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Anticryptococcal cytotoxicity of murine non-adherent cells is perforin and non-perforin mediated

Ljubica Pavlina Petkovi ^1,*
Miha Skvar ²
Andreja Kopitar ²
Vladimir Kotnik ²
Saa Simi ²
Branka Wraber ²
Tadeja Matos ²
Alojz Ihan ²
Valentina Arsi Arsenijevi ³

     ¹ Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Zaloka 4, Ljubljana, Slovenia, 1000, Slovenia
² Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia
³ Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade, Dr Subotia 1, 11 000 Belgrade, Serbia

The encapsulated fungal pathogen Cryptococcus neoformans is a significant agent of life-threatening infections, particularly in people with suppressed cell-mediated immunity. The cellular cytotoxicity against C. neoformans infection is mainly mediated by NK and T cells, but effector mechanisms are not well understood. The objective of this study was (i) to determine whether prior exposure to the cryptococcal antigens enhances anticryptococcal activity of cytotoxic cells in mice and (ii) the contribution of perforin- and nonperforin-mediated cytotoxicity of NK and T cells in growth inhibition of C. neoformans. Our data showed that in vitro exposure of nonadherent (NA) spleen mononuclear cells from nonimmunized mice to heat-killed C. neoformans strain Cap67 unencapsulated mutant of B3501 (Ag1) or its supernatant (Ag2) demonstrated higher anticryptococcal activity. This effector mechanism can be enhanced further after immunization with either Ag1 or Ag2. There is a synergistic effect of immunization and in vitro incubation of the NA cells with the same antigens. Concanamycin A (CMA) and strontium chloride (SrCl2) inhibition assays were performed to clarify the contribution of perforin- and nonperforin-mediated anticryptococcal cytotoxicity of NA cells in these events. Treatment with these inhibitors demonstrated that anticryptococcal cytotoxicity of nonprimed NA cells was primarily perforin mediated. Anticryptococcal activity of the NA cells obtained from immunized mice after in vitro incubation with cryptococcal antigens was both perforin and nonperforin mediated. Taken together these data demonstrate that in mice a nonperforin-mediated pathway of anticryptococcal cytotoxicity can be induced by immunization. Further research is needed to examine their potential role for human vaccines strategies and/or therapies.

Key words: Cryptococcus neoformans, immunization, NK cells, NA cells