In vitro effector activity of Pneumocystis murina-specific T-cytotoxic-1 CD8+ T cells: role of granulocyte-macrophage colony-stimulating factor.

Pneumocystis jiroveci is an opportunistic pathogen of particular importance since the onset of the AIDS epidemic (19, 22). Chemoprophylaxis of human immunodeficiency virus (HIV)-infected patients at high clinical risk for Pneumocystis pneumonia and the introduction of highly active antiretroviral therapy have contributed to a reduced incidence of Pneumocystis pneumonia (19). Despite the success of these clinical interventions, Pneumocystis pneumonia remains one of the most common opportunistic pneumonias and the most common life-threatening infectious complication in HIV-infected patients (19). The first-line agent for effective therapy and chemoprophylaxis remains trimethoprim-sulfamethoxazole, and corticosteroids represent an important adjunctive agent in patients with hypoxemia (21). However, treatment failures, high rates of adverse drug reactions, drug intolerance to first-line antimicrobials, and drug resistance represent formidable challenges to the management and treatment of AIDS-related P. carinii pneumonia (19, 21). The recruitment of CD4+ T cells into lung tissue is requisite for efficient host defense against Pneumocystis spp. (13, 24). The role of CD8+ T cells in Pneumocystis pneumonia is less clear (4, 7, 10, 14, 31). Our laboratory has examined host defense against P. murina in mice specifically depleted of CD4+ T lymphocytes, which mimics the immunologic defect of HIV infection (13, 24). We have demonstrated that gamma interferon (IFN-γ) overexpression using adenovirus-mediated gene transfer (AdIFN) results in the clearance of P. murina in the absence of CD4+ T cells (14, 18). The mechanism for the clearance of P. murina with AdIFN involves CD8+ T cells, as depletion of this T-cell subset abrogated the effect of AdIFN on P. murina clearance (14). AdIFN resulted in the pulmonary recruitment of P. murina-specific T-cytotoxic-1 (Tc1)-like CD8+ T cells, defined by high levels of endogenous IFN-γ production. These cells have in vitro effector activity against P. murina in a macrophage coculture assay and affect the clearance of the organism upon adoptive transfer into P. murina-infected scid mice (18). Moreover, CD8+ T cells without a Tc1 phenotype, from mice treated with a control adenovirus vector, lacked in vitro effector activity against P. murina and upon adoptive transfer into P. murina-infected scid mice resulted in significantly worse lung injury (18). To gain a better understanding of the potential mechanisms by which Tc1 CD8+ T cells have effector activity against P. murina, we generated and expanded Tc1 and Tc2 CD8+ T cells specific for P. murina. Polarized CD8+ T-cell populations were tested directly against P. murina or in macrophage coculture assays. Tc1-polarized CD8+ T cells were found to elaborate significantly higher levels of IFN-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF) in antigen recall assays and lower levels of interleukin-4 (IL-4) than Tc2 CD8+ T cells. Moreover, P. murina-specific Tc1 CD8+ T cells had significantly greater effector activity against P. murina, but only in the presence of macrophages. Conditioned medium from antigen-stimulated Tc1 CD8+ T cells also augmented macrophage-mediated killing of P. murina, suggesting that a soluble factor may be responsible for the increase in macrophage-mediated killing of P. murina by Tc1 CD8+ T cells. Among the cytokines differentially expressed between Tc1 CD8+ T cells with effector activity and Tc2 CD8+ T cells without effector activity were IFN-γ and GM-CSF. Antibody neutralization studies demonstrated that GM-CSF secretion by Tc1 cells is critical for effector activity in vitro. Moreover, recombinant GM-CSF was more potent at increasing macrophage-mediated killing of P. murina than IFN in peritoneal macrophages, resident alveolar macrophages, and monocyte chemoattractant protein 1 (MCP-1)-elicited alveolar macrophages. Taken together, these data suggest that the antigen-specific release of GM-CSF may be critical for host defense against P. murina.