Investigation of host-parasite relationships involving the parasitic form of Coccidioides immitis has been difficult because, previously, spherules and endospores have not been grown continuously in tissue culture medium without detectable formation of hyphae. Arthroconidia were harvested from mycelial cultures and inoculated into tissue culture flasks which contained RPMI 1640 medium supplemented with 10% calf serum and N-Tamol (Rohm & Haas Co., Philadelphia, Pa.). Flasks were purged with 5% C02, sealed, and placed on a reciprocating shaker at 35°C. Hyphae which arose during incubation were removed by filtration. Arthroconidia readily converted to the spherule-endospore form within 12 days. Six days after complete conversion, spherules and endospores were transferred to RPMI 1640 without N-Tamol. The spheruleendospore cycle was maintained in tissue culture medium for 84 days without the formation of detectable hyphae.

The recent development of a method for culturing the parasitic form of Coccidioides immitis by using conditions compatible with the growth of lymphoid cells has enabled us to investigate the role of natural killer (NK) cells in defense against this pathogenic fungus. Pure cultures of spherules and endospores were grown in RPMI 1640 which contained 10% calf serum. Single cell suspensions of young spherules and endospores were incubated in the presence of freshly isolated human peripheral blood lymphocytes (PBL). After a 4-hr incubation, the colony-forming ability of the fungus was significantly reduced. Leu-11 is a monoclonal antibody that binds to the Fc receptor of NK cells. When PBL were incubated in the presence of this monoclonal antibody and complement, the colony-forming ability of C. immitis was not reduced, indicating that the effector cell involved in reduction of colony-forming units is also recognized by the Leu-11 monoclonal antibody. Classical NK activity can be enhanced by preincubation with interferon; the inhibitory activity of the PBL which are responsible for the reduction in colony-forming units of C. immitis is similarly enhanced by pretreatment with interferon. When PBL are incubated in the presence of young spherules and endospores for 24 hr, the cellfree supernatants will kill U937 target cells. In addition to stimulating the release of NK cytotoxic factor, C. immitis is susceptible to inactivation when incubated in the presence of factors released by PBL which have been incubated in the presence of either K562 or C. immitis. Other evidence reported by this laboratory demonstrates that C-reactive protein is present on the surface of NK cells and that antibody to this molecule blocks NK-mediated killing of standard tumor cell targets. Pretreatment with anti-C-reactive protein also blocks the ability of PBL to inhibit the colony-forming capacity of this fungus. These data suggest that the cell that inhibits the in vitro growth of the pathogenic fungus, C. immitis, is an NK cell.