CD4+ T cells play a dominant role in protection against New World leishmaniasis induced by vaccination with the P-4 amastigote antigen

Abstract

Immunodepletion studies of P-4-vaccinated mice indicate that CD4+ and not CD8+ T cells are critical for protection against Leishmania pifanoi (Leishmania mexicana complex). Although a moderate CD8+ T-cell response is elicited by vaccination, CD4+ T cells are the dominant responding population in vitro and at the cutaneous site of infection. These protective T cells produce gamma interferon (IFN-gamma), macrophage migration inhibitory factor (MIF), and tumor necrosis factor/lymphotoxin (TNF/LT), each of which significantly contributed to intracellular parasite destruction in vitro. These results indicate that a singular CD4+ T-cell response (IFN-gamma, MIF, and/or LT/TNF) can provide protection against New World cutaneous leishmaniasis.

FIG. 1.

Evaluation of the contribution of CD4 and CD8 T cells to protection induced by vaccination with the P-4 antigen. (A) BALB/c mice (10/group) were immunized three times at biweekly intervals with purified P-4 antigen along with P. acnes as an adjuvant. Six weeks after the last immunization, depletion of CD4⁺ and CD8⁺ T cells was performed as described previously (2). FACS analyses of uninfected mice indicated that 98% of CD4 T cells and 96% of CD8 T cells had been depleted. Mice were then infected with 10⁶ late log L. pifanoi promastigotes. Disease progression was monitored by measurements of lesion development, which is expressed as a ratio of the size of the infected right foot to that of the uninfected left foot. Data shown are average values ± standard errors and are representative of two independent experiments. (Using a Student t test: *, P < 0.02 in comparison to P-4 vaccinated; **, P < 0.002 for P-4 vaccinated and CD8-depleted P-4 vaccinated in comparison to adjuvant control.) (B) T-cell depletion and parasite challenge were performed as indicated for Fig. 1A. However, mice were immunized either twice (2ximm) or three times (P-4) with P-4 antigen together with P. acnes. Groups of mice that were vaccinated twice with P-4 were depleted of either CD4⁺ or CD8⁺ T cells; depleted mice are denoted as 2x CD4depl and 2x CD8depl, respectively. Lesion sizes were measured at different time points after infection, as indicated. Ten mice were used per group. Data shown are averaged values ± standard errors for each group. (**, P < 0.002, and *, P < 0.02, compared to adjuvant control; $, P = 0.22, comparison of 2× P-4-vaccinated/ CD4-depleted mice to adjuvant control mice; P = 0.28, 2× P-4-vaccinated CD8-depleted mice compared to P-4-vaccinated mice). (C) Parasite burden analyses of lesions from the site of infection of 3×- P-4-vaccinated and nonvaccinated (adjuvant control) BALB/c mice (from experiment shown in A). The numbers of lesion parasites were determined by limiting-dilution assay at the indicated time points after infection. Data shown are means for six mice per group. Student t test: *, P < 0.02; **, P < 0.001; #, P > 0.05 (when compared to adjuvant control); $, P < 0.001, comparison of P-4-vaccinated and CD4-depleted P-4 vaccinated mice. Note: P = 0.3 when CD8-depleted P-4 vaccinated mice are compared to P-4-vaccinated mice (all three time points).

FIG. 2.

mRNA levels of cytokines in draining lymph nodes. Lymph node cells from control (P. acnes-immunized) and P-4-immunized mice were isolated after 4 weeks of infection and cultured for 72 h in the presence of P-4 antigen. mRNA expression in the cells for different cytokines of interest were determined by RNase protection assay. Intensity for each band (representing a cytokine) was measured by a densitometer. Data were normalized to the level of glyceraldehyde-3-phosphate dehydrogenase expressed in each sample. Each value represents the density of the cytokine relative to corresponding glyceraldehyde-3-phosphate dehydrogenase density, considered to be 100. These results are representative of three experiments.

FIG. 3.

Cytokine levels produced in response to infection in vaccinated and control mice. The IL-2 (A), IL-4 (B), IFN-γ (C), and TNF-α/LT (D) levels in spleen cell culture supernatants of control and P-4-vaccinated mice. Splenic T lymphocytes were isolated from different groups of mice after 4 weeks of infection and cultured with P-4 antigen as described previously (15). IL-2, IL-4, and IFN-γ levels in the supernatants were measured by enzyme-linked immunosorbent assay. Data shown are means of different cytokine levels in the supernatants from P-4 antigen-containing cultures in three duplicate wells. TNF/LT levels in the spleen culture supernatants were evaluated using a cytotoxicity assay employing MTT and the L929 cell line. The TNF/LT concentration required to achieve 50% cytotoxicity was considered 1 unit. These results are representative of three independent determinations. Student t test: *, P ≤ 0.01; **, P ≤ 0.001 (compared to adjuvant controls).

FIG. 4.

Cytokine regulation of macrophage-mediated L. pifanoi killing. Shown are the results of macrophage-Leishmania killing assays. Isolated T cells from either P. acnes-vaccinated or P-4 plus P. acnes-vaccinated BALB/c mice were incubated together with L. pifanoi-infected peritoneal exudate macrophages. Antibodies (anti-IFN-γ, anti-TNF-α/LT, and/or anti-MIF) were added to the cultures, as indicated, to a final concentration of 10 μg/ml for each antibody. After 72 h, macrophages were washed, dried, fixed with methanol, and stained with 4′,6′-diamidino-2-phenylindole. The number of parasites per 100 macrophages and the percentage of infected macrophages were determined microscopically. Results are the averaged values of three independent experiments. Student t test: **, P ≤ 0.01; *, P ≤ 0.05 (compared to T cells from P-4-vaccinated mice); in A, P = 0.5, and in B, P = 0.1, when adjuvant control is compared with combination of anti-IFN-γ, anti-TNF-α/LT, and anti-MIF.