Progress was achieved in the following areas:A. Host Resistance And Immune Deviation In TCR Transgenic Mice Infected With T. gondii Resistance to T. gondii is dependent on the host IFN-gamma response. While NK cells are thought to be the main source of the cytokine in acute infection, several lines of evidence suggest that IFN-gamma production by CD4+ T lymphocytes also plays an important role in controlling early parasite growth. To evaluate whether this function is due to nonspecific as opposed to T cell receptor (TCR)-dependent stimulation by the parasite, we examined the resistance to T. gondii infection of pigeon cytochrome C transgenic (PCC-Tg) mice in which all CD4+ T lymphocytes are unreactive with the protozoan. These animals exhibited only temporary control of acute infection. Intracellular cytokine staining revealed that, in contrast to infected non-transgenic controls, infected PCC-Tg animals failed to develop IFN-gamma-producing CD4+ T cells. Nevertheless, when acutely infected transgenic mice were primed by PCC injection, the lymphokine responses restimulated by the Ag in vitro displayed a strong Th1 bias. These findings argue that while T. gondii cannot trigger IFN-gamma production by CD4+ T cells in the absence of TCR ligation, the pathogen is able to nonspecifically promote Th1 responses against non-parasite antigens, an effect which may explain its immunostimulatory properties. B. Requirement for persistent IL-12 and IL-10 in host resistance to T. gondii.Our previous work has indicated that IL-12 is required for IFN-gamma-dependent control of acute infection while IL-10 is needed to dampen this response to prevent immunopathology. To determine whether continuous IL-12 production is necessary once immunity is established, IL-12 K0 mice were given injections of the cytokine for 14d after T. gondii inoculation. In contrast to KO mice not given the cytokine that died within 2 wk, the IL-12-inoculated mice maintained IFN-gamma mediated control of infection for a full 45 d but then succumbed. In the converse experiment, IL-12 production was dampened in IL-10 KO mice by """"""""paralyzing"""""""" dendritic cell function with injected parasite Ag (see last year?s report). These animals, in contrast to non-paralyzed IL-10 KO mice which died of acute infection, survived for 30d but then succumbed during the following 2-3 wks. Together these findings indicate that while IL-12 and IL-10 are both critical for survival of acute toxoplasmosis, they are also unexpectedly necessary for long-term host protection during chronic infection.C. Association of TNFR1/LT- alpha -dependent chemokine expression with T. gondii-induced dendritic cell recruitment in vivo.We have previously shown that after i.v. injection of STAg, a soluble extract of T. gondii tachyzoites, dendritic cells (DC) are recruited into T cell areas of mouse spleen where they produce IL-12. We have now demonstrated that this DC response is associated with the induction of mRNAs for the chemokines MIP-1 alpha, MIP-1 beta, RANTES, MCP-1, IP-10 and lymphotactin. Expression of the genes for these chemokines in mouse spleen is detected by 1 hr following STAg injection and is maintained for at least 5 additional hr. That the observed chemokine expression is functional is suggested by the finding that supernatants from STAg- stimulated splenocytes induce the migration of enriched population of splenic DC in microchemotaxis chambers. Further evidence for the role of chemokine expression in DC migration came from studies in TNFR1 and LT-alpha deficient mice. These animals show impaired recruitment of DC into T cell areas of mouse spleen following STAg injection and fail to express all chemokines assayed except for MIP-1 alpha. We are now attempting to identify the specific chemokines and chemokine receptors responsible for DC migration as well as investigate the role of TNFR1/LT in chemokine synthesis and DC recruitment.D. Malaria infection induces viral expression in HIV-1 transgenic miceTo test the capacity of malaria parasites to trigger the expression of HIV-1, transgenic mice carrying complete DNA copies of the HIV genome were infected with Plasmodium chabaudi chabaudi (AS). Expression of HIV-1 was monitored by measuring the p24 HIV core protein in plasma and in supernatants of splenocyte cultures from infected animals. Spleen cells recovered at 10d post-infection (1 d after peak parasitemia) showed a dramatic elevation in p24 production (over 500-fold the level detected in control cultures from non-infected transgenic animals). By 15d post- infection, however, in vitro p24 production returned almost to baseline levels. Smaller increases in p24 were observed in plasma of the same P. chabaudi-infected transgenic mice with peak levels again occurring at 10d. Nevertheless, elevated viral expression was not observed during later recrudescence of the infection. These findings demonstrate the capacity of Plasmodium spp. to induce immune activation of HIV in vivo and suggest that if this phenomenon occurs in malaria infected humans it is probably restricted to the acute phase of the disease.
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