VCAM-1/α4β1 Integrin Interaction Is Crucial for Prompt Recruitment of Immune T Cells into the Brain during the Early Stage of Reactivation of Chronic Infection with Toxoplasma gondii To Prevent Toxoplasmic Encephalitis

Reactivation of chronic infection with Toxoplasma gondii can cause life-threatening toxoplasmic encephalitis in immunocompromised individuals. We examined the role of VCAM-1/α4β1 integrin interaction in T cell recruitment to prevent reactivation of the infection in the brain. SCID mice were infected and treated with sulfadiazine to establish a chronic infection. VCAM-1 and ICAM-1 were the endothelial adhesion molecules detected on cerebral vessels of the infected SCID and wild-type animals. Immune T cells from infected wild-type mice were treated with anti-α4 integrin or control antibodies and transferred into infected SCID or nude mice, and the animals received the same antibody every other day. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Expression of mRNAs for CD3δ, CD4, CD8β, gamma interferon (IFN-γ), and inducible nitric oxide synthase (NOS2) (an effector molecule to inhibit T. gondii growth) and the numbers of CD4⁺ and CD8⁺ T cells in the brain were significantly less in mice treated with anti-α4 integrin antibody than in those treated with control antibody at 3 days after sulfadiazine discontinuation. At 6 days after sulfadiazine discontinuation, cerebral tachyzoite-specific SAG1 mRNA levels and numbers of inflammatory foci associated with tachyzoites were markedly greater in anti-α4 integrin antibody-treated than in control antibody-treated animals, even though IFN-γ and NOS2 mRNA levels were higher in the former than in the latter. These results indicate that VCAM-1/α4β1 integrin interaction is crucial for prompt recruitment of immune T cells and induction of IFN-γ-mediated protective immune responses during the early stage of reactivation of chronic T. gondii infection to control tachyzoite growth.     

CD8+-T-Cell Immunity against Toxoplasma gondii Can Be Induced but Not Maintained in Mice Lacking Conventional CD4+ T Cells

T-cell immunity is critical for survival of hosts infected with Toxoplasma gondii. Among the cells in the T-cell population, CD8(+) T cells are considered the major effector cells against this parasite. It is believed that CD4(+) T cells may be crucial for induction of the CD8(+)-T-cell response against T. gondii. In the present study, CD4(-/-) mice were used to evaluate the role of conventional CD4(+) T cells in the immune response against T. gondii infection. CD4(-/-) mice infected with T. gondii exhibited lower gamma interferon (IFN-gamma) messages in the majority of their tissues. As a result, mortality due to a hyperinflammatory response was prevented in these animals. Interestingly, T. gondii infection induced a normal antigen-specific CD8(+)-T-cell immune response in CD4(-/-) mice. No difference in generation of precursor cytotoxic T lymphocytes (pCTL) or in IFN-gamma production by the CD8(+)-T-cell populations from the knockout and wild-type animals was observed. However, the mutant mice were not able to sustain CD8(+)-T-cell immunity. At 180 days after infection, the CD8(+)-T-cell response in the knockout mice was depressed, as determined by pCTL and IFN-gamma assays. Loss of CD8(+)-T-cell immunity at this time was confirmed by adoptive transfer experiments. Purified CD8(+) T cells from CD4(-/-) donors that had been immunized 180 days earlier failed to protect the recipient mice against a lethal infection. Our study demonstrated that although CD8(+)-T-cell immunity can be induced in the absence of conventional CD4(+) T cells, it cannot be maintained without such cells.     

Gamma interferon-dependent temporary resistance to acute Toxoplasma gondii infection independent of CD4+ or CD8+ lymphocytes.

It is well established that resistance to acute primary Toxoplasma gondii infection is mediated by a gamma interferon (IFN-gamma)-dependent mechanism. The present in vivo experiments were undertaken to investigate the cellular basis for this resistance. We show here that immunocompetent T. gondii-infected C57BL/6 (B6) mice treated with anti-IFN-gamma or with anti-Thy-1 or anti-asialo-GM1 antibodies die sooner than infected mice treated with antibodies that deplete both CD4+ and CD8+ T lymphocytes. Thy-1+ CD4- CD8- cells accumulated in the peritoneal cavities of B6 mice during the early stages of an intraperitoneal infection but did not accumulate in sham-infected control mice, and substantial numbers of Thy-1+ CD4- CD8- cells were recovered from the peritoneal cavities of infected B6 mice treated with antibodies that depleted CD4+ and CD8+ lymphocytes. Depletion of Thy-1+ cells reduced IFN-gamma to undetectable levels, whereas depletion of CD4+ and CD8+ cells did not reduce IFN-gamma levels. Thus T. gondii infection in immunocompetent B6 mice elicits Thy-1+ CD4- CD8- cells which either produce protective IFN-gamma themselves or control its production by other cells. It is likely that the function of these Thy-1+ CD4- CD8- cells is to control T. gondii tachyzoites during the early stages of primary infection before specific CD4(+)- and/or CD8(+)-dependent immunity develops.     

Monocyte adhesion to endothelium in simian immunodeficiency virus-induced AIDS encephalitis is mediated by vascular cell adhesion molecule-1/alpha 4 beta 1 integrin interactions.

Because the mechanisms associated with recruitment of monocytes to brain in AIDS encephalitis are unknown, we used tissues from rhesus monkeys infected with simian immunodeficiency virus (SIV) to examine the relative contributions of various adhesion pathways in mediating monocyte adhesion to endothelium from encephalitic brain. Using a modified Stamper and Woodruff tissue adhesion assay, we found that the human monocytic cell lines, THP-1 and U937, and the B cell line, Ramos, preferentially bound to brain vessels from monkeys with AIDS encephalitis. Using a combined tissue adhesion/immunohistochemistry approach, these cells only bound to vessels expressing vascular cell adhesion molecule-1 (VCAM-1). Furthermore, pretreatment of tissues with antibodies to VCAM-1 or cell lines with antibodies to VLA-4 (CD49d) inhibited adhesion by more than 70%. Intercellular adhesion molecule-1 (ICAM-1)/beta 2 integrin interactions were not significant in mediating cell adhesion to the vasculature in encephalitic simian brain using a cell line (JY) capable of binding rhesus monkey ICAM-1. In addition, selectin-mediated interactions did not significantly contribute to cell binding to encephalitic brain as there was no immunohistochemical expression of E-selectin and P-selectin in either normal or encephalitic brain, nor was there a demonstrable adhesive effect from L-selectin using L-selectin-transfected 300.19 cells on simian encephalitic brain. These results demonstrate that using the tissue adhesion assay, THP-1, U937, and Ramos cells bind to vessels in brain from animals with AIDS encephalitis using VCAM-1/alpha 4 beta 1 integrin interactions and suggest that VCAM-1 and VLA-4 may be integral for monocyte recruitment to the central nervous system during the development of AIDS encephalitis.     

CD4+ T cells are not essential for control of early acute Cryptosporidium parvum infection in neonatal mice

Cryptosporidiosis is an important diarrheal disease of humans and neonatal livestock caused by Cryptosporidium spp. that infect epithelial cells. Recovery from Cryptosporidium parvum infection in adult hosts involves CD4(+) T cells with a strong Th1 component, but mechanisms of immunity in neonates are not well characterized. In the present investigation with newborn mice, similar acute patterns of infection were obtained in C57BL/6 wild-type (WT) and T and B cell-deficient Rag2(-/-) mice. In comparison with uninfected controls, the proportion of intestinal CD4(+) or CD8(+) T cells did not increase in infected WT mice during recovery from infection. Furthermore, infection in neonatal WT mice depleted of CD4(+) T cells was not exacerbated. Ten weeks after WT and Rag2(-/-) mice had been infected as neonates, no patent infections could be detected. Treatment at this stage with the immunosuppressive drug dexamethasone produced patent infections in Rag2(-/-) mice but not WT mice. Expression of inflammatory markers, including gamma interferon (IFN-γ) and interleukin-12p40 (IL-12p40), was higher in neonatal WT mice than in Rag2(-/-) mice around the peak of infection, but IL-10 expression was also higher in WT mice. These results suggest that although CD4(+) T cells may be important for elimination of C. parvum, these cells are dispensable for controlling the early acute phase of infection in neonates.     

Sialomucin CD43 regulates T helper type 17 cell intercellular adhesion molecule 1 dependent adhesion,apical migration and transendothelial migration

T helper type 17 lymphocytes (Th17 cells) infiltrate the central nervous system (CNS), induce inflammation and demyelination and play a pivotal role in the pathogenesis of multiple sclerosis. Sialomucin CD43 is highly expressed in Th17 cells and mediates adhesion to endothelial selectin (E-selectin), an initiating step in Th17 cell recruitment to sites of inflammation. CD43^−/− mice have impaired Th17 cell recruitment to the CNS and are protected from experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis. However, E-selectin is dispensable for the development of EAE, in contrast to intercellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1). We report that CD43^−/− mice have decreased demyelination and T-cell infiltration, but similar up-regulation of ICAM-1 and VCAM-1 in the spinal cord, compared with wild-type (WT) mice, at the initiation of EAE. CD43^−/− Th17 cells have impaired adhesion to ICAM-1 under flow conditions in vitro, despite having similar expression of LFA-1, the main T-cell ligand for ICAM-1, as WT Th17 cells. Regardless of the route of integrin activation, CD43^−/− Th17 cell firm arrest on ICAM-1 was comparable to that of WT Th17 cells, but CD43^−/− Th17 cells failed to optimally apically migrate on immobilized ICAM-1-coated coverslips and endothelial cells, and to transmigrate under shear flow conditions in an ICAM-1-dependent manner. Collectively, these findings unveil novel roles for CD43, facilitating adhesion of Th17 cells to ICAM-1 and modulating apical and transendothelial migration, as mechanisms potentially responsible for Th17 cell recruitment to sites of inflammation such as the CNS.     

Cerebral leucocyte infiltration in lupus-prone MRL/MpJ-fas lpr mice--roles of intercellular adhesion molecule-1 and P-selectin

The autoimmune disease which affects MRL/MpJ-fas(lpr) mice results in cerebral leucocyte recruitment and cognitive dysfunction. We have previously observed increased leucocyte trafficking in the cerebral microcirculation of these mice; however, the types of leucocytes recruited have not been analysed thoroughly, and the roles of key endothelial adhesion molecules in recruitment of these leucocytes have not been investigated. Therefore the aim of this study was to classify the phenotypes of leucocytes present in inflamed brains of MRL/MpJ-fas(lpr) mice, and dissect the roles of endothelial adhesion molecules in their accumulation in the brain. Immunohistochemical analysis revealed significant leucocyte infiltration in the brains of 16- and 20-week-old MRL/MpJ-fas(lpr) mice, affecting predominantly the choroid plexus. Isolation of brain-infiltrating leucocytes revealed that lymphocytes and neutrophils were the main populations present. The CD3(+) lymphocytes in the brain consisted of similar proportions of CD4(+), CD8(+) and CD4(-)/CD8(-)[double negative (DN)] populations. Assessment of MRL/MpJ-fas(lpr) mice deficient in endothelial adhesion molecules intercellular adhesion molecule-1 (ICAM-1) or P-selectin indicated that cerebral leucocyte recruitment persisted in the absence of these molecules, with only minor changes in the phenotypes of infiltrating cells. Together these data indicate that the brains of MRL/MpJ-fas(lpr) mice are affected by a mixed leucocyte infiltrate, of which the unusual DN lymphocyte phenotype contributes a substantial proportion. In addition, endothelial adhesion molecules ICAM-1 and P-selectin, which modulate survival of MRL/MpJ-fas(lpr) mice, do not markedly inhibit leucocyte entry into the central nervous system.     

Increased disease severity of parasite-infected TLR2-/- mice is correlated with decreased central nervous system inflammation and reduced numbers of cells with alternatively activated macrophage phenotypes in a murine model of neurocysticercosis

In a murine model for neurocysticercosis (NCC), intracranial inoculation of the helminth parasite Mesocestoides corti induces multiple Toll-like receptors (TLRs), among which TLR2 is upregulated first and to a relatively high extent. Here, we report that TLR2(-/-) mice displayed significantly increased susceptibility to parasite infection accompanied by increased numbers of parasites in the brain parenchyma compared to infection in wild-type (WT) mice. This coincided with an increased display of microglial nodule formations and greater neuropathology than in the WT. Parasite-infected TLR2(-/-) brains exhibited a scarcity of lymphocytic cuffing and displayed reduced numbers of infiltrating leukocytes. Fluorescence-activated cell sorter (FACS) analyses revealed significantly lower numbers of CD11b(+) myeloid cells, γδ T cells, αβ T cells, and B cells in the brains of parasite-infected TLR2(-/-) mice. This correlated with significantly reduced levels of inflammatory mediators, including tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), CCL2, CCL3, and interleukin-6 (IL-6) in the central nervous system (CNS) of TLR2(-/-) mice. As TLR2 has been implicated in immune regulation of helminth infections and as alternatively activated macrophages (AAMs) are thought to play a profound regulatory role in such infections, induction of AAMs in infected TLR2(-/-) mice was compared with that in WT mice. Parasite-infected WT brains showed larger numbers of macrophages/microglia (CD11b(+) cells) expressing AAM-associated molecules such as YM1, Fizz1 (found in inflammatory zone-1 antigen), and arginase 1 than TLR2(-/-) brains, consistent with a protective role of AAMs during infection. Importantly, these results demonstrate that TLR2-associated responses modulate the disease severity of murine NCC.     

Counter-protective role for interleukin-5 during acute Toxoplasma gondii infection

The role of interleukin-5 (IL-5) during Toxoplasma gondii infection was investigated by comparing disease progression in IL-5 gene deficient (IL-5-/-) mice and their wild-type (WT) counterparts on a C57BL/6 background. IL-5-/- mice infected orally with T. gondii were less susceptible to infection than WT mice as demonstrated by reduced mortality rates. Consistent with this data, orally infected IL-5-/- mice had less severe pathological changes in their small intestines than WT mice at 8 days postinfection. At this time, splenocytes and mesenteric lymph node cells derived from IL-5-/- mice produced levels of IL-12, interferon-gamma (IFN-gamma), IL-4, IL-10, and nitric oxide (measured as nitrite) similar to those derived from WT mice when stimulated with Toxoplasma lysate antigen. However, peak serum IL-12 and IFN-gamma levels (at days 6 and 8, respectively) were significantly higher in IL-5-/- mice than in WT mice. In addition, WT mice but not IL-5-/- mice had raised levels of eosinophils in their peripheral blood between days 5 and 8 following infection. Oral administration of N omega-nitro-L-arginine methyl (from day 4 postinfection) increased mortality rates in both IL-5-/- and WT mice, indicating a protective role for nitric oxide during the early stages of oral T. gondii infection. In comparison with oral infection, no difference in mortality was observed between IL-5-/- and WT mice following intraperitoneal infection with T. gondii, with all mice surviving until 35 days postinfection. Similarly, no significant differences were observed in the severity of the meningitis, perivascular cuffing, or number of microglial nodules or parasites in the brains of intraperitoneally infected mice. Together, these results demonstrate a detrimental role for IL-5 during the early stage of oral infection with T. gondii which is associated with increased small-intestine pathology, eosinophilia, and reduced plasma IL-12 and IFN-gamma levels.     

Profound effect of the absence of IL-4 on T cell responses during infection with Schistosoma mansoni.

T cell responses of interleukin (IL)-4(-/-) and wild-type (WT) mice infected with the helper T cell 2 (Th2) response-inducing pathogen Schistosoma mansoni were compared. As expected, given the important role of IL-4 in Th2 response induction, the absence of IL-4 resulted in diminished Th2 responses, apparent as reduced production of IL-4, -5, and -10 by CD4(+) cells isolated from the spleens of infected IL-4(-/-) mice. Surprisingly, these cells produced significantly less interferon (IFN)-gamma and proliferated less than did those from infected WT mice after T cell receptor ligation. CD8(+) cells isolated from infected IL-4(-/-) mice also produced less IFN-gamma than WT CD8 cells, although there was no difference in the proliferative responses of these cell populations. After infection, spleens of infected IL-4(-/-) mice did not enlarge to the same extent as those of WT mice, and attrition of the CD8(+) cell population within this lymphoid organ was noted. Taken together, the data indicate that in addition to inhibiting Th2 response development, the lack of IL-4 during schistosomiasis significantly affects additional aspects of T cell responses.     

The CD40/CD40 ligand interaction is required for resistance to toxoplasmic encephalitis

Since the CD40/CD40 ligand (CD40L) interaction is involved in the regulation of macrophage production of interleukin 12 (IL-12) and T-cell production of gamma interferon (IFN-gamma), effector cell functions associated with resistance to Toxoplasma gondii, the role of CD40L in immunity to this parasite was assessed. Infection of C57BL/6 mice with T. gondii results in an upregulation of CD40 expression on accessory cell populations at local sites of infection as well as in lymphoid tissues. Splenocytes from C57BL/6 mice infected with T. gondii for 5 days produced high levels of IL-12 and IFN-gamma when stimulated with toxoplasma lysate antigen, and blocking CD40L did not significantly alter the production of IFN-gamma or IL-12 by these cells. Similar results were observed with splenocytes and mononuclear cells isolated from the brains of chronically infected mice. Interestingly, although CD40L(-/-) mice infected with T. gondii produced less IL-12 than wild-type mice, they produced comparable levels of IFN-gamma but succumbed to toxoplasmic encephalitis 4 to 5 weeks after infection. The inability of CD40L(-/-) mice to control parasite replication in the brain correlated with the ability of soluble CD40L, in combination with IFN-gamma, to activate macrophages in vitro to control replication of T. gondii. Together, these results identify an important role for the CD40/CD40L interaction in resistance to T. gondii. However, this interaction may be more important in the control of parasite replication in the brain rather than the generation of protective T-cell responses during toxoplasmosis.     

CD8α(+) dendritic cells are the critical source of interleukin-12 that controls acute infection by Toxoplasma gondii tachyzoites

CD8α(+) dendritic cells (DCs) are important in?vivo for cross-presentation of antigens derived from intracellular pathogens and tumors. Additionally, secretion of interleukin-12 (IL-12) by CD8α(+) DCs suggests a role for these cells in response to Toxoplasma gondii antigens, although it remains unclear whether these cells are required for protection against T.?gondii infection. Toward this goal, we examined T.?gondii infection of Batf3(-/-) mice, which selectively lack only lymphoid-resident CD8α(+) DCs and related peripheral CD103(+) DCs. Batf3(-/-) mice were extremely susceptible to T.?gondii infection, with decreased production of IL-12 and interferon-γ. IL-12 administration restored resistance in Batf3(-/-) mice, and mice in which IL-12 production was ablated only from CD8α(+) DCs failed to control infection. These results reveal that the function of CD8α(+) DCs extends beyond a role in cross-presentation and includes a critical role for activation of innate immunity through IL-12 production during T.?gondii infection.     

Experimental cerebral malaria develops independently of caspase recruitment domain-containing protein 9 signaling

The outcome of infection depends on multiple layers of immune regulation, with innate immunity playing a decisive role in shaping protection or pathogenic sequelae of acquired immunity. The contribution of pattern recognition receptors and adaptor molecules in immunity to malaria remains poorly understood. Here, we interrogate the role of the caspase recruitment domain-containing protein 9 (CARD9) signaling pathway in the development of experimental cerebral malaria (ECM) using the murine Plasmodium berghei ANKA infection model. CARD9 expression was upregulated in the brains of infected wild-type (WT) mice, suggesting a potential role for this pathway in ECM pathogenesis. However, P. berghei ANKA-infected Card9(-/-) mice succumbed to neurological signs and presented with disrupted blood-brain barriers similar to WT mice. Furthermore, consistent with the immunological features associated with ECM in WT mice, Card9(-/-) mice revealed (i) elevated levels of proinflammatory responses, (ii) high frequencies of activated T cells, and (iii) CD8(+) T cell arrest in the cerebral microvasculature. We conclude that ECM develops independently of the CARD9 signaling pathway.     

A GRA1 DNA vaccine primes cytolytic CD8(+) T cells to control acute Toxoplasma gondii infection

Protective immunity against Toxoplasma gondii is known to be mediated mainly by T lymphocytes and gamma interferon (IFN-gamma). The contribution of CD4(+) and CD8(+) T-lymphocyte subsets to protective immune responses against T. gondii infection, triggered by a GRA1 (p24) DNA vaccine, was assessed in this study. In vitro T-cell depletion experiments indicated that both CD4(+) and CD8(+) T-cell subsets produced IFN-gamma upon restimulation with a T. gondii lysate. In addition, the GRA1 DNA vaccine elicited CD8(+) T cells that were shown to have cytolytic activity against parasite-infected target cells and a GRA1-transfected cell line. C3H mice immunized with the GRA1 DNA vaccine showed 75 to 100% protection, while 0 to 25% of the mice immunized with the empty control vector survived challenge with T. gondii cysts. In vivo T-cell depletion experiments indicated that CD8(+) T cells were essential for the survival of GRA1-vaccinated C3H mice during the acute phase of T. gondii infection, while depletion of CD4(+) T cells led to an increase in brain cyst burden during the chronic phase of infection.     

Host resistance and immune deviation in pigeon cytochrome c T-cell receptor transgenic mice infected with Toxoplasma gondii

Resistance to Toxoplasma gondii has been shown to be mediated by gamma interferon (IFN-gamma) produced by NK, CD4(+), and CD8(+) T cells. While studies of SCID mice have implicated NK cells as the source of the cytokine in acute infection, several lines of evidence suggest that IFN-gamma production by CD4(+) T lymphocytes also plays an important role in controlling early parasite growth. To evaluate whether this function is due to nonspecific as opposed to T-cell receptor (TCR)-dependent stimulation by the parasite, we have examined the resistance to T. gondii infection of pigeon cytochrome c transgenic (PCC-Tg) Rag-2(-/-) mice in which all CD4(+) T lymphocytes are unreactive with the protozoan. When inoculated with the ME49 strain, PCC-Tg animals exhibited only temporary control of acute infection and succumbed by day 17. Intracellular cytokine staining by flow cytometry revealed that, in contrast to infected nontransgenic controls, infected PCC-Tg animals failed to develop IFN-gamma-producing CD4(+) T cells. Moreover, the CD4(+) lymphocytes from these mice showed no evidence of activation as judged by lack of upregulated expression of CD44 or CD69. Nevertheless, when acutely infected transgenic mice were primed by PCC injection, the lymphokine responses measured after in vitro antigen restimulation displayed a strong Th1 bias which was shown to be dependent on endogenous interleukin 12 (IL-12). The above findings argue that, while T. gondii-induced IL-12 cannot trigger IFN-gamma production by CD4(+) T cells in the absence of TCR ligation, the pathogen is able to nonspecifically promote Th1 responses against nonparasite antigens, an effect that may explain the immunostimulatory properties of T. gondii infection.     

Deficient Humoral Responses Underlie Susceptibility to Toxoplasma gondii in CD4-Deficient Mice

Resistance to infection with Toxoplasma gondii was studied in mice lacking CD4 expression. Such mice developed more brain cysts and survived for a shorter time than did wild-type controls after peroral infection with ME49 cysts. After immunization with the ts-4 strain of T. gondii, CD4-deficient mice exhibited impaired resistance to a challenge infection with virulent RH tachyzoites. Thus, deficient CD4 expression increases the susceptibility of mice to a primary peroral T. gondii infection with cysts and impairs their ability to be successfully vaccinated. CD8(+) T cells from blood or spleens of Toxoplasma-infected, CD4-deficient mice expressed markers of activation at frequencies similar to those of infected wild-type mice. Production of IFN-gamma in vitro was moderately depressed, and levels of Toxoplasma-specific immunoglobulin G2a in serum were substantially lower than in wild-type mice. Administration of Toxoplasma-immune serum to ts-4-vaccinated CD4-deficient mice significantly improved their resistance to RH challenge. Also, the survival of CD4-deficient mice chronically infected with ME49 was significantly prolonged by administration of immune serum. These results demonstrate that in addition to CD8(+) T cells and IFN-gamma, which are known to be critical for resistance, CD4(+) cells also contribute significantly to protection against chronic T. gondii infections and against challenge infections with highly virulent tachyzoites in immunized mice via their role as helper cells for production of isotype-switched antibodies.     

Importance of CD8(+)Vbeta8(+) T cells in IFN-gamma-mediated prevention of toxoplasmic encephalitis in genetically resistant BALB/c mice.

In our attempt to identify a major T cell population(s) that recognizes protective Toxoplasma gondii antigens and produces interferon-gamma (IFN-gamma) for prevention of toxoplasmic encephalitis (TE), we found T cell receptor Vbeta8(+) cells to be the most frequent IFN-gamma-producing population infiltrated into the brain of T. gondii-infected BALB/c mice genetically resistant to the disease. To examine the role of IFN-gamma production by this T cell population for resistance, we transferred Vbeta8(+) immune T cells purified from spleens of infected BALB/c and IFN-gamma(/) mice into infected, sulfadiazine-treated, athymic nude mice. After discontinuation of sulfadiazine treatment, control nude mice that had not received any T cells and animals that had received Vbeta8(+) T cells from IFN-gamma(/) mice all died because of reactivation of infection (TE). In contrast, animals that had received the cells from BALB/c mice survived. Thus, IFN-gamma production by Vbeta8(+) T cells plays an important role in prevention of TE in these animals. When Vbeta8(+) immune T cells were divided into CD4(+) and CD8(+) subsets, a potent protective activity was observed only in the CD8(+) subset, whereas a combination of both subsets provided greater protection than did the CD8(+)Vbeta8(+) population alone. These results indicate that the CD8(+) subset of Vbeta8(+) T cells is a major afferent limb of IFN-gamma-mediated resistance of BALB/c mice against TE, although the CD4(+) subset of the T cell population works additively or synergistically with the CD8(+)Vbeta8(+) population.     

MEK kinase 1 is a negative regulator of virus-specific CD8(+) T cells

MEK kinase 1 (MEKK1) is a potent JNK-activating kinase, a regulator of T helper cell differentiation, cytokine production and proliferation in vitro. Using mice deficient for MEKK1 activity (Mekk1(DeltaKD)) exclusively in their hematopoietic system, we show that MEKK1 has a negative regulatory role in the generation of a virus-specific immune response. Mekk1(DeltaKD) mice challenged with vesicular stomatitis virus (VSV) showed a fourfold increase in splenic CD8(+) T cell numbers. In contrast, the number of splenic T cells in infected WT mice was only marginally increased. The CD8(+) T cell expansion in Mekk1(DeltaKD) mice following VSV infection is virus-specific and the frequency of virus-specific T cells is significantly higher (more than threefold) in Mekk1(DeltaKD) as compared to WT animals. Moreover, the hyper-expansion of T cells seen in Mekk1(DeltaKD) mice after VSV infection is a result of increased proliferation, since a significantly higher percentage of virus-specific Mekk1(DeltaKD) CD8(+) T cells incorporated BrdU as compared to virus-specific WT CD8(+) T cells. In contrast, similar levels of apoptosis were detected in Mekk1(DeltaKD) and WT T cells following VSV infection. These results strongly suggest that MEKK1 plays a negative regulatory role in the expansion of virus-specific CD8(+) T cells in vivo.     

Intestinal intraepithelial lymphocytes prevent pathogen-driven inflammation and regulate the Smad/T-bet pathway of lamina propria CD4+ T cells

Intraepithelial lymphocytes (IEL) play a key role in gut homeostasis and are critical effector cells preventing the inflammatory intestinal lesions induced in mice following oral infection with Toxoplasma gondii. In this intestinal inflammatory model, CD4(+) T lymphocytes from the lamina propria (LP) synergize with the infected enterocytes to secrete pro-inflammatory chemokines and cytokines. In this study, we assessed the mechanisms accounting for the ability of IEL to modulate the inflammatory activity of these cells. Adoptive transfer of IEL purified from wild-type mice, or CD154-,CD95L- or IL-10-deficient mice infected with T. gondii completely impairs the development of the lethal ileitis in recipient mice orally infected with T. gondii.Compared with unprimed IEL isolated from naive mice, the CD8 alpha beta TCR alpha beta subset of primed IEL, isolated from T. gondii-infected mice, secretes increased amount of TGF-beta. IEL interact with the LP CD4(+) T lymphocytes, down-regulate their production of inflammatory cytokines such as IFN-gamma and reduce their proliferative activity. These effects are linked to the secretion of TGF-beta and are correlated with a shift in the balance between Smad7/T-bet down-regulation and Smad2/Smad3 up-regulation in LP CD4(+) T lymphocytes.