Down-regulation of the immune response in aging individuals puts this population at a potential risk against infectious agents. In-depth studies conducted in humans and mouse models have demonstrated that with increasing age, the T cell immune response against pathogens is compromised and response to vaccinations is subdued. In the present study, using a mouse model, we demonstrate that older animals exhibit greater susceptibility to Encephalitozoon cuniculi infection, and their ability to evoke an Ag-specific T cell response at the gut mucosal site is reduced. The dampening of T cell immunity was due to the defective priming by the dendritic cells (DC) isolated from the mucosal tissues of aging animals. When primed with DC from younger mice, T cells from older animals were able to exhibit an optimal Ag-specific response. The functional defect in DC from older mice can be attributed to a large extent to reduced IL-15 message in these cells, which can be reversed by addition of exogenous IL-15 to the cultures. IL-15 treatment led to optimal expression of costimulatory molecules (CD80 and CD86) on the surface of older DC and restored their ability to prime a T cell response against the pathogen. To our knowledge, this is the first report which demonstrates the inability of the DC population from aging animals to prime a robust T cell response against an infectious agent. Moreover, the observation that IL-15 treatment can reverse this defect has far-reaching implications in developing strategies to increase vaccination protocols for aging populations.

A CD8؉ T-cell response is critical for protection against Encephalitozoon cuniculi infection. However, the factors responsible for the generation of CD8 ؉ T-cell immunity during E. cuniculi infection and the cytokines involved in this process have not been identified. In the present study, we demonstrated that p40-deficient animals, which are unable to produce interleukin-12 (IL-12), have a serious defect in expansion of the CD8 ؉ T-cell response which compromises the survival of an infected host. Adoptive transfer of CD8 ؉ T cells from immunocompetent donors protected SCID mice infected with E. cuniculi, whereas administration of CD8 ؉ T cells from p40 ؊/؊ mice failed to protect infected SCID mice. In vitro dendritic cell (DC) cultures from knockout mice pulsed with E. cuniculi spores were unable to develop a robust CD8؉ T-cell immune response. Addition of exogenous IL-12 or transfer of CD8 ؉ T cells that were initially primed with DC from p40 ؊/؊ animals to DC cultures from immunocompetent mice (directly or via transwells) led to optimal expansion of these cells. This IL-12-mediated reinstatement of CD8 ؉ T-effector immunity was independent of gamma interferon (IFN-␥) as addition of antibody to the cultures failed to have an effect. These studies demonstrated that IL-12 plays a predominant role in the expansion of effector CD8 ؉ T-cell immunity against E. cuniculi, which is critical for host survival. These findings are very important for understanding the protective immune mechanisms needed to protect an immunocompromised host against an opportunistic infection and can be extended to other microsporidial pathogens.

CD8؉ T-cell immunity has been shown to play an important role in the protective immune response against Encephalitozoon cuniculi. Although earlier studies suggest that dendritic cells (DC) are important for the induction of this response, the factors responsible for initiation of the dendritic cell response against this pathogen have not been evaluated. In the current study, we demonstrate that E. cuniculi infection causes strong Toll-like receptor 4 (TLR4)-dependent dendritic cell activation and a blockade of this molecule reduces the ability of DC to prime an antigen-specific CD8 ؉ T-cell response. Pretreatment of DC with anti-TLR4 antibody causes a defect in both in vitro and in vivo CD8 ؉ T-cell priming. These findings, for the first time, emphasize the contribution of TLR4 in the induction of CD8 ؉ T-cell immunity against E. cuniculi infection.Microsporidia are small obligate intracellular parasites that, until recently, were thought to be protozoans; however, evidence now suggests that they are related to fungi (15,17). Microsporidia can infect a vast number of species of vertebrates and invertebrates; of the 150 genera of microsporidia, however, only 7 have been found to infect humans (13). Severe infections have been reported predominantly for immunocompromised patients, such as patients with HIV and organ transplant recipients (2,7,23,37). Acute infections have also been reported in travelers and the elderly (26, 27), and there is evidence of colonization of healthy, nonsymptomatic patients (34).Due to the prevalence of opportunistic microsporidian infections associated with the HIV-AIDS pandemic, recent research has focused on the host's immune response to these pathogens. Early animal studies showed that cellular immunity was necessary to protect SCID mice from a lethal Encephalitozoon cuniculi challenge. Moreover, depletion of CD8 ϩ T cells caused mice to succumb to intraperitoneal (i.p.) E. cuniculi infection (21), and previous studies in our laboratory have shown that cytotoxic lymphocytes play a major role in protection against this effect (20,21).Recent reports from our laboratory have demonstrated that dendritic cells (DC) play an important role in the priming of the immune response against E. cuniculi (31, 32). T cells incubated with E. cuniculi-pulsed DC exhibited antigen-specific characteristics, specifically gamma interferon (IFN-␥) production, cytotoxicity, and proliferation (31, 32). In order to mount an immune response against a foreign pathogen, DC must first recognize the pathogen to initiate an appropriate response. One key method of recognition is through Toll-like receptors (TLRs), which were first discovered in Drosophila in response to infection with fungal pathogens (24). However, specific TLR molecules involved in DC activation during E. cuniculi infection have not been identified previously. We evaluated the upregulation of specific molecules involved in activation of the DC response after E. cuniculi infection. Different TLR molecules were tested, and TLR4 expression was found ...

Microsporidiosis poses a problem for immunocompromised individuals including patients with HIV infection as well as those with organ transplantation. Recent reports from Africa have suggested that microsporidiosis with diarrhea is an independent risk factor for malnutrition in children. Previous studies from our laboratory have demonstrated that CD8 + T cells are an essential component of protective immunity against the microsporidium Encephalitozoon cuniculi. Mutant mice lacking this T cell subset or cytotoxic function are unable to clear the infection and ultimately succumb to the disease. However, information regarding the antigens involved in the elicitation of CD8 + T cell response is not available. In this study, we report that immunization of animals with Encephalitozoon hellem polar tube protein 1 (rEhPTP1) induces a strong T cell response in vaccinated animals. Splenic dendritic cells pulsed with rEhPTP1 are able to induce E. cuniculi specific CD8 + T cell response with no effect on the CD4 + T cell subset. This is the first report identifying a protein capable of inducing CD8 + T cell immunity, which is conserved in other microsporidial species of human importance.