Both Th1 and Th2 cytokines affect the ability of monoclonal antibodies to protect mice against Cryptococcus neoformans

Abstract

Variable-region-identical mouse immunoglobulin G1 (IgG1), IgG2b, and IgG2a monoclonal antibodies to the capsular polysaccharide of Cryptococcus neoformans prolong the lives of mice infected with this fungus, while IgG3 is either not protective or enhances infection. CD4+ T cells are required for IgG1-mediated protection, and CD8+ T cells are required for IgG3-mediated enhancement. Gamma interferon is required for both effects. These findings revealed that T cells and cytokines play a role in the modulation of cryptococcal infection by antibodies and suggested that it was important to more fully define the cytokine requirements of each of the antibody isotypes. We therefore investigated the efficacy of passively administered variable-region-identical IgG1, IgG2a, IgG2b, and IgG3 monoclonal antibodies against intravenous infection with C. neoformans in mice genetically deficient in interleukin-12 (IL-12), IL-6, IL-4, or IL-10, as well as in the parental C57BL/6J strain. The relative inherent susceptibilities of these mouse strains to C. neoformans were as follows: IL-12(-/-) > IL-6(-/-) > C57BL/6J approximately IL-4(-/-) >> IL-10(-/-). This is consistent with the notion that a Th1 response is necessary for natural immunity against cryptococcal infection. However, none of the IgG isotypes prolonged survival in IL-12(-/-), IL-6(-/-), or IL-4(-/-) mice, and all isotypes significantly enhanced infection in IL-10(-/-) mice. These results indicate that passive antibody-mediated protection against C. neoformans requires both Th1- and Th2-associated cytokines and reveal the complexity of the mechanisms through which antibodies modulate infection with this organism.

FIG. 1

Survival of MAb-treated C57BL/6J mice infected with C. neoformans. Mice were given an intraperitoneal injection of SCID mouse ascites fluid containing either 1 mg of the anti-GXM 3E5 IgG3, one of its in vitro switch variants of the IgG1, IgG2b, or IgG2a isotype, or 1 ml of NSO cell SCID mouse ascites fluid as a control and then 24 h later were infected intravenously with 10⁶ C. neoformans CFU. Administration of IgG1, IgG2b, or IgG2a was significantly protective compared to the control (P < 0.006). Animals treated with IgG3 fared worse than control mice, but this did not reach statistical significance (P = 0.09). Mean survival in days by treatment group: NSO cell ascites fluid, 41; IgG1, 63; IgG2a, 75; IgG2b, 62; IgG3, 36.

FIG. 2

(A) Survival of untreated cytokine-deficient mice and the parental C57BL/6J mice infected intravenously with 2.5 × 10⁶ C. neoformans CFU. IL-12^−/− and IL-6^−/− mice were more susceptible to infection than control mice (P < 0.0001 and P < 0.02, respectively), while IL-10^−/− mice were very resistant to cryptococcal challenge (P < 0.0001). IL-4^−/− mice resembled the parental C57BL/6J mice (P = 0.4). The experiment was terminated on day 140 postinfection, when there was still 40% survival in the IL-10^−/− mice. Mean survival in days by mouse strain: IL-12^−/−, 12; IL-6^−/−, 15; C57BL/6J, 19; IL-4^−/−, 21; IL-10^−/−, 79. (B) Serum GXM levels measured on day 14 postinfection from mice in panel A. By this time, two mice in the IL-6^−/− group and seven in the IL-12^−/− group had died. Horizontal lines, mean serum concentrations. In comparison to C57BL/6J, only IL-10^−/− mice had significantly different levels of circulating GXM (P < 0.003).

FIG. 3

Survival of MAb-treated IL-12^−/− and IL-6^−/− mice. (A) IL-12^−/− mice were infected with 10⁵ C. neoformans CFU. NSO cell ascites fluid-treated C57BL/6J given the same inoculum are also shown so that the effect of this lower inoculum on the wild-type mice can be seen. Passive-antibody administration was neither protective nor enhancing compared to the control (P > 0.2). Mean survival in days by treatment group: NSO cell ascites fluid, 60; IgG1, 50; IgG2a, 55; IgG2b, 56; IgG3, 52; C57BL/6J mice treated with NSO cell ascites fluid, 116. (B) Survival of MAb-treated IL-6^−/− mice infected with 10⁶ C. neoformans CFU. 3E5 IgG2a was not protective and 3E5 IgG3 enhanced infection in comparison to the control (P = 0.23 and 0.0009, respectively). Mean survival in days by treatment group: NSO cell ascites fluid, 26; IgG2a, 23; IgG3, 18; C57BL/6J mice treated with NSO cell ascites fluid, 36.

FIG. 4

Survival of MAb-treated IL-4^−/− mice infected with 10⁶ C. neoformans CFU. The antibody was neither protective nor enhancing compared to the control (P > 0.2). At this lower inoculum, control IL-4^−/− mice were more resistant to infection than C57BL/6J mice (P = 0.03). Mean survival in days by treatment group: NSO cell ascites fluid, 55; IgG1, 46; IgG2a, 42; IgG2b, 45; IgG3, 54; C57BL/6J mice treated with NSO cell ascites fluid, 41.

FIG. 5

(A) Survival of MAb-treated IL-10^−/− mice infected with 5 × 10⁶ C. neoformans CFU. All antibodies greatly enhanced cryptococcal infection compared to the control (P > 0.006). Mean survival in days by treatment group: NSO cell ascites fluid, 50; IgG1, 17; IgG2a, 22; IgG2b, 19; IgG3, 20; C57BL/6J mice treated with NSO cell ascites fluid, 15. (B) Survival of IL-10^−/− mice treated as above but given a lower inoculum of 10⁶ C. neoformans CFU. Again, all antibodies greatly enhanced cryptococcal infection compared to the control (P > 0.002). Mean survival in days by treatment group: NSO cell ascites fluid, 114; IgG2a, 42; IgG2b, 47; IgG3, 55; C57BL/6J mice treated with NSO cell ascites fluid, 37.

FIG. 6

Liver pathology in IL-10^−/− mice 17 days after infection with C. neoformans. (A) H&E staining of the livers of IL-10^−/− mice in the control (NSO cell ascites fluid) group showed granulomatous cryptococcal lesions that contained macrophages with a mixture of other leukocytes. (B) Mucicarmine staining showed small numbers of organisms in these lesions (arrow) and demonstrated pink cytoplasmic staining of macrophages, which is consistent with phagocytosis of cryptococcal capsular polysaccharide. (C) In contrast, H&E staining of sections from C57BL/6J mice showed smaller, less-organized inflammatory responses to cryptococcal infection. (D) Mucicarmine staining demonstrated extracellular yeast in sinusoids (arrow) without associated inflammation. Magnification (all panels), ×400.

FIG. 7

Phagocytic indices of peritoneal macrophages from C57BL/6J and IL-10^−/− mice pretreated overnight without (A) or with (B) recombinant murine IL-10 (2 ng/ml) and then incubated with either IgG1, IgG2a, IgG2b, or IgG3 3E5 MAbs to GXM and heat-killed C. neoformans for 4 h. This experiment was repeated three times, once with alveolar macrophages, with similar results.