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Russo, D. M., Chakrabarti, P., and Higgins, A. Y. 1999. Leishmania: Naive human T cells sensitized with promastigote antigen and IL-12 develop into potent Th1 and CD8(+) cytotoxic effectors. Experimental Parasitology 93, 161-170. The differentiation of naive human T cells into Leishmania-specific Th1 or cytotoxic effector cells was examined by sensitizing T cells in vitro with dead Leishmania antigen in the presence or absence of IFN-gamma or IL-12. These Leishmania-specific T cell lines proliferated and produced cytokines in response to challenge with autologous Leishmania-infected macrophages. Sensitization in the presence of IL-12 or IFN-gamma induced Leishmania-specific human Th1 responses, with IL-12 inducing more potent Th1 responses. However, IL-12-induced Th1 responses were IFN-gamma dependent. T cell lines exhibited Th2 or Th0 phenotypes when primed in the absence of cytokines. Only T cell lines primed in the presence of IL-12 contained high percentages of CD8(+) cells. These cells lysed autologous Leishmania-infected but not uninfected macrophages in an MHC-dependent manner. Thus, this in vitro sensitization system can be used to delineate the conditions for optimally priming human Leishmania-specific effector cells.

Most studies of human T-cell responses in tuberculosis have focused on persons with either active disease or latent infection. Although this work has been critical in defining T-cell correlates of successful versus failed host containment, little is known about the development ofMycobacterium-specific T-cell responses in uninfected persons. To explore this issue, naive T cells from uninfected donors were sensitized in vitro with avirulentMycobacterium tuberculosis-infected autologous macrophages. T-cell lines primed in this manner proliferated and produced cytokines after challenge with mycobacterial antigens. Of 11 such lines, 8 were high Th1 responders, 2 were low Th1 responders, and 1 was a Th2 responder. Furthermore, similar patterns and magnitudes of proliferative and cytokine responses were seen whenMycobacteriuminfection-primed lines were challenged with recombinant antigen 85 (Ag85) proteins. The addition of interleukin 12 (IL-12) during the initial sensitization increased the magnitude of Th1 responses; however, antibody to IL-12 did not eliminate Th1 responses, suggesting that additional factors contributed to the differentiation of these cells. Finally, in the presence of IL-12, recombinant Ag85B was able to prime naive T cells for Th1 responses upon challenge withMycobacterium-infected macrophages or Ag85B. Therefore, under the appropriate conditions, priming with whole bacteria or a subunit antigen can stimulateMycobacterium-specific Th1 effector cell development. Further definition of the antigens and conditions required to drive naive human T cells to differentiate into Th1 effectors should facilitate the development of an improved tuberculosis vaccine.

Studies of human disease suggest that naturally acquired immunity is the predominant outcome of Leishmania infection. Normally protective immune mechanisms activated during asymptomatic or self-healing infections may be minimal in patients who develop disease. To explore early immune responses, an in vitro model of human Leishmania infection was developed in which naive T cells were sensitized with Leishmania-infected macrophages. An analysis of Leishmania-specific cytokine production by these T cell lines revealed that most individuals developed Th1 or Th0 responses early after infection. Infected macrophages from Th1 responders produced interleukin-12. Th0 responders who produced little or no endogenous interleukin-12 could be converted to the Th1 phenotype by addition of interleukin-12 during priming. Finally, infection-sensitized T cells specifically lysed Leishmania-infected macrophages. Thus, this in vitro model system can be used to delineate protective human immune responses against Leishmania induced early after infection.

The Plasmodium yoelii murine model was used to test several combinations of blood-stage antigens and adjuvants for the ability to induce immunity to blood-stage malaria. Upon fractionation of whole blood-stage antigen into soluble and insoluble components, only the particulate antigens (pAg) induced protective immune responses. Of a number of adjuvants tested, Quil A was the most effective. Immunization with pAg plus Quil A induced solid protection against nonlethal and lethal P. yoelii challenge infection. Analysis of cytokine production revealed mRNA for Th1-type cytokines (interleukin 2 [IL-2] and gamma interferon) as well as Th2-type cytokines (IL-4 and IL-10) in the spleens of both protected and susceptible animals. The data suggested that the protective pAg response was associated with the earlier production of cytokine mRNA with a Th2 phenotype somewhat favored. Immunization of B-cell-deficient JHD mice indicated that the protection against P. yoelii induced by pAg immunization was B cell dependent. Although immunization with pAg plus Quil A increased the levels of antigen-specific antibodies of all four immunoglobulin G (IgG) isotypes, protection correlated most closely with the presence of IgG1 and IgG2b antibodies. Sera from pAg-plus-Quil A-immunized animals recognized only a limited subset of six to eight distinct P. yoelii antigens, primarily associated with the pAg fraction. These results provide the basis for the identification and characterization of potential vaccine antigens, selected solely for their ability to immunize against blood-stage malaria.

Leishmaniasis is a major public health problem in several areas of the world, occurring in large areas of the Middle East, Africa, and Latin America. The Leishmania complex is a group of closely related parasites that occupy a wide variety of ecologic niches and cause a spectrum of clinical disease. Infection is initiated by the bite of a sandfly, which injects the motile form, called promastigotes. Once inside the mammalian host, Leishmania are obligatory intramacrophage parasites, multiplying in these cells as nonmotile amastigotes. Basic biologic aspects of the parasite are thoroughly reviewed elsewhere (1,2).

The development of genetically engineered subunit vaccines has renewed and intensified efforts to immunize third world populations against currently uncontrolled diseases including malaria; however, the success of such vaccines will depend in large part upon efforts to enhance their immunogenicity. It is likely that subunit vaccines will require potent adjuvants in order to be effective. While considerable work has been aimed at enhancing antibody responses to peptide antigens, little is know regarding the potentiation of antigen specific cell-mediated immune responses. We have utilized a delayed type hypersensitivity (DTH) model to investigate cell-mediated immune responses, including those parameters which affect the induction, magnitude and duration of responsiveness. We examined the potency of R32tet32, a subunit vaccine directed against the sporozoite stage of Plasmodium falciparum, to induce and elicit DTH responses in mice and found that these responses were potentiated significantly through the use of selected lymphokines and immunomodulators.

The regulation of delayed-type hypersensitivity (DTH) to soluble antigens derived from blood-stage parasites was investigated. DTH responses to soluble blood-stage malarial antigen were induced by subcutaneous (sc) sensitization in the flanks and elicited by ear challenge with the same antigen 6 days later. Adoptive transfer studies revealed that T cells of the L3T4+ phenotype were mediating this response. When a high dose of malarial antigen was injected intravenously (iv) prior to sc sensitization, immunosuppression of DTH resulted. The degree of immunosuppression was dependent on the dose of antigen injected iv and the time at which it was administered prior to sc sensitization. Immunosuppression was antigen-specific and mediated by Lyt-2+ splenic T cells.