Dysregulated production of interleukin-10 (IL-10) and IL-12 by peripheral blood lymphocytes from human immunodeficiency virus-infected individuals is associated with altered proliferative responses to recall antigens.

The loss of immune function following infection with human immunodeficiency virus (HIV) may result from altered production of immunoregulatory cytokines such as interleukin-10 (IL-10) and IL-12. In this study, we analyzed IL-10 and IL-12 production by mitogen-stimulated peripheral blood mononuclear cells (PBMC) from HIV+ individuals and correlated their levels with proliferative responses to the recall antigens HIV p25 and influenza virus. We report two distinct groups of HIV+ patients. One group produced small amounts of IL-10, had PBMC that proliferated in response to recall antigens, and demonstrated enhanced recall antigen-induced proliferation upon addition of anti-IL-10 antibodies and/or IL-12. Conversely, the second group produced high levels of IL-10, had PBMC that failed to proliferate to recall antigens, and did not demonstrate enhanced proliferation upon addition of anti-IL-10 antibodies and/or IL-12. Mitogen-stimulated PBMC from both groups produced significantly lower levels of IL-12 than did those from HIV- controls. Analysis of the source of the IL-10-producing cell subset in PBMC demonstrated that in HIV+ individuals, IL-10 is produced by monocytes, while in HIV- controls, it is produced by both T cells and monocytes. Taken together, our results suggest that monocytes from HIV+ individuals secrete decreased amounts of IL-12, a Th1-type cytokine, which may lead to the development of Th2-type responses characterized by high IL-10 secretion and immune dysfunction.     

HIV I infection of dendritic cells

Dendritic cells (DC) from human peripheral blood are susceptible to productive and probably to latent infection with HIV-I. Infection of DC also occurs in vivo since in HIV-seropositive individuals Langerhans' cells of the skin and DC from peripheral blood, (in preparation) are infected. In peripheral blood 3-25% of DC, identified as large, low-density cells lacking monocyte markers, are infected as judged by in situ hybridization with an HIV probe. This contrasts with the lower proportion (< 0.2%) of other cells infected. DC exposed to HIV in vitro or in vivo fail to present other antigens or mitogens to stimulate T cells. This functional defect in infected DC is not blocked by the presence of soluble CD4 antigen and occurs in the absence of T cell infection suggesting a block at the level of the antigen-presenting cell itself. Infection, depletion and dysfunction of DC in HIV seropositive patients is already present in asymptomatic individuals and this precedes the appearance of T cell defects. We speculate that loss of functional DC may be a fundamental defect leading to a block in recruitment of resting T cells into immune responses. In contrast to the HIV-induced impairment of antigen presentation by DC, these cells were potent stimulators of responses to the HIV antigens themselves. Normal DC infected with HIV in vitro stimulated primary proliferative and cytotoxic T cell responses (in preparation). These were produced in cells from individuals expressing a range of different MHC types but the cytotoxic cells, once produced, killed autologous but not allogeneic, infected T cell blasts. Primary response to viral peptides can also be produced suggesting that this system may be useful for identifying immunogenic epitopes of HIV using cells from sero-negative, non-immunocompromised individuals.     

Natural controlled HIV infection: preserved HIV-specific immunity despite undetectable replication competent virus

Long-term non-progressive HIV infection, characterized by low but detectable viral load and stable CD4 counts in the absence of antiviral therapy, is observed in about 5% of HIV-infected patients. Here we identified four therapy na?ve individuals who are strongly seropositive for HIV-1 but who lack evidence of detectable HIV p24 antigen, plasma RNA, and proviral DNA in routine diagnostic testing. With an ultrasensitive PCR, we established that frequencies of pol proviral DNA sequences were as low as 0.2-0.5 copies/10(6) PBMC. HIV could not be isolated using up to 30x10(6) patient PBMC. One individual was heterozygous for CCR5 Delta32, but CCR5 expression on CD4+ T cells was normal to high in all four individuals. In vitro R5 and X4 HIV-1 susceptibility of CD8-depleted PBMC of all study subjects was significantly lower than the susceptibility of CD8-depleted PBMC of healthy blood donors. All individuals expressed protective HLA-B*58s alleles and showed evidence of HIV-specific cellular immunity either by staining with HLA-B*57 tetramers folded with an HIV RT or gag peptide or after stimulation with HIV-1 p24 gag, RT, or nef peptides in ELIspot analysis. HIV-specific CD4+ T helper cells were demonstrated by proliferation of CD4+ T cells and intracellular staining for IL-2 and IFNgamma after stimulation with an HIV-gag peptide pool. Sera of all individuals showed antibody-mediated neutralization of both R5 and X4 HIV-1 variants. These data implicate that very low-level antigen exposure is sufficient for sustained HIV-specific immunity and suggest the possibility of a multi-factorial control of HIV infection.     

四聚体技术检测人外周血巨细胞病毒抗原特异性CTL

为深入了解抗病毒免疫机制,探索人在健康状态及巨细胞病毒(CMV)急性感染时抗原特异性细胞毒T淋巴细胞(CTL)数量的动态变化。以CMV抗原肽、HLA-A*0201重链和轻链制备CMV四聚体;分离并检测12名健康被检者外周血单个核细胞(PBMC)中CMV抗原特异CTL数量;PBMC体外培养建立CTL细胞系,于末次刺激的不同时相进行四聚体染色,流式细胞仪(FACS)检测抗原特异CTL数量。结果发现9名被检者PBMC中均检出抗原特异CTL;细胞系中CMV特异性CTL数量急剧增多,细胞系与PBMC相比,差异有显著的统计学意义(P<0.001);研究结果提示,9名被检者感染过CMV,血液中存在少量免疫记忆性T细胞,当再次遭遇同一抗原后发生克隆扩增。     

Analysis of the proliferative responses to peptides in individuals with vigorous Gag protein-specific proliferation

Proliferative responses to recombinant HIV proteins in infected individuals may represent a correlate of protection from disease progression. In this study, the proliferative responses to HIV p24, p55 and gp120 were evaluated in infected subjects. Whereas, vigorous proliferative responses directed at the Gag proteins were detected in several individuals, Env-specific proliferation was observed in only one subject. Epitope mapping using overlapping peptides demonstrated proliferative responses of PBMC to Gag peptides. Responses were broadly directed at multiple peptides in some subjects. Although several of the peptides that induced proliferative responses also contain CTL epitopes potentially relevant in the particular individuals, many additional Gag T cell epitopes were present in each subject. This finding may be relevant for the design and testing of HIV candidate vaccines.     

Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials

Interferon-gamma (IFN-gamma) ELISpot and intracellular cytokine staining (ICS) assays are routinely employed in clinical HIV vaccine trials to identify antigen-specific T cells in cryopreserved peripheral blood mononuclear cells (PBMC). Several parameters involved in blood collection, processing and shipping may influence immunological function of the resulting cells, including anticoagulant type, time from venipuncture to PBMC isolation/cryopreservation, method of PBMC isolation and procedure for sample shipping. We examined these parameters in single and multiple site studies, and found the length of time from venipuncture to cryopreservation is the most important parameter affecting performance of T cells in immunological assays. Comparing blood processed at 24 h after venipuncture with that processed within 8 h, we observed on average a modest reduction in PBMC viability ( approximately 8% decrease), a greater loss in cell recovery ( approximately 32%), and between 36-56% loss in IFN-gamma T cell frequencies by ELISpot assay. We also describe three cold shipping methods that maintain immunological function in appropriately cryopreserved PBMC. These data indicate that cryopreservation of PBMC should occur within 8 h of venipuncture for optimal performance. This narrow window for specimen processing has important implications in selecting and monitoring clinical sites with laboratory capacity to perform these procedures in future clinical trials.     

Peripheral blood mononuclear cells from individuals infected with human T-cell lymphotropic virus type 1 have a reduced capacity to respond to recall antigens

Evidence indicates that human T-cell lymphotropic virus type 1 (HTLV-1) infection leads to chronic immunosuppression and a greater susceptibility to infectious diseases. Spontaneous in vitro proliferation of peripheral blood mononuclear cells (PBMC) is an important immunological feature of HTLV-1-infected individuals. However, the association between spontaneous proliferation and immunosuppression is not clear. In this study, we evaluated the cellular immune responses of PBMC from 58 asymptomatic HTLV-1-infected individuals with PBMC showing or not showing spontaneous proliferation. Individuals with PBMC that spontaneously proliferated had increased proportions of CD4 T cells expressing CD45RO and dramatically reduced responses to recall antigens. In addition, frequencies of positive responses to recall antigens were also decreased in HTLV-infected individuals without spontaneous proliferation of PBMC. There was a polyclonal expansion of multiple T-cell receptor Vbeta families of CD4+ T lymphocytes in patients with spontaneous proliferation. We observed that HTLV-1 induced an immunosuppression characterized by a decrease in the stimulation index to a recall antigen, even in individuals who did not present spontaneous proliferation. On the other hand, only patients with PBMC presenting spontaneous proliferation showed polyclonal activation and increased proportion of CD4 T cells expressing CD45RO.     

Preservation of lymphocyte immunophenotype and proliferative responses in cryopreserved peripheral blood mononuclear cells from human immunodeficiency virus type 1-infected donors: implications for multicenter clinical trials. The ACTG Immunology Advanced Technology Laboratories

Human immunodeficiency virus type 1 (HIV-1) infection results in impaired immune function that can be measured by changes in immunophenotypically defined lymphocyte subsets and other in vitro functional assays. These in vitro assays may also serve as early indicators of efficacy when new therapeutic strategies for HIV-1 infection are being evaluated. However, the use of in vitro assays of immune function in multicenter clinical trials has been hindered by their need to be performed on fresh specimens. We assessed the feasibility of using cryopreserved peripheral blood mononuclear cells (PBMC) for lymphocyte immunophenotyping and for lymphocyte proliferation at nine laboratories. In HIV-1-infected patients with moderate CD4(+) lymphocyte loss, the procedures of density gradient isolation, cryopreservation, and thawing of PBMC resulted in significant loss of CD19(+) B cells but no measurable loss of total T cells or CD4(+) or CD8(+) T cells. No significant changes were seen in CD28(-) CD95(+) lymphocytes after cell isolation and cryopreservation. However, small decreases in HLA-DR(+) CD38(+) lymphocytes and of CD45RA(+) CD62L(+) were observed within both the CD4(+) and CD8(+) subsets. Fewer than 10% of those specimens that showed positive PBMC proliferative responses to mitogens or microbial antigens lost their responsiveness after cryopreservation. These results support the feasibility of cryopreserving PBMC for immunophenotyping and functional testing in multicenter AIDS clinical trials. However, small changes in selected lymphocyte subsets that may occur after PBMC isolation and cryopreservation will need to be assessed and considered in the design of each clinical trial.     

Loss of T cell responses following long-term cryopreservation

Although cryopreservation of peripheral blood mononuclear cells (PBMC) is a commonly used technique, the degree to which it affects subsequent functional studies has not been well defined. Here we demonstrate that long-term cryopreservation has detrimental effects on T cell IFN-gamma responses in human immunodeficiency virus (HIV) infected individuals. Long-term cryopreservation caused marked decreases in CD4(+) T cell responses to whole proteins (HIV p55 and cytomegalovirus (CMV) lysate) and HIV peptides, and more limited decreases in CD8(+) T cell responses to whole proteins. These losses were more apparent in cells stored for greater than one year compared to less than six months. CD8(+) T cell responses to peptides and peptide pools were well preserved. Loss of both CD4(+) and CD8(+) T cell responses to CMV peptide pools were minimal in HIV-negative individuals. Addition of exogenous antigen presenting cells (APC) did not restore CD4(+) T cell responses to peptide stimulation and partially restored T cell IFN-gamma responses to p55 protein. Overnight resting of thawed cells did not restore T cell IFN-gamma responses to peptide or whole protein stimulation. A selective loss of phenotypically defined effector cells did not explain the decrement of responses, although cryopreservation did increase CD4(+) T cell apoptosis, possibly contributing to the loss of responses. These data suggest that the impact of cryopreservation should be carefully considered in future vaccine and pathogenesis studies. In HIV-infected individuals short-term cryopreservation may be acceptable for measuring CD4(+) and CD8(+) T cell responses. Long-term cryopreservation, however, may lead to the loss of CD4(+) T cell responses and mild skewing of T cell phenotypic marker expression.     

A diffusible lymphokine produced by CD8+ T lymphocytes suppresses HIV replication.

Peripheral blood CD8+ T lymphocytes from human immunodeficiency virus (HIV)-infected individuals suppress replication of HIV in peripheral blood mononuclear cells (PBMC). This anti-viral activity appears to be mediated in part by a diffusible factor. Production of this lymphokine varies among infected individuals and may reflect the intrinsic ability of an individual's CD8+ cells to control HIV infection. In some cases in which factor activity is not apparent, contact of the CD8+ cells with infected CD4+ cells can produce for suppression of virus replication. These observations could lead to approaches for enhancing anti-viral responses in HIV-infected individuals.     

Impacts of HIV infection on Vgamma2Vdelta2 T cell phenotype and function: a mechanism for reduced tumor immunity in AIDS

HIV infection causes rapid and lasting defects in the population of Vgamma2Vdelta2 T cells. To fully describe the impact of HIV, we examined PBMC samples from HIV+ patients receiving highly active antiretroviral therapy, who had displayed prolonged viral control and CD4 counts above 300 cells/mm3. We observed lower frequencies of CD27-/CD45RA- Vgamma2Vdelta2 cells in HIV+ individuals when compared with controls, coupled with an increased proportion of CD45RA+ cells. These changes were common among 24 HIV+ patients and were not related to CD4 cell count or viral RNA burden. Vgamma2 cells from HIV+ individuals had lower expression of Granzyme B and displayed reduced cytotoxicity against Daudi targets after in vitro stimulation. There was increased expression of FasR (CD95) on Vgamma2 cells from HIV+ PBMC that may be a mechanism for depletion of Vgamma2 cells during disease. In addition to the well-characterized defects in the Vgamma2 repertoire and functional responses to phosphoantigen, the proportion of CD27-/CD45RA- Vgamma2Vdelta2 T cells after isopentenyl pyrophosphate stimulation was reduced sharply in HIV+ donors versus controls. Thus, HIV infection has multiple impacts on the circulating Vgamma2Vdelta2 T cell population that combine to reduce the potential effector activity in terms of tumor cytotoxicity. Changes in Vgamma2Vdelta2 T cells, along with concomitant effects on NK and NKT cells that also contribute to tumor surveillance, may be important factors for elevating the risk of malignancy during AIDS.     

Analysis of nonhuman primate peripheral blood mononuclear cells for susceptibility to HIV-1 infection and HIV coreceptor expression

HIV-1 infection of nonhuman primates does not lead to the acquired immunodeficiency syndrome seen in humans. The basis for this lack of disease progression in these animals is still unknown. In this study, primary nonhuman primate peripheral blood mononuclear cells (PBMC) were tested for their susceptibility to in vitro infection by several different primary HIV-1 isolates representing distinct subtypes or clades. None of the five HIV-1 subtypes tested were able to readily establish an infection in chimpanzee or baboon PBMC, as determined by p24 antigen capture assays. To address the mechanism of in vitro resistance to HIV-1 infection, PBMC were analyzed for HIV coreceptor mRNA expression and cell surface expression. Flow cytometry analysis of the nonhuman primate PBMC demonstrated that they do express CD4, CCR3, CCR5, and CXCR4 on their cell surface. Therefore, the level of restriction in the virus replication cycle does not appear to lie at the point of entry in these cells.     

Preferential activation of peripheral blood V gamma 9+ gamma/delta T cells by group A, B and C but not group D or F streptococci.

Previous studies have established that inactivated mycobacteria are potent and selective activators of V gamma 9+/V delta 2+ human gamma/delta T cells. Here we have analysed the proliferative response of human gamma/delta T cells to five serologically distinct groups of streptococci. While heat-inactivated streptococci of all five serogroups tested (A, B, C, D and F) induced a strong proliferative response in peripheral blood mononuclear cells (PBMC), only groups A, B and C elicited a selective activation of V gamma 9+ gamma/delta T cells in 10 (serogroup B) or 11 (serogroups A and C) of 11 tested healthy individuals. In striking contrast, groups D and F streptococci failed to activate gamma/delta T cells in nine of 11 donors and induced only a weak gamma/delta T cell response in two additional individuals. Depletion of V gamma 9+ T cells before culture completely eliminated all gamma/delta T cell responses to streptococci. These data indicate that groups A, B and C (but not D or F) streptococci can be included in the growing list of selective ligands for V gamma 9+/V delta 2+ human gamma/delta T cells.     

Effect of Pre-S1 protein on the long-term cultures of human lymphocytes after hepatitis B immunization.

Long-term cultured T-cells, reactive to Pre-S1 protein, were developed from peripheral blood mononuclear cells (PBMC) of individuals after recovery from hepatitis B infection and of vaccine recipients by in vitro Pre-S1 protein stimulation in the presence of IL-2. The proliferative responses to Pre-S1 protein and functional activities of cultured T-cells were characterized.     

HIV I Infection of Dendritic Cells

Dendritic cells (DC) from human peripheral blood are susceptible to productive and probably to latent infection with HIV-I [18, 29]. Infection of DC also occurs in vivo since in HIV-seropositive individuals Langerhans’ cells of the skin [16] and DC from peripheral blood ([17], in preparation) are infected. In peripheral blood 3–25% of DC, identified as large, low-density cells lacking monocyte markers, are infected as judged by in situ hybridization with an HIV probe. This contrasts with the lower proportion (<0.2%) of other cells infected. DC exposed to HIV in vitro or in vivo fail to present other antigens or mitogens to stimulate T cells [29, 38, 41]. This functional defect in infected DC is not blocked by the presence of soluble CD4 antigen and occurs in the absence of T cell infection suggesting a block at the level of the antigen-presenting cell itself. Infection, depletion and dysfunction of DC in HIV seropositive patients is already present in asymptomatic individuals and this precedes the appearance of T cell defects. We speculate that loss of functional DC may be a fundamental defect leading to a block in recruitment of resting T cells into immune responses.

In contrast to the HIV-induced impairment of antigen presentation by DC, these cells were potent stimulators of responses to the HIV antigens themselves. Normal DC infected with HIV in vitro stimulated primary proliferative and cytotoxic T cell responses ([52], in preparation). These were produced in cells from individuals expressing a range of different MHC types but the cytotoxic cells, once produced, killed autologous but not allogeneic, infected T cell blasts. Primary response to viral peptides can also be produced suggesting that this system may be useful for identifying immunogenic epitopes of HIV using cells from sero-negative, non-immunocompromised individuals.     

Discordant cellular and humoral immune responses to cytomegalovirus infection in healthy blood donors: existence of a Th1-type dominant response

Previous studies have documented discordant cellular and humoral immune responses to subjects exposed to HIV-1, and that the nature of such responses may determine susceptibility and resistance to disease. We determined whether there is a spectrum of cellular versus humoral immunodominant responses to cytomegalovirus (CMV) infection. Blood samples from 50 healthy blood donors were tested for anti-CMV IgG antibodies and for proliferative responses of peripheral blood mononuclear cells (PBMC) to CMV antigens. Four patterns of immune responses to CMV were found: no detectable response (30%, Ab(-)/Tc(-)), anti-CMV IgG only (28%, Ab(+)/Tc(-)), both anti-CMV IgG and T lymphocyte proliferation to CMV antigens (18%, Ab(+)/Tc(+)), and, interestingly, T lymphocyte proliferation to CMV only (24%, Ab(-)/Tc(+)). To determine whether these immunodominant phenotypes correlate with the ability of PBMC to secrete IL-2 and IFN-gamma in response to CMV antigens, we found that a greater percentage of individuals with a T cell proliferative response to CMV antigens (Ab(-)/Tc(+) and Ab(+)/Tc(+)) responded with increased IL-2 (P = 0.001) and IFN-gamma levels (P = 0.002), compared to those without a proliferative response (Ab(-)/Tc(-) and Ab(+)/Tc(-)). Our data therefore demonstrate that different individuals exhibit different immunodominant patterns of response to CMV. In particular, some individuals who are exposed to CMV fail to develop an antibody response but do develop cellular immunity. Whether these different patterns predict susceptibility or resistance to CMV-induced disease remains to be determined.     

Whole blood assay for assessment of the mixed lymphocyte reaction

When lymphocytes from genetically different individuals are mixed together in tissue culture blast transformation occurs, a reaction known as the mixed lymphocyte reaction (MLR). The MLR is a clinically relevant in vitro assay where lymphocytes from one individual (effector, E) are incubated with the lymphocytes of another individual (stimulator, S) which have been previously rendered incapable of blast transformation by gamma-irradiation. We have standardised a whole blood (WB) MLR assay where the E lymphocytes were provided by 20 microl of WB and the S lymphocytes were provided by irradiated peripheral blood mononuclear cells (PBMCs) either as a mixed pool of 20 donor PBMCs or as single donor PBMC. The optimum number of S lymphocytes needed was comparatively higher than in the standard PBMC MLR: the optimum calculated E:S ratio was 1:20 compared to a E:S ratio of 1:1 or 3:2 in the standard PBMC MLR. In ten normal individuals the WB/PBMC MLR was similar to the standard PBMC/PBMC MLR. As a clinical example, the WB/PBMC MLR proliferative capacity of 13 patients with malignant mesothelioma was no different from the proliferative capacity of their age-sex matched controls. This standardised WB/PBMC MLR assay is a simple and more practical assay than the standard MLR assay and can be incorporated easily in clinical studies with biological end-points.     

Antigen-presentation by macrophages but not by dendritic cells in human immunodeficiency virus (HIV) infection.

Dendritic cells (DC) have a potent antigen-presenting capacity for recruiting resting T cells into immune responses. They also promote expansion of already activated memory T cells. By contrast, macrophages (M phi) are only effective in stimulating memory responses. Infection and depletion of DC occur in human immunodeficiency virus (HIV)-infected individuals and recruitment of T cells into primary responses is blocked. Here comparisons between DC and M phi in stimulating secondary T-cell responses in HIV infection were made. Adherent M phi, and DC isolated by a new method, were separated from peripheral blood of patients in different stages of HIV infection and from uninfected controls and added to allogeneic lymphocytes in mixed leucocyte reactions (MLR). Some were pulsed with influenza virus or tetanus toxoid and used to stimulate autologous T cells. Responses were measured from uptake of [3H]thymidine in 20 microliters hanging drop cultures. DC, but not M phi, from normal individuals stimulated MLR but both populations stimulated secondary responses to recall antigens. DC from all HIV seropositive individuals caused little or no stimulation of any lymphocyte responses. However, M phi from HIV seropositive asymptomatic individuals and those with persistent generalized lymphadenopathy stimulated responses to recall antigens. There was no stimulation using cells from acquired immune deficiency syndrome (AIDS) patients. Blocked DC but not M phi function may underlie progressive immunological non-responsiveness in HIV infection. Without recruitment of resting T cells, loss of memory T cells may be cumulative; failure of secondary activation (e.g. by M phi) would lead to lost T-cell activity. Identification and circumvention of the defect in DC could offer new therapeutic approaches.     

Leishmanin skin test lymphoproliferative responses and cytokine production after symptomatic or asymptomatic Leishmania major infection in Tunisia

Resistance to Leishmania parasite infection requires the development of a cellular immune response that activates macrophage leishmanicidal activity. In this study we have investigated the lymphoproliferative responses and in vitro cytokine production of peripheral blood mononuclear cells (PBMC) from individuals living in an endemic area for L. major infection in Tunisia. The results were compared with the DTH reaction of the leishmanin skin test (LST). Sixty-seven individuals were included in the study: 22 persons (age range 9-60 years) who developed, 2 years before the present study, a parasitologically confirmed localized cutaneous leishmaniasis (LCL) that healed spontaneously, and 45 individuals (age range 18-20 years) born and living in the same area, with no previous history of LCL. LST was positive (skin induration > or = 5 mm) in 20/22 cured cases of LCL and in 75% of healthy individuals without history of LCL. LST+ individuals expressed vigorous Leishmania-specific lymphoproliferative responses associated with in vitro production of interferon-gamma (IFN-gamma) but not IL-4. Interestingly, IL-10 was detected in parallel with the highest levels of IFN-gamma in PBMC supernatants from 3/20 cured LCL and 8/25 individuals without history of LCL. Our results showed a 98% concordance between the DTH reaction assessed by LST and the in vitro proliferative assay induced by soluble leishmanial antigens. Moreover, proliferative assays as well as cytokine analysis did not show any significant difference of the immune memory to parasite antigens developed by patients who had overt cutaneous leishmaniasis and those who had apparently asymptomatic infection.     

Distinct innate and acquired immune responses to Leishmania in putative susceptible and resistant human populations endemically exposed to L. (Viannia) panamensis infection

Mechanisms of constitutive and acquired susceptibility/resistance to Leishmania Viannia panamensis (L. (V ) p.) were investigated in endemically exposed human populations presenting either recurrent disease (putative susceptible) or subclinical infection (clinically resistant). Cutaneous delayed type hypersensitivity response to leishmanin was significantly lower among individuals experiencing recurrent leishmaniasis than among those whose skin test converted without developing the disease. Monocyte derived macrophages from individuals with recurrent disease were more permissive in vitro to the entry of parasites than macrophages from subclinically infected individuals. In vitro proliferation of CD4 and CD8 T lymphocytes in response to intracellular amastigotes was significantly lower among individuals with a history of recurrent disease compared with subclinically infected individuals. Linear regression analyses revealed a strong direct relationship between the production of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 by peripheral blood mononuclear cells (PBMC) from resistant (subclinically infected) individuals and no correlation in the production of these cytokines by PBMC from individuals who experienced recurrent disease. The results provide evidence of differences in the innate and acquired responses to Leishmania according to the outcome of the natural infection. These findings support the feasibility of identifying the immunological bases of innate and acquired resistance through studies in naturally exposed human populations.