CD28 Exerts Protective and Detrimental Effects in a Pulmonary Model of Paracoccidioidomycosis

T-cell immunity has been claimed as the main immunoprotective mechanism against Paracoccidioides brasiliensis infection, the most important fungal infection in Latin America. As the initial events that control T-cell activation in paracoccidioidomycosis (PCM) are not well established, we decided to investigate the role of CD28, an important costimulatory molecule for the activation of effector and regulatory T cells, in the immunity against this pulmonary pathogen. Using CD28-deficient (CD28^−/−) and normal wild-type (WT) C57BL/6 mice, we were able to demonstrate that CD28 costimulation determines in pulmonary paracoccidioidomycosis an early immunoprotection but a late deleterious effect associated with impaired immunity and uncontrolled fungal growth. Up to week 10 postinfection, CD28^−/− mice presented increased pulmonary and hepatic fungal loads allied with diminished production of antibodies and pro- and anti-inflammatory cytokines besides impaired activation and migration of effector and regulatory T (Treg) cells to the lungs. Unexpectedly, CD28-sufficient mice progressively lost the control of fungal growth, resulting in an increased mortality associated with persistent presence of Treg cells, deactivation of inflammatory macrophages and T cells, prevalent presence of anti-inflammatory cytokines, elevated fungal burdens, and extensive hepatic lesions. As a whole, our findings suggest that CD28 is required for the early protective T-cell responses to P. brasiliensis infection, but it also induces the expansion of regulatory circuits that lately impair adaptive immunity, allowing uncontrolled fungal growth and overwhelming infection, which leads to precocious mortality of mice.It has long been appreciated that cellular immunity is the most important resistance mechanism against fungal infections (14, 36, 64). CD4⁺ and CD8⁺ T-cell subpopulations have been described to have a fundamental role in the control of fungal growth, and disease severity is also controlled by regulatory T (Treg) cells, which prevent tissue pathology by controlling excessive inflammatory reactions (25, 45, 46, 65). Similar to other deep mycoses, the severity of paracoccidioidomycosis (PCM), the most severe pulmonary mycosis in Latin America, is controlled by cellular immunity and cytokine-activated phagocytes that are able to kill Paracoccidioides brasiliensis, the etiological agent of this infection (10, 20, 30, 60, 61). In humans and in murine models of PCM, resistance to the disease is associated with the secretion of gamma interferon (IFN-γ) and other type 1 cytokines, whereas impaired Th1 immunity and the prevalent secretion of Th2 cytokines correlate with a systemic and progressive disease (2, 6, 39, 59, 76). Studies with CD4⁺ and CD8⁺ T-cell-deficient mice revealed that both T-cell subsets are involved in the protective immunity against P. brasiliensis infection and indicated the prominent role of CD8⁺ T cells (3, 21, 25). Besides the prevalent Th2 immunity, recent investigations have described alternative mechanisms underlying T-cell dysfunction in humans and experimental PCM. Increased apoptosis and overexpression of Fas and FasL in T cells suggest that activation-induced cell death (AICD) is a mechanism that controls T-cell expansion during the active disease (13, 19). In addition, the increased expression of CTLA-4 and the expansion of Treg cells were associated with severe patterns of the disease (24, 45, 46, 56). Thus, in addition to cytokine imbalance, other regulatory mechanisms can actively participate in the unresponsiveness of T cells in P. brasiliensis-infected hosts.Optimal activation, proliferation, and cytokine production by antigen-specific T cells require two distinct signals from dendritic cells or other antigen-presenting cells (APCs). After T-cell receptor (TCR) occupancy by the antigen epitope/major histocompatibility (MHC) complex (first signal), a second signal is mediated by costimulatory molecules (43, 63), such as CD28 on T cells and their counter-receptors CD80 (B7-1) and CD86 (B7-2) expressed by APCs (1, 34). Soluble molecules, such as cytokines and chemokines, also participate in the activation process, which drives and controls T-cell numbers and fates (1). CD28 enhances the TCR-triggered activation of naïve T cells, promotes interleukin-2 (IL-2) secretion and prevents T-cell anergy (1, 37). Alternatively, CD28-independent T-cell activation can occur if a strong and sustained antigen-specific signal is available (40, 81). Like CD28, two other molecules, cytotoxic T-lymphocyte antigen-4 (CTLA-4) and mouse inducible costimulatory molecule (ICOS), are selectively expressed by T cells, but the expression of these molecules depends on previous cell activation (50, 71). More recently, evidence has emerged that CD28 family members are also crucial regulators of natural and induced regulatory (CD4⁺CD25⁺Foxp3⁺) T cells (9). These cells are induced in the thymus and in the periphery, respectively, and control self-tolerance and the activation of several components of innate and adaptive immunity (68). Treg cells can suppress immune responses through the production of immunosuppressive cytokines (mainly IL-10 and transforming growth factor β [TGF-β]), through the induction of the apoptosis of effector T cells and through the modification of the functional properties of antigen-presenting cells (70, 78).Immunoprotection against microorganisms has been shown to be either CD28 dependent or independent. CD28-deficient (CD28^−/−) mice are highly susceptible to infection with Salmonella enterica serovar Typhimurium due to the poor ability of these mice to secrete IFN-γ (51). During some viral and parasitic infections, CD28 was shown to be required to mediate CD8⁺ T-cell immunoprotection (8, 53). In contrast, CD28^−/− mice infected with Mycobacterium bovis or Listeria monocytogenes control the bacterial burden and develop cell-mediated immunity (35, 52). In primary and opportunistic fungal infections, CD28 costimulation controls protective immunity, the expansion and function of regulatory T cells, and the intensity of inflammatory reactions (5, 54, 55, 66, 84).Because CD28 is critical for T-cell activation in fungal infections, we investigated its role in a murine model of P. brasiliensis infection. We show that CD28 costimulation exerts contrasting roles in pulmonary PCM. Early in infection, CD28 expression results in efficient adaptive immunity that is able to control fungal growth. Late in infection, however, this costimulatory molecule induces significant expansion of regulatory T cells, diminished immunity, and uncontrolled fungal growth that eventually leads to the death of the mice. In contrast, the absence of CD28 costimulation results in impaired T-cell immunity, which appeared to be compensated by the absence of Treg cell expansion. This weak but persistent immunity was able to partially control fungal growth, organize granulomatous lesions, and guarantee the enhanced survival of the mice, suggesting the relative protection conferred by CD28-independent mechanisms.