Cutting Edge: Small Molecule CD40 Ligand Mimetics Promote Control of Parasitemia and Enhance T Cells Producing IFN-γ during Experimental Trypanosoma cruzi Infection

Mohammed Habib, Magali Noval Rivas, Mustapha Chamekh, Sébastien Wieckowski, Weimin Sun, Alberto Bianco, Nathalie Trouche, Olivier Chaloin, Hélène Dumortier, Michel Goldman, Gilles Guichard, Sylvie Fournel and Bernard Vray
J Immunol June 1, 2007, 178 (11) 6700-6704; DOI: https://doi.org/10.4049/jimmunol.178.11.6700

Abstract

Host resistance to Trypanosoma cruzi infection depends on a type 1 response characterized by a strong production of IL-12 and IFN-γ. Amplifying this response through CD40 triggering results in control of parasitemia. Two newly synthesized molecules (<3 kDa) mimicking trimeric CD40L (mini CD40Ls-1 and -2) bind to CD40, activate murine dendritic cells, and elicit IL-12 production. Wild-type but not CD40 knockout mice exhibited a sharp decrease of parasitemia and mortality when inoculated with T. cruzi mixed with miniCD40Ls. Moreover, the immunosuppression induced by T. cruzi infection was impaired in mice treated with miniCD40Ls, as shown by proliferation of splenic lymphocytes, percentage of CD8+ T cells, and IFN-γ production. Mice surviving T. cruzi infection in the presence of miniCD40L-1 were immunized against a challenge infection. Our results indicate that CD40L mimetics are effective in vivo and promote the control of T. cruzi infection by overcoming the immunosuppression usually induced by the parasites.

Trypanosoma cruzi, Chagas’ disease etiological agent, is a hemoflagellate parasitic protozoa that infects humans and other mammals where it multiplies as amastigotes within various cells. Human disease can be mimicked by experimental infection of BALB/c mice displaying an acute phase with parasitemia and mortality, followed by a chronic phase during which parasites become undetectable in peripheral blood while persisting in tissues and inducing pathological manifestations (1).

CD40L (or CD154) belongs to the TNF superfamily and is mainly expressed by activated CD4+ T lymphocytes, whereas its cognate receptor CD40 is expressed by APC such as dendritic cells (DC).3 CD40L-CD40 interaction plays a major role in immunity against intracellular pathogens such as Leishmania amazonensis and T. cruzi (2, 3, 4) by promoting an effective type 1 response. We have previously reported that 1) coinjection of CD40L-transfected 3T3 fibroblasts and T. cruzi trypomastigotes dramatically reduced both parasitemia and mortality of T. cruzi-infected mice through a cascade involving IL-12, IFN-γ, and NO (3), and 2) CD40L gene-transfected T. cruzi trypomastigotes induced very low parasitemia and no mortality when compared with the wild-type strain (Y strain) or to the Y strain transfected with the pTEX vector alone. Furthermore, the proliferative capacity and the secretion of IFN-γ were preserved in spleen cells (SC) from CD40L-infected mice compared with controls (4).

By taking advantage of the 3-fold symmetry of homotrimeric CD40L, we have recently designed small trivalent molecules (<3 kDa) that can bind to CD40 and reproduce similar functional properties of soluble CD40L (5). In this study, we have investigated whether these small molecules namely miniCD40Ls −1 and −2 (5) can induce a protective immune response against experimental T. cruzi infection in mice.

Materials and Methods

MiniCD40Ls

MiniCD40Ls −1 and −2 (C1 and C2) were designed as previously described (5) by attaching a short CD40-binding motif (Lys143-Gly-Tyr-Tyr 146) to each arm of a trimeric C3-symmetric circular peptide architecture cyclo(Lys-D-Ala)3 for C1 and cyclo(β3-HLys) for C2 via an appropriate spacer (amino hexanoic acid residue for C1 and C2). Compounds 3 and 4 (C3 and C4), which correspond, respectively, to the circular core of C1 and to the CD40-binding motif attached to the spacer, do not bind to CD40 and were used as controls.

T. cruzi trypomastigotes, lysates, and mice infection

Two strains (Tehuantepec strain and Y strain) were used (4). Trypomastigote lysate was obtained by six freeze/thaw cycles and then adjusted to 20 μg/ml (6, 7). BALB/c, C57BL/6, and C57BL/6 CD40 knockout mice were inoculated i.v. with 1 × 103 trypomastigotes/mouse (Tehuantepec strain) in 0.9% NaCl or mixed with C1, C2, C3, or C4 at the indicated concentrations. Mice (n = 5) inoculated with trypomastigotes (Tehuantepec strain) mixed with C1 at day 0 were inoculated with a different strain of T. cruzi (Y strain 1 × 103 trypomastigotes/mouse) at day 117. Parasitemia was monitored by counting the trypomastigotes in blood samples every week. Survival rates were determined daily.

Cytotoxicity of miniCD40Ls

The possible cytotoxicity of miniCD40Ls on parasite infectivity was tested by investigating the capacity of T. cruzi trypomastigotes incubated with C1, C2, C3, or C4 to infect Vero cells and mouse peritoneal macrophages (MPM) (4). Briefly, Vero cells (5 × 105 cells/well) and MPM (2 × 105 cells/well) were cultured in the presence of trypomastigotes (10:1, parasite:cell ratio), then incubated with C1, C2, C3, or C4 (200 μg/100 μl in 0.9% NaCl) for 8 h at 37°C. After 16 h, the cultures were washed to remove free parasites. For Vero cells, trypomastigotes released were counted on a Thoma chamber over 21 days. For MPM, cultures were fixed with methanol and stained with Giemsa. At least 200 MPM were microscopically counted to determine the percentage of infected cells and the mean of amastigotes per infected cell (4).

Immunophenotyping and intracellular detection of IFN-γ

SC were either labeled (8) directly or after stimulation with T. cruzi lysate (20 μg/ml) for 56 h at 37°C in the presence of brefeldin A (10 μg/ml; 3 h). FITC-conjugated anti-CD3ε chain for CD3+, allophycocyanin-conjugated L3T4/RM4-5 for CD4+, PerCP-conjugated Ly-2, 53-6.7 for CD8+, and PE-conjugated anti-IFN-γ were from BD Pharmingen. Flow cytometry was performed using FACSCalibur (BD Biosciences).

SC cultures were cultured in the presence of T. cruzi lysate (20 μg/ml) for 96 h. Cells were pulsed with 2.5 μCi/well of tritiated thymidine (MP Biomedicals) during the last 16 h of culture (4). [3H]Thymidine uptake was measured using an automated scintillation counter (Packard Microplate scintillation counter; Packard Instrument).

Culture and maturation of DC line D1 or bone marrow-derived DC

A total of 2 × 105 cells/ml was cultured for 24 h in fresh medium containing various inducers (9). Cells were harvested with PBS containing 2 mM EDTA and stained with anti-mouse PE-conjugated CD54, CD80, and CD86 and anti-mouse FITC-conjugated CD40 (BD Pharmingen). Cells were analyzed by flow cytometry with a FACSCalibur using the CellQuest 3.3 software (BD Biosciences). Data were processed with the WinMDI 2.8 freeware (J. Trotter; The Scripps Research Institute).

ELISA

IFN-γ measurement in SC supernatants was performed after 80 h of stimulation with T. cruzi lysates (7), according to the manufacturer’s instructions (CytoSets; BioSource International). IL-12 secretion in D1 cell supernatants, after 48 h of stimulation, was evaluated by sandwich ELISA (5) using Ab from BD Pharmingen and polyvinyl plates (BD Falcon).

Statistical analysis

Data were compared using the Mann-Whitney U test. The survival analyses were obtained with Kaplan-Meier curves and Gehan’s generalized Wilcoxon test.

Results and Discussion

MiniCD40Ls-1 and -2 decrease T. cruzi infection and induce survival of inoculated mice

To investigate the potential effects of trimeric molecules mimicking CD40L homotrimer, we inoculated BALB/c mice with trypomastigotes (Tehuantepec strain, 1 × 103 parasites/mouse) alone or mixed with C1 or C2 or with the control molecules C3 or C4. Inoculation in the presence of C1 or C2 (100 μg/mouse) induced a significant decrease in parasitemia at days 14, 21, 28, and 35 postinfection. Eighty percent of mice treated with C1 or C2 were still alive at day 42 postinfection in comparison with 20% in the absence of miniCD40L or in the presence of C3 or C4 (Fig. 1⇓).

FIGURE 1.
FIGURE 1.

Inoculation with T. cruzi trypomastigotes mixed with C1 or C2 results in decreased parasitemia and mouse survival. Mice were inoculated i.v. with trypomastigotes (1 × 103/mouse) mixed with 100 μg in 100 μl of C1 (n = 34) or C2 (n = 8) or C3 (n = 7) or C4 (n = 28) or with NaCl 0.15 M (n = 41) at day 0. The numbers of trypomastigotes in blood, as well as the percentage of surviving mice (insert), were measured at various days postinfection. For parasitemia, the p values obtained with C1 or C2 vs C4 or NaCl were statistically significant; p < 0.0009 at days 14, 21, and 28 and C1 or C2 vs C3, p < 0.01 at days 14 and 28. For the mortality, statistically significant differences were obtained with C1 or C2 vs C4 or NaCl, p < 0.0001 and C1 or C2 vs C3, p < 0.008. No significant differences were obtained for C1 vs C2 or C4 or C3 vs NaCl both in parasitemia or mortality. Data are means of three independent or one representative experiments; *, p < 0.05, **, p < 0.01, and ***, p < 0.001.

To investigate the possible cytotoxic effect of miniCD40Ls on trypomastigotes, Vero cells and MPM were incubated with trypomastigotes mixed with miniCD40Ls. In this setting, trypomastigote infection was not decreased (data not shown).

Interestingly, a second injection of C1, 7 days after the initial inoculation, improved the effect of the ligand (Table I⇓). Trypomastigotes were injected (1 × 103 parasites/mouse) in the presence of 100, 10, or 1 μg/mouse C1. On day 7, a second injection using the same concentrations was performed. The maximal protective effect was obtained with 100 μg/mouse, but a significant decrease of parasitemia was already observed with 10 and 1 μg/mouse C1. These results indicate that C1 and C2 induce a protective immune response against T. cruzi infection. Furthermore, CD40-deficient mice inoculated with T. cruzi mixed with C1 (100 μg) exhibited very high parasitemia and succumbed to infection compared with wild-type mice (data not shown). This suggests that the protective capacity of miniCD40Ls peptides depends on CD40 interaction in vivo.

View this table:
Table I.

Efficacy of injection number and dose of C1 on T. cruzi infectiona

We have previously shown that both C1 and C2 bind to CD40 and display agonistic activity on immunocompetent cells (5). Therefore, one might hypothesize that an effective immune response against the parasite originates from activation of immunocompetent cells by miniCD40Ls at the onset of T. cruzi infection. Moreover, we have demonstrated that immune responses were more effective when a second injection of miniCD40Ls was administered on day 7 postinfection, demonstrating the importance of cellular recall for the induction of this effective immune response. Interestingly, live parasites have to be inoculated i.v. together with C1 (or C2) to trigger a protective immune response.

Inoculation of T. cruzi in the presence of miniCD40Ls induces a “memory response”

To investigate whether infection in the presence of miniCD40Ls could induce persistent protective immunity, we performed a challenge infection in surviving mice treated by C1 mixed with T. cruzi (Tehuantepec strain) with a different strain (Y strain). Whereas 100% of control mice died 20 days after inoculation, mice previously inoculated with C1 did not show significant parasitemia and survived (Fig. 2⇓). These data show that T. cruzi inoculation in the presence of miniCD40Ls induces not only an effective immune response against T. cruzi but also the development of a “memory response” effective against infection by a different strain of T. cruzi.

FIGURE 2.
FIGURE 2.

Inoculation of T. cruzi in the presence of miniCD40Ls induces a “memory response.” Mice treated with C1 that survived to T. cruzi (Tehuantepec strain) inoculation were infected with a different strain of T. cruzi (Y strain). They all survived the challenge infection (no detectable parasitemia) while 100% of the control mice died at 20-day postinfection.

The immunosuppression induced by T. cruzi infection is impaired in mice treated with miniCD40Ls

As for many other pathogens, the evolutionary adaptation of T. cruzi allows the parasite to evade the immune system and to induce dramatic immunodeficiency (6, 7, 10). We have previously reported a depletion of both CD4+ and CD8+ T cells at the peak of infection with T. cruzi that is believed to account for high parasitemia and mortality (7). To investigate whether treatment with miniCD40Ls could impair this immunosuppression, mice were sacrificed at days 21, 28, and 35 postinfection. SC were harvested, and the number of CD4+ and CD8+ T cells, as well as their IFN-γ production, was evaluated. As expected, in the absence of treatment, infection by T. cruzi was associated with a decrease in the percentage of splenic CD4+ and CD8+ T cells (Fig. 3⇓, A and B). Although these cells displayed a transitory production of IFN-γ (Fig. 3⇓, C and D), they were not able to induce an effective immune response. Treatment with C1 prevented the decrease of CD8+ T cells at day 35 postinfection. Moreover, intra- and extracellular increases of IFN-γ was observed in C1-treated mice. This increase was particularly significant in the CD8+ T cell population. Taken together, these results show that miniCD40Ls significantly induce an immune response. However, to investigate whether this response was directed against T. cruzi, SC were cultured in the presence of T. cruzi lysate. In this condition, proliferation and IFN-γ production was increased in SC from C1-treated mice (Fig. 3⇓, E and F). These data suggest that miniCD40L treatment overcomes the immunosuppressive effect of T. cruzi by inducing a type 1 immune response that enhances CD8+ T cell activation. Previous studies showed the pivotal role of CD8+ T cells in protective immunity (11). Strong evidence pointed toward the important role of other immunocompetent cells such as CD4+ and NK cells in immune protection against infection (12). Based on the protective capacity of miniCD40Ls peptides, one may speculate on the role of these potent immunocompetent cells through CD40-CD40L signaling.

FIGURE 3.
FIGURE 3.

The immunosuppression induced by T. cruzi infection is impaired in mice treated with C1. Mice were infected at day 0 with T. cruzi in 0.15 M NaCl (▪, n = 25) and mixed with C4 (▤, n = 22) or C1 (□, n = 18). Noninfected mice were used as controls (▥, n = 5). At days 0, 7, 28, and 35 postinfection, SC were harvested, and the percentage of CD3+CD4+ (A) and CD3+CD8+ (B) lymphocytes producing IFN-γ, as well as the percentage of CD3+CD4+ (C) and CD3+CD8+ lymphocytes (D), were evaluated by flow cytometry. A portion of the SC were also incubated with T. cruzi lysate (E and F), and the proliferative response (E; as cpm after [3H]thymidine incorporation) and IFN-γ production (F; measured by ELISA) were evaluated after 96 h. Data are means ± SEM of two independent experiments; *, p < 0.05 and **, p < 0.01 (Mann-Whitney U test).

MiniCD40Ls’ effect on T. cruzi infection can be mediated by DC maturation

CD40L-induced CD8+ T cell activation is mediated by DC maturation and IL-12 production (3, 13). Like soluble recombinant CD40L, C1 and C2 induced maturation of D1 cells, a DC line that matures upon CD40 ligation (14), as shown by both increased IL-12 production and up-regulation of CD80, CD86, CD54, and CD40 expression (Fig. 4⇓). Similar effects were also observed with bone marrow-derived DC (data not shown).

FIGURE 4.
FIGURE 4.

MiniCD40L-induced IL-12 production and DC maturation. A, IL-12p40/p70 secretion by D1 cells upon treatment with C1, C2, C3, and C4. LPS (10 μg · ml−1) served as a control; *, p > 0.1, **, 0.05 > p > 0.03, and ***, p < 0.02. B, Increased expression of CD80, CD86, CD54, and CD40 in C1- and C2-treated D1 cells (mCD40L, soluble recombinant CD40L).

Overall, these results are in line with two other reports establishing that CD40 ligation facilitates the control of T. cruzi infection through a cascade involving IL-12 and IFN-γ (3, 4). Early production of IL-12 during T. cruzi infection is critical in directing differentiation of Th precursors toward a Th1 phenotype that produces high levels of IFN-γ. Therefore, induction of DC maturation by miniCD40Ls could be responsible for high T CD8+ IFN-γ production, which is important in the control of T. cruzi replication in acute infection.

Although cells exposed to live T. cruzi trypomastigotes produced IL-12, optimal induction of this cytokine by DC generally requires two signals (15, 16). The first signal originates from the parasite via activation of pattern recognition receptors such as members of the TLR family. Indeed, MyD88-deficient mice exhibited impaired production of proinflammatory cytokines and showed enhanced parasitemia and mortality upon challenge with trypomastigotes compared with wild-type mice. Several T. cruzi molecules known to stimulate the synthesis of proinflammatory cytokines by macrophages have been shown recently to function as TLR2 (GPI anchors) (17) or TLR4 (glycoinositolphospholipid) agonists (18). The second signal required for IL-12 production by DC involves CD40 triggering (19). This article suggests that miniCD40Ls induce a persistent immune response by providing this second signal at an early stage after T. cruzi inoculation. This is the first demonstration that rationally designed CD40L mimetics such as C1 and C2 are effective in vivo. Recent data showed that TLR agonists synergize with CD40L for DC activation, IL-12 production, and CD8+ T cell expansion (20). This might provide a rationale for the use of miniCD40Ls in combination with TLR2 or TLR4 ligands purified from the parasite or with other TLR agonists such as imidazoquinolines (TLR7 and TLR8), unmethylated oligonucleotides (TLR9), or poly(deoxyinosinic-deoxycytidylic acid) (TLR3). Studies aimed at determining the effects of concomitant delivery of miniCD40Ls and such TLR agonists on protective immunity against intracellular pathogens are currently under investigation.

Acknowledgments

We acknowledge the help of A. Le Moine and J. Johnson (Institute for Medical Immunology, Gosselies, Belgium), J.-P. Briand, J. Hoebeke, and S. Muller (Unité Propre de Recherche 9021 Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France), and C. Decaestecker, M. Libin, and I. Mazza (Université Libre de Bruxelles, Brussels, Belgium).

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by grants from the Centre de Recherche Interuniversitaire en Vaccinologie (to M.H. and B.V.), by the Centre National de la Recherche Scientifique, the “Ministère de la Recherche” (Action Concertee Incitative “Jeunes Chercheurs”) and “La Ligue contre le Cancer, Région Alsace” (to G.G., N.T., and S.F.). S.W. and W.S. were supported, respectively, by a grant from the “Ministère de la Recherche” and the “Fondation pour la Recherche Médicale.”

  • ↵2 Address correspondence and reprint requests to Prof. Bernard Vray, Laboratoire d’Immunologie Expérimentale (CP 615), Faculté de Médecine, Université Libre de Bruxelles, 808 Route de Lennik, 1070 Brussels, Belgium; E-mail address: bvray{at}ulb.ac.be or Prof. Sylvie Fournel, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, Laboratoire d’Immunologie et Chimie Thérapeutiques, Strasbourg, France; E-mail address: S.Fournel{at}ibmc.u-strasbg.fr

  • ↵3 Abbreviations used in this paper: DC, dendritic cell; MPM, mouse peritoneal macrophage; SC, spleen cell.

  • Received May 19, 2006.
  • Accepted April 2, 2007.

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