Leishmania donovani p36(LACK) DNA Vaccine Is Highly Immunogenic but Not Protective against Experimental Visceral Leishmaniasis

The acquisition of immunity following subclinical or resolved infection with the intracellular parasite Leishmania donovani suggests that vaccination could prevent visceral leishmaniasis (VL). The LACK (Leishmania homolog of receptors for activated C kinase) antigen is of interest as a vaccine candidate for the leishmaniases because of its immunopathogenic role in murine L. major infection. Immunization of mice with a truncated (24-kDa) version of the 36-kDa LACK antigen, delivered in either protein or DNA form, was found previously to protect against cutaneous L. major infection by redirecting the early T-cell response away from a pathogenic interleukin-4 (IL-4) response and toward a protective Th1 response. The amino acid sequence of the Leishmania p36(LACK) antigen is highly conserved, but the efficacy of this vaccine antigen in preventing disease caused by strains other than L. major has not been determined. We investigated the efficacy of a p36(LACK) DNA vaccine against VL because of the serious nature of this form of leishmaniasis and because it was unclear whether the LACK vaccine would be effective in a model where there was not a dominant pathogenic IL-4 response. We demonstrate here that although the LACK DNA vaccine induced a robust parasite-specific Th1 immune response (IFN-gamma but not IL-4 production) and primed for an in vivo T-cell response to inoculated parasites, it did not induce protection against cutaneous or systemic L. donovani challenge. Coadministration of IL-12 DNA with the vaccine did not enhance the strong vaccine-induced Th1 response or augment a protective effect.     

T-cell response in human leishmaniasis

In the present communication we provide evidence for the existence of a Th1/Th2 dichotomy in the T-cell response to Leishmania antigens in human leishmaniasis. Our data suggest that the pattern of IL-4 and IFN-gamma response is polarised in these patients. Lymphocytes from individuals recovered from cutaneous leishmaniasis (CL) responded by IFN-gamma production following stimulation with Leishmania antigens whereas cells from patients recovered from visceral leishmaniasis (VL) showed a mixed pattern of IFN-gamma and IL-4 responses. The cells producing these cytokines were predominantly CD4+. Furthermore, IL-10 plays an important role in the development of post kala azar dermal leishmaniasis (PKDL) from VL. The balance between the parasitic-specific T-cell response plays an important regulatory role in determining the outcome of Leishmania infections in humans.     

Control of Leishmania infantum Infection Is Associated with CD8+ and Gamma Interferon- and Interleukin-5-Producing CD4+ Antigen-Specific T Cells

Visceral leishmaniasis is a severe and lethal disease caused by the protozoan parasites of the genus Leishmania. In areas where leishmaniasis is endemic, most infected individuals control the infection and remain asymptomatic; chemotherapy of visceral leishmaniasis restores some immunity which protects against relapses. In the present study, Leishmania-specific T-cell clones were established from six asymptomatic and five cured patients. Cytokines production by these clones was analyzed. A large fraction of the parasite-specific T-cell clones from asymptomatic patients were CD8(+) and produced high amounts of gamma interferon (IFN-gamma). Most CD4(+) T-cell clones from two asymptomatic subjects exhibited an unusual phenotype: production of high levels of IFN-gamma low levels of interleukin-4, (IL-4), but high levels of IL-5. In contrast, only few parasite-specific CD8(+) T-cell clones were obtained from cured patients after chemotherapy; moreover, CD4(+) T-cell clones from these patients exhibited an heterogeneous profile of cytokines from Th1-like to Th2-like phenotypes. These results point to CD8(+) T cells and to IL-5- and IFN-gamma-producing CD4(+) T cells as possible contributors to human resistance to Leishmania infection. They should stimulate new immunological approaches in the control of this disease.     

Functional heterogeneity among CD4+ T-cell clones from blood and skin lesions of leprosy patients. Identification of T-cell clones distinct from Th0, Th1 and Th2.

In the present study we examined the functional properties of T-cell clones reactive with Mycobacterium leprae and other mycobacterial antigens. Clones isolated from the skin lesions and blood of leprosy patients across the spectrum were exclusively CD4+CD8- and expressed the alpha beta T-cell receptor. Substantial heterogeneity in the production of cytokines, in particular interleukin-4 (IL-4), was observed, although no striking correlation with clinical status was apparent. A variety of patterns of cytokine secretion distinct from those of T-helper type-1 (Th1) Th2 or Th0, as defined in murine studies, was evident. Most noteworthy was a large number of clones from skin which secreted neither IL-2 nor IL-4, but large amounts of tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma). Clones isolated from the blood of leprosy patients had a more restricted cytokine secretion profile, and appeared to resemble more closely previously described patterns, including those of high level production of IL-2 and/or IL-4. Virtually all clones, from either skin or blood, produced high levels of IFN-gamma, and thus many clones were IL-4 and IFN-gamma co-producers. The pattern of cytokine production by skin-derived T-cell clones was significantly affected by the in vitro activation status of the cells. Cells enriched in activated blasts tended to produce more IL-4 than small resting cells. In addition, the production of IFN-gamma by skin T-cell clones after < or = 10 weeks of culture was strikingly distinct from that of these clones after 5 months of culture. IL-4 and IFN-gamma co-producing clones shifted to a Th2-like pattern with much less IFN-gamma secretion, whereas non-IL-4-producing clones secreted much higher levels of IFN-gamma after prolonged culture, and became much more Th1-like. However, there was still no correlation between clinical status and pattern of cytokines produced. These results imply that a high fraction of T cells exists in leprosy lesions that is distinct from or that has not yet fully matured into Th1 or Th2 cells.     

Interleukin-27R (WSX-1/T-cell cytokine receptor) gene-deficient mice display enhanced resistance to leishmania donovani infection but develop severe liver immunopathology

The interleukin-27 (IL-27)/T-cell cytokine receptor (TCCR) pathway plays an important role in development of protective immunity against cutaneous leishmaniasis caused by Leishmania major. In this study, we analyzed the role of IL-27/TCCR pathway in the host defense against visceral leishmaniasis (VL) by monitoring the course of L. donovani infection in TCCR-deficient C57BL/6 (TCCR-/-) mice. TCCR-/- mice mounted a robust inflammatory response, produced high levels of pro-inflammatory cytokines, and developed severe liver pathology after L. donovani infection that eventually resolved. Interestingly, L. donovani-infected TCCR-/- mice controlled the parasite growth in their organs significantly faster than similarly infected TCCR+/+ mice. Adoptive cell transfer and cell depletion studies revealed that CD4(+) T cells were involved in mediating liver immunopathology and controlling L. donovani growth in TCCR-/- mice. These results indicate that the IL-27/TCCR pathway is not essential for the induction of protective Th1 response during VL but is involved in mediating susceptibility to L. donovani. Additionally, the data demonstrate that although the IL-27/TCCR interaction limits the severity of liver inflammation during VL by controlling CD4(+) T-cell activity, it is not required for the resolution of hepatic immunopathology.     

Th1 and Th2 cell-associated cytokines in experimental visceral leishmaniasis.

In experimental Leishmania donovani infection in BALB/c mice, initial susceptibility gives way to T-cell-dependent acquired resistance and eventual control over visceral infection. Since various cytokines appear to underlie the host response to Leishmania infection, we examined infected liver tissue for gene expression of cytokines associated with Th1 (gamma interferon [IFN-gamma] and interleukin-2 [IL-2]) and Th2 cells (IL-4 and IL-10). By Northern (RNA) blot analysis, only IFN-gamma mRNA expression was detected in livers of infected euthymic mice. To determine whether activation of Th1 cells develops selectively in this model, qualitative PCR analysis was used. These results indicated that mRNAs for IFN-gamma, IL-2, IL-4, and IL-10 were all induced by L. donovani infection. The potentially negative Th2 cell-associated response did not appear to play a functional role, however, since resistance was acquired, anti-IL-4 monoclonal antibody treatment did not accelerate control over visceral infection, and serum immunoglobulin E levels remained low. As judged by PCR analysis, IL-4 and IL-10 mRNAs were also expressed under three other conditions without apparent effect: in naive euthymic mice treated with IL-2, which induces leishmanicidal activity; in rechallenged immune mice, which resist reinfection; and in nude mice, which fail to control L. donovani. These results suggest that, like other Leishmania species, L. donovani infection may trigger a potentially suppressive Th2 cell-associated cytokine response. However, in T-cell-intact mice able to control L. donovani, this response either is insufficient to influence outcome or more likely is overshadowed by the Th1 cell response.     

IL-15 in human visceral leishmaniasis caused by Leishmania infantum

Interleukin (IL)-15 is a recently discovered cytokine with the ability to stimulate the proliferation activity of Th1 and/or Th2 lymphocytes. Here, we investigated the involvement of IL-15 in the immune response to Leishmania infantum infection by studying patients with visceral leishmaniasis (VL). We found that IL-15 is produced by leishmanial antigen (LAg)-stimulated peripheral blood mononuclear cells (PBMC) from active VL patients at a significantly higher level than those produced by cells from healed VL subjects or healthy controls. A significant increase in IL-15 serum blood levels was also observed in acute VL patients compared with healed ones. Furthermore, recombinant IL-15 had an appreciable effect in vitro in reducing IL-4 and increasing the production of IL-12 in response to LAg, but it was ineffective in altering the production of interferon-gamma (IFN-gamma). The production of endogenous IL-15 in acute VL patients appeared to be insufficient to activate both IFN-gamma and IL-12, as attested by the absence of modification of these two cytokines by neutralization experiments in the presence of anti-IL-15 monoclonal antibodies (MoAB). On the contrary, the neutralization of IL-15 increased IL-4 production. Together, these results indicate that endogenous IL-15 plays a role in the suppression of Th2-type cytokines, even though it does not enhance the production of Th1 cytokines in acute VL patients. Since IL-15, in the presence of anti-IL-4 MoAb, caused a further increase in IL-12 production and led to a significant production of IFN-gamma, one of its indirect effects on Th1 cell activation could be due to the latter's effect on Th2 cytokines such as IL-4. Therefore, our observations indicate that there is a potential for IL-15 to augment the T-cell response to human intracellular pathogens.     

T Cells Cloned from Human Rheumatoid Synovial Membrane Functionally Represent the Th 1 Subset

The presence of activated T cells in the synovial membrane of patients with rheumatoid arthritis (RA) suggests a role for these cells in the pathogenesis of the disease. Recent evidence indicates that human T cells may fall into functional categories dependent on their cytokine profile and cytotoxic capacity. The human Th1 subset is cytolytic and produces high levels of IFN-gamma whereas the Th2 type of T cell produces IL-4. In order to investigate whether Th1 or Th2 type cells are present in the inflammatory synovial membrane in RA, a panel of synovial membrane derived T-cell clones (n = 19) was generated and studied functionally. Anti-CD3-induced cytotoxicity assays were performed to demonstrate the cytotoxic potential of clones. Except for two, all clones were cytolytic in this test. Clone cells were activated to initiate cytokine production and assessment of the cytokine levels showed that all clones produced large amounts of IFN-gamma (18 out of 19 clones: over 50,000 pg/ml) whereas IL-4 was absent or present in minimal amounts (17 out of 19 clones: less than 1000 pg/ml). The production of IL-1, IL-2 and IL-6 was variable. The functional characteristics of the clones studied indicate that they may resemble the Th1 subtype of T cells. Our data suggest a relation between Th1-type functions the chronic inflammation characteristic of RA.     

Identification of Leishmania donovani antigens stimulating cellular immune responses in exposed immune individuals

Human visceral leishmaniasis (VL), also known as kala azar (KA) in India, is a systemic progressive disease caused by Leishmania donovani. In VL, Th1 responses correlate with recovery from and resistance to disease and resolution of infection results in lifelong immunity against the disease. However, recent data suggest an important role for interleukin (IL)-10 in maintaining the resistant state. We evaluated whole cell extract (WE) and 11 antigenic fractions [F1-F11, molecular weight (MW) range of 139-24.2 kDa] from L. donovani (2001 strain, a fresh field isolate from Bihar), for their ability to induce in vitro T cell proliferation and production of interferon (IFN)-gamma, interleukin (IL)-12, IL-10 and IL-4 by peripheral blood mononuclear cells (PBMCs) of exposed immune individuals (14 patients with history of VL, 10 household endemic contacts) and 20 non-endemic healthy controls. Twenty-one of 24 exposed individuals and no healthy controls showed proliferative response to WE. Whole-extract activated IFN-gamma, IL-12, IL-10 levels were higher in the exposed group than in controls; IL-4 was not detectable in any of the samples. Among 21 responders to WE, frequent proliferative responses were seen to fractions F1-F4 (MW > 64.2 kDa) and none to fractions F5-F11; fractions F1-F11 stimulated comparable levels of IFN-gamma and IL-12 while IL-10 levels were higher in response to F5-F11 compared to F1-F4. These data demonstrate the presence of immunostimulatory antigens in the high MW fractions of whole L. donovani antigen. However, these fractions do not stimulate a Th1 response and produce variable amounts of IFN-gamma and the regulatory cytokine, IL-10. Hence, these high MW immunostimulatory fractions need to be evaluated in greater depth for their possible role as protective antigens.     

Autoreactive T cell clones isolated from normal and autoimmune-susceptible mice exhibit lymphokine secretory and functional properties of both Th1 and Th2 cells

Recent studies have suggested the existence of two mutually exclusive subpopulations of T helper (Th) cells in the murine immune system, called Th1 which produces interleukin (IL)-2 and interferon (IFN)-gamma but not IL-4 and Th2 which secretes IL-4 and IL-5 but not IL-2. Also, functionally, Th1 cells generally activate the macrophages and mediate delayed-type hypersensitivity whereas Th2 cells provide help efficiently to B cells. In the present study, we investigated the lymphokine secretory properties of two well-characterized autoreactive (self-Ia reactive) T cell clones isolated from normal DBA/2 mice and autoimmune-susceptible MRL-lpr/lpr mice. It was observed that both the autoreactive T cell clones, following activation, produced IL-2, IL-4, and IFN-gamma. They induced hyper-Ia expression and cell proliferation in syngeneic B cells as well as activated the macrophages to exhibit tumoristatic properties. Both clones could also induce T-T network interaction in which syngeneic naive CD4+ T cells responded directly to stimulation with autoreactive T cell clones. The T-T interaction was demonstrable in 1-month-old MRL-lpr/lpr mice prior to the onset of the autoimmune disease but not in 6-month-old mice having lymphadenopathy and autoimmune disease. Unlike Th1 and Th2 cells which upon antigenic stimulation respond to exogenous IL-2 and IL-4, the autoreactive T cell clones responded only to IL-2 but not to IL-4. Our data suggest the existence of a unique subset of immunoregulatory CD4+ Th cells having the lymphokine secretory and functional properties of both the murine Th1 and Th2 subsets.     

Human Th1 and Th2 lymphocytes: their role in the pathophysiology of atopy

In human beings, as in mice, two distinct patterns of cytokine secretion have been defined among CD4+ helper T-cell clones. Human type 1 helper (Th1), but not type 2 helper (Th2), cells produce interleukin-2 (IL-2), gamma-interferon (IFN-gamma), and tumor necrosis factor-beta, whereas Th2, but not Th1, cells secrete IL-4 and IL-5, but not IL-2 or IFN-gamma. Other cytokines, such as IL-3, IL-6, GM-CSF, or TNF-alpha, are produced by both Th1 and Th2 cells. Th0 cells, a third Th subset, show combined production of Th1- and Th2-type cytokines. The different cytokine patterns are associated with different functions. In general, Th2 cells provide an excellent helper function for B-cell antibody production, particularly of the IgE class. On the other hand, Th1 cells are responsible for delayed type hypersensitivity reactions and are cytolytic for autologous antigen-presenting cells, including B cells. Most allergen- or helminth-antigen-specific human CD4+ T-cell clones exhibit a Th2 phenotype, whereas most clones specific for bacterial antigens show a Th1 profile. Allergen-specific Th2 cells seem to play a crucial role in atopy. These cells induce IgE production via IL-4 and favor the proliferation, differentiation, and activation of eosinophils via IL-5. In addition, Th2-derived IL-3 and IL-4 are mast-cell growth factors that act in synergy, at least in vitro. Recent evidence indicates that allergen-specific Th2 cells are selectively enriched in tissues affected by allergic inflammation, such as the bronchial mucosa of subjects with allergic asthma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis.

The T cell response to antigens from Leishmania major promastigotes was investigated in peripheral blood mononuclear cells from Sudanese individuals with a history of cutaneous leishmaniasis (CL), Sudanese individuals with positive DTH reaction in the leishmanin skin test but with no history of skin lesions, and in Danes without known exposure to Leishmania parasites. Proliferation and production of interferon-gamma (IFN-gamma) and IL-4 in antigen-stimulated cultures was measured. Lymphocytes from individuals with a history of CL proliferated vigorously and produced IFN-gamma after stimulation with either a crude preparation of L. major antigens or the major surface protease gp63. These cultures produced no or only little IL-4. Also cells from leishmanin skin test-positive donors with no history of CL produced IFN-gamma and no IL-4 in response to L. major antigens. Cells from the unexposed Danes were not activated by gp63. The cells from Danish donors produced either IFN-gamma or IL-4, but not both cytokines after incubation with the crude preparation of L. major antigens. The data show that the T cell response to Leishmania antigens in humans who have had uncomplicated CL or subclinical L. major infection is an IFN-gamma-producing Th1-like response.     

The method of deriving human T-cell clones alters the proportion of IL-10-producing cells.

It is now well documented that the mode of primary stimulation in the mouse determines the ratio of Th1-, Th0- or Th2-type T cells obtained. In this paper we determine whether driving and cloning human peripheral blood mononuclear cells (PBMC) non-specifically with interleukin-2 (IL-2) and/or IL-4 and mitogen, will differentially select T cells for IL-10 production. The presence of IL-2 during culture (with or without IL-4) yielded predominantly Th1-type clones (81% of clones with IL-2 alone and 77% with IL-2 + IL-4) and a low frequency of Th0-type clones (19% of clones with IL-2 and 10% of clones with IL-2 + IL-4), whereas IL-4 alone increased the yield of the IL-4 producing Th0 (35%) clones. The proportions of IL-2-producing clones did not alter with treatment; however, the combination of IL-2 + IL-4 altered the proportions of IL-10-producing clones. 47% of IL-2-cultured cells and 51% of IL-4-cultured cells produced IL-10 respectively, whereas only 22% of clones driven with IL-2 + IL-4 produced detectable levels of IL-10. Thus in the preliminary experiments described here we have demonstrated that the cytokines used to initially drive out human T cells and maintain the T-cell growth can skew the Th1/Th2/Th0 cytokine profiles of the clones obtained from mitogen-stimulated cultures. Moreover and unexpectedly the proportion of IL-10-producing cells is altered. These results are important in interpreting analysis of the cytokine profiles of human T cells and raise questions concerning how we should derive T-cell clones for a cytokine analysis that truly reflects the in vivo situation.     

Cellular profile of cytokine production in a patient with visceral leishmaniasis: gammadelta+ T cells express both type 1 cytokines and interleukin-10

The cytokine profile of CD4+, CD8+ T cells, gammadelta+ T cells and natural killer (NK) cells (CD94+CD3-) was studied in a patient with visceral leishmaniasis (VL). The otherwise healthy, human immunodeficiency virus-negative patient acquired the disease in Tuscany, Italy. Diagnosis was made by demonstration of high concentrations of antibodies against Leishmania antigens in serum. Flow cytometry for the detection of intracellular interferon-gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-6, IL-10, IL-13 and tumour necrosis factor (TNF)-alpha expression in peripheral blood mononuclear cells stimulated with phorbol 12-myristate 13-acetate and ionomycin was performed, followed by treatment with liposomal amphotericin B. CD4+ cells were identified as major cytokine-expressing cells, capable of producing both type 1 and type 2 cytokines. A high frequency of IL-4- and IL-13-expressing CD8+ cells was noted. NK cells and gammadelta+ T cells, thought to be involved in innate host defences against Leishmania, expressed IFN-gamma and TNF-alpha. Ten per cent of gammadelta+ T cells expressed IL-10, predominantly together with IFN-gamma, suggesting additional immune-regulatory roles for this T-cell subset in VL.     

T helper (h)1/Th2 and Leishmania: paradox rather than paradigm

Studies on Leishmania major have been largely responsible for the characterisation of the Th1/Th2 paradigm of healing/non-healing associated with intracellular infection. IFN-gamma and IL-4 were identified respectively as the counter-regulatory Th1 and Th2 cytokines promoting resistance and susceptibility to infection. While resistance against infection remains largely associated with an IL-12 induced type-1 response studies using in particular gene-deficient mice have questioned the paramount role of IL-4 in the non-healing disease and implicated several alternative candidates. Indeed IL-4 has been shown to have no exacerbatory role in murine visceral leishmaniasis while its contribution to the progression of cutaneous disease has been clearly shown to be influenced not only by the parasite species but also the mouse strain used. Furthermore, it is now well established that not only can Th2 responses be induced independently of IL-4 but IL-4 under certain circumstances can prime for IL-12 production and a type-1 response. Clearly a reappraisal of our current understanding of the role of IL-4 and the Th2 response in the immunobiology of leishmaniasis is required and this review seeks to address this issue.     

Kinesin motor domain of Leishmania donovani as a future vaccine candidate

Visceral leishmaniasis (VL) is one of the important parasitic diseases, with approximately 350 million people at risk. Due to the nonavailability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. The present study was carried out to examine the immunological potential of kinesin protein from the microtubule locus of Leishmania donovani as a suitable vaccine candidate. In silico analysis of this region revealed clusters of major histocompatibility complex class I and II binding epitopes in its motor domain region. A recombinant protein was expressed from this region and named rLvacc. The antigenicity and immunogenicity studies of this protein by Western blot analysis revealed that rLvacc is strongly recognized by sera from acute VL patients. To evaluate its immunogenicity, peripheral blood mononuclear cells from cured VL patients were separated, and a lymphocyte proliferation assay was carried out in the presence of rLvacc. After lymphocyte proliferation, the pooled culture supernatant was assayed for anti-rLvacc antibody titers using an enzyme-linked immunosorbent assay. The results showed that immunoglobulin G2 (IgG2) subtype antibodies were predominant, while IgG1 subtype antibodies were produced in very low titers. On the basis of these ex vivo preliminary findings, its immunogenicity was studied in BALB/c mice. Vaccination with the DNA construct generated a good cellular immune response with significant increases in gamma interferon and interleukin-2 (IL-2) cytokine levels (Th1), but no increase in IL-4 levels (Th2). Taken together, our findings suggest the kinesin motor domain region of L. donovani as a potential vaccine candidate against visceral leishmaniasis.     

Induction and abrogation of LACK reactive cells in the evolution of human leishmaniasis.

Peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis patients with ongoing Leishmania aethiopica infection and individuals cured/under treatment from L. infantum or L. donovani infection were stimulated in vitro with LACK, the Leishmania homologue of receptors for activated C kinase. The LACK protein is conserved in related leishmanial species and is expressed both in the promastigote and amastigote stages of Leishmania. Our results show that LACK induced marked NK and some CD8+ cell proliferation in PBMC from cutaneous leishmaniasis patients with active disease. These responses were coupled with high levels of IFN-gamma and IL-10 production. At the concentration tested, the proliferative responses to freeze-thawed Leishmania antigen (Ft-Leish) were higher, while the levels of IFN-gamma were consistently lower than that of LACK. Although cells from individuals cured of leishmaniasis could respond to whole Leishmania lysate by proliferation and IFN-gamma production, there was no evident response to LACK. Ethiopian controls tested at the same time also showed LACK induced proliferation with IFN-gamma and IL-10 responses. Thus LACK reactivity in terms of proliferation and cytokine induction were present in cells from some healthy donors and most of the patients with active lesions, while this response was absent in individuals cured of L. infantum or L. donovani leishmaniasis. Since cure from leishmaniasis often results in life-long protection, and active but not cured patients showed in vitro responses to LACK stimulation, questions arose as to how this highly immunodominant molecule functions during human leishmanisasis. Some possible mechanisms are discussed.     

Immunoregulatory effects of N9-benzyl- and N7-benzyl-8-bromoguanines

In this study we investigated the effects of two guanine derivatives, 9-benzyl- (I) and 7-benzyl-8-bromoguanines (II) on the proliferation of human T-cell leukemia and T-cell lymphoma, normal human peripheral blood mononuclear cells (PBMC), and mouse Th1 (pGL10) and Th2 (D10.G4.1) clones. We also assessed their effects on cytokine production (IL-3, IL-10 and IFN-gamma) in PBMC, T-cell lymphoma, HUT78 (IL-2), and murine Th1 (IL-2) and Th2 (IL-4 and IL-5) clones. These compounds were synthesize as analog of known inhibitors of purine nucleoside phosphorylase (PNP) 8-amino-9-benzylguanine. These compounds suppressed proliferation of human leukemia MOLT-4 cells, human cutaneous lymphoma HUT78 cells and normal PMBC. Compound II was a significantly more potent inhibitor than compound I. Exogenous recombinant human IL-2 reversed the anti-proliferative effects of both compounds on HUT78 cells. These compounds had low toxicity to human EBV-transformed B-lymphocytes. Both compounds suppressed the production of IL-2 by activated human HUT78 cells, IFN-gamma by PBMC and did not affect IL-3 and IL-10 production in PBMC. Compound I inhibited anti-CD3-activated IL-2 secretion from the murine Th1 clone. The murine Th2 clone was less sensitive to both compounds as compared with Thl. The production of IL-4 and IL-5 by this clone was not suppressed. Thus, it has been shown that not only 9-substituted guanines but also their 7-isomers selectively inhibit T-cell functions and both selectively inhibit Th1-related cytokines secretion.     

Identification of Th0 cells responding to measles virus

Mechanisms involved in the induction of immunity to measles virus (MV) are not well understood. In the present study, we assessed proliferation, interferon (IFN)-gamma, and interleukin (IL)-4 production of MV-specific T cells after secondary in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from human donors. Such secondary stimulation resulted in responses substantially higher than after primary in vitro exposure. Most study participants produced both IFN-gamma and IL-4 after secondary in vitro stimulation. Patterns of secondary in vitro responses that use genetically disparate antigen-presenting cells were consistent with T-cell recognition restricted to human leukocyte antigen class II molecules. Limiting dilution analyses indicated that precursor frequencies of cytokine secreting and proliferating cells ranged from about 0.001% to 0.1% among fresh PBMCs. Split-well analyses of limiting dilution cultures suggested that virtually all putative T-cell clones produced either IFN-gamma alone or both IFN-gamma and IL-4. Intracytoplasmic flow cytometric analysis of polyclonal MV-specific secondary in vitro responding T cells revealed a similar pattern of cytokine expression. These results suggest that memory T cells responding in vitro to MV generate cells that produce either IFN-gamma alone (and resemble Th1-like cells) or secreted both IFN-gamma and IL-4 (resembling Th0-like cells) in vitro with few cells expressing a Th2-like pattern.