Highlighting the Relevance of CD8+ T Cells in Filarial Infections

The T cell immune responses in filarial infections are primarily mediated by CD4+ T cells and type 2-associated cytokines. Emerging evidence indicates that CD8+ T cell responses are important for anti-filarial immunity, however, could be suppressed in co-infections. This review summarizes what we know so far about the activities of CD8+ T cell responses in filarial infections, co-infections, and the associations with the development of filarial pathologies.


HELPER T CELLS IN ANTI-FILARIAL IMMUNITY AND IMMUNOREGULATION: AN OVERVIEW
T cell-mediated immune responses are major components of the anti-filarial immunity, as per evidence from experimental animal models and antigen stimulation studies (7)(8)(9). This T cellmediated response has been characterized by activities of both T helper 1 and T helper 2 cytokines, depending on the stage of parasite/infection (10)(11)(12). At the interplay of the Th1/Th2 activities are regulatory T cells (Treg) which may be simultaneously active during the filarial infections (13,14). Thus, several scenarios of helper T cell responses could be observed. First, the responses can be dominated by strong Th1 cytokines with diminished Th2 cytokine activity as a result of poor regulation by Treg. Another scenario could involve a dominant type 2 cytokine response with a marginal activity of Th1, due to immunoregulatory activities. There could also be well-balanced Th1/Th2 responses when Treg activities are sufficient. The activities of primed CD4 + T cells involved in Onchocerca microfilariae clearance in infected mice are dominated by Th2 responses (15). In the early stages of filarial infection, an increased expression of Th1 cytokines, particularly triggered by antigenpresenting cells have been reported (7). This suggests that the initial responses against infective stage larva involve proinflammatory responses induced by innate components although the primary responses against the parasites are of Th2 phenotype. A large body of evidence shows that the interactions with host innate cells involve toll-like receptors (16)(17)(18). The Wolbachia induces dendritic cell activation and IFN-g secretion which are correlated with increased TLR2 expression (19). Studies with O. volvulus keratitis-infected mice demonstrated that IFN-g increased expression of TLR2 on corneal macrophages which triggered the production of TNF-a, interleukin-6 (IL-6), IL-1a, and IL-1b in macrophages (17). Moreover, TLR2 is involved in dendritic cell activation, IFN-g secretion, and neutrophil recruitment (17,18). In addition to TLR2, the Wolbachia induces innate immune responses through TLR4 and TLR6 and involves MyD88 (18,20). The W. bancrofti microfilaria sheath protein directly interacts with TLR4 to mediate macrophage proinflammatory responses (IL-6, TNF-a, and IL-1b) via NF-kB activation (20,21).
Reports by Mukherjee et al. (22) demonstrated that W. bancrofti sheath antigen promotes maturation and activation of dendritic cells by directly interacting with TLR4. In the same study, the matured dendritic cells promoted type-1 cytokines and regulatory T cells (Treg) responses while proportions of Th2, Th17, IL-4, and IL-17A were low (22). While the low Th2 responses could be a characteristic of the sheath antigen, there is a possibility of immunoregulatory activities by Treg. Studies have shown that the Th2 responses to B. malayi adult worm antigen are enhanced upon abrogation of Treg (23). Elsewhere, depletion of Treg promoted Th1 responses without affecting Th2 proportions (24). The Wolbachia surface protein promotes pro-inflammatory responses in mice by increasing the production of Th17 cells while decreasing the levels of Treg (25,26). In addition, neutralizing CD25 and GITR in B. malayi L3-infected mice elevated Th17 and IFN-g levels while reducing IL-10 (26). Figure 2 summarizes the mediation of cytokine responses through antigen-presenting cells during filarial infections. This interplay of the type-1 and type-2 CD4 + T cell responses in anti-filarial immunity and the immunomodulatory actions of Treg have been extensively reviewed elsewhere (13,14).
Here, we discuss the relevance of CD8 + T cells in the immune responses against filarial parasites, based on documented evidence from animal models and human subjects. We also discuss the roles of CD8 + T cells in the development of filarial pathologies and their possible involvement in immunomodulatory mechanisms during filarial co-infections.

Evidence From Studies Involving Humans
There is currently insufficient evidence from human studies which clarify the mechanisms of CD8 + T cell activities in anti-filarial immunity. However, available data from studies in human subjects demonstrate the elevated levels of CD8 + T cells during filarial infections, thus, depicts potential roles in the immune responses against filarial parasites or immunopathology. Kalinkovich and colleagues examined a group of helminth-infected Ethiopian immigrants in Israel and reported elevated CD8 + T cells in this infected group (27). Moreover, W. bancrofti-infected individuals reportedly had increased frequencies of activated CD8 + T cells compared to non-infected subjects (28). From a cohort of 12 African adults with ocular onchocerciasis, Chan et al. found that levels of CD8 + T cells were significantly elevated in the infected group (29). There are additional reports that CD8 + T cells may have regulatory roles during the induction of ocular inflammation by O. volvulus antigen-specific CD4 + T cells (30).
Filarial antigen stimulation studies suggest that type 2 cytokines mediate the cytotoxic activities of CD8 + T cells against filarial parasites. In individuals with patent infections, CD8 + T cells expressing antigen-specific type 1 cytokines (IFN-g, TNF-a, and IL-22) were diminished, while levels of type 2 cytokine (IL-4, IL-9, IL-13, and IL-21)-expressing CD8 + T cells were elevated (31). This may suggest that CD8 + T cell responses in patent LF infections are mediated by antigen-specific type 2 cytokines (31,32). Notwithstanding, treatment of the filarial infection changes the CD8 + T cell cytokine secretion pattern, thus, the regulation of these patterns of CD8 + T cell cytokine release is dependent on antigen presence or disease state (31,32). The activities of CD8 + are modulated by IL-10 depending on the stage of infection (31,32). CD8 + T cells expressing IL-24 and IL-19 were increased while IL-26 expressing CD4 + T cells were diminished in asymptomatic infected individuals compared to individuals presenting with pathology (32). These regulation of parasite antigen-specific IL-19 and IL-24 expressing CD8 + T cells are more dependent on the stage of the infection than on IL-10, IL-1b, and IL-23 as observed in the regulation of CD4 + T cell response (31)(32)(33). Filaria antigenspecific type-1 cytokines (TNF-a, IFN-g, IL-22) expressing CD8 + T cells were reportedly lower in asymptomatic infected individuals, relative to individuals presenting with pathology (31). This evidence supports the notion that CD8 + T cell responses in filarial infections may be mediated by type 2 cytokines while filarial pathologies are driven by type 2 responses, as a result of continuous antigen presence and immunomodulation ( Figure 3).

Evidence From Studies With Animal Models
An early description of T cell-mediated immunity in experimental filaria excluded contributions from CD8 + T cells (34). b 2 -microglobulin deficient mice, which lack MHC class I molecules and therefore do not elicit CD8 + T cell cytotoxicity, were able to establish a resistance to the infective parasites of B. malayi (34). Several in vivo immunization studies, however, also suggested possible CD8 + T cell cytotoxic activities during filarial infections. The B. malayi trehalose-6-phosphate phosphatase antigen induced high levels of CD8 + T cells as part of the host antigen-specific cellular immune responses (35). Immunization of mice with B. malayi Wolbachia recombinase A resulted in a significant expansion of CD8 + T cells in the host spleen (36). Studies in TCR transgenic mice have also shown that the O. volvulus antigens OvALT-2 and OvNLT-1 exert suppressive effects on CD8 + T cell proliferation and cytokine release (37). Recently, heterologous prime-boost vaccination administration of B. malayi heavy chain myosin induced enhanced CD8 + T cell expansion in mouse spleen (38). Although CD8 + T cell levels are elevated during filarial infections, it may be that CD8 + T cell-mediated immunity is most relevant at the initial stages of the infections when the parasite is establishing itself in the host. Babu and Nutman explored the events of host immunity that occur in the early stages following infection with live B. malayi L3 parasites and reported that efficient CD8 + T cell responses are noticed at the very early stages of infection (7). Studies with Onchocerca lienalis-infected mice model demonstrated the contributions of T cell-mediated immunity against microfilariae, which is dominated by CD8 + T cells at the initial stages of the infection (15). The filaria-specific CD8 + T cells expressing cytokines during filarial infections are summarized in Table 1.

CD8 + T CELLS AND ASSOCIATIONS WITH FILARIAL PATHOLOGY
The degenerating filaria and its endosymbiont promotes proinflammatory responses which drive the pathologies associated with the disease. The interactions between the microfilariae (Mf) and host macrophages result in the activation of proinflammatory macrophage subtype which induces a proinflammatory response (22,40) aimed at promoting microfilariae clearance (7,41). However, the macrophage proinflammatory responses executed by IL-6, TNF-a, IL-1b, especially to dead parasites contribute to immunopathology (41).
The proportions of CD8 + T cells appear to vary during asymptomatic infections and pathological infections, suggesting that the activities of CD8 + T cells in filarial infections are dependent on the stage of the infection. In terms of cellular subsets, individuals presenting with chronic filarial pathologies have elevated levels of activated CD8 + T lymphocytes when compared to individuals with Mf only (42). Further analysis of patient sera confirmed the elevation of soluble CD8 + T cells in individuals with chronic LF pathology than those at an asymptomatic Mf stage (43). Differential gene expression analysis of blood samples from Loa loa-infected individuals FIGURE 3 | The possible roles of CD8 + in filarial immunity. The protective effects of CD8 + T cells are mediated by type-2 cytokines and upon the persistence of the filaria antigens, switch to pro-inflammatory type-1 responses which ultimately contribute to filarial pathology.
originating from Cameroon, Gabon, and Nigeria have defined the functional annotation of gene expression profiles in CD8 + T cells (44). The responses to microfilaria antigens by CD8 + T cells in endemic individuals were characterized by inflammatorydriven attacks and cell death (44). Furthermore, studies in Loa loa-infected rhesus monkeys suggest that CD8 + T cells may have a role in the hypo-responsiveness to antigens of Loa loa parasites (45). Studies in B. malayi-infected monkeys also showed that levels of CD8 + T cells were increased in symptomatic models than in asymptomatic ones (46). CD8 + T cells expressing type 1 cytokine responses have been associated with clinical filarial pathologies (2, 31). IFN-g-and TNF-a-producing CD8 + T cells are known to promote filarial pathology particularly through the stimulation of lymphangiogenic growth factors and inflammatory cell migration (47)(48)(49). These observations provide preliminary evidence on the involvement of CD8 + T cells in filarial pathology. The pathophysiological onset of filarial infections is believed to be driven at the cellular level via inappropriate regulation of CD8 + T cell activation in an increased pro-inflammatory-mediated fashion (31,32), as summarized in Figure 3.

ROLE OF CD8 + T CELLS IN FILARIAL CO-INFECTION
Helminths are known to be master modulators of host immune responses (50). The immunomodulatory benefits of helminths are being exploited for therapeutic purposes against inflammatory bowel diseases and autoimmunity (51). The filarial immuno-regulatory influence on disease progression and control has been demonstrated with several conditions, including filarial-plasmodium co-infection (52), filarialmycobacterium co-infection (53), filarial-virus co-infections (54) and allergic reactions (55).

Filarial-Virus Co-Infection
Given that both filaria and HIV infections compromise the host immune system, one would anticipate that their co-existence would affect immune responses. It is established that type-1 responses mediate immunity to viruses (56), however, filarial parasites mostly induce a type 2-dependent immunity in their hosts and are capable of regulating these responses to enhance their survival. The suppression of virus-specific CD8 + T cell cytotoxicity in HIV-Schistosoma mansoni co-infected mice models, with a corresponding delay in viral clearance, has been reported previously (57). The Schistosoma-induced reduction in cytotoxic T lymphocyte responsiveness to HIV antigens was markedly due to diminished induction of IFN-g and IL-2, either through direct IL-10 activities or parasite-induced immunomodulation of innate cells (57). Similar findings in norovirus-Trichinella co-infected mice have been reported, where the modulation of viral-specific CD8 + T cell responses was observed (58). This inhibition of antiviral immunity was associated with innate immunomodulatory mechanisms during virus-helminth co-infection (58). Thus, the efficiency of viral immune responses can be suppressed during helminth-virus coinfections, at least for S. mansoni and Trichinella spiralis.
Although the specific immunomodulatory influence filarial parasites have on HIV co-infections in vivo is not clearly established, several studies have provided insights into the situation. Nielsen et al. (59) found no influence of HIV-W.  L. sigmodontis infection suppressed the function of CSPspecific CD8 + T cells remains. Dietze et al. (54) studied the immunomodulatory effects of filaria-virus co-infection using an Litosomodes sigmodontis-Friend virus (FV) co-infected mice model. Both filaria-specific and viral-specific humoral responses were diminished, but no effect was found on CD8 + T cell response to FV infections. While the viral infection did not affect the worm burden, the L. sigmodontis infection resulted in increased viral loads due to virus-specific antibody response suppression (54). According to Gopinath et al. (61), the in vivo immunological interactions of filaria-HIV co-infections could be properly modeled if cells of individuals with pre-existing filarial infections are used rather than inducing the filarial-immunity with antigens. By infecting CD8 + T cell-depleted peripheral blood mononuclear cells from filaria-infected individuals with HIV strains, an indication of increased susceptibility to HIV infections were observed (61).

Filaria-Plasmodium Co-Infection
In addition to their co-endemicity in particular regions, malaria parasites and some filarial parasites share similar transmission vectors. Several studies have explored the possible alterations in host immune responses as a result of filaria-plasmodium coinfection (62,63). Filarial parasites are well known to induce immunosuppressive mechanisms in their human hosts by impairing the production of pro-inflammatory cytokines through the activities of Treg and related cytokines, IL-10 and TGF-b (13,31,64). Under the hypothesis that the induced immunosuppression by filarial alters the pro-inflammatory onset of cerebral malaria (CM), Specht et al. (52) studied the development of CM in malaria-infected-murine models with previous filaria (L. sigmodontis) infection. CD8 + T cells are key players in the progression of CM (65). In the co-infected model, CD8 + T cell sequestration into the brain was significantly reduced, which was corroborated with the observed reduction in CM pathology (52). The filaria-induced IL-10 suppresses type-1 cytokines which are known promoters of CM pathologies. In addition, IL-10 mediates the accumulation of CD8 + T cells in the spleen, thus, reducing its sequestration into the brain (52). Preexisting filarial infections in mice models can suppress the production and functioning of CD8 + T cells induced by anti-Plasmodium vaccination, although the implementation of a heterologous prime/boost immunization regime could stop the observed interference (39).

Filaria-Mycobacteria Co-Infection
Similar to malaria, tuberculosis and filarial infections share a common endemic hotspot. Recent studies have explored the immunoregulatory effect on filaria-mycobacteria co-infections (66,67), however, the precise influence on TB outcome and progression remains open for further investigations. The influence of filarial infections on Mycobacterium tuberculosis and Plasmodium falciparum infections have been extensively discussed elsewhere (68). Immunity in helminth infections is mostly mediated by Th2 immune responses, whereas immune responses to M. tuberculosis require type-1 cytokines (69). CD8 + T cells are known inducers of Th1 and Th17 associated cytokines, which are critical components of host immunity against TB (70). It is also well established that filarial parasites can modulate CD8 + T cells. The Mycobacterial antigen-specific CD8 + T cell responses were down-modulated by the occurrence of W. bancrofti infection in active pulmonary TB patients (53). Both CD4 + and CD8 + Th1/Th17 cytokines were diminished in the co-infected mice model, as a result of filaria-induced IL-10 modulation of the pro-inflammatory responses against the mycobacterium (53).

Filarial Infections and Sepsis
In the initial phases of septic attacks, microorganisms mount a robust pro-inflammatory immunity, a stage described as systemic inflammatory response syndrome (SIRS) (71,72). The compensatory anti-inflammatory response syndrome (CARS) phase immediately follows, which involves an increase in antiinflammatory molecules and apoptosis of activated immune cells with a corresponding decrease in pro-inflammatory agents (73,74). This dampens the strength of host adaptive immunity, allowing possible infections with opportunistic microbes (74). Moreover, CD8 + T cell cytotoxicity is reduced during the CARS phase (75). Filaria parasites are known to down-modulate CD8 + T cell responses and trigger the production of anti-inflammatory cytokines like IL-10 (50). Thus, the alteration of T-cell responses during the SIRS and CARS phases in Escherichia coli-induced septic mice model with chronic L. sigmodontis infection has been recently explored (76). Interestingly, the weakening of CD8 + T cell cytotoxicity through sepsis attack was not promoted by chronic filarial infection (76).

HOW FILARIAL PARASITES COULD RE-PROGRAM CD8 + T CELLS
The transcriptional factors that regulate CD8 + T cell phenotypes and functions are important, particularly during chronic infections (44). How filarial parasites influence the differentiation and subsequent proliferation of naive CD8 + T cells is not clear and remain to be established. However, like all other forms of CD8 + T cell activation, the presence of a suitable peptide with an MHC class I molecule drives CD8 + T cell proliferation and differentiation via cross-presentation, given that the filarial parasites are extracellular. An effective CD8 + T cell challenge has been implicated in the clearance against viral infections (77) and helminth infections (78) and similar mechanisms could be applied to filarial parasites. Given that filarial pathogens live long in their host, sustained antigen stimulation may alter the differentiation program of CD8 + T cells and render them exhausted as previously documented in other infection scenarios (77,78). These exhausted CD8 + T cells are functionally poor since they have reduced lethal impact against their targets. In essence, this could be a possible scenario in filarial infections, where some compromises could be achieved to reduce tissue damage since filarial worms generally survive for at least 10 years in the host. Exhausted CD8 + T cells are predominant in chronic infections (77,79).
Experimental investigation of the functional relevance of regulatory CD8 + T cells during infection has advanced, following growth in phenotypic characterization of CD8 + T cells subtypes. Activated CD8 + T cells release granzyme B, which plays a seminal role during cytotoxic functions. More recently, granzyme B in the regulatory T cell compartment has been found to mediate the suppression of antigen-specific CD8 + T cells in viral infection (77). In the same study, increased numbers of antigen-specific CD8 + T cells in the lungs of granzyme Bdeficient mice were observed, suggesting that granzyme B in Tregs cells regulates either the initiation of antigen-specific CD8 + T cells responses or the down-modulation of terminal effector cells or a combination of both. Further, granzyme B has been implicated in enhancing susceptibility during the filarial nematode, L. sigmodontis infection (78). Additionally, results from field studies also suggest the suppressive role of granzyme B in human onchocerciasis (79). In this study, the authors observed a high expression of granzyme B and Foxp3 Tregs in the generalized hyporeactive than the hyperreactive individuals (79).
Although the underlying mechanisms dictating CD8 + T cell in the presence of filarial antigens is unknown, the fact that increased expression of granzyme B has been associated with immunomodulatory factors such as FoxP3 and TGF-b during filarial infections are good indicators of some immunological cross-talk with CD8 + T cell serving as a key player. Thus, we speculate a possible interplay between CD8 + T cells and granzyme B during filarial infection in a regulatory fashion among individuals with patent infection. Soluble CD8 (sCD8) released upon activation of CD8 + T cells has been documented to reflect filarial disease severity (42,43).
The developmental stages of filarial parasites are complex and are known to regulate several aspects of the cell-mediated immune responses mounted by the host. Emerging evidence shows that CD8 + T cells contribute to immune responses against the filaria parasites at the early stages of infection, but are highly regulated to favor parasite survival (31). Given this, a very plausible therapeutic approach would be to prime potential prophylactic vaccine candidates to induce sufficient CD8 + T cells and associated memory responses which can have detrimental effects on the establishment of the parasites (15). Identifying CD8 + T cell epitopes within filarial antigens could be achieved through immunoinformatics predictions and experimental validations or by screening a library of peptides spanning the complete antigen sequence (83). Recently, our group identified a CD8 + T cell peptide ( 146 KPWENFMRV 154 ) within onchocystatin, as part of a multi-epitope vaccine candidate for onchocerciasis, which demonstrates promising potential based on bioinformatics analysis (98). For most defined T cell antigens, the peptide sequences are usually similar to the corresponding sequences in related filarial parasites which may ensure cross-reaction (98)(99)(100).
To accelerate the introduction of filaria vaccines into clinical practice, there is the need to enhance the immunogenicity of potential candidates by boosting their ability to induce CD8 + T cell responses. Vaccine development against filarial infections is focused on multi-antigenic or cocktail vaccine formulations, and their cytotoxic mechanisms can be enhanced by conjugating peptides to adjuvants such as TLR ligands (101).

CONCLUDING REMARKS
The current understanding of CD8 + T cell-mediated immunity against filarial parasites suggests a type 2-dependent immunity. The CD8 + T cell cytotoxic activities appear to be effective in the early stages of filarial infections. Available evidence from animal models and human studies show that CD8 + T cells are involved in the immunomodulatory mechanisms that drive the pathogenesis of filarial infections. The mechanistic role of CD8 + T cells in filarial pathology could be associated with its poor regulation as a result of filaria-driven suppression, thus, promoting the secretion of pro-inflammatory agents. In the future, the ability to manipulate the CD8 + T cell response could have major implications, especially towards developing vaccines against filarial infections. Understanding the dynamics of the CD8 + T cells response to infections would help define manipulations that would be optimal for establishing protection against these complex extracellular nematodes. Of note, the recent advances regarding the importance of blocking T cellderived cytokines or receptors, for instance, provide compelling evidence towards the development of potential therapeutics (102), which could be explored in filarial infections. Here, we have discussed evidence regarding the potential protection of CD8 + T cells against filarial infections as well suppression of same during co-infections; thus, suggesting a need for further studies in the future.

AUTHOR CONTRIBUTIONS
Conceptualization: AK. Writing -Original Draft: AK, EA, and KK. Writing -Review & Editing: AK, EA, KK, and SA. All authors contributed to the article and approved the submitted version.

ACKNOWLEDGMENTS
We are indebted to the staff of the Kwarteng Lab at Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR) for their unflinching support in the preparation of this manuscript.