**4. Peripheral blood leukocytes as potential biomarkers of disease activity**

A biomarker is defined as a characteristic that can be objectively measured and evaluated and serves as an indicator of normal biological processes, pathological processes or pharmacological reactions to therapy. An ideal biomarker is characterized by high sensitivity and specificity as well as a simple, cost effective, reproducible, and non-invasive detection method [6].

In this section we synthesize and integrate the most relevant data regarding the characteristics of the selected immune cells that could be considered as IFN-β treatment-related biomarkers. The main goal of this work is an attempt to help researchers to perform a good assessment of immune cells in future studies. The presented data is a result of a compilation of several studies and findings.

#### **4.1 Antigen-presenting cells**

Antigen presenting cells (APCs) are considered key players in the immune surveillance of CNS and, at the same time, they are critically involved in the pathogenesis of CNS autoimmune diseases. They are a morphologically and functionally diverse group of cells that links the innate and adaptive immune responses. These cells are specialized in the presentation of antigens to lymphocytes, particularly T cells. Included among such cells are dendritic cells (DCs), monocytes and macrophages (derived from monocytes that migrated from the blood stream to tissues). B lymphocytes that specifically capture antigens via their clonally expressed membrane immunoglobulin can also function efficiently as APCs to T cells [24].

#### *4.1.1 Dendritic cells*

In humans, DCs comprise two major subsets: plasmacytoid DCs (pDCs) and myeloid (mDCs). Through nucleic acid-sensing, pDCs activate toll-like receptors (TLR), such as TLR7 and TLR9, rapidly producing type I IFN. mDCs are dedicated APCs that have a characteristic dendritic morphology, express high levels of MHC class II molecules and recognize pathogen-derived lipids, proteins and nucleic-acids by TLR2, TLR4 and TLR3 respectively [25].

The DCs subsets may be helpful as biomarker between remission and relapse of RRMS patients treated with IFN-β. The circulating mDCs subset reduces in remission and increase in relapse RRMS patients. On the other hand, the pDCs frequency are maintain across the different phases of disease. Usually, these subsets present a low frequency in systemic circulation, so the mDCs/pDCs ratio is a good representative of the alteration observed in the DCs subsets. The mDCs/pDCs decreases in remission RRMS patients and is re-established in relapse RRMS patients, constituting a potential peripheral biomarker [26, 27].

The involvement of DCs in MS arises from studies that demonstrate the abundant presence of these cells in the inflamed CNS lesions and in the CSF of MS patients [23].

One of the immunomodulatory effects of IFN-β in the EAE model is the reduction in antigen presentation, particularly myelin-specific antigens, leading to reduced T-cell responses [25, 28]. In contrast with these effects, in remission phase it was observed that the DCs subsets increase the expression of HLA-DR and decrease in the relapse phase. The variation in HLA-DR expression is more evident in the mDCs subset. The same subset reduce the mRNA gene expression of CX3CR1; fractalkine is known to be upregulated and released in response to proinflammatory stimuli and induces adhesion, chemoattraction, and activation of leukocytes [27].

The activation status of the mDCs subset could discriminate between RRMS phases. This subset shown a highest activated status in remission than in relapse phase, through the increased HLA-DR expression and a reduced migratory capability, since reduce the mRNA gene expression of CX3CR1.

*Peripheral Biomarkers in Multiple Sclerosis Patients Treated with Interferon-Beta DOI: http://dx.doi.org/10.5772/intechopen.99006*

#### *4.1.2 Monocytes*

Monocytes represent a heterogeneous population of primary immune effector cells with distinct phenotypical and functional characteristics; their differential roles in steady-state immune surveillance and the pathogenesis of human CNS disease are poorly understood [20].

The differential expression of CD14 (part of the receptor for lipopolysaccharide) and CD16 (also known as FcγRIII) allows monocytes to be segregated into three subsets. The major subset designated "classical" monocytes (CD14++CD16− , cMo), corresponds to 80–90% of circulating monocytes. CD16 expressing monocytes are divided into a named "intermediate" monocyte (CD14++CD16+ , iMo) and a subset classified as "non-classical" monocytes (CD14+ CD16++, ncMo); each of these subsets corresponds to 5–10% of circulating monocytes [21, 29].

Patients with MS display high levels of monocyte-secreted inflammatory molecules in serum compared to healthy individuals, demonstrating a role for peripheral monocytes in the progression of the disease. Increased levels of serum tumor necrosis factor (TNF) α and β have been reported in MS relapse. Monocytes and microglia are known to act as major effectors in the demyelinating process through direct interaction and the production of proinflammatory cytokines and mediators (e.g., IL-1b, nitric oxide). CD16+ monocytes may contribute to the breakdown of the BBB by facilitating T cell trafficking into the CNS [20, 24, 30].

Research performed on monocyte pool in RRMS patients is scarce and ambiguous. A recent work achieved a significant decrease of the ncMo subset in both phases of RRMS patients, although in a higher extension in remission patients [27].

The frequency of monocytes subsets does not allow us to identify different phases of RRMS, but the HLA-DR expression could constitute a potential important biomarker between remission and relapse phases. A significant increase in HLA-DR expression in all monocyte subsets in the remission group when compared with healthy and relapse groups, has been described [27]. IFN-β enhances HLA-DR expression in circulating monocytes, but inside the CNS, one prominent model is based on the observation that IFN-β inhibits the IFNγ upregulation of MHC class II molecules on cell surface of macrophages and glial cells and therefore diminishes antigen presentation [28]. In the periphery, Kantor et al. report that the increase of MHC Class II expression in monocytes induced by IFN-β may contribute to the positive immunomodulatory effect in MS [31]. These findings were reinforced by the observation that when IFN-β-stimulated monocytes were used to stimulate autologous T cells, there was an increased secretion of anti-inflammatory cytokine IL-13 [32].

### **4.2 T cells**

#### *4.2.1 CD4+ and CD8+ T cells*

T cells are central regulators of the adaptive immune response, they help B lymphocytes to produce antibodies and secrete cytokines that provide efficient protection against pathogens. Distinct T helper (Th) cell subsets, producing one or more lineage-defining cytokines and expressing master transcription factors and homing receptors. Th subsets are differentiated from naive CD4+ T cells in response to a specific class of pathogenic microorganisms and to the cytokine milieu. This occurs in peripheral lymph nodes by mature DCs that present pathogen-derived peptides associated to MHC class II. With the involvement of their costimulatory molecules, DCs promote T cell proliferation and produce polarizing cytokines. In turn T cell was differentiated in distinct Th cell subsets, such as Th1, Th2, Th17, regulatory T (T reg) and T follicular helper (Tfh) [33].

The CD4+ T cells have been the most studied in the pathogenesis of MS, although CD8+ T cells are the dominant lymphocyte population in all stages of disease and lesions of MS patients. Naive CD8+ T cells follow a similar differentiation programme of CD4+ T cells [34, 35].

Th1 cells are described as being the pathogenic subset of T cells, whereas Th2 cells are reported to exert inhibitory effects [5]. Previous studies have pointed to a reduction in pro-inflammatory capability promoted by IFN-β therapy, consisting of a reduction of the expression of Th1-induced cytokines while enhancing Th2 responses [18]. Concerning the T cytotoxic (Tc) subsets, it has been reported the same behavior, in remission a downregulation of pro-inflammatory Tc1 responses and up-regulation of anti-inflammatory Tc2 with a beneficial effect on disease activity [36]. This dichotomy Th1, Th2 subsets and Tc1, Tc2 subsets could contribute to discriminate between remission and relapse phases.

The identification of Th17 cells helped to resolve some in adequacies of the original Th1/Th2 concept that had dominated T cell immunology research filed for almost 20 years. For a long time, it was thought that the IL-12/IFNγ pathway and Th1 cells were central to the development of autoimmune disease [37].

Both Th1 and Th17 cells have been implicated in the initiation and progression of disease in RRMS and its experimental model EAE [19]. The link between Th17 cells, IL-17 and MS relapses comes from the observation that in humans, Th17 cells are able to cross the BBB in MS lesions, enhancing neuroinflammation. In vitro studies have revealed that IL-17 blocks the differentiation and reduces the survival of oligodendrocyte lineage cells. In EAE model, it has been suggested that Th17 cells interact directly with neurons, forming antigen-independent, immune, synapselike contacts [7, 38].

It is assumed that the inhibition of Th17 cells in RRMS patients attenuates the disease, however conflicting data have been published. Axtell et al. reported that IFN-β treatment effectively blocked disease symptoms in mice with EAE induced with Th1 cells. Otherwise, in EAE induced with Th17 cells the IFN-β treatment worsened disease [19].

In RRMS patients, it is not clear whether a more specific blockade of the Th17 pathway has beneficial effects in MS patients. Treatment with an antibody directed against IL-12p40 and therefore neutralizing both IL-12 and IL-23 did not result in a significant reduction of disease activity [39].

A meta-analysis pointed out several limitations across studies that assess the levels of peripheral Th17 cells and serum Th17-related cytokines. Like the severities of the disease and clinical subtypes in MS patients; the disease duration from relapse; and that the MS treatments were not consistent; and it was postulated that most studies selected MS patients with high disease activity. There were differences in experimental methods between studies and a lack of detailed standardized methods to identify the Th17 cells and Th17-related cytokines [40].

A recent in vivo study observed an increased frequency of circulating Th17 and Tc17 cells, accompanied by increased serum levels of IL-17 in remission RRMS patients treated with IFN-β [41]. This contradiction underlines the need to clarify the role of the IL-17-producing T cells in RRMS patients.

It has been demonstrated that a significant proportion of Th17 cells convert into IFN-γ-producing T cells and have chemokine receptors from both Th17 and Th1 subtypes, referred as Th17.1 cells. The enhanced potential of Th17.1 cells to infiltrate the CNS was supported by their predominance in CSF of early MS patients and their preferential transmigration across human brain endothelial layers [42, 43]. In remission RRMS patients, it was observed that Th17 and Tc17 cells exhibited a higher degree of Th1 plasticity since there were higher frequencies of those cells simultaneously producing intracellular IL-17 and IL-2 or IFNγ or TNFα [41].

#### *Peripheral Biomarkers in Multiple Sclerosis Patients Treated with Interferon-Beta DOI: http://dx.doi.org/10.5772/intechopen.99006*

Another subset of T cells, the Tregs, are characterized by high expression of CD25 and the transcription factor *Foxp3*, which is critical for their development, lineage commitment, and regulatory functions. Tregs are a very heterogeneous population with suppressive functions that maintain tolerance to harmless food/ self-antigens and prevent autoimmune disease. Numerous studies have identified Tregs as important immunoregulators in many inflammatory and autoimmune disease conditions including asthma, MS, and type-I diabetes [37, 44].

In MS patients, both reduced or normal frequency of Tregs was observed. Libera et al. described a significant decrease in Treg cells in remission RRMS patients [45]. Haas et al. state that the frequency of Treg cells was normal in MS patients but with a lower suppressive function on autoreactive T cells [46]. Venken et al. described that RRMS patients treated with IFN-β showed restored naive Treg numbers as compared with age- and disease-duration-matched untreated patients [47].

Recently identified, the Tfh subset expresses the chemokine receptor CXCR5 as well as CD279 [48], is specialized in helping B cells to produce antibodies in the face of antigenic challenge and plays a crucial role in orchestrating the humoral arm of adaptive immune responses. Tfh cells have the unique ability to migrate into follicles in secondary lymphoid organs where they colocalize with B cells to deliver contact-dependent and soluble signals that support survival and differentiation of the latter cells. There is no complete and thorough understanding of how naïve Th cells differentiate into mature Tfh [49, 50].

Tfh cell levels are elevated in the blood of MS patients and this population is positively correlated with the progression of disability. One potential mechanism through which Tfh cells can contribute to disease is promoting the inflammatory B-cell activities, suggesting that Tfh cells cooperate with Th17 cells to induce inflammatory B cell responses in the CNS and increase disease severity [49].

The increased frequencies of Th1 cells, activated Tfh- and B-cells parallel findings from pathology studies, along with the correlation between activated Tfh- and B-cells, suggest a pathogenic role of systemic inflammation in progressive MS [51].

A similar frequency of Tfh cells between RRMS patients and healthy subjects was reported. However, this subset tend to exhibit a more proinflammatory activity, since higher frequencies of TNF-α<sup>+</sup> Tfh cells have been observed [41]. It is well known that Tfh cells play an important role in T/B interactions in germinal centres (GC) and one potential mechanism through which Tfh cells can contribute to MS is in promoting inflammatory B-cell activities [49]. The Tfh subset and others follicular like T cells subsets, like Treg/follicular cells, are promising targets in the study of T cells in pathophysiology of MS.

## *4.2.2* γδ *T cells*

γδ T cells develop in the thymus together with αβ T cells but rearrange a different TCR, consisting of a TCR-γ and TCR-δ chain. One of the most striking characteristics of γδ T cells is their inherent ability to secrete pro-inflammatory cytokines very rapidly, which influences adaptive immunity, they carry out immediate effector functions as well as mounting a memory response upon microbial reinfection. This fast response can be explained by γδ T cells exiting the thymus already with the functional competence to produce cytokines with no need of APCs cells [52, 53].

In MS, their potential importance is increased by the finding that γδ T cells accumulate in demyelinating CNS MS plaques; these cells show evidence of oligoclonal expansion indicating a local response to currently unknown antigens. γδ T cells have been shown to be present in both MS lesions and in CSF, and sequencing studies have shown that the major γδ T subsets present in the lesion differ from those in the CSF, suggesting specific functions for these cells in lesion development. In more chronic lesions, γδ T cells may become the most prevalent type of T cell in the lesion. γδ T cells isolated from the CNS can be expanded but only in patients with relapse disease, not chronic MS patients, suggesting that these cells may have differential roles during various phases of the disease [54, 55].

The frequency, the migratory pattern, the activation status of γδ T cells in RRMS patients are unclear. Between remission and relapse RRMS patients, the γδ terminally differentiated effector memory T cells (TEMRA) and the CCR5+ γδ TEMRA decrease in relapse when compared with remission RRMS patients [56], constituting a good biomarker between phases of the disease. Probably as a result of the migratory pattern describe for this phase of MS, preferentially toward RANTES and MIP-1α, whose expression is increased during relapses [57, 58].

The decrease of Eomesodermin and granzyme B mRNA expression in CD27<sup>−</sup> γδ T cells suggests a reduction in the cytotoxic potential of the circulating pool of γδ T cells, particularly in relapsing RRMS patients [56].

### **4.3 B lymphocytes**

The most consistent immunodiagnostic feature and hallmark immunologic finding in MS patients is the presence of oligoclonal bands (OCB) in the CSF and their absence in peripheral circulation. Consequently, the pathogenic function of B cells in MS has been traditionally associated with antibody production. However, B cells have three putative biological roles: production of proinflammatory or regulatory cytokines, function as APCs and antibody production [59].

In MS, the memory B cells, plasmablasts and plasma cells preferentially cross the BBB and migrate into the CNS, where they dominate the B cell pool and exert different effector functions. B cells seem to be abnormally polarized toward a more proinflammatory phenotype [60].

More recent, somatic hypermutation studies have demonstrated that identical B cell clones can be shared between the CNS and the periphery in individual patients. These studies provide evidence of bidirectional trafficking of distinct B cell clones (both into and out of the CNS). The patterns suggest that B cells can travel back and forth across the BBB and commonly re-enter GC (in the meninges or cervical lymph nodes) to undergo further somatic hypermutations. These findings change our view of lymphocytic surveillance of CNS tissue and underline that B-cell trafficking is an important topic for future research and therapy strategies [60–62]. This news about recirculation of B cells through the BBB alters the perception of the role of B cells in MS.

B cells are released in the peripheral blood, recirculate between the secondary lymphoid tissues, and dying after a few days. According to phenotypic profile of B cell subsets, which also reflects their functional abilities and behavior, four major maturation-associated subsets can be identified in the human peripheral blood: immature/transitional, naive, memory and plasmablast [63].

In remission RRMS patients submitted to IFN-β, the percentage of immature/ transitional B cells increases. This increase can be seen as an attempt to increase anti-inflammatory cytokines. Meanwhile, a decrease in the proportion of circulating class-switched memory B cells was reported [64, 65].

The relapsing RRMS patients exhibited distinct changes in B cell subsets homeostasis, resulting in a decrease in the total population of B cells, including a decrease of the immature/transitional and naïve B cell subsets when compared with remission RRMS patients. On the other hand, the plasmablast B cell subset presented an increase in relapse RRMS patients. The ratio between immature/transitional B cells and plasmablasts can thus be considered as a potential biomarker between phases of RRMS patients. The remission RRMS patients and the healthy subjects presented a similar ratio, and the relapse RRMS patients present a decreased ratio [66].

*Peripheral Biomarkers in Multiple Sclerosis Patients Treated with Interferon-Beta DOI: http://dx.doi.org/10.5772/intechopen.99006*

According to the new and recent data about the recirculation of B cells in RRMS, it seems that the increase of plasmablasts in circulation of relapsing episodes may be due to a migration of these cells from cervical lymph nodes and/or from B cell aggregates described in the meninges of MS patients to the blood marrow in an attempt to promote the immune response [67].
