Cryptococcal capsular polysaccharide utilizes an antigen-presenting cell to induce a T-suppressor cell to secrete TsF

Journalof Medical& VeterinaryMycology 1996, 34, 19-30 Accepted 27 July 1995 Cryptococcal capsular polysaccharide utilizes an antigen-presenting cell to induce a T-suppressor cell to secrete TsF R. B L A C K S T O C K Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Introduction The capsular polysaccharide of Cryptococcusneoformans is a glucuronoxylomannan (GXM) and is known to induce a T suppressor cell (Ts) response [1,2]. Ts cells induced by GXM secrete an antigen-specific soluble suppressor factor (TsF) which has several different activities. These activities have been described previously [3,4]. This TsF is functionally identical to the TsF which inhibits contact sensitivity to the hapten trinitrophenyl, TNP, also known as picryl [3 5]. The anti-capsule TsF can trigger two separate suppressor pathways which release nonspecific suppressor factors [3,4]. These later factors can inhibit contact sensitivity, delayed hypersensitivity and granulomatous responses in an antigen non-specific manner [3-6]. The ability of the anti-capsular Ts response to inhibit DTH and granulomatous inflammation induced by non-capsular cryptococcal antigens is a mechanism Correspondence: Dr R. Blackstock, Department of Pediatrics, University of Oklahoma Health Sciences Center, PO Box 26901, Oklahoma City, OK 73190, USA. Fax: 405-271-3265. © 1996 ISHAM which contributes to the organism's ability to evade these T-helper 1 (Thl) responses that contribute to protection in cryptococcosis [7,8]. For this reason, an understanding of the cellular and molecular mechanisms of the Ts response is important to future attempts to inhibit this response in the infected patient. From previous studies normal spleen cells and antigen were known to be required before TsF was secreted by an inducible T-T hybridoma [9]; however, the requirement of normal spleen cells had never been examined in detail. Because GXM is a polysaccharide antigen, it seemed unlikely that the function of the spleen cells was to present the antigen via the exogenous antigen-processing route where peptides of protein antigens are incorporated into the antigen-binding groove of class II major histocompatibility complex (MHC) molecules [10]. Antigen specific CD8 + suppressor cells are not restricted by class I MHC molecules as are cytotoxic T cells. Nonetheless, the possibility that antigen-presenting cells were required which might utilize different stimulatory mechanisms could not be ruled out. It is also possible that the polysaccharide A T-T hybridoma (F6.6.2) which secretes a T-suppressor factor (TsF) specific for cryptococcal capsular polysaccharide (glucuronoxylomannan, GXM) was tested to determine if antigen-presenting cells (APC) were necessary for activation of the hybridoma to secrete TsF. Normal, syngeneic spleen cells were required along with G X M before TsF could be detected in culture supernatants. Ts cells did not secrete TsF unless the APC were obtained from mice which were identical at the 'so-called' I-J sublocus as defined by the difference between B10.A(3R) and B10.A(5R) mice. The APC was adherent and could be depleted from spleen cell suspensions by treatment with anti-I-J and complement but not anti-I-A and complement. Additionally, treatment with anti-T cell serum or anti-immunoglobulin and complement did not remove the APC function of the spleen cell population. A role for I-E antigens in the function of the APC was determined by blocking antigen presentation to the suppressor cell with anti-I-E antibodies. The polysaccharide was associated with splenic adherent cells as extensive washing of the APC after incubation with G X M did not eliminate the antigen presenting function of the population. ~[~ Blackstock may stimulate the Ts response because of a lack of co-stimulation by the APC. Currently, very little is known about the role of APC in the immune response to polysaccharide antigens. This investigation was undertaken to determine if the requirement for spleen cells was genetically restricted and to obtain information regarding the phenotype of the cell(s) which were required to elicit anti-GXM TsF secretion by an inducible T-T hybridoma suppressor cell. Animals C57B1/6J, C3HeB/FeJ, B10.A(3R) and B10.A(5R) male mice were purchased from the Jackson Laboratories (Bar Harbor, Maine). C3H/HeNHsD mice were obtained from Harlan Sprague Dawley (Indianapolis, IN). The mice were 8 weeks old on arrival and were used in experiments at 9-14 weeks of age. The animals were housed in the University of Oklahoma Health Sciences Center Animal Facility which is approved by the American Association for the Accreditation of Laboratory Animal Care. Animals were maintained in accordance with the National Research Council Guidelines (DHSS publication no (NIH) 85-23, revised, 1985). Reagents Sterile water for irrigation was supplied by American McGaw, Irvine, CA. Antibiotic-antimycotic was purchased from Grand Island Biological, Grand Island, NY. Whittaker, MA Bioproducts, Walkersville, MD, was the source of Dulbecco's phosphate buffered saline. Hybricare medium was purchased from the American Type Culture Collection, Rockville, MD. Iron supplemented newborn calf serum was obtained from Hyclone, Logan, UT. Hybri-sure foetal calf serum was purchased from Hazelton Research Products, Lexana, KS. Brewer's thioglycollate medium and mycological agar were purchased from Difco Laboratories, Detroit, MI. Rabbit complement H2 was supplied by Pel-Freeze, Brown Deer, WI. Fungal strains An encapsulated stain of C. neoformans, NU-2, and a non-encapsulated strain, M7, have been described previously [11]. The yeast cells were maintained by subculture on Mycological agar (Difco). When used in phagocytic experiments, M7 was harvested from the surface of 72 h Sabouraud glucose agar cultures grown at 25 °C. The yeast cells were suspended in hybricare medium Antibodies Anti-I-J k was purchased from Accurate Chemical (Westbury, NY). Anti-I-J b was obtained from the Transplantation Immunology Branch, National Institutes of Health (Bethesda, MD). These mouse alloantisera have been used previously and were shown to eliminate suppressor cells from spleen cell suspensions of appropriate mouse strains at a concentration of l:10 in the presence of rabbit complement [12]. Monoclonal anti-I-A k and anti-I-A b were purchased from Accurate Chemical. The antisera were pretested for specificity on spleen cell populations of C57B1/6J (H-2 b) and C3HeB/FeJ (H-2 k) by the ability to lyse cells in the presence of rabbit complement. Anti-mouse T cell serum was purchased from Accurate Chemical and anti-mouse immunoglobulin (polyvalent) was purchased from Cappel Laboratories (Durham, NC). Fluorescein conjugated goat anti-mouse immunoglobulins was obtained from Cappel. Antigens The capsular polysaccharide (GXM) of C. neoformans was prepared as described previously [2]. It is the same antigen which was called solub (...truncated)