Preliminary evaluation of whole-blood gamma interferon release for clinical assessment of cellular immunity in patients with active coccidioidomycosis

Assessment of the cellular immune response in coccidioidomycosis has epidemiologic and prognostic importance. Measurement of delayed-type hypersensitivity to skin testing has been used in the past to determine cellular immunity in coccidioidomycosis. However, no skin tests are currently available in the United States. Assay of gamma interferon (IFN-gamma) release in whole blood in response to incubation with antigen has been used to assess cellular immunity in tuberculosis. We used a similar assay using the coccidioidal antigen preparation T27K to measure the in vitro cellular immune responses among a cohort of 69 subjects with active coccidioidomycosis. IFN-gamma release was bimodal, with concentrations above and below 5 IU/ml. Using multivariate logistic regression, underlying disease and disseminated or chronic pulmonary coccidioidomycosis was significantly associated with the release of IFN-gamma at a concentration of <5 IU/ml (P = 0.02 or 0.05, respectively). In addition, the release IFN-gamma concentration was <5 IU/ml in all subjects with a clinical severity score of > or =6 (P = 0.02). The release IFN-gamma concentration correlated with expression of CD69 on T lymphocytes in an in vitro assay using T27K as the antigen (Spearman's rho = 0.59; P < 0.01). These results suggest that the IFN-gamma release assay with T27K as the antigen may be a useful clinical test for assessing cellular immunity in patients with active coccidioidomycosis.     

Flow cytometric assessment of human peripheral blood mononuclear cells in response to a coccidioidal antigen.

The in vitro responses of peripheral blood mononuclear cells (PBMC) from healthy immune and non-immune donors were assessed by flow cytometry after incubation with the coccidioidal antigen toluene spherule lysate (TSL). After 120 h of incubation with 100 microg ml(-1) of TSL, expression of the activation markers CD69, CD25 and human leukocyte antigen-DR were all significantly increased in CD3+ lymphocytes from immune donors compared to non-immune donors (P < 0.03 for all). No differences in the surface expression of the costimulatory molecules CD28, CD152 or CD154 was seen between immune and non-immune donors after either 24 or 120 h of TSL incubation, nor were differences detected in the expression of the B7 ligands CD80 or CD86 on CD14+ monocytes. The percent of CD3+ lymphocytes expressing intracellular interferon-gamma (IFN-gamma) was significantly increased in immune compared to non-immune donors and was further increased by the addition of 10 ng ml(-1) of human recombinant interleukin (IL)-12 (P < 0.05 for both). Both CD4+ and CD8+ lymphocytes contributed to IFN-gamma production. These data indicate that coccidioidal antigen stimulation of lymphocytes from healthy immune donors leads to specific expression of activation molecules and production of intracellular IFN-gamma. Addition of IL-12 leads to a significant recruitment of cells producing IFN-gamma among immune donors.     

In vitro whole-blood analysis of cellular immunity in patients with active coccidioidomycosis by using the antigen preparation T27K

Measurement of cellular immunity in human coccidioidomycosis has important diagnostic and prognostic implications. The coccidioidin skin test has been the standard for the measurement of this, but it is not available in the United States. We examined the utility of measuring surface expression of CD69 on T lymphocytes in whole blood incubated with the coccidioidal antigen preparation T27K as an alternative to the skin test. Seventy donors with active coccidioidomycosis were studied. The mean fluorescent intensity (MFI) of CD69 expression on CD3 lymphocytes in response to T27K was 28.61 +/- 1.77, significantly greater than the control response of 11.45 +/- 0.78 (P < 0.001). The MFI CD69 response to T27K above that for the control (MFI CD69 above control) was 6.35 +/- 2.18 for seven subjects with disseminated coccidioidomycosis who were studied within 5 months of diagnosis. This was significantly below the value of 20.17 +/- 3.17 for 18 subjects with pulmonary coccidioidomycosis studied within 5 months of diagnosis and the value of 19.58 +/- 2.91 for 27 subjects with disseminated coccidioidomycosis studied after 5 months of diagnosis (for both, P < 0.05). There was an inverse correlation between coccidioidal clinical score and MFI CD69 above control for all 34 subjects with disseminated coccidioidomycosis (r = 0.362; P = 0.036) but not for the 36 subjects with pulmonary disease (r < 0.001; P = 0.993). Among 30 subjects for whom data were available, there was a highly significant association between the MFI CD69 above control and the supernatant concentrations of gamma interferon, interleukin-2 (IL-2), and tumor necrosis factor alpha (for all, P < 0.001), but not for IL-4, IL-5, or IL-10. These data indicate that in vitro assessment of CD69 expression on T lymphocytes by using T27K may be a useful measure of cellular immune response among subjects with active coccidioidomycosis.     

In Vitro Whole-Blood Analysis of Cellular Immunity in Patients with Active Coccidioidomycosis by Using the Antigen Preparation T27K

Measurement of cellular immunity in human coccidioidomycosis has important diagnostic and prognostic implications. The coccidioidin skin test has been the standard for the measurement of this, but it is not available in the United States. We examined the utility of measuring surface expression of CD69 on T lymphocytes in whole blood incubated with the coccidioidal antigen preparation T27K as an alternative to the skin test. Seventy donors with active coccidioidomycosis were studied. The mean fluorescent intensity (MFI) of CD69 expression on CD3 lymphocytes in response to T27K was 28.61 ± 1.77, significantly greater than the control response of 11.45 ± 0.78 (P < 0.001). The MFI CD69 response to T27K above that for the control (MFI CD69 above control) was 6.35 ± 2.18 for seven subjects with disseminated coccidioidomycosis who were studied within 5 months of diagnosis. This was significantly below the value of 20.17 ± 3.17 for 18 subjects with pulmonary coccidioidomycosis studied within 5 months of diagnosis and the value of 19.58 ± 2.91 for 27 subjects with disseminated coccidioidomycosis studied after 5 months of diagnosis (for both, P < 0.05). There was an inverse correlation between coccidioidal clinical score and MFI CD69 above control for all 34 subjects with disseminated coccidioidomycosis (r = 0.362; P = 0.036) but not for the 36 subjects with pulmonary disease (r < 0.001; P = 0.993). Among 30 subjects for whom data were available, there was a highly significant association between the MFI CD69 above control and the supernatant concentrations of gamma interferon, interleukin-2 (IL-2), and tumor necrosis factor alpha (for all, P < 0.001), but not for IL-4, IL-5, or IL-10. These data indicate that in vitro assessment of CD69 expression on T lymphocytes by using T27K may be a useful measure of cellular immune response among subjects with active coccidioidomycosis.     

In vitro modulation of proliferation and cytokine production by human peripheral blood mononuclear cells from subjects with various forms of coccidioidomycosis.

Using peripheral blood mononuclear cells (PBMC) from individuals with or without coccidioidal delayed-type hypersensitivity (DTH), we examined and attempted to modulate the in vitro responses of PBMC from various donors to the coccidioidal antigen toluene spherule lysate (TSL). Among healthy DTH-positive donors, 100 ng of human recombinant interleukin-10 (IL-10) per ml suppressed both PBMC proliferation (P = 0.01) and gamma interferon (IFN-gamma) and IL-12 production (for both, P < 0.05). In vitro proliferation and production of IFN-gamma and IL-12 by PBMC were significantly higher in DTH-positive donors with active coccidioidomycosis than in healthy, nonimmune controls (P < 0.05) but not in active DTH-negative donors with or without human immunodeficiency virus infection (for both, P > 0.05). Human recombinant IL-12 increased IFN-gamma production by PBMC from active, DTH-positive donors (P = 0.01) but not by PBMC from DTH-negative groups. For healthy DTH-positive donors, the median antigen-reactive cell frequency per 10(5) PBMC was 3.7, compared to 1.7 in DTH-negative donors with active coccidioidomycosis (P = 0.03). These data indicate that the in vitro TSL response is highly dependent on coccidioidal DTH. Not only do PBMC from individuals with DTH appear to respond to TSL, but their response can be modulated in vitro with either IL-10 or IL-12. On the other hand, PBMC from DTH-negative individuals do not respond in vitro to TSL and their response is not modulable, suggesting a lack of antigen response.     

Polyfunctional T Lymphocytes Are in the Peripheral Blood of Donors Naturally Immune to Coccidioidomycosis and Are Not Induced by Dendritic Cells

Coccidioidomycosis is a fungal infection endemic in the southwestern United States that is increasing in incidence. While cellular immunity correlates with protection from clinical illness, the precise elements of that response are undefined. Using the coccidioidal antigen preparation T27K and multiparametric flow cytometry, the in vitro frequency of polyfunctional T lymphocytes in the peripheral blood of naturally immune healthy donors and those who were nonimmune was determined. Polyfunctional CD4 lymphocytes, defined as producing intracellular interleukin 2 (IL-2), gamma interferon (IFN-γ), and tumor necrosis factor alpha simultaneously, had a frequency of 137 per 400,000 events among peripheral blood mononuclear cells (PBMC) of immune donors compared to 11 per 400,000 PBMC from nonimmune donors (P = 0.03). When monocyte-derived mature dendritic cells pulsed with T27K (mDC_T27K) were used for antigen presentation, the frequency of polyfunctional CD4 T lymphocytes did not significantly increase for either group, although mDC_T27K did significantly increase the concentrations of IL-2 and IFN-γ released by PBMC from nonimmune donors (P = 0.02). After in vitro stimulation with T27K, polyfunctional CD4 and CD8 lymphocytes of PBMC from immune donors had a mixture of low- and high-expression CCR7 cells, suggesting both effector and central memory, compared with predominantly high-expression CCR7 cells when PBMC were incubated with the mitogen phytohemagglutinin (P = 0.03). These data demonstrate the presence of polyfunctional T lymphocytes in the peripheral blood of individuals with coccidioidal immunity and suggest a model for the in vitro testing of vaccine candidates for coccidioidomycosis.Coccidioidomycosis is a major mycosis endemic in the southwestern United States whose incidence is increasing (7, 10). Infection usually occurs when airborne coccidioidal arthroconidia are inhaled and lodge in the terminal alveoli of the lung (20). In approximately 60% of cases, infection is completely asymptomatic and is manifested only by a cellular immune response, such as a delayed-type hypersensitivity (DTH) reaction to coccidioidal antigens. Smith and colleagues initially demonstrated that DTH is the hallmark of coccidioidal infection and that a lack of this response in otherwise healthy individuals indicates an absence of infection (30). Moreover, this cellular immune response appears to be long-lived and protective (15). In vitro studies have suggested that control of infection is associated with the release of T-helper type 1 (Th1) cytokines, such as interleukin-2 (IL-2) and gamma interferon (IFN-γ), from peripheral blood mononuclear cells (PBMC) (1-5, 13).Beyond the measurement of DTH and the release of Th1 cytokines by PBMC, little is known of the precise elements of immune protection in human coccidioidomycosis. Defining these elements is critical for the development of a protective vaccine (28). Using multiparametric flow cytometry, Darrah and colleagues (14) have presented murine data demonstrating that immunization with a live Leishmania vaccine results not only in strong protection against rechallenge but also in the production of lung, lymph node, and splenic CD4 T lymphocytes containing intracellular IL-2, IFN-γ and tumor necrosis factor alpha (TNF-α). The majority of these polyfunctional CD4 T lymphocytes also express small amounts of surface CCR7, suggesting that they are of the effector memory phenotype. These results have been interpreted to suggest that the quality of the T-lymphocyte response, as manifested by a polyfunctional cytokine expression, is a marker for protective cellular immunity. Since publication of this work, polyfunctional T lymphocytes have been identified in the peripheral blood of humans with Mycobacterium tuberculosis infection (9), as well as a variety of viral infections (11, 17, 18, 22).In work previously performed, mature monocyte-derived dendritic cells pulsed with coccidioidal antigen were shown to activate lymphocytes from nonimmune healthy donors as well as lymphocytes from donors with extrathoracic dissemination and anergy (24). In that study, the mature dendritic cells (mDC) generated were functionally and phenotypically normal. Stimulation in this model was accomplished using T27K, an antigen preparation derived from Coccidioides posadasii that specifically induces the release of Th1 cytokines from PBMC from healthy immune donors (3-5) and correlates with DTH responses (6).In the present study, we wished to determine if polyfunctional T lymphocytes occur in human coccidioidomycosis. We used individuals who had acquired their infection naturally and compared them to donors without infection who were not immune. Infection and immunity were determined based on the in vitro release of Th1 cytokines by PBMC in response to incubation with the coccidioidal antigen preparation T27K. As noted above, the expression of specific coccidioidal cellular immunity indicates infection and long-lived immunity, while the absence of this in a healthy individual indicates no prior infection and no immunity. We have previously demonstrated that in vitro measurement of cellular immunity using T27K correlates significantly with DTH to coccidioidin (6).In addition, we wished to see if the lymphocyte activation that occurs with incubation with mDC pulsed with coccidioidal antigen is similar to the cytokine profile observed among cells obtained from naturally immune donors. Finally, we attempted to ascertain the memory phenotype of antigen-stimulated lymphocytes from naturally immune donors. In all of this work, we used the antigen preparation T27K, since it has consistently been shown to induce specific Th1 responses from coccidioidal antigen immune individuals (3-5).     

Cellular immune suppressor activity resides in lymphocyte cell clusters adjacent to granulomata in human coccidioidomycosis

The in situ immunologic response in human coccidioidomycosis remains undefined. To explore this further, pulmonary necrotizing coccidioidal granulomata were examined using immunohistochemical staining for lymphocyte subsets and for the cytokines interleukin-10 (IL-10) and gamma interferon (IFN-gamma). Discrete perigranulomatous lymphocytic clusters were seen in eight of nine tissues examined. In these tissues, T lymphocytes (CD3+) significantly outnumbered B lymphocytes (CD20+) in the mantle area of the granulomata (P = 0.028), whereas the clusters were composed of roughly equal numbers of T and B lymphocytes. While the number of cells in the mantle expressing IL-10 was similar to those in the perigranulomatous clusters, there were significantly more cells expressing IFN-gamma in the mantle than in the clusters (P = 0.037). Confocal microscopy revealed that CD4+ T lymphocytes and B lymphocytes are associated with IL-10 production. CD4+CD25+ T lymphocytes were identified in the perigranulomatous clusters but were not associated with IL-10 production. This is the first report noting perigranulomatous lymphocyte clusters and IL-10 in association with human coccidioidal granulomata and suggests that down-regulation of the cellular immune response is occurring within coccidioidal granulomata.     

Delayed-type hypersensitivity, in vitro T-cell responsiveness and risk of active coccidioidomycosis among HIV-infected patients living in the coccidioidal endemic area.

The prognostic importance of specific and general tests of immune function were examined among a cohort of 170 subjects infected with human immunodeficiency virus type-1 (HIV), living in an area endemic for the fungal infection coccidioidomycosis. Using the proportional hazards model and multivariate analysis, lack of expression of coccidioidal delayed-type hypersensitivity (DTH) was found to be dependent on anergy in response to the non-coccidioidal antigens mumps, Trichophyton and Candida (relative hazard 4.2, 95% CI 1.8-9.8, P=0.001). Among subjects with CD4 lymphocyte counts >/=250 microl-1 on entry into the study, the in vitro lymphocyte transformation (LT) response to the coccidioidal antigen toluene spherule lysate was 4967+/-1652 (mean counts per minute (c.p.m.)+/-SEM) in subjects with coccidioidal DTH compared with 136+/-222 in those with negative DTH (P<0.001). However, amongst those whose CD4 count was <250 microl-1, LT responses were low and there was no significant difference based on coccidioidal DTH (P=0.965). Using the proportional hazards model and multivariate analysis, only a CD4 count <250 microl-1 was prognostically associated with the development of either active coccidioidomycosis or AIDS. These data indicate that immunodeficiency, particularly a CD4 lymphocyte count <250 microl-1, is the most important factor in the lack of expression of specific immunity to coccidioidomycosis and in the development of active coccidioidomycosis among HIV-infected individuals living in the coccidioidal endemic area.     

Preliminary Evaluation of Whole-Blood Gamma Interferon Release for Clinical Assessment of Cellular Immunity in Patients with Active Coccidioidomycosis

Assessment of the cellular immune response in coccidioidomycosis has epidemiologic and prognostic importance. Measurement of delayed-type hypersensitivity to skin testing has been used in the past to determine cellular immunity in coccidioidomycosis. However, no skin tests are currently available in the United States. Assay of gamma interferon (IFN-γ) release in whole blood in response to incubation with antigen has been used to assess cellular immunity in tuberculosis. We used a similar assay using the coccidioidal antigen preparation T27K to measure the in vitro cellular immune responses among a cohort of 69 subjects with active coccidioidomycosis. IFN-γ release was bimodal, with concentrations above and below 5 IU/ml. Using multivariate logistic regression, underlying disease and disseminated or chronic pulmonary coccidioidomycosis was significantly associated with the release of IFN-γ at a concentration of <5 IU/ml (P = 0.02 or 0.05, respectively). In addition, the release IFN-γ concentration was <5 IU/ml in all subjects with a clinical severity score of ≥6 (P = 0.02). The release IFN-γ concentration correlated with expression of CD69 on T lymphocytes in an in vitro assay using T27K as the antigen (Spearman's rho = 0.59; P < 0.01). These results suggest that the IFN-γ release assay with T27K as the antigen may be a useful clinical test for assessing cellular immunity in patients with active coccidioidomycosis.     

Intracellular production of type I and type II cytokines during HIV-1 progression in Thai patients

A type I to type II cytokine switch on cells of the immune system has been suggested as a critical step in the etiology of HIV infection. In this study, type I and type II cytokine production of both CD4+ and CD8+ T cells activated by superantigen were investigated in 10 healthy donors and 39 HIV-1 infected patients. Patients were divided into 3 groups based on their CD4 count (< 200, 200-500, > 500 cells/microl). Whole blood from each subject was activated by staphylococcal enterotoxin B (SEB) and anti-CD28. Intracellular cytokine stainings for proinflamatory cytokine (TNF-alpha), type I cytokines (IFN-gamma and IL-2) and type II cytokines (IL-4 and IL-5) in CD4+ and CD8+ T lymphocytes were determined by flow cytometer. Type I cytokine (IFN-gamma) expression in CD4+ T cells co-expressing with CD69 were significantly increased in HIV infected patients, particularly in patients with CD4 counts < 200 and 200-500 cells/microl (means +/- S.D. of 20.7 +/- 18.7% and 10.5 +/- 5.9%, respectively) when compared with 4.8 +/- 1.8% in the normal group (p < 0.05). But IL-2 production in both groups of patients was significantly lower than the normal (3.8 +/- 2.6% and 3.2 +/- 1.4% in patients with < 200, 200-500 cells/microl, and 5.9 +/- 1.5% in the normal group) (p < 0.05). For type II cytokines, there was no difference in all groups of subjects when IL-4 was determined. However, IL-5 production was significantly higher in patients with a CD4 count < 200 cells/microl (0.6 +/- 0.5%) than that in the normal group (0.1 +/- 0.1%) (p < 0.005). CD8+ T cells also showed higher IFN-gamma production in patients with a CD4 count < 200 cells/microl (11.9 +/- 4.7%) and 200-500 cells/microl (12.0 +/- 4.3%) than the normal group (5.3 +/- 2.5%) (p < 0.005). In contrast, IL-2 production in CD8+ T cells was low in these HIV infected patients (0.3 +/- 0.2%, 0.3 +/- 0.2%, and 0.3 +/- 0.4% in patients with < 200, 200-500, and > 500 cells/microl, respectively), which was significantly different compared to the control group (1.2 +/- 0.8%) (p < 0.05). For type II cytokines, only IL-4 production in patients with a CD4 count < 200 cells/microl (0.1 +/- 0.1%) was significantly reduced when compared to the other groups (p < 0.05). This study shows that although HIV infection alters the production of both type I and type II cytokines, it does not induce a polarized type I or type II state in the course of HIV-1 progression in Thai patients.     

Effect of dendritic cells on flow cytometric quantification of cytomegalovirus-specific, interferon-gamma-producing T cells

Flow cytometric measurement of intracellular cytokines in T cells exposed to antigen is a widely used method for quantification of an antigen-specific T-cell response. As the frequency of antigen-specific T cells is often very low, any improvement in signal to noise ratio is of great importance. Thus, in this study, the ability of antigen-pulsed dendritic cells (DCs) to increase the number of antigen-specific, interferon-gamma (IFN-gamma)-producing CD4+ T cells measurable both in fresh peripheral blood and in reconstituted frozen blood mononuclear cell (MNC) samples was evaluated. Cytomegalovirus (CMV) was used as antigen in a 10 h assay, using cells from both CMV-seropositive and -seronegative donors. When reconstituted frozen samples were analysed, the general response towards CMV lysate in CMV-seropositive donors was 23-86% lower compared to the corresponding fresh blood samples. Antigen-pulsed DCs could not improve the sensitivity of the intracellular cytokine-detection assay when fresh peripheral blood samples were used. Interestingly, however, the addition of CMV lysate-pulsed DCs to cryopreserved MNC samples substantially increased the frequency of specifically induced IFN-gamma-producing cells to a level comparable to the frequency found in the corresponding fresh blood samples.     

T lymphocyte interferon-gamma production induced by Plasmodium falciparum antigen is high in recently infected non-immune and low in immune subjects.

Interferon (IFN) alpha and gamma were measured by radio-immunoassays in supernatants from cultures of peripheral blood mononuclear cells (PBMC) or purified T cell subsets incubated with either Plasmodium falciparum schizont-enriched malaria antigen (mAg), uninfected red blood cells (RBC) or pokeweed mitogen (PWM). Cell donors were 24 clinically immune, healthy African adult native residents of a P. falciparum-endemic region, Haut-Ogooué, Gabon, and seven non-immune, European temporary residents with a history of a single to a few malaria infections during the previous 1 to 9 months. When PBMC were cultured in medium alone or with RBC antigen no or low titres of IFN-gamma were detected. PBMC proliferation and IFN-gamma production observed in the presence of mAg were dose dependent and significantly correlated. When cultured with mAg, PBMC from non-immune Europeans produced significantly higher levels of IFN-gamma than did PBMC from clinically immune Africans. No such difference was found when PBMC were cultured with PWM. The mAg-induced IFN-gamma production was due mainly to CD4+ T cells and was not enhanced by CD8+ T cell depletion. No IFN-alpha was detected in culture supernatants. Thus, P. falciparum antigens are able to induce in vitro production of IFN-gamma by CD4+ T cells; however, in this sample, individuals considered to be clinically resistant to malaria were low producers of IFN-gamma.     

The quantitative and qualitative defect of CD4+ CD45RO+ memory-type T cells are involved in the abnormality of TH1 immunity in atopic dermatitis patients.

Peripheral blood mononuclear cells (PBMCs) obtained from atopic dermatitis (AD) patients produced low levels of IFN-gamma in response to Dermatophagoides farinae antigen (Der f Ag) plus IL-2 or OKT3 MoAb in contrast with PBMCs obtained from healthy donors. The reduced IFN-gamma production in AD patients' T cells appeared to be derived from the defect of CD4+ T cells but not CD8+ T cells. Indeed, from the cytoplasmic staining analysis of cytokines, it was demonstrated that the frequency of IFN-gamma producing CD4+ T cells (TH1 cells) in AD patients was markedly lower than that of healthy donors. From the phenotypic analysis using flow cytometry, it was also found that the number of CD4+ CD45RO+ memory type T cells was significantly reduced in AD patients compared with that of healthy donors. In addition to quantitative defect of memory type CD4+ T cells, functional defect of CD4+ CD45RO+ memory type T cells was also demonstrated in AD patients. Enriched CD4+ CD45RO+ T cells obtained from AD patients, who exhibited greatly reduced delayed-type hypersensitivity (DTH) response in tuberculin test, showed no significant TH1 immunity in terms of IFN-gamma production by stimulation with OKT3 MoAb or purified protein derivative (PPD). Thus, the immunological abnormality of TH1 immunity in AD patients appeared to be induced in concomitant with both the quantitative and qualitative defect of memory type CD4+ T cells.     

The mannose receptor mediates the cellular immune response in human coccidioidomycosis

Mannose is the predominant monosaccharide in the coccidioidal antigen preparation T27K. Mannan and anti-CD206 antibody significantly decreased the surface expression of mannose receptor (MR) on adherent peripheral blood mononuclear cells and reduced the interleukin-2 (IL-2) release induced by T27K. These data suggest that MR mediates IL-2 release by T27K.     

Characterization of a cytotoxic CD57+ T cell subset from patients with pulmonary tuberculosis

We investigated the proportion, phenotype, and cytotoxicity of CD8+CD57+ and CD57- T cells in peripheral blood from 20 tuberculosis (TB)-patients and 20 healthy tuberculin skin test-positive donors. Our results showed an increase in CD8+CD57+ T cells from TB-patients as compared with those from age-matched healthy donors (p<0.0001). CD8+CD57+ T cells from TB-patients expressed CD69, perforin, granzyme-A, and a CD28-CD62L-CD161- phenotype without recognition for the alpha-galactosylceramide-CD1d complex. This cell subset also expressed TNF-alpha and IFN-gamma, under phorbol-myristate-acetate/ionomycin stimulation. Interestingly, the cytotoxicity against autologous monocytes was higher in CD57- cells from TB-patients and donors than their CD57+ counterparts, in the presence of Mycobacterium tuberculosis H37Rv culture filtrate. However, only CD8+CD57+ T cells from TB-patients exhibited spontaneous cytotoxicity against monocytes in the absence of antigen. Our results suggest that CD8+CD57+ T cells are a subset of effector cells that could be helpful to evaluate the cell-mediated immune response to M. tuberculosis.     

CD4+-T-cell counts, spontaneous apoptosis, and Fas expression in peripheral blood mononuclear cells obtained from human immunodeficiency virus type 1-infected subjects.

We examined the relationships among CD4+-T-cell counts, spontaneous apoptosis, and Fas expression among peripheral blood mononuclear cells obtained from human immunodeficiency virus type 1 (HIV-1)-infected patients. After 2 days of incubation, propidium iodide DNA staining and flow cytometry revealed that peripheral blood mononuclear cells from subjects with the lowest CD4+-cell numbers (0 to 99/microl; n = 20) showed the highest frequency of apoptosis: 22.4% +/- 2.7% (mean +/- standard error) versus 13.8% +/- 1.2% and 12.7% +/- 1.4% among peripheral blood mononuclear cells obtained from patients with 100 to 499 CD4+ cells/microl (n = 19) and >500 CD4+ cells/microl (n = 17), respectively. Each of these means differed significantly from the mean frequency of apoptosis (6.3% +/- 0.7%) of peripheral blood mononuclear cells obtained from HIV-1-seronegative controls (P < 0.001, Student's t test). After incubation, the percentage of peripheral blood mononuclear cells expressing Fas antigen was increased for the HIV-1-infected subjects, and this was most evident for patients with more advanced disease. Among patients with fewer than 100 CD4+ cells/microl, 64.4% +/- 5.4% of peripheral blood mononuclear cells were Fas+, as opposed to 25.8% +/- 3.0% and 14.5% +/- 1.7% Fas+ cells among patients with more than 100 CD4+ cells/microl and healthy controls, respectively (P < 0.05 for each group comparison). Interestingly, in all populations, most apoptotic cells did not express Fas. Thus, apoptosis and Fas expression are increased in incubated peripheral blood mononuclear cells obtained from HIV-1-infected patients and these phenomena are enhanced as disease progresses.     

Interleukin-15 production by monocyte-derived dendritic cells and T cell proliferation in HIV-infected patients with discordant response to highly active antiretroviral therapy

A discordant response to highly active antiretroviral therapy (HAART) occurs when CD4 T cell counts are stable or increased over time despite persistently detectable HIV-RNA levels. In order to identify immunological factors affecting discordant treatment responses, a total of 27 HIV-infected patients were studied: (a) 10 naive patients (mean CD4+ = 101.5 cells/microl; mean HIV-RNA = 4.8 log10 copies/ml); (b) seven responder patients (mean CD4+ = 908.9 cells/microl); and (c) 10 discordant patients (mean CD4+ = 396.1 cells/microl; mean HIV-RNA = 5.4 log10 copies/ml). Five healthy blood donors were included as HIV-seronegative controls. The following parameters were evaluated: interleukin (IL)-15 production by monocyte-derived dendritic cells (MDDC) after stimulation with lypopolysaccaride (LPS) and Candida albicans; recall and HIV-1-specific antigen lymphocyte proliferation (LP). Increased levels of IL-15 production by MDDC after stimulation with LPS and C. albicans were found both in discordant patients and responder patients. Conversely, a strong reduction of IL-15 levels was observed in naive patients. Discordant patients developed positive LP responses to C. albicans and HIV-1 p24. LP in response to C. albicans and HIV-1 p24 was also positive in responder patients. Decreased LP response was found in naive patients. In conclusion, HIV-infected patients with discordant viro-immunological responses to HAART present increased levels of IL-15 production by MDDC and enhanced recall and HIV-1-specific antigen LP responses, suggesting an improvement in indices of immune function.     

Simultaneous production of interleukin 2, interleukin 4 and interferon-gamma by activated human blood lymphocytes

The production of interleukin 2 (IL 2), IL 4 and interferon-gamma (IFN-gamma) by in vitro activated unselected human blood mononuclear cells was studied at a single-cell level. Individual lymphokine-synthesizing cells were identified by intracellular immunofluorescent staining using cytokine-specific monoclonal or polyclonal antibodies. Cultures from adult blood donors revealed a biphasic kinetic production pattern for IL 2 and IFN-gamma with peaks occurring 4-6 and 24-30 h after initiation of the cultures. Approximately 20%-40% of the lymphocytes produced IL 2 and IFN-gamma. In contrast, only 1%-3% of the lymphocytes synthesized IL 4 with maximal frequency after 6 h of culture. CD4+ as well as CD8+ T cells contributed to the synthesis of all three lymphokines studied. CD4+CD45R- T cells were the major producers of IL 2 and IL 4, while CD8+CD45R- T cells were the most common phenotype of IFN-gamma-synthesizing cells. By performing two-color immunofluorescence studies we observed that among IL 4-producing cells every second one made simultaneously IL 2 and every fourth one made IFN-gamma. Mononuclear cells from umbilical cord blood could be stimulated to make IL 2 to the same extent as cells from adult blood donors. No IL 4 production and a strikingly reduced frequency of IFN-gamma producers were noted in cell cultures from neonates. IL 2, IL 4 and IFN-gamma accumulated in the Golgi system, which resulted in a characteristic morphology of the staining, eliminating problems with evaluation of background signals.     

Detection of CD8+ T cells sensitized to BK virus large T antigen in healthy volunteers and kidney transplant recipients

BK virus (BKV) infections after renal transplantation are increasingly recognized. Development of immune monitoring strategies against BKV requires definition of antigenic epitopes. Hence, T cells from HLA-A02-positive healthy subjects and kidney transplant recipients were stimulated by BKV lysate pulsed on mature autologous dendritic cells and screened against four different T antigen peptides or against BKV lysate. IFN-gamma production was measured by ELISPOT assays. The peptide BKV362-371 (MLTERFNHIL) was naturally processed and recognized by five of six healthy subjects (39 +/- 11 IFN-gamma spots/100,000 cells) and five of seven kidney transplant recipients (21 +/- 12 IFN-gamma spots). Less frequent and weaker CD8+ T-cell responses were detected against three other peptides. Thus, BKV large T antigen is a target for CD8+ T-cell immunity. T-antigen-specific T-cytotoxic cells circulate in healthy blood donors, implying that transient expression of T antigen presumably occurs at sites of viral latency and helps maintain a constant pool of circulating CD8+ T memory cells.     

Cytokine Profiles from Antigen-Stimulated Whole-Blood Samples among Patients with Pulmonary or Nonmeningeal Disseminated Coccidioidomycosis

The outcome of coccidioidomycosis depends on a robust specific cellular immune response. A T-helper type 1 (Th1) cellular immune response has been previously associated with resolution of clinical illness. However, the precise elements of this response and whether cytokines not involved with the Th1 response play a role in coccidioidomycosis are not known. Whole-blood samples were obtained from subjects with active coccidioidomycosis and controls and incubated for 18 h with T27K, a coccidioidal antigen preparation. The supernatant was then assayed for gamma interferon (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), IL-4, IL-6, IL-10, and IL-17A. A total of 43 subjects, 16 with acute pneumonia, 9 with pulmonary sequelae of nodules and cavities, and 18 with nonmeningeal disseminated coccidioidomycosis, were studied. Compared to concentrations in healthy immune and nonimmune donors, the median concentration of IL-17A was significantly higher in those with active coccidioidomycosis (for both, P < 0.01). In addition, IL-6 concentrations were higher while IL-2 and IFN-γ concentrations were significantly lower in those with nonmeningeal disseminated disease diagnosed within 12 months than in those with acute pneumonia (for all, P < 0.05). The cytokine profile among patients with active coccidioidomycosis is distinct in that IL-17A is persistently present. In addition, those with nonmeningeal disseminated disease have an increased inflammatory cytokine response and diminished Th1 responses that modulate over time.