Naive human T cells develop into Th1 effectors after stimulation with Mycobacterium tuberculosis-infected macrophages or recombinant Ag85 proteins.

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 of Mycobacterium-specific T-cell responses in uninfected persons. To explore this issue, naive T cells from uninfected donors were sensitized in vitro with avirulent Mycobacterium 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 when Mycobacterium infection-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 with Mycobacterium-infected macrophages or Ag85B. Therefore, under the appropriate conditions, priming with whole bacteria or a subunit antigen can stimulate Mycobacterium-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.

Leishmania: naive human T cells sensitized with promastigote antigen and IL-12 develop into potent Th1 and CD8(+) cytotoxic effectors.

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.

Naive human T cells develop into Th1 or Th0 effectors and exhibit cytotoxicity early after stimulation with Leishmania-infected macrophages.

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.

Intravesical therapy with vinorelbine tartrate: antitumor activity in orthotopic murine cell carcinoma of the bladder.

OBJECTIVES: To ascertain intravesical vinorelbine tartrate (VNR) antitumor activity against MB-49, a murine transitional cell carcinoma of the bladder (TCC), in an in vivo setting. MATERIALS AND METHODS: C57B1/6J female mice were intravesically implanted with 5 x 10(4) MB-49 cells and treated locally with VNR. Tumor incidence and volume analyses, as well as survival studies were carried out. RESULTS: Tumor incidence was significantly lower in VNR-treated mice (48%, n = 23) than in controls (84%, n = 19), as evaluated sixteen days after MB-49 orthotopic inoculation. Intravesical tumor volume was also significantly smaller in treated mice respect to controls (median [range]: 0.5 [0.4 to 61.8] mm.3 versus 47.7 [4.2 to 179.7] mm.3 respectively, p < 0.001 Kruskal-Wallis test). Median survival duration of the animals treated with VNR was 68 [21 to 68] days, and was significantly greater (p = 0.01, Kruskal-Wallis test) than that of untreated controls (18 [16 to 20] days). CONCLUSION: Intravesical VNR treatment demonstrated an evident antitumor effect against the TCC model assayed. The results obtained suggest a potential use of VNR as intravesical treatment for superficial TCC following transurethral bladder tumor resection to prevent recurrence or retard tumor growth.

Protective immunity against Plasmodium yoelii malaria induced by immunization with particulate blood-stage antigens.

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.

Exposure to vinorelbine inhibits in vitro proliferation and invasiveness of transitional cell bladder carcinoma.

PURPOSE: To study the effect of vinorelbine (VNR) on in vitro cell proliferation, invasiveness, cell adhesion to substrate, cell motility and metalloproteinase secretion of MB-49, a murine transitional cell carcinoma of the bladder (TCC). MATERIALS AND METHODS: The colorimetric MTS assay, which depends upon viable versus nonviable mitochondria, was used to evaluate the effect of graded concentrations of VNR on in vitro MB-49 cell growth. Chemoinvasion and cell motility were studied in TCC cells exposed for 24 hours to a noncytotoxic dose of VNR, through their ability to migrate across Matrigel-coated or Type IV collagen-coated 8-microns. pore filters. Zymographic studies in gelatin-embedded polyacrylamide gels were done to investigate gelatinolytic activity in conditioned media from treated and untreated MB-49 cells. RESULTS: Vinorelbine inhibited MB-49 cell growth in a dose-dependent manner (IC(50)40 ng./ml.). In vitro cell invasive capacity of MB-49 cells pretreated for 24 hours with VNR at noncytotoxic doses (1 and 10 ng./ml.) was significantly lower than that of untreated cells. The decreased invasion of VNR-treated cells was not accompanied by a diminished adhesion to Matrigel or type IV collagen nor by a significant reduced secretion of gelatinolytic metalloproteinases. Instead, motility of MB-49 cells exposed to noncytotoxic concentrations of VNR was inhibited in a dose-response fashion similar to that of invasion. CONCLUSION: Vinorelbine proved to be an effective drug to inhibit tumor cell growth and invasion in a transitional cell bladder carcinoma model. The results obtained would justify preclinical studies to evaluate the effectiveness of VNR as a potential treatment of TCC.

IL-10 mediates susceptibility to Trypanosoma cruzi infection.

IL-10 has been shown to inhibit some aspects of macrophage activation, including the in vitro IFN-gamma-mediated intracellular killing of the protozoan parasite Trypanosoma cruzi. We have previously shown that genetically susceptible mice produced more IL-10 during T. cruzi infection than resistant mice, suggesting an association between IL-10 production and disease susceptibility. In the present study, such an association was documented. IL-10 mRNA was present in the spleens of susceptible C57BL/6 mice, but not in resistant (C57BL/6 x DBA/2) F1 mice, as early as 2 days after infection with T. cruzi. In susceptible mice, IL-10 mRNA was found in enriched populations of splenic T cells and peritoneal macrophages by 4 days after infection. By 14 days after infection, IL-10 mRNA was detected in enriched populations of splenic T cells and peritoneal macrophages, as well as by splenic B cells and macrophages. In SCID mice infected with T. cruzi, IL-10 mRNA was detected in peritoneal cells 2 days after infection. The IL-10 mRNA production was not abolished by treatment with anti-asialo GM-1 Ab before infection, which is consistent with its production by macrophages. Finally, the role of endogenous IL-10 production in the susceptibility to T. cruzi infection was demonstrated by the protection of highly susceptible C57BL/6 mice against acute disease and death from T. cruzi by the administration of neutralizing anti-IL-10 mAb. This study demonstrated an important and perhaps essential role of IL-10 in mediating in vivo susceptibility to T. cruzi infection.

Antigen-reactive gamma delta T cells in human leishmaniasis.

The importance of Ag-specific gamma delta T lymphocytes in human immune responses to pathogenic organisms is unknown. In the present study the expression of gamma delta TCR on T lymphocytes from patients with cutaneous, mucosal, or visceral leishmaniasis was examined. All of these patient groups had elevated levels of gamma delta T cells in peripheral blood. Patients' gamma delta T cells included CD8+ as well as null cells. The percentage of T cells expressing gamma delta TCR was increased significantly by stimulation in vitro with certain parasite Ag. T-cell lines generated by stimulation with promastigote lysates of Leishmania amazonensis or L. braziliensis typically contained 25 to 60% gamma delta T cells. In contrast, two immunodominant surface Ag of L. amazonensis, gp63 and gp42, did not expand gamma delta T cells from infected patients, although both Ag elicited strong alpha beta T-cell responses. gamma delta T cells isolated from a Leishmania-specific T-cell line responded to stimulation with promastigote lysate. Of particular interest, gamma delta T cells from PBMC of a patient with mucosal leishmaniasis responded to stimulation with a recombinant 70 kDa heat shock protein of L. chagasi. This study demonstrated that several clinical forms of leishmaniasis induced elevated numbers of gamma delta T cells that responded specifically to Leishmania Ag in vitro. Therefore, this component of the T-cell response to Leishmania may impact the outcome of clinical disease.

Analysis and partial epitope mapping of human T cell responses to Trypanosoma cruzi cysteinyl proteinase.

Human infection with Trypanosoma cruzi (Chagas' disease) is usually accompanied by humoral and cellular immune responses to GP57/51, a major antigen that was recently identified as a prominent cysteinyl proteinase (cruzipain). The PBMC responses of 11 chronic chagasic patients and the properties of anti-cruzipain T cell lines are reported herein. GP57/51, isolated from Y strain epimastigotes (n-cruzipain) or the recombinant protein expressed in E. coli (r-cruzipain), elicited proliferative responses of variable intensity from the patient's PBMC. T cell lines were then generated using each of these antigens. These lines, which always carried the CD4+ phenotype, were reciprocally stimulated by n-cruzipain or r-cruzipain, the responses to the former being usually stronger. The analysis of cytokine production suggested that Th1-like subsets dominate the patient's responses: IFN-gamma was consistently induced on stimulation with either n-cruzipain or r-cruzipain. In contrast, IL-4 was present in very small concentrations or was undetectable. We then sought to define T cell epitopes of cruzipain using synthetic peptides spanning portions of the central (catalytic) domain and COOH-terminal extension. From a panel of 11 peptides, only one 33 mer peptide (P214) elicited a strong proliferative response on anti-cruzipain T cell lines, the intensity being comparable to that induced by r-cruzipain. Conversely, T cell lines started with P214 were responsive to either n-cruzipain or r-cruzipain, the proliferative responses again being accompanied by IFN-gamma production, but not IL-4. Interestingly, P214 is located in a conserved region of the catalytic domain of cruzipain, hence may propitiate opportunities for cross-recognition of other members of the papain superfamily. Fine epitope mapping should reveal whether structurally similar regions of host thiol-cathepsins can be potential targets for cross-reactive T cell responses during chronic human infection.

Interleukin 10 production correlates with pathology in human Leishmania donovani infections.

We have found that an important Th2 cytokine, IL-10, is produced by tissues from patients acutely infected with Leishmania donovani. In all individuals tested, IL-10 mRNA production was increased in lymph nodes taken during acute disease over that observed in postacute samples. In contrast, both pre- and posttreatment lymph nodes had readily detected mRNA for IFN-gamma and IL-2. A down-regulating effect of IL-10 on leishmania-induced proliferative responses was demonstrated when Hu rIL-10 was added to cultures of PBMC from clinically cured individuals. PBMC from individuals with acute visceral leishmaniasis responded to stimulation with leishmania lysate by producing IL-10 mRNA. Simultaneously cultured PBMC collected from the same patients after successful chemotherapy produced no detectable IL-10 mRNA after leishmania antigen stimulation. Neutralizing anti-IL-10 mAb added to PBMC from patients with acute visceral leishmaniasis markedly increased the proliferative response to leishmania lysate. Finally, we observed mRNA for IL-10 and IFN-gamma concurrently in a lesion from a patient with post-kala-azar dermal leishmaniasis (PKDL). These results indicate the production of IL-10 during L. donovani infection, and suggest a role for this cytokine in the regulation of immune responsiveness during visceral leishmaniasis.

Mapping human T cell epitopes in leishmania gp63. Identification of cross-reactive and species-specific epitopes.

Both a conserved surface metalloprotease of leishmania, gp63 as well as certain gp63-derived peptides, have been shown to have immunoprophylactic potential in mouse models of leishmaniasis. In addition, PBMC from individuals with cutaneous, mucosal, or cured visceral leishmaniasis respond in vitro to both native and rgp63. In this report, we mapped human T cell epitopes within gp63. T cells from leishmaniasis patients responded in vitro to certain peptides of gp63 by proliferation and IFN-gamma production. One peptide, (PT7), stimulated cells from all individuals tested (n = 7). Anti-PT7 T cell lines derived from PBMC of a mucosal leishmaniasis patient contained a heterogeneous population of cells which responded by proliferation and IFN-gamma production to in vitro stimulation with Leishmania promastigote lysate. Another peptide (PT1) derived from Leishmania chagasi gp63 stimulated PBMC from an L. chagasi patient although the corresponding Leishmania major-derived peptide did not. Both L. major PT7 and L. chagasi PT1 were able to induce anti-Leishmania-specific T cell lines from normal human PBMC. These T cell lines responded to in vitro stimulation with promastigote lysate indicating that both peptides were immunogenic for naive T cells in vitro. In conclusion, both antigenic and immunogenic gp63 peptide sequences have been defined, some appearing to be conserved among Leishmania species and at least one that appears to be species specific.

Characterization of responses of normal human T cells to Trypanosoma cruzi antigens.

The stimulation of normal human PBMC by Trypanosoma cruzi Ag was analyzed. PBMC showed significant in vitro proliferation in response to parasite lysate (Tct), with stimulation indices ranging from 10 to 400, peaking at 6 to 7 days. The cells stimulated with Tct produced significant levels of IL-2. To determine which cells proliferated in response to Tct, PBMC were separated into T- and B-enriched cell populations. Purified T cells, but not B cells, proliferated strongly to Tct. The T cell response required APC and was processing dependent. T cell lines generated against Tct proliferated in response to parasite lysate only in the presence of autologous APC and produced IL-2, IL-6, and IFN-gamma but not IL-4 in response to PMA plus ionomycin. Although there were a significant number of CD45Ra+ cells, the majority of the cells in these T cell lines were CD45Ro+. The V beta usage of Tct-responding T cells was heterogeneous, with most V beta genes represented among the responding cells. An immunodominant repeat Ag (TcD) and a ribosomal phosphoprotein (P0) of T. cruzi elicited strong proliferative responses in all subjects tested. These data indicate the presence of T cell-stimulatory Ag in Tct, characterized by nonpreferential usage of the V beta gene families. The strong stimulation of normal human PBMC by Tct may contribute to immunologic alterations seen in T. cruzi infection.

Stimulation of human T lymphocytes by Leishmania lipophosphoglycan-associated proteins.

Lipophosphoglycan (LPG) is a glycoconjugate present on the surface of Leishmania promastigotes that has been reported to promote intracellular survival of these parasites, to protect mice against leishmaniasis, and to elicit T cell responses in infected mice and humans. We investigated whether LPG and its components could elicit proliferative responses and cytokine secretion from leishmaniasis patient PBMC. LPG prepared by standard methods (LPG-1) stimulated patients T cells to proliferate and secrete IFN-gamma. LPG was fractionated into several components. An LPG-1-specific T cell line was shown to respond to the core region but not to the repeating saccharide units. LPG-1 was fractionated to yield an LPG-free- associated protein complex and an LPG-2 fraction that was more than 95% depleted of associated protein. The ability of LPG-2 to stimulate T cells was significantly decreased over that of LPG-1. In contrast, LPG-AP stimulated T cell proliferation and IFN-gamma production. Therefore, proteins associated with LPG were effective in eliciting patient T cell responses, whereas the glycolipid enriched moiety was weakly effective or ineffective at stimulating these responses.

Cytokine activation of human macrophages infected with HIV-1 to inhibit intracellular protozoa.

Peripheral blood mononuclear cells (PBMCs) from HIV-seronegative donors were infected in vitro with HIV-1. Infection was monitored by cytopathology, supernatant p24 antigen, and by immunocytochemical staining. After 14 days in culture, approximately 70-90% of the cells became infected with HIV, as indicated by cell fusion and immunostaining for virus. At this time, recombinant HuIFN-gamma was added to the cultures, followed by infection 24 h later with the intracellular protozoan parasites Toxoplasma gondii, Trypanosoma cruzi, or Leishmania chagasi. Percentages of intracellular parasites were determined at various points thereafter. Using a system capable of detecting both virus and parasite infection, we determined that (a) cells infected with HIV were capable of ingesting and/or being infected by each of these parasitic protozoa, (b) HIV-infected macrophages could be activated to inhibit the replication of all three parasites following treatment with IFN-gamma, and (c) cultures of HIV-infected macrophages could respond to IFN-gamma with increased oxidative burst activity. The degree of parasite infection or inhibition observed in infected cells was not significantly different from that observed in non-HIV-infected cells. From these observations, we concluded that HIV-1 infection does not render macrophages unresponsive to IFN-gamma activation for microbicidal activity.

Human T cell responses to gp63, a surface antigen of Leishmania.

gp63, an abundant and conserved leishmania cell surface protein, has been implicated in the ability of these parasitic protozoa to infect macrophages in vitro and has shown potential as a protective immunogen in mice. However, little is known regarding human immune responses to this glycoprotein Ag. In this study, human T lymphocyte responses to Leishmania amazonensis native gp63 and to recombinant gp63 (rgp63) produced in Escherichia coli were evaluated in individuals with active or cured cutaneous, mucosal or visceral leishmaniasis. Both native and rgp63 elicited strong proliferative responses in all patients tested. In addition, IFN-gamma was produced in response to stimulation with both forms of the protein. T cell lines generated from PBMC by stimulation with native or rgp63 were phenotypically similar, and proliferated and produced IFN-gamma in response to stimulation with both forms of the molecule. These results suggest that gp63 is a strong T cell immunogen and that the recombinant and native forms can elicit the same type of T cell response from infected patients. In order to compare the immunogenic properties of these two forms of gp63, PBMC from naive (uninfected) donors were sensitized in vitro with native or rgp63. T cell lines generated against rgp63 proliferated in response to rgp63, but failed to proliferate in response to native gp63 or to promastigote lysate. Thus, rgp63 was effective in eliciting T cell responses from patients with active or cured leishmania infection, but did not effectively induce T cell responses under the conditions used.

Characterization of a membrane antigen of Leishmania amazonensis that stimulates human immune responses.

To investigate human immune responses to defined leishmania Ag we have begun to characterize biochemically and immunologically, an abundant 42-kDa surface Ag of Leishmania amazonensis, a causative agent of human leishmaniasis. We have shown that this Ag, La gp42, is expressed on the surface of L. amazonensis promastigotes, being anchored to the membrane by a glycosyl-phosphatidylinositol moiety. As demonstrated by lectin blotting studies, La gp42 is glycosylated, binding both Con A and wheat germ agglutinin. Immunologically, La gp42 is strongly recognized by sera from patients with different forms of leishmaniasis as well as by patients with Chagas' disease. In addition, we show that purified La gp42 stimulates the proliferation of human T lymphocytes obtained from several leishmaniasis patients. Finally, the N-terminal sequence of La gp42 was obtained and a serologically cross-reactive 42-kDa protein with a homologous sequence was identified in Leishmania major.

In vitro responses to Leishmania antigens by lymphocytes from patients with leishmaniasis or Chagas' disease.

T cell responses are correlated with recovery from and resistance to leishmaniasis. Antigens of Leishmania chagasi were evaluated by determining their ability to elicit in vitro proliferation and cytokine production in peripheral blood lymphocytes and in T cell lines and clones from patients with histories of leishmaniasis or Chagas' disease. Antigens tested were selected by their reactivity with patient antibodies. Several of the antigens induced proliferative responses in peripheral blood lymphocytes from patients recovered from visceral or cutaneous leishmaniasis or with chronic Chagas' disease. Two purified glycoproteins, 30 and 42 kD, were consistently among the most effective in eliciting high proliferative responses and IL-2 production. Lymphocytes from a recovered visceral leishmaniasis patient were used to produce T cell lines against either the 30- or 42-kD antigen. Each of the lines responded to both of these antigens as well as to crude leishmania lysate. CD4+ T cell clones specific for either or both of these antigens were also isolated from a visceral leishmaniasis patient. In contrast, rabbit antisera produced against these two antigens were not crossreactive. Both antigens were effective in inducing the production of IFN-gamma from T cell lines from both leishmaniasis and Chagas' disease patients. These studies demonstrate the potential for defining parasite antigens with broad immunostimulatory capabilities.

Cell-mediated immune responses to vaccine peptides derived from the circumsporozoite protein of Plasmodium falciparum.

T cell epitopes residing within vaccine candidate peptides have been identified by delayed-type hypersensitivity (DTH) responses in mice. The recombinant sporozoite vaccine candidate, R32tet32, contains at least two T epitopes, one located within the repeat region and another in the tet tail. When C57BL/6 (H-2b) and BALB/c (H-2d) mice were sensitized intradermally with R32tet32 or the truncated protein R32LR emulsified in CFA and challenged 5 days later with R32tet32, only H-2b mice recognized a T epitope located within the major repeat sequence (NANP) and encoded by four or less repeats. H-2d mice responded solely to the T epitope located on the tet tail. Ear swelling was maximal at 48 h and revealed a histologic pattern characteristic of DTH. CD4+ T cell lines derived from immunized animals demonstrated the ability to mediate local DTH, proliferate, and secrete lymphokines in response to stimulation with Ag. High dose i.v. administration of R32tet32 in C57BL/6 and BALB/c mice before intradermal sensitization with R32tet32 revealed that DTH responses were suppressed only in BALB/c mice. Further experiments localized the suppressive determinant to the tet tail. Collectively, these data indicate that DTH may prove to be a useful method to characterize the biologic activity of T epitopes, furthermore they suggest that candidate vaccine peptides should be tested for suppressive activity before inclusion in a vaccine.