Previously, NK cells have been reported to kill the opportunistic fungal pathogen Cryptococcus neoformans through a perforin-dependent mechanism; however, the receptor and signaling involved are unknown. In this report we sought to identify the signaling pathways activated and required for direct perforin-mediated killing of microbes. In this study, using the NK-like cell line YT and primary peripheral blood NK cells, it is demonstrated that YT cells kill C. neoformans and that the killing is accompanied by the activation of PI3K. We demonstrate that inhibition of either the catalytic subunit (using a pharmacological inhibitor) or the α-regulatory subunit (using small interfering RNA knockdown) of PI3K significantly inhibited the killing of C. neoformans. Downstream of PI3K, ERK1/2 was activated in a PI3K-dependent fashion and was required for cryptococcal killing. Furthermore, we demonstrate that perforin release from YT cells can be detected by 4 h after contact of the YT cells with C. neoformans and that the release of perforin is blocked by pharmacological inhibition of either PI3K or ERK1/2. Defective degranulation is rooted in the inability to polarize perforin-containing granules toward the target. Finally, we demonstrate that PI3K-ERK1/2-dependent signaling is activated and required for the killing of C. neoformans by primary NK cells. Taken together, these data identify a conserved PI3K-ERK1/2 pathway that is used by NK cells during the direct killing of C. neoformans and demonstrate that the pathway is essential in the formation and activation of the microbicidal mechanism.

NK cells, in addition to possessing antitumor and antiviral activity, exhibit perforin-dependent microbicidal activity against the opportunistic pathogen Cryptococcus neoformans. However, the factors controlling this response, particularly whether the pathogen itself provides an activation or rearming signal, are largely unknown. The current studies were performed to determine whether exposure to this fungus alters subsequent NK cell anticryptococcal activity. NK cells lost perforin and mobilized lysosome-associated membrane protein 1 to the cell surface following incubation with the fungus, indicating that degranulation had occurred. Despite a reduced perforin content during killing, NK cells acquired an enhanced ability to kill C. neoformans, as demonstrated using auxotrophs that allowed independent assessment of the killing of two strains. De novo protein synthesis was required for optimal killing; however, there was no evidence that a soluble factor contributed to the enhanced anticryptococcal activity. Exposure of NK cells to C. neoformans caused the cells to rearm, as demonstrated by increased perforin mRNA levels and enhanced loss of perforin when transcription was blocked. Degranulation alone was insufficient to provide the activation signal as NK cells lost anticryptococcal activity following treatment with strontium chloride. However, NK cells regained the activity upon prolonged exposure to C. neoformans, which is consistent with activation by the microbe. The enhanced cytotoxicity did not extend to tumor killing since NK cells exposed to C. neoformans failed to kill NK-sensitive tumor targets (K562 cells). These studies demonstrate that there is contact-mediated microbe-specific rearming and activation of microbicidal activity that are necessary for optimal killing of C. neoformans.

No abstract

Granulysin is a cytolytic effector molecule used by lymphocytes to kill tumor and microbial cells. Regulation of granulysin production is complex. A significant delay (5 days) following stimulation of CD4+ T cells with IL-2 occurs before granulysin is produced. Unfortunately, the mechanisms responsible for this delay are unknown. We have recently demonstrated that granulysin-mediated killing of Cryptococcus neoformans by CD4+ T cells is defective during HIV infection. This is because CD4+ T cells from HIV-infected patients fail to produce granulysin in response to IL-2 activation. The present studies examined the mechanism of delayed production of granulysin and the mechanism of the defect in HIV patients. We demonstrate that IL-2 initially requires both STAT5 and PI3K activation to increase expression of IL-2Rβ, produce granulysin, and kill C. neoformans. The increased expression of IL-2Rβ precedes granulysin, and preventing the increased expression of IL-2Rβ using small interfering RNA knockdown abrogates granulysin expression. Moreover, following the increased expression of IL-2Rβ, blocking subsequent signaling by IL-2 using IL-2Rβ-specific blocking Abs abrogates expression of granulysin. Finally, CD4+ T cells from HIV-infected patients, who are defective in both STAT5 and PI3K signaling, fail to express IL-2Rβ and fail to produce granulysin. These results suggest that IL-2 signals via PI3K and STAT5 to increase expression of IL-2Rβ, which in turn is required for production of granulysin. These results provide a mechanism to explain the “late” production of granulysin during normal T cell responses, as well as for defective granulysin production by CD4+ T cells in HIV-infected patients.

NK cell cytotoxicity requires two positive signals for killing of tumors. Activation receptors induce polarization of the microtubule organization center and degranulation, while leukocyte function-associated antigen (LFA)-1 is required for conjugate formation and actin polymerization and under some circumstances may be sufficient for NK cell cytotoxicity. Although the receptor for direct killing of fungi is not known, CD18, the beta2 chain of LFA-1, binds components of the capsule and cell wall of the opportunistic pathogen Cryptococcus neoformans, namely the polysaccharides glucoronoxylomannan and galactoxylomannan. Herein, we also demonstrate that LFA-1 was concentrated in regions of the NK cell surface interacting with C. neoformans. Consequently, there was compelling evidence to hypothesize that NK cells would also use LFA-1 to recognize and kill C. neoformans. Using a combination of NK cell lines that did or did not express LFA-1 or by using a CD18-specific functional blocking antibody, we confirm that NK cell anti-tumor activity is critically dependent upon the expression of LFA-1. Duplicating the events of tumor cytotoxicity, NK cells form conjugates with cryptococcal targets, rearrange the cell cytoskeleton to develop an NK immunologic synapse and release perforin-containing granules; however, each of these events occurred independently of LFA-1. Furthermore, NK cell-mediated killing of C. neoformans was detectable in both NK cells pre-treated with CD18-blocking antibodies and in NK cells lacking cell surface LFA-1 expression. These results demonstrate that in the absence of LFA-1 expression, NK cells are fully capable of recognizing a target (C. neoformans) and retain all of the events required for cytotoxicity.