*3.2.1. Lymphocytes*

The major subpopulations of lymphocytes are CD4+ T cells (Th1, producing IFN-γ and TNFα; Th2, secreting IL-4, IL-5, and IL-13; and Th17, producing IL-17 and IL-22) and CD8+ T cells. Antigen-presenting cells submit *Leishmania* spp. antigens to CD4+ T cells via MHC class II, and because the agent is an intracellular parasite, there may be presentation via MHC class I with activation of CD8+ T cells as well [69].

The role of CD4+ T cells in the response to visceral leishmaniasis (VL) has not been fully elucidated. Research carried out so far points to a mixed response (Th1/Th2) during infection [84, 85]. It is reported, however, that control of infection depends on Th1 cells that activate macrophages, promoting elimination of intracellular parasites [86], whereas Th2 cells direct the immune system toward humoral response and negatively regulate cellular immunity, promoting Th1 cell anergy [87].

CD8+ cells constitute a significant population in cellular immunity against canine visceral leishmaniasis (CVL), outnumbering CD4+ cells in the dermis [87]. They play an important role in resistance to infection. Guerra et al. [88] associated phenotypic changes with tissue parasitism in the spleen and skin of infected dogs. They noticed that the high frequency of CD8+ circulating lymphocytes is directly related to low splenic parasitism and that there is a negative correlation between CD8+ T cells with skin parasite density, indicating that this cell type relates to resistance against LVC [88].

The regulatory role of FOXP3+ CD4+ T cells in canine VL has not been fully elucidated; however, reduction of Treg cell percentage in peripheral blood of infected dogs has been observed [89]. Silva et al. [90] reported increased production of IL-10 by splenic Treg cells of dogs with LV, along with decrease in the total number of T cells when compared to healthy dogs. The findings suggest that Treg cells are a major source of IL-10 in the spleen and participate in the modulation of immune response, while a small percentage of these cells in infected dogs may be related to persistent immune activation [90].

T-cell exhaustion (CD4+ and CD8+ cells) in peripheral blood of dogs with LV, followed by reduction of the expression of cytokines (such as IFN-γ), was recently demonstrated. This phenomenon, called cell exhaustion, is mainly mediated by high expression of programmed death protein ("programmed cell death 1," PD-1) and may be related to the strong immunosuppression observed in advanced stages of the disease, corresponding to increase in symptomatology of VL [86].

The role of B cells in CVL is unclear. However, increases in CD21+ B cell, CD4+ T cell, and CD8+ T cell levels are frequently reported, as for the clinical asymptomatic form. Lower frequency of B cells and monocytes in the bloodstream is an important marker of severe disease, while increased levels of CD8 + T cells appear to be the most important phenotypic feature for asymptomatic clinical presentation [74]. Among symptomatic animals, decrease in CD21+ Bcell count associates with decreased CD4+ T cells, which does not occur with asymptomatic or disease-free control animals, suggesting that the decay of immunity in leishmaniasis may be related to decrease in the CD4+ T-cell population [91].

Although the relationship between a pattern of anti-*Leishmania* humoral response and resistance or susceptibility to LV is not well defined [92], immunoglobulin profile may appear as a biomarker for monitoring clinical prognosis and tissue parasitic density, as it is associated with the progression of clinical signs and increase of parasites in lymphoid organs [73]. Reis et al. reported increase in IgG1 levels associated with the asymptomatic clinical presentation and poor tissue parasitic load, while the symptomatic clinical form would be characterized by high parasitic load and high levels of anti-*Leishmania* IgG, IgG2, IgM, IgA, and IgE [73, 74]. Teixeira-Neto et al. reported similar results, with symptomatic dogs demonstrating high levels of anti-*Leishmania* IgG2 antibodies, whereas the asymptomatic showed higher IgG1 titles, when compared to IgG2 [93]. Production of IFN-γ is associated with the production of IgG1 [94], which may indicate a Th1 response profile.
