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.

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-␥). 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-␥ 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.Coccidioidomycosis is a fungal infection of increasing importance in the southwest United States (4). In the vast majority of cases, infection is acquired by inhalation of the fungus. Two-thirds of individuals who become infected are asymptomatic, their only evidence of infection being the expression of delayed-type hypersensitivity in response to a coccidioidal skin test. A variety of studies have indicated that protective immunity in human coccidioidomycosis is associated with an appropriate cellular immune response. Smith and colleagues demonstrated that persistent expression of delayed-type hypersensitivity presaged a good outcome, while failure to develop this response was associated with disseminated disease (20). More recently, in vitro studies have shown that peripheral blood T lymphocytes from individuals with well-controlled infection produce gamma interferon (IFN-␥) in response to coccidioidal antigen, while cells from individuals with disseminated disease do not (1). Failure of T lymphocytes to react to coccidioidal antigen in vitro is associated with a poorer clinical score among those with disseminated disease (2).The hallmark of a robust T-lymphocyte response is the granuloma (10), and the formation of necrotizing granulomata as a response to pulmonary coccidioidomycosis has long been associated with a strong cellular immune response and control of coccidioidal disease (6). However, the precise immunological events associated with this response have been largely unexplored. Modlin and colleagues, in work published in 1985 (13), described a distinct pattern of cellular response in a single human pulmonary granuloma. In this pattern, CD4 ϩ lymphocytes were dispersed throughout the granuloma, while CD8 ϩ lymphocytes...

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.The mannose receptor (MR) is found on the surface of macrophages and dendritic cells, can bind terminal mannoses found on fungi and other pathogens (6), and has been shown to mediate the in vitro cellular immune response to fungal antigens (7). We used mannan, an MR ligand (11), and anti-CD206 (␣CD206), a monoclonal antibody directed against MR (4), to block the surface expression of MR on adherent peripheral blood mononuclear cells (PBMC) and examined the effect of this on the cellular immune response induced by T27K, a glycosylated coccidioidal antigen preparation (1-3).PBMC, derived from the blood of healthy human donors of known coccidioidal immunity, were resuspended in RPMI 1640 (GIBCO, Grand Island, Mich.) with 10% autologous serum, added to 35-mm flat-bottom wells (Falcon, Becton Dickinson Labware), and incubated at 37°C in 95% air-5% CO 2 . For the first 30 min, mannan (from Sacchyromyces cerevisiae; Sigma Chemical Company, St. Louis, Mo.) or ␣CD206 (from clone 19.2; BD Biosciences Pharmingen, San Diego, Calif.) was added to wells. In some experiments, immunoglobulin G1 (IgG1) (no. 555748; BD Biosciences Pharmingen), the isotype of ␣CD206, was used. Control wells received nothing. After 30 min, 10-g/ml T27K was added to cells and further incubated for 72 h. Adherent cells were removed and incubated with fluorescein isothiocyanate (FITC)-labeled ␣CD206 or FITC-labeled IgG1 for 30 min at 22°C in the dark. Cell viability just prior to flow cytometry was Ͼ90%, as determined by trypan blue exclusion. A gate was set around viable nonlymphocytes and 4,000 events were collected. MR surface expression was measured as the ratio of the geometric mean fluorescent intensity (MFI) of samples stained with FITC-labeled ␣CD206 divided by the geometric MFI of cells stained with labeled isotype. Interleukin-2 (IL-2) and gamma interferon (IFN-␥) concentrations in harvested supernatant were measured using a flow cytometry-based immunoassay (CBA; BD Immunosciences, San Jose, Calif.) or by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minn.).Monosaccharide analysis of T27K was performed by the Glycotechnology Core Resource of the University of California at San Diego by using high-pH anion-exchange chromatography with pulsed amperometric detection after protein denaturization and desalting (A. Datta, personal communication). The Wilcoxon signed-rank test for paired data was employed for statistical analysis. All work was approved by the Human Subjects Protection Program of the University of Arizona.To assess mannan blocking of MR, 3.0 ϫ 10 6 PBMC in 2 ml of RPMI 1640 with 10% autologous serum were incubated for 72 h with or without 3 mg of mannan/ml. Subsequent...