A Lower Dose of Infection Generates a Better Long-Term Immune Response against Toxoplasma gondii.

Toxoplasma gondii, an obligate intracellular pathogen, induces a strong immune response in the infected host. In the encephalitis model of infection, long-term protective immunity is mediated by CD8 T cells, with the CD4 T cell population providing important help. Most of the immune studies have used a 10- to 20-cyst dose of T. gondii, which leads to T cell dysfunctionality during the late phase of chronic infection and increases the chances of reactivation. In the current study, we compared the immune response of mice orally infected with either 2 or 10 cysts of T. gondii. During the acute phase, we demonstrate that the lower dose of infection generates a reduced number of CD4 and CD8 T cells, but the frequency of functional CD4 or CD8 T cells is similar in animals infected with two different doses. However, Ag-experienced T cells (both CD4 and CD8) are better maintained in lower dose-infected mice at 8 wk postinfection, with an increase number functional cells that exhibit lower multiple inhibitory receptor expression. In addition to better long-term T cell immunity, animals infected with a lower dose display reduced inflammation manifested by lesser Ag-specific T cell and cytokine responses during the very early stage of the acute infection. Our studies suggest a previously unappreciated role of dose-dependent early programming/imprinting of the long-term CD4/CD8 T cell response during T. gondii infection. These observations point to the need for an in-depth analysis of how early events shape long-term immunity against this pathogen.

Immune Response to Microsporidia.

Microsporidia are a group of pathogens, which can pose severe risks to the immunocompromised population, such as HIV-infected individuals or organ transplant recipients. Adaptive immunity has been reported to be critical for protection, and mice depleted of T cells are unable to control these infections. In a mouse model of infection, CD8 T cells have been found to be the primary effector cells and are responsible for protecting the infected host. Also, as infection is acquired via a peroral route, CD8 T cells in the gut compartment act as a first line of defense against these pathogens. Thus, generation of a robust CD8 T-cell response exhibiting polyfunctional ability is critical for host survival. In this chapter, we describe the effector CD8 T cells generated during microsporidia infection and the factors that may be essential for generating protective immunity against these understudied but significant pathogens. Overall, this chapter will highlight the necessity for a better understanding of the development of CD8 T-cell responses in gut-associated lymphoid tissue (GALT) and provide some insights into therapies that may be used to restore defective CD8 T-cell functionality in an immunocompromised situation.

Complex and Multilayered Role of IL-21 Signaling during Thymic Development.

Unlike IL-7, which is known to be critical for T cell thymic development, the role of IL-21 in this process is still controversial. IL-21 has been shown to accelerate thymic recovery in mice treated with glucocorticoids and revives the peripheral T cell pool in aged animals. However, mice with a defect in IL-21 signaling exhibit normal thymic cellularity, challenging the importance of this cytokine in the thymic developmental process. Using mixed bone marrow chimeric mice, our studies describe a multilayered role for IL-21 in thymopoiesis. In this system, IL-21R-deficient cells are unable to compete with wild-type populations at different stages of the thymic development. Using a mixed bone marrow chimeric animal model, IL-21 seems to be involved as early as the double-negative 1 stage, and the cells from the knockout compartment have problems transitioning to subsequent double-negative stages. Also, similar to IL-7, IL-21 seems to be involved in the positive selection of double-positive lymphocytes and appears to play a role in the migration of single-positive T cells to the periphery. Although not as critical as IL-7, based on our studies, IL-21 plays an important complementary role in thymic T cell development, which, to date, has been underrecognized.

Downregulated IL-21 Response and T Follicular Helper Cell Exhaustion Correlate with Compromised CD8 T Cell Immunity during Chronic Toxoplasmosis.

CD8 T cells are important for maintaining the chronicity of Toxoplasma gondii infection. In a T. gondii encephalitis susceptible model, we recently demonstrated that CD4 T cells play an essential helper role in the maintenance of the effector response and CD8 T cell dysfunctionality was linked to CD4 T cell exhaustion. However, CD4 T cells are constituted of different subsets with various functions and the population(s) providing help to the CD8 T cells has not yet been determined. In the present study, T follicular helper cells (Tfh), which are known to be essential for B cell maturation and are one of the main sources of IL-21, were significantly increased during chronic toxoplasmosis. However, at week 7 p.i., when CD8 T cells are exhausted, the Tfh population exhibited increased expression of several inhibitory receptors and levels of IL-21 in the serum were decreased. The importance of IL-21 in the maintenance of CD8 T cells function after T. gondii infection was further demonstrated in IL-21R KO mouse model. Interestingly, while CD8 T cells from both knockout (KO) and wild-type mice expressed similar levels of PD-1, animals with defective IL-21 signaling exhibited lower polyfunctionality than wild-type controls. This reduced polyfunctional ability observed in CD8 T cells from KO mice was associated with a significant increase in other inhibitory receptors like Tim-3, LAG-3, and 2B4. Furthermore, the animals exhibited greater signs of Toxoplasma reactivation manifested by the reduced number of cysts and increased expression of tachyzoite (replicative form of the parasite) specific genes (SAG1 and ENO2) in the brain. Also, IL-21R KO mice displayed a higher frequency of tachyzoite-infected monocytes in the blood and spleen. Our findings suggest the importance of Tfh and IL-21 during chronic toxoplasmosis and establish a critical role for this cytokine in regulating CD8 T cell dysfunction by preventing the co-expression of multiple inhibitory receptors during chronic parasitic infection.

IL-21 Is Important for Induction of KLRG1+ Effector CD8 T Cells during Acute Intracellular Infection.

Microsporidia, a latent opportunistic infection associated with mild inflammation, is characterized by a strong CD8 T cell response, which has been shown to be CD4 T cell dependent. In this manuscript, we demonstrate that CD4 help is provided via IL-21 production, a common γ-chain cytokine closely related to IL-2. The peak of IL-21 expression, observed during the acute infection, is associated with an elevated IL-21(+) CD4 T subset, and these cells bear a phenotypic resemblance to T follicular helper cells. We observed that, during per-oral microsporidial infection, IL-21 was critical for the generation of an optimal effector CD8 T cell immunity. Sharply decreased effector KLRG1(+) CD8 response was observed in IL-21R knockout mice, and although these cells exhibited reduced functional properties, they retained the ability to proliferate. The role of IL-21 in the generation of CD8 effectors was cell intrinsic, as stronger defects were observed in the IL-21-deficient compartment from the bone marrow chimeric mice (IL-21R knockout/wild-type). These findings are different from those reported for viral infections in which IL-21 has been primarily associated with the generation and maintenance of CD8 memory response. To the best of our knowledge, this report demonstrates a critical role for IL-21 in the generation of a primary effector CD8 T cell response to an infectious disease model.

Interleukin-12-producing CD103+ CD11b- CD8+ dendritic cells are responsible for eliciting gut intraepithelial lymphocyte response against Encephalitozoon cuniculi.

Microsporidia, which belong to the kingdom Fungi, are important opportunistic pathogens in HIV-infected populations and organ transplant recipients that are often associated with a broad range of symptoms, such as diarrhea, nephritis, and encephalitis. Natural infection occurs via the oral route, and as a consequence, gut immunity plays an important role in restricting the dissemination of these pathogens. Studies from our laboratory have reported that the pathogens induce a rapid intraepithelial lymphocyte (IEL) response important for host protection. Although mucosal dendritic cells (DC) are likely involved in triggering an antigen-specific IEL response, the specific subset(s) responsible has yet to be identified. Toward this goal, we demonstrate a very important role for mucosal CD11b(-) CD8(+) DC in the initiation of an antigen-specific IEL in vivo. Effectively, after Encephalitozoon cuniculi infection, CD11b(-) CD8(+) DC were activated in the lamina propria (LP) and acquired the ability to process retinoic acid (RA). However, this subset did not produce interleukin 12 (IL-12) but upregulated CD103, which is essential for migration to the mesenteric lymph nodes (MLN). Interestingly, CD103(+) CD11b(-) CD8(+) DC in the MLN, in addition to processing RA, also secreted IL-12 and were responsible for gut imprinting specificity on mucosal CD8 T cells. To the best of our knowledge, this is the first report describing the importance of MLN CD103(+) CD11b(-) CD8(+) DC isolated from infected animals in the generation of an IEL response against a live pathogen.

Effector CD8 T cell immunity in microsporidial infection: a lone defense mechanism.

Microsporidia is a group of pathogens, which can pose severe risks to the immunocompromised population such as HIV-infected individuals. The expertise to diagnose these pathogens is limited and therefore their prevalence is believed to be much higher than what is currently known. In a mouse model of infections, it has been reported that CD8 T cells are the primary effector cells responsible for protecting the infected host. As the infection is acquired via per-oral route, CD8 T cells in the gut compartment apparently act as a first line of defense against the pathogens. Thus, generation of a robust CD8 T cell response that exhibits polyfunctional ability is critical for host survival. In this review, we describe the effector CD8 T cells generated during microsporidial infection and underline the factors that may be essential for the elicitation of protective immunity against this understudied but significant pathogen. Overall, this review will highlight the necessity for a better understanding of the development of the CD8 T cell response in gut associated lymphoid tissue (GALT) and provide some insights into therapies that may be used to restore defective CD8 T cell functionality in an immunocompromised situation.

Intrinsic TGF-ß signaling promotes age-dependent CD8+ T cell polyfunctionality attrition.

Advanced age is associated with immune system deficits that result in an increased susceptibility to infectious diseases; however, specific mediators of age-dependent immune dysfunction have not been fully elucidated. Here we demonstrated that aged mice exhibit poor effector CD8+ T cell polyfunctionality, primarily due to CD8+ T cell-extrinsic deficits, and that reduced CD8+ T cell polyfunctionality correlates with increased susceptibility to pathogenic diseases. In aged animals challenged with the parasite Encephalitozoon cuniculi, effector CD8+ T cell survival and polyfunctionality were suppressed by highly elevated TGF-ß1. Furthermore, TGF-ß depletion reduced effector CD8+ T cell apoptosis in both young and aged mice and enhanced effector CD8+ T cell polyfunctionality in aged mice. Surprisingly, intrinsic blockade of TGF-ß signaling in CD8+ T cells was sufficient to rescue polyfunctionality in aged animals. Together, these data demonstrate that low levels of TGF-ß1 promote apoptosis of CD8+ effector T cells and high TGF-ß1 levels associated with age result in both CD8+ T cell apoptosis and an altered transcriptional profile, which correlates with loss of polyfunctionality. Furthermore, elevated TGF-ß levels are observed in the elderly human population and in aged Drosophila, suggesting that TGF-ß represents an evolutionarily conserved negative regulator of the immune response in aging organisms.

T cell exhaustion in protozoan disease.

Protozoan parasites cause severe morbidity and mortality in humans worldwide, especially in developing countries where access to chemotherapeutic agents is limited. Although parasites initially evoke a robust immune response, subsequent immunity fails to clear infection, ultimately leading to the chronic stage. This enigmatic situation was initially addressed in chronic viral models, where T cells lose their function, a phenomenon referred to as 'exhaustion'. However, recent studies demonstrate that this paradigm can be extended to protozoan diseases as well, although with notable differences. These studies have revealed that T cell responses generated against Toxoplasma gondii, Plasmodium sp., and Leishmania sp. can become dysfunctional. This review discusses T cell exhaustion in parasitic infection, mechanisms of development, and a possible role in disease outcome.

Optimal CD8 T-cell response against Encephalitozoon cuniculi is mediated by Toll-like receptor 4 upregulation by dendritic cells.

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.

Purified PTP1 protein induces antigen-specific protective immunity against Encephalitozoon cuniculi.

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.

Lack of interleukin-12 in p40-deficient mice leads to poor CD8+ T-cell immunity against Encephalitozoon cuniculi infection.

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-gamma) 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.

Aging mice exhibit a functional defect in mucosal dendritic cell response against an intracellular pathogen.

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.

IFN-gamma-producing dendritic cells are important for priming of gut intraepithelial lymphocyte response against intracellular parasitic infection.

The importance of intraepithelial lymphocytes (IEL) in immunoprotection against orally acquired pathogens is being increasingly recognized. Recent studies have demonstrated that Ag-specific IEL can be generated and can provide an important first line of defense against pathogens acquired via oral route. However, the mechanism involved in priming of IEL remains elusive. Our current study, using a microsporidial model of infection, demonstrates that priming of IEL is dependent on IFN-gamma-producing dendritic cells (DC) from mucosal sites. DC from mice lacking the IFN-gamma gene are unable to prime IEL, resulting in failure of these cells to proliferate and lyse pathogen-infected targets. Also, treatment of wild-type DC from Peyer's patches with Ab to IFN-gamma abrogates their ability to prime an IEL response against Encephalitozoon cuniculi in vitro. Moreover, when incubated with activated DC from IFN-gamma knockout mice, splenic CD8(+) T cells are not primed efficiently and exhibit reduced ability to home to the gut compartment. These data strongly suggest that IFN-gamma-producing DC from mucosal sites play an important role in the generation of an Ag-specific IEL response in the small intestine. To our knowledge, this report is the first demonstrating a role for IFN-gamma-producing DC from Peyer's patches in the development of Ag-specific IEL population and their trafficking to the gut epithelium.

CCR5 is essential for NK cell trafficking and host survival following Toxoplasma gondii infection.

The host response to intracellular pathogens requires the coordinated action of both the innate and acquired immune systems. Chemokines play a critical role in the trafficking of immune cells and transitioning an innate immune response into an acquired response. We analyzed the host response of mice deficient in the chemokine receptor CCR5 following infection with the intracellular protozoan parasite Toxoplasma gondii. We found that CCR5 controls recruitment of natural killer (NK) cells into infected tissues. Without this influx of NK cells, tissues from CCR5-deficient (CCR5-/-) mice were less able to generate an inflammatory response, had decreased chemokine and interferon gamma production, and had higher parasite burden. As a result, CCR5-/- mice were more susceptible to infection with T. gondii but were less susceptible to the immune-mediated tissue injury seen in certain inbred strains. Adoptive transfer of CCR5+/+ NK cells into CCR5-/- mice restored their ability to survive lethal T. gondii infection and demonstrated that CCR5 is required for NK cell homing into infected liver and spleen. This study establishes CCR5 as a critical receptor guiding NK cell trafficking in host defense.

Lack of IL-15 results in the suboptimal priming of CD4+ T cell response against an intracellular parasite.

IFN-gamma-producing CD4+ T cells, although important for protection against acute Toxoplasma gondii infection, can cause gut pathology, which may prove to be detrimental for host survival. Here we show that mice lacking IL-15 gene develop a down-regulated IFN-gamma-producing CD4+ T cell response against the parasite, which leads to a reduction in gut necrosis and increased level of survival against infection. Moreover, transfer of immune CD4+ T cells from WT to IL-15-/- mice reversed inhibition of gut pathology and caused mortality equivalent to levels of parental WT mice. Down-regulated CD4+ T cell response in the absence of IL-15, manifested as reduced antigen-specific proliferation, was due to defective priming of the T cell subset by dendritic cells (DCs) of these animals. When stimulated with antigen-pulsed DCs from WT mice, CD4+ T cells from IL-15-/- mice were primed optimally, and robust proliferation of these cells was observed. A defect in the DCs of knockout mice was further confirmed by their reduced ability to produce IL-12 upon stimulation with Toxoplasma lysate antigen. Addition of exogenous IL-15 to DC cultures from knockout mice led to increased IL-12 production by these cells and restored their ability to prime an optimal parasite-specific CD4+ T cell response. To our knowledge, this is the first demonstration of the role of IL-15 in the development of CD4+ T cell immunity against an intracellular pathogen. Furthermore, based on these observations, targeting of IL-15 should have a beneficial effect on individuals suffering from CD4+ T cell-mediated autoimmune diseases.

NK cells help to induce CD8(+)-T-cell immunity against Toxoplasma gondii in the absence of CD4(+) T cells.

CD8(+) T-cell immunity plays an important role in protection against intracellular infections. Earlier studies have shown that CD4(+) T-cell help was needed for launching in vivo CD8(+) T-cell activity against these pathogens and tumors. However, recently CD4(+) T-cell-independent CD8 responses during several microbial infections including those with Toxoplasma gondii have been described, although the mechanism is not understood. We now demonstrate that, in the absence of CD4(+) T cells, T. gondii-infected mice exhibit an extended NK cell response, which is mediated by continued interleukin-12 (IL-12) secretion. This prolonged NK cell response is critical for priming parasite-specific CD8(+) T-cell immunity. Depletion of NK cells inhibited the generation of CD8(+) T-cell immunity in CD4(-/-) mice. Similarly neutralization of IL-12 reduces NK cell numbers in infected animals and leads to the down-regulation of CD8(+) T-cell immunity against T. gondii. Adoptive transfer of NK cells into the IL-12-depleted animals restored their CD8(+) T-cell immune response, and animals exhibited reduced mortality. NK cell gamma interferon was essential for cytotoxic T-lymphocyte priming. Our studies for the first time demonstrate that, in the absence of CD4(+) T cells, NK cells can play an important role in induction of primary CD8(+) T-cell immunity against an intracellular infection. These observations have therapeutic implications for immunocompromised individuals, including those with human immunodeficiency virus infection.