516 J. Parasitol., 90(3), 2004, pp. 516–523  American Society of Parasitologists 2004 CHALLENGE OF TRYPANOSOMA CRUZI CHRONICALLY INFECTED MICE WITH TRYPOMASTIGOTES ACTIVATES THE IMMUNE SYSTEM AND REDUCES SUBPATENT PARASITEMIA LEVELS Claudio R. F. Marinho, Karina R. B. Bastos, Luiz R. Sardinha, Marcos G. Grisotto, Maria Regina D’Impe ´rio Lima, and Jose M. Alvarez Department of Immunology, Instituto de Cie ˆ ncias Biome ´ dicas, Universidade de Sa ˜o Paulo, Av. Prof. Lineu Prestes, 1730, Sa ˜o Paulo, SP CEP-05508-900, Brazil. e-mail: marinho@usp.br ABSTRACT: Challenge of 1-yr Trypanosoma cruzi chronically infected mice with trypomastigotes results in a consistent reduction of parasite dissemination that correlates with spleen activation and increase in the anti–T. cruzi effector immune mechanisms. That is, parasite challenge results not only in elimination of the inoculum but also in a drastic decrease in basal subpatent parasitemia levels as revealed by transferring blood samples to immunosuppressed mice. Parasite elimination correlated with (1) a brief and intense burst in the ability of spleen cells to produce interferon-, (2) an increase in total IgG2a-producing spleen cells, (3) higher parasite-specific IgG2a serum levels, and (4) an accumulation of non–B, non–T class II + cells in the spleen. Furthermore, challenged, chronically infected mice had increased numbers of B, CD4 + , and CD8 + large spleen cells. Besides reinforcing the activation of protective Th1 effector mechanisms, challenge with T. cruzi also induced Th2 effector molecules, such as interleukin (IL)-10 and IL-4, and IL-4–dependent IgG1. Our results are the first evidence that the immune system of T. cruzi chronically infected mice can be optimized in its ability to restrict parasite dissemination, opening the possibility that therapeutic vaccination could be used to reduce the parasite load and pathology of patients with chronic Chagas’ disease. An important feature of the human natural infection by Try- panosoma cruzi or its experimental murine models is the lack of spontaneous cure. Despite the fact that a potent anti–T. cruzi immune response is generated, allowing the control of parasit- emia at the end of the acute phase, a small number of persistent T. cruzi in the tissues resist the immune system effector mech- anisms. From these sites, and for the lifetime of the host, par- asites will occasionally gain access to the blood, where they can be detected by indirect methods such as xenodiagnosis, sub- inoculation, or polymerase chain reaction (PCR) (Brener, 1962; Segura, 1987; Avila et al., 1991; Vago et al., 1996). Many different cell types and soluble molecules participate in the control of T. cruzi parasites. Mice lacking B cells, helper, or cytotoxic T cells (Rodrigues et al., 1981; Russo et al., 1988; Tarleton, 1990; Tarleton et al., 1992; Rottemberg et al., 1995) and mice expressing poor interferon- (IFN-), interleukin (IL)- 12, tumor necrosis factor  (TNF-), or granulocyte-macro- phage colony–stimulating factor activities are highly suscepti- ble to infection (Reed, 1988; Torrico et al., 1991; Petray et al., 1993; Aliberti et al., 1996; Olivares-Fontt et al., 1996; Santos- Lima et al., 1997). In contrast, infected IL-10KO mice show reduced numbers of tissue and circulating parasites and in- creased production of IFN-, IL-12, TNF-, and NO (Abraham- sohn and Coffman, 1996; Hunter et al., 1997). The major pro- tective role of IFN- suggests that parasite control is dependent on activation of the Th1 pathway of immune response. IFN- not only activates macrophages to destroy ingested parasites (Wirth et al., 1985) but also induces Ig-isotype switch to cy- tophilic and complement-fixing IgG2a antibodies (Snapper and Paul, 1987) and upregulates the expression of high-affinity Fc receptors (FcRI) on macrophages, which increase parasite clearance (Umekita et al., 1988). At the chronic phase of the disease, the activation of the immune system by T. cruzi must be limited by the amount of available parasites or parasite antigens and by the immunoreg- ulatory mechanisms that control the immune response. There- Received 8 August 2003; revised 5 September 2003; accepted 9 Sep- tember 2003. fore, if the antigen level is kept relatively constant, a situation that may occur if the parasite is relatively safe in its intracellular location, the activation of the immune system must reach a plateau. In this equilibrium state, the immune system could be underoptimized for effective parasite destruction, although its activation might be sufficient to restrain the dissemination and expansion of the parasite. The aim of this study was to determine whether the equilib- rium between parasite and activation of the immune system established at the chronic phase can be modified to increase the efficiency of parasite destruction. With this purpose in mind, we evaluated the changes in parasitemia level and activation of the spleen compartment in chronically infected mice after chal- lenge with trypomastigotes. Mice chronically infected with the Y strain of T. cruzi were used in the present study because in this infection model, subpatent parasitemia can be indirectly quantified by transferring blood samples from chronically in- fected to immunosuppressed mice. This subinoculation tech- nique allows the detection of a single parasite in the blood of infected mice (Alvarez et al., 1991) and, compared with PCR, it offers the advantage of ensuring that only live parasites are considered. MATERIALS AND METHODS Mice and parasites Six- to 8-wk-old A/J female mice were obtained from our animal facilities (Biote ´rio de Camundongos Isoge ˆnicos, ICB/USP, Sao Paulo, Brazil). Trypanosoma cruzi parasites of the Y strain were maintained by weekly passages in A/J mice. Infection model, specific chemotherapy treatment, and challenge of chronically infected mice with live trypomastigotes Mice were infected i.p. with 1,000 blood forms of T. cruzi parasites. At day 6, infected mice or control (noninfected) mice were treated with a single oral dose of benznidazole (Rochagan, Roche, Sao Paulo, Brazil) of 1 g/kg of body weight to interrupt the ascending parasitemia curves and to avoid mortality. Mice were then left unmanipulated for over a year, at which time they were challenged i.v. with 5  10 6 trypomas- tigotes of the Y strain. Seven or 12 days after challenge, challenged mice, unchallenged mice, and uninfected mouse controls were screened for parasitemias and immunological studies.