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Three antigens were compared for their ability to detect immune responses in C57Bl/6 mice sensitized to Cryptococcus neoformans. Elicitation of responses in vitro was greatest with a urea extract antigen, followed in efficiency by an alkali extract and a soluble capsular polysaccharide preparation. The reactivity paralleled the protein content of the preparation.

An alkali-soluble, water-soluble cell wall fraction of Blastomyces dermatitidis, designated B-ASWS, was evaluated as an antigen for detecting in vivo (skin tests) and in vitro migration inhibition factor (MIF) production and lymphocyte transformation (LT) responses in Blastomyces-infected guinea pigs. The biological activity of B-ASWS was compared with that of blastomycin KCB-26. The superiority of B-ASWS, in terms of its sensitivity and specificity, was evident in in vivo and in vitro assays. Skin tests responses were obtained in 21 of the 24 Blastomyces-infected guinea pigs, whereas only one of the 14 Histoplasma-infected guinea pigs were significantly greater than those obtained using cell populations from Histoplasma-infected or noninfected guinea pigs. The con-MIF and LT in peritoneal exudate cells and lymph node cells of homologuosly infected animals. In each biological system, the response of the Blastomyces-infected guinea pigs were significantly greater than those obtained using cell populations from Histoplasma-infected or non-infected guinea pigs. The contrasting efficacy of B-ASWS as compared with blastomycin KCB-26, suggests that the cell wall antigen will be a useful tool for detecting cell-mediated immune responses in blastomycosis.

Soluble extracts of Cryptococcus neoformans were examined for their ability to induce a macrophage-regulatory T-suppressor cell known to appear in the spleens of mice infected with cryptococci. Suppressor cells were induced by injection of extracts of encapsulated or thinly encapsulated strains of cryptococci. Dose-response analysis showed that as little as 25 micrograms of soluble capsular polysaccharide antigen could induce significant suppressor cell activity, with maximum suppression occurring at a dose of 100 micrograms. The suppressor cells appeared within 1 week of injection of antigen and persisted for at least 2 months. Suppressor cells were induced in animals given tolerogenic doses of levan, human gamma globulin, and soluble capsular polysaccharide antigen. When these same antigens were administered in immunogenic form, no suppressor cell activity was detected. Therefore, the suppressive mechanism was common to models of immunologic tolerance and was not unique to cryptococcal disease or cryptococcal capsular polysaccharide antigen. The phagocytosis-inhibiting lymphokine produced by the suppressor cell population completely inhibited the phagocytic activity of only a portion of peritoneal exudate cells. Other macrophages in the population were not totally inhibited but exhibited a reduction in the number of yeast cells engulfed.

An alkali-soluble water-soluble extract of Blastomyces dermatitidis yeast-phase cell walls was tested for its ability to elicit a response in lymphocytes isolated from the peripheral blood of Blastomyces-infected guinea pigs. Sequential preparations of the antigen were reproducible and specific in the in vitro lymphocyte transformation assay. Cross-reactivity of the antigen was not evident in lymphocyte transformation assays on lymphocytes obtained from Histoplasma-infected guinea pigs or from animals sensitized with complete Freund adjuvant. Fractionation of the antigen was accomplished on an isoelectric-focusing column, using a sucrose density gradient support. Components were assayed for activity in skin testing and lymphocyte transformation. Comparison of column fractions to the whole antigen showed greater response to the whole antigen in in vivo and in vitro assays.