Quantitative, not qualitative, differences in CD8(+) T cell responses to Theiler's murine encephalomyelitis virus between resistant C57BL/6 and susceptible SJL/J mice

Theiler's murine encephalomyelitis virus (TMEV) infection of the CNS induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis. However, it is not yet clear what immunological parameters determine the susceptibility of SJL/J mice compared to resistant mice. We have here compared the TMEV-specific CD8(+) T cell responses in highly susceptible SJL/J mice with those of highly resistant C57BL/6 mice. Our results clearly indicate that the levels of initial responses of infiltrating CD8(+) T cells to viral capsid proteins are higher in resistant C57BL/6 mice compared to susceptible SJL/J mice. However, the level of virus-specific CD8(+) T cells was much more rapidly reduced in resistant C57BL/6, resulting in a higher CD8(+) T cell level in SJL/J mice later in viral infection. The activation states, cytokine production, as well as the cytolytic function of the CD8(+) T cells were similar to each other in these mice. These results suggest that an initial induction of a vigorous CD8(+) T cell response to TMEV is critically important for the resistance to virally induced demyelinating disease.     

Pro-inflammatory functions of astrocytes correlate with viral clearance and strain-dependent protection from TMEV-induced demyelinating disease

Intracerebral infection of susceptible strains of mice, e.g. SJL/J, with Theiler's murine encephalomyelitis virus (TMEV) leads to a persistent CNS infection accompanied by development of a chronic-progressive inflammatory CNS autoimmune demyelinating disease which is clinically and pathologically similar to human multiple sclerosis. In contrast, resistant strains of mice, e.g. C57BL/6 (B6), effectively clear TMEV from the CNS and do not develop demyelinating disease. Although CD8(+) T cells are crucial for viral clearance in B6 mice, SJL mice also mount potent CD8(+) T cell responses against virus, thus the reason for the viral persistence in the CNS in these mice is unclear. Here, we examined innate anti-viral responses of CNS-resident astrocytes as a potential determinant of viral persistence and disease susceptibility. We demonstrate that B6 astrocytes produce significantly higher levels of cytokines, chemokines and adhesion molecules in response to TMEV infection, or stimulation with IFN-gamma and TNF-alpha or poly I:C than SJL mice. In addition, TMEV more effectively induces MHC I molecules on B6 astrocytes than SJL, corresponding with an increased ability to activate TMEV-specific CD8(+) T cells directly ex vivo. These results suggest that enhanced anti-viral responses of B6 astrocytes contribute to the ability of these mice to clear TMEV from the CNS and therefore to their resistance to the development of autoimmune demyelinating disease.     

Virus expanded regulatory T cells control disease severity in the Theiler's virus mouse model of MS

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) serves as virus-induced model of chronic progressive multiple sclerosis. Infection of susceptible SJL/J mice leads to life-long CNS virus persistence and a progressive autoimmune demyelinating disease mediated by myelin-specific T cells activated via epitope spreading. In contrast, virus is rapidly cleared by a robust CTL response in TMEV-IDD-resistant C57BL/6 mice. We investigated whether differential induction of regulatory T cells (Tregs) controls susceptibility to TMEV-IDD. Infection of disease-susceptible SJL/J, but not B6 mice, leads to rapid activation and expansion of Tregs resulting in an unfavorable CNS ratio of Treg:Teffector cells. In addition, anti-CD25-induced inactivation of Tregs in susceptible SJL/J, but not resistant B6, mice results in significantly decreased clinical disease concomitant with enhanced anti-viral CD4(+), CD8(+) and antibody responses resulting in decreased CNS viral titers. This is the first demonstration that virus-induced Treg activation regulates susceptibility to autoimmune disease differentially in susceptible and resistant strains of mice and provides a new mechanistic explanation for the etiology of infection-induced autoimmunity.     

A critical role for virus-specific CD8 CTLs in protection from Theiler's virus-induced demyelination in disease-susceptible SJL mice

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) is a relevant mouse model of multiple sclerosis. Infection of susceptible SJL/J mice leads to life-long CNS virus persistence and development of a chronic T cell-mediated autoimmune demyelinating disease triggered via epitope spreading to endogenous myelin epitopes. Potent CNS-infiltrating CD8⁺ T cell responses to TMEV epitopes have previously been shown to be induced in both disease-susceptible SJL/J and resistant C57BL/6 mice, in which the virus is rapidly cleared. Specific tolerization of SJL CD8⁺ T cells specific for the immunodominant TMEV VP3_159-166 epitope has no effect on viral load or development of clinical TMEV-IDD, but adoptive transfer of activated CD8⁺ VP3_159-166-specific T cell blasts shortly after TMEV infection to boost the early anti-viral response leads to clearance of CNS virus and protection from subsequent TMEV-IDD. These studies have important implications for vaccine strategies and treatment of chronic infections in humans.     

Identification of capsid epitopes of Theiler's virus recognized by CNS-infiltrating CD4+ T cells from virus-infected C57BL/6 mice

Intracerebral infection of Theiler's murine encephalomyelitis virus (TMEV) induces immune-mediated demyelinating disease in some mouse strains but not in others. We report here for the first time two new predominant capsid epitopes (VP4(21-40) and VP2(201-220)) recognized by CD4+ T cells from virus-infected resistant C57BL/6 mice based on IFNgamma ELISPOT assay utilizing a 20-mer peptide library covering the entire capsid proteins. Further experiments by IFNgamma ELISPOT and flow cytometry for intracellular IFNgamma production using truncated peptides indicated that the epitope regions recognized by CNS-infiltrating CD4+ T cells are VP4(25-38) and VP2(206-220), respectively. No apparent reduction in the T cell response to these viral epitopes is seen in the CNS of IL-12- and ICAM-1-deficient C57BL/6 mice compared to those in control C57BL/6 mice, suggesting that T cell response to TMEV in the CNS is largely insensitive to the absence of these proinflammatory cytokine and adhesion molecules. Therefore, these newly defined CD4+ T cell epitopes are likely to provide an important tool to investigate the role of CD4+ T cell responses in H-2b-bearing congenic strains.     

Differential interaction of dendritic cells with Rickettsia conorii: impact on host susceptibility to murine spotted fever rickettsiosis

Spotted fever group rickettsioses are emerging and reemerging infectious diseases, some of which are life-threatening. In order to understand how dendritic cells (DCs) contribute to the host resistance or susceptibility to rickettsial diseases, we first characterized the in vitro interaction of rickettsiae with bone marrow-derived DCs (BMDCs) from resistant C57BL/6 (B6) and susceptible C3H/HeN (C3H) mice. In contrast to the exclusively cytosolic localization within endothelial cells, rickettsiae efficiently entered and localized in both phagosomes and cytosol of BMDCs from both mouse strains. Rickettsia conorii-infected BMDCs from resistant mice harbored higher bacterial loads compared to C3H mice. R. conorii infection induced maturation of BMDCs from both mouse strains as judged by upregulated expression of classical major histocompatibility complex (MHC) and costimulatory molecules. Compared to C3H counterparts, B6 BMDCs exhibited higher expression levels of MHC class II and higher interleukin-12 (IL-12) p40 production upon rickettsial infection and were more potent in priming na?ve CD4(+) T cells to produce gamma interferon. In vitro DC infection and T-cell priming studies suggested a delayed CD4(+) T-cell activation and suppressed Th1/Th2 cell development in C3H mice. The suppressive CD4(+) T-cell responses seen in C3H mice were associated with a high frequency of Foxp3(+) T regulatory cells promoted by syngeneic R. conorii-infected BMDCs in the presence of IL-2. These data suggest that rickettsiae can target DCs to stimulate a protective type 1 response in resistant hosts but suppressive adaptive immunity in susceptible hosts.     

Depletion of CD8(+) T cells in vivo impairs host defense of mice resistant and susceptible to pulmonary paracoccidioidomycosis

Using a pulmonary model of infection, we demonstrated previously that A/Sn and B10.A mice are, respectively, resistant and susceptible to Paracoccidioides brasiliensis infection. Employing the same experimental model, we examined herein the role of CD8(+) T cells in the course of paracoccidioidomycosis. Treatment with anti-CD8 monoclonal antibodies caused a selective depletion of pulmonary and splenic CD8(+) T cells in both mouse strains. The number of pulmonary CD4(+) T cells and immunoglobulin-positive cells was independent of the number of CD8(+) T cells. In susceptible mice, the loss of CD8(+) T cells by in vivo treatment with anti-CD8 monoclonal antibodies impaired the clearance of yeasts from the lungs and increased the fungal dissemination to the liver and spleen. The same treatment in resistant mice increased fungal dissemination to extrapulmonary tissues but did not alter the pulmonary fungal load. Furthermore, CD8(+) T-cell depletion did not modify delayed-type hypersensitivity reactions of A/Sn mice but increased these reactions in B10.A mice. The production of P. brasiliensis-specific antibodies by resistant and susceptible mice depleted of CD8(+) T cells was similar to that of mice given control antibody. Histopathologically, depletion of CD8(+) T cells did not disorganize the focal granulomatous lesions developed by both mouse strains. These results indicate that CD8(+) T cells are necessary for optimal clearance of the fungus from tissues of mice infected with P. brasiliensis and demonstrate more prominent protective activity by those cells in the immune responses mounted by susceptible animals.     

IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells

CD25(+)CD4(+) regulatory T cells inhibit the activation of autoreactive T cells in vitro and in vivo, and suppress organ-specific autoimmune diseases. The mechanism of CD25(+)CD4(+) T cells in the regulation of experimental autoimmune encephalomyelitis (EAE) is poorly understood. To assess the role of CD25(+)CD4(+) T cells in EAE, SJL mice were immunized with myelin proteolipid protein (PLP)(139-151) to develop EAE and were treated with anti-CD25 mAb. Treatment with anti-CD25 antibody following immunization resulted in a significant enhancement of EAE disease severity and mortality. There was increased inflammation in the central nervous system (CNS) of anti-CD25 mAb-treated mice. Anti-CD25 antibody treatment caused a decrease in the percentage of CD25(+)CD4(+) T cells in blood, peripheral lymph node (LN) and spleen associated with increased production of IFN-gamma and a decrease in IL-10 production by LN cells stimulated with PLP(130-151) in vitro. In addition, transfer of CD25(+)CD4(+) regulatory T cells from naive SJL mice decreased the severity of active EAE. In vitro, anti-CD3-stimulated CD25(+)CD4(+) T cells from naive SJL mice secreted IL-10 and IL-10 soluble receptor (sR) partially reversed the in vitro suppressive activity of CD25(+)CD4(+) T cells. CD25(+)CD4(+) T cells from IL-10-deficient mice were unable to suppress active EAE. These findings demonstrate that CD25(+)CD4(+) T cells suppress pathogenic autoreactive T cells in actively induced EAE and suggest they may play an important natural regulatory function in controlling CNS autoimmune disease through a mechanism that involves IL-10.     

Interferon-γ confers resistance to experimental allergic encephalomyelitis

In experimental allergic encephalomyelitis (EAE), T cells infiltrate the central nervous system (CNS) and induce inflammation. These CD4⁺ T cells secrete interferon (IFN)-γ, levels of which correlate with disease severity, and which is proposed to play a key role in disease induction. Many strains of mice are resistant to EAE. We have studied the effect of deletion of IFN-γ on the ability to induce EAE in resistant BALB/c-backcrossed mice. As expected, only 0–6 % of BALB/c or BALB/c-backcrossed mice developed EAE when immunized with myelin basic protein in adjuvant. Strikingly, abrogation of IFN-γ expression by targeted disruption of the IFN-γ gene (GKO mice) converted them to a susceptible phenotype. As many as 71 % of these IFN-γ-deficient mice developed EAE, a frequency comparable to that seen with the susceptible SJL/J strain. In addition, EAE was of unusually high severity in mice lacking IFN-γ. Immunological characteristics of disease in IFN-γ-deficient mice were comparable to those seen in susceptible (SJL/J) mice with EAE, including perivascular infiltration in the CNS and order-of-magnitude increases for both CD3 γ chain and TNF-α mRNA levels in the spinal cord. We thus demonstrate that lack of IFN-γ converts an otherwise EAE-resistant mouse strain to become susceptible to disease. Therefore, in BALB/c mice, IFN-γ confers resistance to EAE.     

Involvement of CD4(+) Th1 cells in systemic immunity protective against primary and secondary challenges with Trypanosoma cruzi

In general, gamma interferon (IFN-gamma)-producing CD4(+) Th1 cells are important for the immunological control of intracellular pathogens. We previously demonstrated an association between parasite-specific induction of IFN-gamma responses and resistance to the intracellular protozoan Trypanosoma cruzi. To investigate a potential causal relationship between Th1 responses and T. cruzi resistance, we studied the ability of Th1 cells to protect susceptible BALB/c mice against virulent parasite challenges. We developed immunization protocols capable of inducing polarized Th1 and Th2 responses in vivo. Induction of parasite-specific Th1 responses, but not Th2 responses, protected BALB/c mice against virulent T. cruzi challenges. We generated T. cruzi-specific CD4(+) Th1 and Th2 cell lines from BALB/c mice that were activated by infected macrophages to produce their corresponding cytokine response profiles. Th1 cells, but not Th2 cells, induced nitric oxide production and inhibited intracellular parasite replication in T. cruzi-infected macrophages. Despite the ability to inhibit parasite replication in vitro, Th1 cells alone could not adoptively transfer protection against T. cruzi to SCID mice. In addition, despite the fact that the adoptive transfer of CD4(+) T lymphocytes was shown to be necessary for the development of immunity protective against primary T. cruzi infection in our SCID mouse model, protective secondary effector functions could be transferred to SCID mice from memory-immune BALB/c mice in the absence of CD4(+) T lymphocytes. These results indicate that, although CD4(+) Th1 cells can directly inhibit intracellular parasite replication, a more important role for these cells in T. cruzi systemic immunity may be to provide helper activity for the development of other effector functions protective in vivo.     

Glycyrrhizin improves the resistance of MAIDS mice to opportunistic infection of Candida albicans through the modulation of MAIDS-associated type 2 T cell responses

Compared with normal mice, MAIDS mice (mice infected with LP-BM5 murine leukemia virus) exhibited an increase up to 100 times greater in susceptibility to infection with Candida albicans. The impaired resistance of MAIDS mice to the infection was recovered to levels observed in normal mice by the administration of glycyrrhizin (GR), an active component of licorice roots. MAIDS mice inoculated with CD4(+) T cells from GR-treated mice were also resistant to C. albicans infection. Normal mice inoculated with CD4(+) T helper type 2 cells (Th2 cells) from MAIDS mice were susceptible to C. albicans infection at the same levels shown in MAIDS mice. The susceptibility of normal mice inoculated with type 2 T cells was reversible by (i) administration of GR and (ii) inoculation of CD4(+) T cells from GR-treated mice and injection of a mixture of mAbs targeted against type 2 cytokines (IL-4 and IL-10). Type 2 cytokines were not detected in sera of MAIDS mice inoculated with CD4(+) T cells from GR-treated mice, while they were present in sera of MAIDS mice treated with saline. These results suggest that, by inducing CD4(+) T cells which suppress type 2 cytokine production by MAIDS-associated Th2 cells, GR improves the resistance of MAIDS mice to C. albicans infection.     

Oral administration of live virus protects susceptible mice from developing Theiler's virus-induced demyelinating disease

Intracerebral infection of susceptible mouse strains with Theiler's murine encephalomyelitis virus (TMEV) results in an immune-mediated demyelinating disease similar to human multiple sclerosis. TMEV infection is widely spread via fecal-oral routes among wild mouse populations, yet these infected mice rarely develop clinical disease. Oral vaccination has often been used to protect the host against many different infectious agents, although the underlying protective mechanism of prior oral exposure is still unknown. To understand the mechanisms involved in protection from demyelinating disease following previous oral infection, immune parameters and disease progression of mice perorally infected with TMEV were compared with those of mice immunized intraperitoneally following intracerebral infection. Mice infected perorally, but not intraperitoneally, prior to CNS viral infection showed lower chronic viral persistence in the CNS and reduced TMEV-induced demyelinating disease. However, a prolonged period of post-oral infection was necessary for effective protection. Mice orally pre-exposed to the virus displayed markedly elevated levels of antibody response to TMEV in the serum, although T cell responses to TMEV in the periphery were not significantly different between perorally and intraperitoneally immunized mice. In addition, orally vaccinated mice showed higher levels of early CNS-infiltration of B cells producing anti-TMEV antibody as well as virus-specific CD4(+) and CD8(+) T cells in the CNS compared to intraperitoneally immunized mice. Therefore, the generation of a sufficient level of protective immune responses appears to require a prolonged time period to confer protection from TMEV-induced demyelinating disease.     

Multiple pathways to induction of virus-induced autoimmune demyelination: lessons from Theiler's virus infection

Infection of SJL mice with wild-type BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) leads to CD4(+)T cell-mediated CNS demyelination characterized by the development of anti-myelin epitope autoimmune responses via epitope spreading during the chronic stage of disease. To exmine the feasibility of virus-encoded mimic epitopes to initiate CNS autoimmunity, we recently developed a molecular mimicry model of virus-induced demyelinating disease wherein a non-pathogenic variant strain of TMEV was engineered to encode a 30-mer peptide encompassing the immunodominant myelin proteolipid protein, PLP139-151, epitope. SJL mice infected intracerebrally with TMEV encoding either the native PLP139-151 determinant or various peptide mimics of the epitope develop an early onset demyelinating disease mediated by activated PLP139-151-specific Th1 cells. The autoimmune nature of this early-onset demyelinating disease is shown by the fact that induction of tolerance to the PLP139-151 peptide prevents clinical disease and associated PLP139-151-specific T cell responses without affecting T cell reactivity to virus epitopes. Most significantly, TMEV encoding a molecular mimic peptide derived from the Haemophilus influenzae bacteria, homologous at only six out of thirteen of the core amino acids, led to CNS disease. These studies provide conclusive evidence that virus-induced myelin-specific autoreactive T cells can be induced by molecular mimicry and provide a useful model to study the disease inducing ability of viruses encoding human-disease-related mimicry peptides.     

CCR2 regulates development of Theiler's murine encephalomyelitis virus-induced demyelinating disease

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a murine model for multiple sclerosis, involves recruitment of T cells and macrophages to the CNS after infection. We hypothesized that CCR2, the only known receptor for CCL2, would be required for TMEV-induced demyelinating disease development because of its role in macrophage recruitment. TMEV-infected SJL CCR2 knockout (KO) mice showed decreased long-term clinical disease severity and less demyelination compared with controls. Flow cytometric data indicated that macrophages (CD45(high) CD11b(+) ) in the CNS of TMEV-infected CCR2 KO mice were decreased compared with control mice throughout disease. CD4(+) and CD8(+) T cell percentages in the CNS of TMEV-infected control and CCR2 KO mice were similar over the course of disease. There were no apparent differences between CCR2 KO and control peripheral immune responses. The frequency of interferon-gamma-producing T cells in response to proteolipid protein 139-151 in the CNS was also similar during the autoimmunity stage of TMEV-induced demyelinating disease. These data suggest that CCR2 is important for development of clinical disease by regulating macrophage accumulation after TMEV infection.     

CD4(+) v(alpha)14 NKT cells play a crucial role in an early stage of protective immunity against infection with Leishmania major

The roles of gamma delta T, NK and NKT cells in an early stage of protective immunity against infection with Leishmania major were investigated. Further, the contribution of these innate cells to the expression of 65 kDa heat shock protein (HSP65) in host macrophages was examined, since we found previously that this expression prevents apoptotic death of infected macrophages and is a crucial step in the acquisition of protective immunity against infection with various obligate intracellular protozoa including L. major. C57BL/6 and DBA/2 mice were found to be resistant against the infection on the basis of the parasite burden in their regional lymph nodes, and to strongly express HSP65 in their macrophages, whereas BALB/c mice were susceptible and barely expressed the HSP65. In those resistant mice, CD4(+) NKT cells prominently increased in their regional lymph node and were the main effector cells at least for an early stage of the protective immunity and for the HSP65 expression, whereas this subset did not increase in susceptible BALB/c mice. Further, neither gamma delta T nor NK cells in resistant mice contributed to those protective immune responses. The NKT cell subset bore CD3, CD4, TCR alpha beta, IL-2R beta and NK1.1 but scarcely asialo-GM(1). Moreover, this effector subset was confirmed to be V(alpha)14 NKT cells by using J(alpha)281(-/-) mice.     

Clearance of Theiler's virus infection depends on the ability to generate a CD8+ T cell response against a single immunodominant viral peptide

Theiler's murine encephalomyelitis virus (TMEV) induces a chronic demyelinating disease in the central nervous system of susceptible mice. Resistance to persistent TMEV infection maps to he D locus of the major histocompatibility complex suggesting a prominent role of antiviral CTL in the protective immune response. Introduction of the D(b) gene into the FVB strain confers resistance to this otherwise susceptible mouse line. Infection of the FVB/D(b) mouse with TMEV provides a model where antiviral resistance is determined by a response elicited by a single class I molecule. Resistant mice of the H-2(b) haplotype mount a vigorous H-2D(b)-restricted immunodominant response to the VP2 capsid protein. To investigate the extent of the contribution of the immunodominant T cell population in resistance to TMEV, FVB/D(b) mice were depleted of VP2-specific CD8(+) T cells by peptide treatment prior to virus infection. Peptide-treated mice were not able to clear the virus and developed extensive demyelination. These findings demonstrate that the D(b)-restricted CD8(+) T cells specific for a single viral peptide can confer resistance to TMEV infection. Our ability to manipulate this cellular response provides a model for investigating the mechanisms mediating protection against virus infection by CD8(+) T cells.     

Differential induction of helper T cell subsets during blood-stage Plasmodium chabaudi AS infection in resistant and susceptible mice.

The induction of T helper cell subsets during the course of non-lethal or lethal blood-stage Plasmodium chabaudi AS infection was investigated using inbred strains of mice which differ in the level of resistance to this intraerythrocytic parasite. Resistant C57Bl/6 mice experience a non-lethal course of infection characterized by moderate levels of both parasitaemia and anaemia and resolution of primary acute infection by 4 weeks, while susceptible A/J mice experience lethal infection with fulminant parasitaemia and severe anaemia. T helper subset function was assessed during infection by determining the kinetics of spleen cell production in vitro of the Th1-derived cytokine, interferon-gamma (IFN-gamma), and of the Th2-derived cytokine, IL-5, using sandwich ELISAs. Spleen cells from resistant C57Bl/6 mice were found to produce high levels of IFN-gamma within 1 week of infection in response to both the mitogen concanavalin A (Con A) and malaria antigen. Furthermore, CD4+ T cells were found to be the source of IFN-gamma while both CD4+ and CD8+ T cells were found to produce IL-5. Decreased IFN-gamma production after day 10 was concomitant with significant production of IL-5 between 2 and 3 weeks post infection. In contrast, spleen cells from susceptible A/J mice produced high levels of IL-5 within the first week of infection. In addition, these animals were found to have high serum levels of IL-5. These results, thus, confirm previous observations that resolution of primary blood-stage P. chabaudi infection occurs by sequential activation of Th1 CD4+ T cells followed by activation of the Th2 subset, and in addition, suggest that induction of a strong Th2 response early in infection may lead to a severe and lethal course of malaria.