Lymphocytes, especially CD4+ T cells, are essential for clearance of the yeast-like organism Cryptococcus neoformans from the infected host. The mechanism(s) by which the lymphocytes facilitate elimination of cryptococci has not been elucidated. It is generally thought, however, that lymphocytes reactive with C. neoformans indirectly function by production of lymphokines to enhance clearance of the organism by natural effector cells such as macrophages. In the present study, we assessed the ability of freshly isolated human lymphocytes to interact directly with C. neoformans and to limit the growth ofthe organism in vitro. We found that large granular lymphocytes (LGL) as well as T cells bound to cryptococcal cells when the lymphocytes were mixed with the cryptococcal cells at a 2:1 ratio. The physical binding interactions of the two lymphocyte populations were different. LGL attached to the cryptococcal cells by many microvilli; T lymphocytes associated with the yeast through broad areas of membrane attached to the cryptococcal cell surface. The two types of lymphocyte interactions did not result in phagocytosis but resulted in direct inhibition of cryptococcal growth, making these lymphocyte interactions with cryptococci distinctly different from interactions of monocytes with cryptococci. With the human natural killer (NK) cell line, NK 3.3, we confirmed that NK cells that were present in the LGL population were capable of limiting the growth of C. neoformans. Through immunoelectron microscopy, human CD3+ lymphocytes were seen attached to cryptococcal cells and by mass cytolysis, human CD3+ lymphocytes were shown to be responsible for inhibition of C. neoformans growth. The direct inhibitory interactions of NK cells and T lymphocytes with cryptococcal cells may be important means of host defense against this ubiquitous organism that frequently causes lifethreatening disease in AIDS patients. (J. Clin. Invest. 1993.

Human natural killer (NK) cells and T lymphocytes can bind to and inhibit the growth of the yeast-like organism Cryptococcus neoformans. Binding of target cells to NK or T cells also has the potential to modulate cytokine production by the effector cells. In this study, we assessed the ability of C. neoformans to modulate NK cell production, or in some cases T-cell production, of granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor alpha (TNF-␣). We found that freshly isolated human NK cells from most individuals make GM-CSF and TNF-␣ constitutively when cultured in vitro. The addition of C. neoformans to T-cell fractions which do not make GM-CSF constitutively did not affect GM-CSF production, but the addition of C. neoformans to NK cell fractions significantly reduced the amounts of GM-CSF produced in most NK cell samples. The reduction in the amount of GM-CSF in C. neoformans-NK cell cocultures could not be attributed to loss of lymphocyte viability or to C. neoformans adsorbing or degrading the cytokine and was dependent on direct contact between the NK cells and cryptococcal cells. GM-CSF was not the only cytokine to be downregulated. TNF-␣ production was also diminished when NK cells were incubated with C. neoformans. The regulation of both cytokines was at the transcriptional level because GM-CSF and TNF-␣ mRNA levels were lower in NK cell samples incubated with C. neoformans than in NK cell samples incubated without C. neoformans. Diminished production of constitutively produced cytokines resulting from the interaction of NK cells with cryptococcal cells has the potential to affect phagocytic cells in the immediate regional environment and to damp the immune response. MATERIALS AND METHODSReagents. The reagents used in these studies were purchased from the following vendors: RPMI 1640 medium and pooled human serum (type AB) from Gibco Laboratories, Grand Island, N.Y.; fetal bovine serum (FBS) from Hy-Clone Laboratories, Inc., Logan, Utah; penicillin, streptomycin, Ficoll-Hypaque, Percoll, and phorbol myristate acetate (PMA) from Pharmacia, Uppsala, Sweden; nylon wool from Robbins Scientific Corp., Sunnyvale, CA.; fluorescein isothiocyanate-conjugated mouse anti-human CD3 monoclonal antibody (immunoglobulin G1 IgG1), phycoerythrin (PE)-conjugated mouse anti-human CD16 monoclonal antibody (IgG1), PE-conjugated mouse anti-human CD14 monoclonal antibody (IgG2), and fluorescein isothiocyanate-conjugated and PE-conju-* Corresponding author. Mailing address: