Hybrid cell vaccination resolves Leishmania donovani infection by eliciting a strong CD8+ cytotoxic T-lymphocyte response with concomitant suppression of interleukin-10 (IL-10) but not IL-4 or IL-13

Abstract

There is an acute dearth of therapeutic interventions against visceral leishmaniasis that is required to restore an established defective cell-mediated immune response. Hence, formulation of effective immunotherapy requires the use of dominant antigen(s) targeted to elicit a specific antiparasitic cellular immune response. We implemented hybrid cell vaccination therapy in Leishmania donovani-infected BALB/c mice by electrofusing dominant Leishmania antigen kinetoplastid membrane protein 11 (KMP-11)-transfected bone marrow-derived macrophages from BALB/c mice with allogeneic bone marrow-derived dendritic cells from C57BL/6 mice. Hybrid cell vaccine (HCV) cleared the splenic and hepatic parasite burden, eliciting KMP-11-specific major histocompatibility complex class I-restricted CD8+ cytotoxic T-lymphocyte (CTL) responses. Moreover, splenic lymphocytes of HCV-treated mice not only showed the enhancement of gamma interferon but also marked an elevated expression of the Th2 cytokines interleukin-4 (IL-4) and IL-13 at both transcriptional and translational levels. On the other hand, IL-10 production from splenic T cells was markedly suppressed as a result of HCV therapy. CD8+ T-cell depletion completely abrogated HCV-mediated immunity and the anti-KMP-11 CTL response. Interestingly, CD8+ T-cell depletion completely abrogated HCV-induced immunity, resulting in a marked increase of IL-10 but not of IL-4 and IL-13. The present study reports the first implementation of HCV immunotherapy in an infectious disease model, establishing strong antigen-specific CTL generation as a correlate of HCV-mediated antileishmanial immunity that is reversed by in vivo CD8+ T-cell depletion of HCV-treated mice. Our findings might be extended to drug-nonresponsive visceral leishmaniasis patients, as well as against multiple infectious diseases with pathogen-specific immunodominant antigens.

FIG. 1.

HCV therapy in L. donovani-infected mice confers sterile protection that is completely abrogated by CD8⁺ T-cell depletion. For vaccination, L. donovani-infected BALB/c mice were treated 60 days postinfection with i.v. injections of any one type of the cells mentioned in Materials and Methods. Therapeutically treated mice were sacrificed 15 days after the last injection. The data represent the means ± the SE for 24 animals per group. Figures in parentheses above the bars indicate percentages of mice showing complete absence of parasites in the spleen or liver as determined by serial dilution assay. (A and B) Comparative dose-response results for mixed cells and electrofused cells (HCV) in L. donovani (AG83)-infected BALB/c mice (2 months postinfection) receiving one to six i.v. injections of unpulsed (mixed) cells or pulsed (hybrid cells) in terms of reduction of parasite burden (bar diagram) and sterile cure (data in parentheses) in the spleen (A) and liver (B). In infected animals, the splenic parasite burden ranged from 4 × 10⁻⁷ ± 0.42 × 10⁻⁷ (at the time of assessing the result of one HCV dose) to 15.4 × 10⁻⁷ ± 0.34 × 10⁻⁷ (at the time of assessing the result of six doses of HCV). Similarly, the hepatic parasite burden in infected mice ranged from 7.6 × 10⁻⁷ ± 1.06 × 10⁻⁷ (at the time of assessing the result of one HCV dose) to 27.7 × 10⁻⁷ ± 1.46 × 10⁻⁷ (at the time of assessing the result of 6 doses of HCV). (C and D) Organ parasite burden of infected BALB/c mice receiving six i.v. injections of either HCV (with or without depletion of CD8⁺ T cells) or other indicated cell types at 5-day interval. At 24 h before each HCV therapy, the mice were treated with 1 mg of anti-CD8 (2.43) antibody for successful depletion of CD8⁺ T cells. Splenic (C) and hepatic (D) parasite burdens of infected BALB/c mice therapeutically treated with the above-mentioned cell types. The percentages of mice showing sterile cure are indicated in parentheses above the bars of the respective groups.

FIG. 2.

HCV elicits a vigorous anti-KMP-11-specific CTL response in L. donovani-infected mice that is completely abrogated by CD8⁺ T-cell depletion. CTL generation was assayed by a standard ⁵¹Cr release assay in mice receiving six doses of HCV (with or without depletion of CD8⁺ T cells) with the other indicated groups. Spontaneous release was <5%. The results are representative of five individual experiments (five animals/group/experiment), and the data represent the mean ± the SE. At both 50:1 and 6:1 E:T ratios, hybrid cell-vaccinated mice showed significant lysis (*, P < 0.0005; and **, P < 0.0001, respectively) with respect to infected controls, while syngeneic KMP-11-transfected APC-vaccinated mice showed statistically significant lysis of targets only at 50:1 (o, P < 0.005) compared to infected controls. Anti-CD8 Abs treated hybrid cell vaccinated mice were unable to generate any effective CTL response as reflected in the figure.

FIG. 3.

HCV-treated mice restore T-cell anergy to SLA that is significantly impaired by CD8 depletion. The results are representative of three individual experiments (five animals/group), and the data represent the means of triplicate wells ± the SE. Proliferation was measured by determining the [³H]thymidine incorporation. At a 5-μg/ml SLA stimulation, the double asterisks represent P < 0.0001 for HCV and the single asterisk represents P = 0.1219 for HCV-treated CD8⁺ T-cell-depleted mice with respect to the infected group.

FIG. 4.

(A) HCV therapy downregulates the IL-10 transcript from splenic CD4⁺ T cells but not IL-4 and IL-13. Real-time PCR analysis was performed to detect the fold change in mRNA expression profiles of IFN-γ (a), along with the Th2 cytokines IL-10 (b), IL-4 (c), and IL-13 (d), in MACS-purified CD4⁺ splenic lymphocytes (5 × 10⁶/ml) of infected, HCV-treated mice (with or without depletion of CD8⁺ T cells) versus normal BALB/c mice in response to 18 h of stimulation with rKMP-11 protein (5 μg/ml). The results are representative of eight individual mice per group analyzed separately, and the data represent the means ± the SE. The expression of a target gene was quantified by using the formula described in Materials and Methods. (B) HCV therapy significantly upregulates GATA-3 along with T-bet. The results of an analysis of the fold change in mRNA expression of the Th1 polarizing transcription factor T-bet (a) and the Th2 polarizing transcription factor GATA-3 (b) in splenic CD4⁺ T lymphocytes of infected, HCV-treated mice versus normal BALB/c mice are shown. The results are representative of eight individual mice per group analyzed separately, and data represent the means ± the SE. (C) Only the IL-10 protein level is significantly reduced but Th1 cytokine IFN-γ and Th2 cytokines IL-4 and IL-13 are elevated after HCV therapy. ELISA measurements of the protein levels of IFN-γ (a), IL-10 (b), IL-4 (c), and IL-13 (d) were done with the supernatants of splenic lymphocytes (5 × 10⁶/ml) depleted of macrophages from mice receiving therapeutic HCV treatment (with or without depletion of CD8⁺ T cells) and from infected mice (four animals/group) stimulated with rKMP-11 protein (5 μg/ml). The results are representative of three experiments, and the data represent the means ± the SE.