IL-10, which in turn decreases the number and activity of circulating immune cells, including B-cells [134, 142]. Transient decreases in IgG levels

were reported in post-stroke patients, which was also associated with increased bacterial infections [19]. Stroke injury is itself a strong stress signal that also activates

the hypothalamic–pituitary axis, resulting in the production of glucocorticoids

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