Modulation of T-cell costimulation as immunotherapy or immunochemotherapy in experimental visceral leishmaniasis

Abstract

CD40 ligand (CD40L)-deficient C57BL/6 mice failed to control intracellular Leishmania donovani visceral infection, indicating that acquired resistance involves CD40-CD40L signaling and costimulation. Conversely, in wild-type C57BL/6 and BALB/c mice with established visceral infection, injection of agonist anti-CD40 monoclonal antibody (MAb) induced killing of approximately 60% of parasites within liver macrophages, stimulated gamma interferon (IFN-gamma) secretion, and enhanced mononuclear cell recruitment and tissue granuloma formation. Comparable parasite killing was also induced by MAb blockade (inhibition) of cytotoxic T lymphocyte antigen-4 (CTLA-4) which downregulates separate CD28-B7 T-cell costimulation. Optimal killing triggered by both anti-CD40 and anti-CTLA-4 required endogenous IFN-gamma and involved interleukin 12. CD40L(-/-) mice also failed to respond to antileishmanial chemotherapy (antimony), while in normal animals, anti-CD40 and anti-CTLA-4 synergistically enhanced antimony-associated killing. CD40L-CD40 signaling regulates outcome and response to treatment of experimental visceral leishmaniasis, and MAb targeting of T-cell costimulatory pathways (CD40L-CD40 and CD28-B7) yields macrophage activation and immunotherapeutic and immunochemotherapeutic activity.

FIG. 1.

Course of L. donovani infection in livers of wild-type C57BL/6 (open circles) and CD40L^−/− (closed circles) mice. Results shown are means ± standard errors of the means (SEM) of values from two experiments with 8 to 10 mice for each time point. P is <0.05 for control versus CD40L^−/− mice at weeks 3, 4, and 8.

FIG. 2.

Histologic response to L. donovani infection in livers of wild-type C57BL/6 and CD40L^−/− mice. (A and B) Four weeks after infection, wild-type C57BL/6 mice (A) show formation of mature granulomas while CD40L-deficient mice (B) show no granuloma formation at parasitized foci (arrows). (C and D) At 8 weeks, granulomas have developed in CD40L-deficient mice but remain heavily parasitized (arrows). Panels A, B, and C, magnification of ×284; panel D, magnification of ×450.

FIG. 3.

Leishmanicidal effect of anti-CD40 and anti-CTLA-4 MAbs in wild-type BALB/c mice. On day 12 after infection, mice were injected once with either control IgG or one of three doses of anti-CD40 or anti-CTLA-4; liver burdens (LDU) were determined on day 21. LDU values for untreated mice on day 14 were used for comparison with day 21 LDU values for treated animals. Treatment of the anti-CD40 group was as follows: rat IgG (0.5 mg; open bar) or MAb at 0.1 mg (diagonally hatched bar), 0.25 mg (closed bar), or 0.5 mg (horizontally hatched bar). Treatment of the anti-CTLA-4 group was as follows: hamster IgG (0.5 mg; open bar) or MAb at 0.1 mg (diagonally hatched bar), 0.3 mg (closed bar), or 0.5 mg (horizontally hatched bar). Results shown are means ± SEM of results from two to three experiments with 7 to 16 mice per group. *, P of <0.05 versus day 14 LDU value and versus day 21 LDU value for IgG-treated controls. **, P of <0.05 versus day 21 LDU value for IgG-treated controls.

FIG. 4.

Extended antileishmanial effects of anti-CD40 and anti-CTLA-4 MAb treatment. Wild-type BALB/c mice were injected once 12 days after infection with 0.25 mg of rat IgG (open circles) or anti-CD40 (closed circles) or 0.5 mg of hamster IgG (open squares) or anti-CTLA-4 (closed squares). Results shown are means ± SEM of results from two experiments with seven to eight mice per time point. P is <0.05 for results with anti-CD40 and anti-CTLA-4 at both days 21 and 28 versus the day 12 LDU value.

FIG. 5.

Serum IFN-γ levels on day 21, after MAb treatment on day 12. Wild-type BALB/c mice were injected once 12 days after infection with either 0.25 mg of rat IgG (horizontally hatched bar) or anti-CD40 (closed bar) or 0.5 mg of hamster IgG (diagonally hatched bar) or anti-CTLA-4 (open bar). Results shown are means ± SEM of results from two experiments with 6 to 10 mice. *, P of ≤0.05 versus day 21 value for IgG-treated controls.

FIG. 6.

Liver histologic responses to anti-CD40 and anti-CTLA-4 MAbs in infected wild-type BALB/c mice. Mice were injected on day 12 with 0.25 mg of rat IgG or anti-CD40 or 0.5 mg of hamster IgG or anti-CTLA-4, and sections were prepared on day 21. (A and C) Infected foci in rat IgG-treated mice show early, developing granulomas. (B and D) In contrast, in anti-CD40-treated mice, parasitized foci are encased by large accumulations of mononuclear cells (arrows). (E and F) Developing granulomas in hamster IgG-treated liver (E) versus more-mature-appearing granulomas in anti-CTLA-4-injected animal (F). Panels A and B, magnification of ×180; panels C to F, magnification of ×284.

FIG. 7.

Histologic reactions in livers of uninfected and infected wild-type BALB/c mice 9 days after a single injection of 0.25 mg of anti-CD40 MAb. Tissue response in uninfected mice (A and C) is modest compared to the pronounced reaction in infected mice (B and D) which were injected on day 12 as described in the legend to Fig. 6. Panels A and B, magnification of ×45; panels C and D, magnification of ×180.

FIG. 8.

Liver histologic response to anti-CD40 on day 21 in granuloma-deficient ICAM-1^−/− and IFN-γ^−/− mice. Mice were injected on day 12 after infection with 0.25 mg of rat IgG or anti-CD40. (A and B) In ICAM-1^−/− mice, IgG had no effect on deficient granuloma formation (arrow) (A), while anti-CD40 induced a granulomatous reaction at parasitized foci (B). (C to E) Rat IgG-treated IFN-γ-deficient mice show no tissue inflammatory response at infected foci (arrows) (C), while anti-CD40-induced granulomas (D) remain heavily parasitized (arrow) (E). Panels A and B, magnification of ×290; panels C and D, magnification of ×184; panel E, magnification of ×460.

FIG. 9.

Treatment with anti-CD40 or anti-CTLA-4 enhances effect of antimony (Sb) in wild-type BALB/c mice. Liver parasite burdens were determined 12 days after infection, and mice were then injected once with either control rat or hamster IgG or suboptimal anti-CD40 (0.1 mg) or anti-CTLA-4 (0.3 mg). Mice either were not treated further or received low-dose Sb on day 14. Liver burdens on day 21 were compared to day 14 levels (1,202 ± 58 LDU; n = 12) to determine percent parasite killing. Results are from two to three experiments with 7 to 12 mice per group. P is <0.05 for results with both anti-CD40 plus Sb and anti-CTLA-4 plus Sb versus (i) day 14 LDU value for untreated mice and (ii) day 21 results with all other indicated treatments.