Rebecca Blackstock

Immunization of mice with an avirulent pseudohyphal form of Cryptococcus neoformans

Mice were immunized with a viable, avirulent strain ofCryptococcus neoformans. Lymphocyte blastogenic assays showed a 10-fold increase in reactivity of sensitized spleen cells, and histopathologic examination revealed marked splenic hyperplasia. Thirty-two days after intravenous inoculation with a virulent strain ofC. neoformans, none of the control animals survived whereas 60 percent of the immunized mice were alive with no clinical evidence of disease. This animal model shows that protective immunity can be established, and once developed, provide a better model for the study of important aspects of immunity in fungal disease.

Pathogenesis of Cryptococcus neoformans is associated with quantitative differences in multiple virulence factors

Two isolates of Cryptococcus neoformans were previously described as being highly divergent in their level of capsule synthesis in vivo and in their virulence for mice. The highly virulent isolate (NU-2) produced more capsule than a weakly virulent isolate (184A) in vitro under tissue culture conditions and in vivo. This investigation was done to determine if there were differences between the two isolates in other factors that might also contribute to virulence. Growth rate was not a factor as NU-2 grew more slowly than 184A. Based on PCR fingerprinting the two isolates were genetically different providing an opportunity to examine differences in multiple virulence traits. Quantitative analysis revealed that NU-2 expressed significantly more melanin and mannitol than did 184A. Although the isolates expressed the same capsular chemotype, NU-2 produced an additional structure reporter group (SRG)under tissue culture conditions that was not present when grown in glucose salts/urea/basal medium (GSU).Capsular polysaccharide SRGs of 184A were unaffected by shifting the growth conditions from GSU to tissue culture conditions. Our results suggest that pathogenesis of a C. neoformans strain is dictated by the quantitative expression of the strains combined virulence traits. Regulators of the expression of these genes may be playing key roles in virulence.

Non-specific immunosuppression by Cryptococcus neoformans infection

Cryptococcus neoformans-infected animals were found to be immunosuppressed when tested by a variety of assays for immune competence. Primary humoral immune responses and delayed-type hypersensitivity reactions to sheep erythrocytes were suppressed in animals which had been infected for two weeks. Lymphocyte proliferation (LP) assays to sRBC stroma were also significantly diminished at two weeks of infection. Spleen cells of infected mice suppressed the LP response of sRBC immunized, normal mice in vitro. At least a part of the suppression could be attributed to a nylon wool non-adherent cell. Suppressor cells continued to be present in spleen cell suspensions following treatment with anti-T cell serum or anti-immunoglobulin and complement. When infected spleen cells were separated by adherence to plastic, both the adherent and non-adherent fractions exhibited suppressive activity. Incubation of infected spleen cells in tissue culture for 48 hr resulted in the elaboration of soluble immunosuppreessive factors into the tissue culture medium. These data indicated that immune suppression in cryptococcosis can occur as a result of infection with Cryptococcus neoformans, and that at least one mechanism involved is the induction of adherent and non-adherent suppressor cells in the spleens of infected mice.

Suppression of responses to cryptococcal antigen in murine cryptococcosis.

Subpopulations of spleen cells responsible for responsiveness and unresponsiveness to cryptococcal antigen in vitro were identified. Lymphocytes which responded in lymphocyte transformation (LT) assays were nylon wool nonadherent and theta antigen positive. These lymphocytes required the presence of an accessory cell which could be supplied by normal peritoneal exudate cells. Spleen cells taken from mice which had been infected for 3 to 15 days were tested to determine their ability to respond to cryptococcal antigen in LT assays. A minimal response was detected at the ninth day of infection. The response of infected spleen cells was attributed to a nonadherent lymphocyte. Nonadherent spleen cells of infected animals had enhanced responses after removal of adherent cells and addition of normal peritoneal exudate cells. Suppressor cells were detected in the spleens of infected mice by the 12th day of infection and thereafter. A nonadherent suppressor cell was identified, but indirect evidence suggested that an adherent cell could also be present in infected spleens.

Presentation of cryptococcal capsular polysaccharide (GXM) on activated antigen-presenting cells inhibits the T-suppressor response and enhances delayed-type hypersensitivity and survival.

A hallmark of infection with Cryptococcus neoformans is depression of the immune system characterized by poor inflammatory responses and loss of delayed‐type hypersensitivity (DTH) and antibody responses. T‐suppressor cell (Ts) responses, elicited by the capsular polysaccharide (GXM) of the organism, are known to develop during infection. This study was undertaken to develop a method to inhibit the anti‐GXM Ts response and thereby study the influence of the Ts response on immune responsiveness and survival in cryptococcosis. Antigen‐presenting cells (APC), elicited with complete Freund’s adjuvant (CFA), were treated in vitro with GXM (GXM–APC). The GXM–APC were injected intravenously into normal mice. These mice were resistant to induction of anti‐GXM Ts cells when soluble GXM was administered in tolerogenic doses or when animals were infected with C. neoformans. Inhibition of the anti‐GXM Ts response was specific to GXM as levan–APC did not inhibit induction of anti‐GXM Ts cells. Inhibition of the anti‐GXM Ts response could not be attributed to increased clearance of GXM due to induction of anti‐GXM antibodies or other mechanisms. Anti‐cryptococcal DTH responses were lost in mice by the second week of infection. However, treatment with GXM–APC, but not levan–APC, allowed mice to maintain their DTH response. GXM–APC pretreatment enhanced survival of infected mice compared with mice pretreated with levan–APC. These results show that GXM–APC induces immune responses that inhibit the induction of Ts responses and enhances DTH responses in infected mice. These responses correlate with enhanced survival after cryptococcal infection.

Comparison of the ease of tolerance induction in thymus and bone marrow of three strains of mice.

Abstract Three strains of mice which vary in their susceptibility to induction of immune tolerance with human gamma-globulin were studied to evaluate the cellular basis for their sensitivity to induction of the unresponsive state. Tolerance induction in BALB/c mice was difficult to establish, while tolerance induction was easily achieved in C57BL/6 and CBF1 (BALB/c × C57BL/6) mice. The degree of unresponsiveness obtained with various tolerogen doses in intact C57BL/6 and CBF1 mice was reflected in the sensitivity of their thymocytes to the production of the unresponsive state. In the BALB/c mouse strain slight immune suppression observed at low tolerogen doses was correlated with bone marrow cell unresponsiveness while significant levels of tolerance observed at a high tolerogen dose was due to suppression of thymus cells. It was apparent that CBF1 mice had inherited both thymus cells and bone marrow cells which exhibited the sensitivity to induction of immune tolerance characteristic of those same cells of their C57BL/6 parent.

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

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 GXM 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 GXM did not eliminate the antigen presenting function of the population.

Functional equivalence of cryptococcal and haptene-specific T suppressor factor (TsF). I. Picryl and oxazolone-specific TsF, which inhibit transfer of contact sensitivity, also inhibit phagocytosis by a subset of macrophages.

Monoclonal and conventional cryptococcal-specific T suppressor factors (TsF) (also called TsFmp) depress phagocytosis by a subset of macrophages, while picryl- and oxazolone-specific TsF depress the passive transfer of contact sensitivity. This paper shows that these haptene-specific TsF also inhibit phagocytosis by a subset of macrophages and, using this assay, that the anti-haptene TsF resemble the anti-cryptococcal TsF in five respects: (i) the need for reexposure to specific antigen to trigger the release of TsF; (ii) genetic restriction in action; (iii) possession of an antigen-binding site; (iv) expression of I-J determinants; and (v) inactivation by reduction and alkylation. Purification of the anti-picryl TsF by sequential affinity chromatography indicates that the inhibition of phagocytosis is due to the TsF itself and not to a TsF-antigen complex. The TsF inhibits phagocytosis by a direct action as macrophages treated with TsF and exposed to antigen do not release a second factor which inhibits phagocytosis. These results and those of the accompanying paper indicate that the anti-cryptococcal and anti-haptene TsF are functionally equivalent, antigen-specific suppressor factors.

Chronic Blepharitis and Pyoderma of the Scalp: An Immune Deficiency State in a Father and Son with Hypercupremia and Decreased Intracellular Killing

Abstract: An immune deficiency state is proposed as the cause of a disorder affecting a father and son with chronic dermatitis, purulent blepharitis with corneal ulceration, and scarring pyodermatous alopecia of the scalp. The results of immunologic investigation revealed abnormal neutrophil function with a variable decrease in intracellular killing, decreased lymphocyte transformation, increased serum IgG and IgE, and elevated serum copper levels. These findings will be compared with previously described immune deficiency disorders.

Functional equivalence of cryptococcal and haptene-specific T suppressor factor (TsF): II. Monoclonal Anti-cryptococcal TsF Inhibits both Phagocytosis by a Subset of Macrophages and Transfer of Contact Sensitivity

Monoclonal anti-cryptococcal TsF (which inhibits phagocytosis by macrophages) and anti-picryl TsF use the same two circuits to block the transfer of contact sensitivity (CS). Both arm macrophages which then release a macrophage suppressor factor (MSF) when exposed to antigen. This MSF depresses the transfer of CS. The evidence suggests that a single molecular species of TsF (MW ca. 70 kDa), which bears an antigen-binding site and I-J determinant, is responsible for MSF production and inhibition of phagocytosis. Anti-cryptococcal TsF also arms the T acceptor cell which then releases nsTsF-1 after triggering with a specific antigen (SCPA). This nsTsF-1, which depresses the transfer of contact sensitivity, was authentic, as shown by its I-J positivity (in contrast to MSF) and its role in the production of nsTsF-2. As anti-picryl TsF also inhibits phagocytosis, it was concluded that anti-cryptococcal TsF, originally detected by the inhibition of phagocytosis, and anti-picryl TsF, originally detected by inhibition of CS, are functionally equivalent.