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. 2018 Nov 19:8:393.
doi: 10.3389/fcimb.2018.00393. eCollection 2018.

Phenotypic and Functional Profiles of Antigen-Specific CD4+ and CD8+ T Cells Associated With Infection Control in Patients With Cutaneous Leishmaniasis

Adriana Egui  1 , Darién Ledesma  1 , Elena Pérez-Antón  1 , Andrés Montoya  2 , Inmaculada Gómez  1 , Sara María Robledo  2 , Juan José Infante  3 , Ivan Darío Vélez  2 , Manuel C López  1 , M Carmen Thomas  1
Affiliations

Phenotypic and Functional Profiles of Antigen-Specific CD4+ and CD8+ T Cells Associated With Infection Control in Patients With Cutaneous Leishmaniasis

Adriana Egui et al. Front Cell Infect Microbiol. .

Abstract

The host immunological response is a key factor determining the pathogenesis of cutaneous leishmaniasis. It is known that a Th1 cellular response is associated with infection control and that antigen-specific memory T cells are necessary for the development of a rapid and strong protective cellular response. The present manuscript reports the analysis of the functional and phenotypic profiles of antigen-specific CD4+ and CD8+ T cells from patients cured of cutaneous leishmaniasis (CL), patients with an active process of cutaneous leishmaniasis, asymptomatic individuals with a positive Montenegro test and healthy donors (HD). Peripheral blood mononuclear cells (PBMCs) from the patients exhibited a lymphoproliferative capacity after stimulation with total soluble protein from either Leishmania panamensis (SLpA) or Leishmania infantum (SLiA) or with a recombinant paraflagellar rod protein-1 (rPFR1). Higher frequencies of antigen-specific TNAIVE cells, mainly following stimulation with rPFR1, were observed in asymptomatic and cured patients than in patients with active cutaneous leishmaniasis, while T cells from patients with active cutaneous leishmaniasis showed a higher percentage of effector memory T cells (TEM for CD4+ T cells and TEMRA for CD8+ T cells). The amount of antigen-specific CD57+/CD8+ TEMRA cells in patients with active cutaneous leishmaniasis was higher than that in cured patients and asymptomatic subjects. Regarding functionality, a more robust multifunctional CD8+ T cell response was detected in cured patients than in those with active cutaneous leishmaniasis. Moreover, cured patients showed a significant increase in the frequency of cells expressing a Th1-type cytotoxic production profile (IFN-γ+/granzyme-B/+perforin+). Patients with an active leishmaniosis process had a significantly higher frequency of CD8+ T cells expressing the inhibitory CD160 and 2B4 receptors than did cured patients. The expression profile observed in cured patients could be indicative of an imbalance toward a CD8+ Th1 response, which could be associated with infection control; consequently, the determination of this profile could be a useful tool for facilitating the clinical follow-up of patients with cutaneous leishmaniasis. The results also suggest a possible exhaustion process of CD8+ T cells associated with the evolution of Leishmania infection.

Keywords: CD8+ and CD4+ T-cells; Leishmania; Th1-cytokines; biomarkers; inhibitory receptors; leishmaniasis; paraflagellar rod protein-1; phenotype.

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Figures

Figure 1
Figure 1
Immunophenotyping of antigen-specific CD4+ and CD8+ memory T cells. Phenotypic characterization of CD4+ (A,C) and CD8+ (B,D) T cells from asymptomatic subjects with a positive Montenegro skin test (Mont +, n = 4), patients cured of cutaneous leishmaniasis (Cured, n = 5) and patients with active cutaneous leishmaniasis (Active CL, n = 4). This profile was determined after stimulation with SLA from L. panamensis (A,B) or with the rPFR1 antigen (C,D). According to CD45RA, CD27, and CCR7 expression, the cells were grouped as TNAIVE (CD8+CD45RA+CCR7+), TEMRA (CD8+CD45RA+CCR7−), TEM (CD8+CD45RA−CCR7−), and TCM (CD8+CD45RA−CCR7+). The boxes (25th−75th percentiles) and whisker plots (using the Tukey's method) show the median frequency and range of the CD4+ and CD8+ T cells. Statistical analyses were carried out using the Mann–Whitney U-test. Statistically significant differences were indicated by *p < 0.05 and **p < 0.01.
Figure 2
Figure 2
Expression of the senescence marker CD57 in CD4+ and CD8+ effector memory T cells. (A) Total number of SLpA-specific CD4+ and CD8+ T cells that expressed the marker CD57+ within the TEMRA and TEM effector memory subpopulations. (B) Total number of rPFR1-specific CD4+ and CD8+ T cells that expressed the marker CD57+ within the TEMRA and TEM effector memory subpopulations. Analyses were carried out in asymptomatic subjects with a positive Montenegro skin test (Mont +, n = 4), patients cured of cutaneous leishmaniasis (Cured, n = 5) and patients with active cutaneous leishmaniasis (Active CL, n = 4). The boxes (25th−75th percentiles) and whisker plots (using Tukey's method) show the median and range of the CD4+ and CD8+ T cells. Statistical analyses were carried out using the Mann–Whitney U-test. Statistically significant differences are indicated by *p < 0.05.
Figure 3
Figure 3
Multifunctional capacity of Leishmania-specific CD4+ and CD8+ T cells. Multifunctional activity of CD4+ (A) and CD8+ (B) T cells from asymptomatic subjects with a positive Montenegro skin test (Mont +, n = 4), patients cured of cutaneous leishmaniasis (Cured, n = 5) and patients with active cutaneous leishmaniasis (Active CL, n = 4). This profile was determined using a five-function assay to simultaneously measure the expression of IFN-γ, IL-2, TNF-α, granzyme B, and perforin after stimulation with SLA from L. panamensis. The color of each portion of the pie charts depicts the number of molecules produced by CD4+ and CD8+ T cells in response to L. panamensis antigens. The arcs of the pie charts represent the proportions of cells expressing each of the analyzed molecules. The percentage of unstimulated CD4+ and CD8+ T cells expressing these molecules (basal response) was subtracted from the value obtained following antigen stimulation.
Figure 4
Figure 4
Functional capacity of Leishmania-specific CD4+ and CD8+ T cells. Cytokine production (IFN-γ, IL-2, and TNF-α) by CD4+ (A) and CD8+ (B) T cells from asymptomatic subjects with a positive Montenegro skin test (Mont +, n = 4), patients cured of cutaneous leishmaniasis (Cured, n = 5) and patients with active cutaneous leishmaniasis (Active CL, n = 4) in response to SLA from L. panamensis. The colors in the pie charts depict the presence of one, two, or three functions. The arcs of the pie charts represent the proportions of cells expressing each of the molecules under study.
Figure 5
Figure 5
Cytotoxic profile of Leishmania-specific CD8+ T cells. Functional activity of CD8+ T cells determined by perforin and granzyme B expression after stimulation with SLA from L. panamensis in asymptomatic subjects with a positive Montenegro skin test (Mont +, n = 4), patients cured of cutaneous leishmaniasis (Cured, n = 5) and patients with active cutaneous leishmaniasis (Active CL, n = 4). The colors in the pie charts depict the proportion of cells expressing each of the analyzed molecules.
Figure 6
Figure 6
Percentage of CD8+ T cells expressing or coexpressing inhibitory receptors. (A) Frequency of CD8+ T cells expressing 2B4, CD160, CTLA-4, PD-1, and TIM-3 in asymptomatic subjects with a positive Montenegro skin test (Mont +, n = 4), patients cured of cutaneous leishmaniasis (Cured, n = 5), patients with active cutaneous leishmaniasis (Active CL, n = 4) and healthy donors (HD, n = 10). (B) Coexpression of 2B4, CD160, CTLA-4, PD-1, and TIM-3 by CD8+ T cells. The boxes (25th−75th percentiles) and whisker plots (using Tukey's method) show the median and range of the frequency of inhibitory receptors on T cells. Statistical analyses were carried out using the Mann–Whitney U-test. Statistically significant differences are indicated by *p < 0.05, **p < 0.01, and ***p < 0.001.
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