Effector CD4+ T-cell involvement in clearance of infectious herpes simplex virus type 1 from sensory ganglia and spinal cords

In primary infection, CD8⁺ T cells are important for clearance of infectious herpes simplex virus (HSV) from sensory ganglia. In this study, evidence of CD4⁺ T-cell-mediated clearance of infectious HSV type 1 (HSV-1) from neural tissues was also detected. In immunocompetent mice, HSV-specific CD4⁺ T cells were present in sensory ganglia and spinal cords coincident with HSV-1 clearance from these sites and remained detectable at least 8 months postinfection. Neural CD4⁺ T cells isolated at the peak of neural infection secreted gamma interferon, tumor necrosis factor alpha, interleukin-2 (IL-2), or IL-4 after stimulation with HSV antigen. HSV-1 titers in neural tissues were greatly reduced over time in CD8⁺ T-cell-deficient and CD8⁺ T-cell-depleted mice, suggesting that CD4⁺ T cells could mediate clearance of HSV-1 from neural tissue. To examine possible mechanisms by which CD4⁺ T cells resolved neural infection, CD8⁺ T cells were depleted from perforin-deficient or FasL-defective mice. Clearance of infectious virus from neural tissues was not significantly different in perforin-deficient or FasL-defective mice compared to wild-type mice. Further, in spinal cords and brains after vaginal HSV-1 challenge of chimeric mice expressing both perforin and Fas or neither perforin nor Fas, virus titers were significantly lower than in control mice. Thus, perforin and Fas were not required for clearance of infectious virus from neural tissues. These results suggest that HSV-specific CD4⁺ T cells are one component of a long-term immune cell presence in neural tissues following genital HSV-1 infection and play a role in clearance of infectious HSV-1 at neural sites, possibly via a nonlytic mechanism.     

Evidence for an Underlying CD4 Helper and CD8 T-Cell Defect in B-Cell-Deficient Mice: Failure To Clear Persistent Virus Infection after Adoptive Immunotherapy with Virus-Specific Memory Cells from μMT/μMT Mice

Adoptive transfer of virus-specific memory lymphocytes can be used to identify factors and mechanisms involved in the clearance of persistent virus infections. To analyze the role of B cells in clearing persistent infection with lymphocytic choriomeningitis virus (LCMV), we used B-cell-deficient μMT/μMT (B−/−) mice. B−/− mice controlled an acute LCMV infection with the same kinetics and efficiency as B-cell-competent (B+/+) mice via virus-specific, major histocompatibility complex (MHC) class I-restricted CD8⁺ cytotoxic T lymphocytes (CTL). CTL from B−/− and B+/+ mice were equivalent in affinity to known LCMV CTL epitopes and had similar CTL precursor frequencies (pCTL). Adoptive transfer of memory cells from B+/+ mice led to virus clearance from persistently infected B+/+ recipients even after in vitro depletion of B cells, indicating that B cells or immunoglobulins are not required in the transfer population. In contrast, transfer of memory splenocytes from B−/− mice failed to clear virus. Control of virus was restored neither by transferring higher numbers of pCTL nor by supplementing B−/− memory splenocytes with LCMV-immune B cells or immune sera. Instead, B−/− mice were found to have a profound CD4 helper defect. Furthermore, compared to cultured splenocytes from B+/+ mice, those from B−/− mice secreted less gamma interferon (IFN-γ) and interleukin 2, with differences most pronounced for CD8 T cells. While emphasizing the importance of CD4 T-cell help and IFN-γ in the control of persistent infections, the CD4 T-helper and CD8 T-cell defects in B−/− mice suggest that B cells contribute to the induction of competent T effector cells.Cytotoxic T lymphocytes (CTL) have in general been associated with the resolution of both acute and chronic viral infections. As first shown by studies of lymphocytic choriomeningitis virus (LCMV) in mice, its natural host, a critical component of immune responses to virus infection is the induction of virus-specific major histocompatibility complex (MHC) class I-restricted CTL (reviewed in reference 14). Evidence that these cells can curtail acute viral infections and clear virus and viral genetic material from sera, peripheral blood leukocytes, and infected tissues came from adoptive transfer of LCMV memory CTL into mice persistently infected with LCMV (1, 25, 33, 47, 53).Studies with humans have correlated the presence of CTL with the control of acute infection and clearance of virus and the absence of CTL activity with persistent viral infections. Hence, humans with genetic deficiencies in the humoral compartment of the immune system but with an intact T-cell compartment overcome most viral infections and display immunological memory when challenged or reinfected with the same virus. For example, agammaglobulinemic children recover from acute measles infection as well as do fully immunocompetent individuals and resist reinfection (23). In contrast, individuals with genetic or acquired defects in the T-cell compartment generally cannot control viral infections. Similarly, activity of CTL specific for hepatitis B virus (HBV) is associated with control of acute HBV infection; in the absence of CTL, HBV persists (39). Additionally, anti-HIV CTL dramatically decrease the load of human immunodeficiency virus (HIV) in infected patients, whereas loss of CTL function is accompanied by regress from a relatively healthy clinical stage to AIDS or rapid development of disease after HIV infection (9, 32). Finally, diminished or missing CTL responses to human cytomegalovirus (HCMV) facilitate HCMV disease in individuals undergoing bone marrow transplantation (40). Adoptive transfer of HCMV MHC-restricted CTL into such patients prevented CMV viremia or CMV disease (55). Thus, understanding the requirements for initiation and maintenance of CTL activity is essential.Earlier, we and others documented the requirement for CD4 T-cell help (5, 16, 29, 48) and gamma interferon (IFN-γ) (48) in maintaining sufficient CTL activity in vivo and resolution of a chronic LCMV infection. Here, we evaluate the role of B lymphocytes in this process. Under the appropriate signals, B lymphocytes can differentiate into plasma cells to function as antibody-secreting cells. Trapping of antibody-antigen complexes as well as processing of antigen and peptide presentation within the MHC complex allows B cells to also function as antigen-presenting cells (APC) to T cells (22). Furthermore, B cells release numerous growth factors and cytokines that regulate immune responses (44).To ascertain the role of B lymphocytes in the clearance of both acute and persistent LCMV infections, we used μMT/μMT B-cell-deficient (B−/−) mice which lack functional B cells and antibody. Earlier studies showed that CD8 T cells from these mice were capable of controlling an acute LCMV infection and that there was no defect in generating CTL precursors (3). Our results confirm and expand these findings. We demonstrate that while adoptive transfer of memory cells from B+/+ mice easily clears infectious virus and viral material in an MHC-matched persistently infected recipient, transfer of similar cells from B−/− mice does not. However, failure to terminate the persistent infection does not result from absence of B cells in the transfer population. Apparently, B−/− mice have a fundamental defect in CD4 helper function as well as a quantitative deficiency in IFN-γ and interleukin 2 (IL-2) preferentially produced by CD8 T cells after LCMV infection. These results emphasize the essential role for CD4 T-lymphocyte help and IFN-γ in achieving CTL activity necessary for clearing a persistent LCMV infection and point to an expanded role for B cells in the development and maintenance of CD4 and CD8 T-cell functions.     

Role of PKR and Type I IFNs in Viral Control during Primary and Secondary Infection

Type I interferons (IFNs) are known to mediate viral control, and also promote survival and expansion of virus-specific CD8⁺ T cells. However, it is unclear whether signaling cascades involved in eliciting these diverse cellular effects are also distinct. One of the best-characterized anti-viral signaling mechanisms of Type I IFNs is mediated by the IFN-inducible dsRNA activated protein kinase, PKR. Here, we have investigated the role of PKR and Type I IFNs in regulating viral clearance and CD8⁺ T cell response during primary and secondary viral infections. Our studies demonstrate differential requirement for PKR, in viral control versus elicitation of CD8⁺ T cell responses during primary infection of mice with lymphocytic choriomeningitis virus (LCMV). PKR-deficient mice mounted potent CD8⁺ T cell responses, but failed to effectively control LCMV. The compromised LCMV control in the absence of PKR was multifactorial, and linked to less effective CD8⁺ T cell-mediated viral suppression, enhanced viral replication in cells, and lower steady state expression levels of IFN-responsive genes. Moreover, we show that despite normal expansion of memory CD8⁺ T cells and differentiation into effectors during a secondary response, effective clearance of LCMV but not vaccinia virus required PKR activity in infected cells. In the absence of Type I IFN signaling, secondary effector CD8⁺ T cells were ineffective in controlling both LCMV and vaccinia virus replication in vivo. These findings provide insight into cellular pathways of Type I IFN actions, and highlight the under-appreciated importance of innate immune mechanisms of viral control during secondary infections, despite the accelerated responses of memory CD8⁺ T cells. Additionally, the results presented here have furthered our understanding of the immune correlates of anti-viral protective immunity, which have implications in the rational design of vaccines.     

Failure of Effector Function of Human CD8+ T Cells in NOD/SCID/JAK3?/? Immunodeficient Mice Transplanted with Human CD34+ Hematopoietic Stem Cells

Humanized mice, which are generated by transplanting human CD34⁺ hematopoietic stem cells into immunodeficient mice, are expected to be useful for the research on human immune responses. It is reported that antigen-specific T cell responses occur in immunodeficient mice transplanted with both human fetal thymus/liver tissues and CD34⁺ fetal cells, but it remains unclear whether antigen-specific T cell responses occur in those transplanted with only human CD34⁺ hematopoietic stem cells (HSCs). Here we investigated the differentiation and function of human CD8⁺ T cells reconstituted in NOD/SCID/Jak3^−/− mice transplanted with human CD34⁺ HSCs (hNOK mice). Multicolor flow cytometric analysis demonstrated that human CD8⁺ T cells generated from the CD34⁺ HSCs comprised only 3 subtypes, i.e., CD27^highCD28⁺CD45RA⁺CCR7⁺, CD27⁺CD28⁺CD45RA⁻CCR7⁺, and CD27⁺CD28⁺CD45RA⁻CCR7⁻ and had 3 phenotypes for 3 lytic molecules, i.e., perforin(Per)⁻granzymeA(GraA)⁻granzymeB(GraB)⁻, Per⁻GraA⁺GraB⁻, and Per^lowGraA⁺GraB⁺. These CD8⁺ T cells failed to produce IFN-γ and to proliferate after stimulation with alloantigens. These results indicate that the antigen-specific T cell response cannot be elicited in mice transplanted with only human CD34⁺ HSCs, because the T cells fail to develop normally in such mice.     

CD8+ T-Cells Expressing Interferon Gamma or Perforin Play Antagonistic Roles in Heart Injury in Experimental Trypanosoma Cruzi-Elicited Cardiomyopathy

In Chagas disease, CD8⁺ T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8⁺ T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8⁺ T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8⁺ T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8⁺ T-cells segregated into IFNγ⁺ perforin (Pfn)^neg or IFNγ^negPfn⁺ cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8⁺Pfn⁺ cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8 ^−/− recipients showed that the CD8⁺ cells from infected ifnγ^−/− pfn ^+/+ donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8⁺ cells from ifnγ ^+/+ pfn ^−/− donors. Moreover, the reconstitution of naïve cd8 ^−/− mice with CD8⁺ cells from naïve ifnγ ^+/+ pfn ^−/− donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ⁺ cells accumulation, whereas reconstitution with CD8⁺ cells from naïve ifnγ ^−/− pfn ^+/+ donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn⁺ cells in the cardiac tissue. Our data support a possible antagonist effect of CD8⁺Pfn⁺ and CD8⁺IFNγ⁺ cells during CCC. CD8⁺IFNγ⁺ cells may exert a beneficial role, whereas CD8⁺Pfn⁺ may play a detrimental role in T. cruzi-elicited heart injury.     

High CD8+ T Cell Activation Marks a Less Differentiated HIV-1 Specific CD8+ T Cell Response that Is Not Altered by Suppression of Viral Replication

Background

The relationship of elevated T cell activation to altered T cell differentiation profiles, each defining features of HIV-1 infection, has not been extensively explored. We hypothesized that anti-retroviral suppression of T cell activation levels would lead to alterations in the T cell differentiation of total and HIV-1 specific CD8+ T cell responses among recently HIV-1 infected adults.

Methodology/Principal Findings

We performed a longitudinal study simultaneously measuring T cell activation and maturation markers on both total and antigen-specific T cells in recently infected adults: prior to treatment; after the initiation of HAART; and after treatment was halted. Prior to treatment, HIV-1 Gag–specific CD8+ T cells were predominantly of a highly activated, intermediate memory (CD27+CD28−) phenotype, while CMV pp65-specific CD8+ T cells showed a late memory (CD27−CD28−), low activation phenotype. Participants with the highest fraction of late memory (CD27−CD28−) HIV-1-specific CD8+ T cells had higher CD4+ T cell counts (rho = +0.74, p = 0.004). In turn, those with the highest fraction of intermediate memory (CD27+ CD28−) HIV-1 specific CD8+ T cells had high total CD8+ T cell activation (rho = +0.68, p = 0.01), indicating poorer long-term clinical outcomes. The HIV-1 specific T cell differentiation profile was not readily altered by suppression of T cell activation following HAART treatment.

Conclusions/Significance

A more differentiated, less activated HIV-1 specific CD8+ T cell response may be clinically protective. Anti-retroviral treatment initiated two to four months after infection lowered T cell activation but had no effect on the differentiation profile of the HIV-1-specific response. Intervention during the first month of acute infection may be required to shift the differentiation phenotype of HIV-1 specific responses to a more clinically favorable profile.     

CD4+ T-Cell Help Is Required for Effective CD8+ T Cell-Mediated Resolution of Acute Viral Hepatitis in Mice

Cytotoxic CD8+ T cells are essential for the control of viral liver infections, such as those caused by HBV or HCV. It is not entirely clear whether CD4+ T-cell help is necessary for establishing anti-viral CD8+ T cell responses that successfully control liver infection. To address the role of CD4+ T cells in acute viral hepatitis, we infected mice with Lymphocytic Choriomeningitis Virus (LCMV) of the strain WE; LCMV-WE causes acute hepatitis in mice and is cleared from the liver by CD8+ T cells within about two weeks. The role of CD4+ T-cell help was studied in CD4+ T cell-lymphopenic mice, which were either induced by genetic deficiency of the major histocompatibility (MHC) class II transactivator (CIITA) in CIITA−/− mice, or by antibody-mediated CD4+ cell depletion. We found that CD4+ T cell-lymphopenic mice developed protracted viral liver infection, which seemed to be a consequence of reduced virus-specific CD8+ T-cell numbers in the liver. Moreover, the anti-viral effector functions of the liver-infiltrating CD8+ T cells in response to stimulation with LCMV peptide, notably the IFN-γ production and degranulation capacity were impaired in CIITA−/− mice. The impaired CD8+ T-cell function in CIITA−/− mice was not associated with increased expression of the exhaustion marker PD-1. Our findings indicate that CD4+ T-cell help is required to establish an effective antiviral CD8+ T-cell response in the liver during acute viral infection. Insufficient virus control and protracted viral hepatitis may be consequences of impaired initial CD4+ T-cell help.     

Theiler’s Virus Infection of Perforin-Deficient Mice

Theiler’s virus, a murine picornavirus, infects the central nervous systems of C57BL/6 mice and is cleared after approximately 10 days by a process which requires CD8⁺ cytotoxic T cells. We used perforin-deficient C57BL/6 mice to test the role of this protein in viral clearance. Perforin-deficient mice died from viral encephalomyelitis between days 12 and 18 postinoculation. They had high levels of viral RNA in their central nervous systems until the time of death. In contrast, viral RNA had disappeared by day 11 postinoculation in wild-type C57BL/6 mice. Cytotoxic T cells can kill infected cells by two main mechanisms: the secretion of the pore-forming protein perforin or the interaction of the Fas ligand with the apoptosis-inducing Fas molecule on the target cell. Our results demonstrate that clearance of Theiler’s virus from the central nervous system in C57BL/6 mice is perforin dependent.     

Lymphocytic choriomeningitis virus infection yields overlapping CD4+ and CD8+ T-cell responses

Activation of CD4⁺ T cells helps establish and sustain other immune responses. We have previously shown that responses against a broad set of nine CD4⁺ T-cell epitopes were present in the setting of lymphocytic choriomeningitis virus (LCMV) Armstrong infection in the context of H-2^d. This is quite disparate to the H-2^b setting, where only two epitopes have been identified. We were interested in determining whether a broad set of responses was unique to H-2^d or whether additional CD4⁺ T-cell epitopes could be identified in the setting of the H-2^b background. To pursue this question, we infected C57BL/6 mice with LCMV Armstrong and determined the repertoire of CD4⁺ T-cell responses using overlapping 15-mer peptides corresponding to the LCMV Armstrong sequence. We confirmed positive responses by intracellular cytokine staining and major histocompatibility complex (MHC)-peptide binding assays. A broad repertoire of responses was identified, consisting of six epitopes. These epitopes originate from the nucleoprotein (NP) and glycoprotein (GP). Out of the six newly identified CD4⁺ epitopes, four of them also stimulate CD8⁺ T cells in a statistically significant manner. Furthermore, we assessed these CD4⁺ T-cell responses during the memory phase of LCMV Armstrong infection and after infection with a chronic strain of LCMV and determined that a subset of the responses could be detected under these different conditions. This is the first example of a broad repertoire of shared epitopes between CD4⁺ and CD8⁺ T cells in the context of viral infection. These findings demonstrate that immunodominance is a complex phenomenon in the context of helper responses.     

Targeted Deletion of FGL2 Leads to Increased Early Viral Replication and Enhanced Adaptive Immunity in a Murine Model of Acute Viral Hepatitis Caused by LCMV WE

Mounting effective innate and adaptive immune responses are critical for viral clearance and the generation of long lasting immunity. It is known that production of inhibitory factors may result in the inability of the host to clear viruses, resulting in chronic viral persistence. Fibrinogen-like protein 2 (FGL2) has been identified as a novel effector molecule of CD4⁺CD25⁺ Foxp3⁺ regulatory T (Treg) cells that inhibits immune activity by binding to FCγRIIB expressed primarily on antigen presenting cells (APC). In this study, we show that infection of mice with Lymphocytic Choriomeningitis Virus WE (LCMV WE) leads to increased plasma levels of FGL2, which were detected as early as 2 days post-infection (pi) and persisted until day 50 pi. Mice deficient in FGL2 (fgl2^−/−) had increased viral titers of LCMV WE in the liver early p.i but cleared the virus by day 12 similar to wild type mice. Dendritic cells (DC) isolated from the spleens of LCMV WE infected fgl2^−/− had increased expression of the DC maturation markers CD80 and MHC Class II compared to wild type (fgl2^+/+). Frequencies of CD8⁺ and CD4⁺ T cells producing IFNγ in response to ex vivo peptide re-stimulation isolated from the spleen and lymph nodes were also increased in LCMV WE infected fgl2 ^−/− mice. Increased frequencies of CD8⁺ T cells specific for LCMV tetramers GP₃₃ and NP₃₉₆ were detected within the liver of fgl2^−/− mice. Plasma from fgl2^−/− mice contained higher titers of total and neutralizing anti-LCMV antibody. Enhanced anti-viral immunity in fgl2^−/− mice was associated with increased levels of serum alanine transaminase (ALT), hepatic necrosis and inflammation following LCMV WE infection. These data demonstrate that targeting FGL2 leads to early increased viral replication but enhanced anti-viral adaptive T & B cell responses. Targeting FGL2 may enhance the efficacy of current anti-viral therapies for hepatotropic viruses.     

Lymphoid-related CD11c+ CD8alpha+ dendritic cells are involved in enhancing herpes simplex virus type 1 latency

The mechanism(s) by which herpes simplex virus type 1 (HSV-1) latency is established in neurons is not known. In this study, we examined the effect of dendritic cells (DCs) on the level of HSV-1 latency in trigeminal ganglia (TGs) of ocularly infected BALB/c and C57BL/6 mice. We found that immunization of wild-type mice with FMS-like tyrosine kinase 3 ligand (Flt3L) DNA, which increases the number of DCs, increased the amount of latency in infected mice. Conversely, depletion of DCs was associated with reduced latency. Latency was also significantly reduced in Flt3L^−/− and CD8^−/− mice. Interestingly, immunization of Flt3L^−/− but not CD8^−/− mice with Flt3L DNA increased latency. Transfer experiments using DCs expanded ex vivo with Flt3L or granulocyte-macrophage colony-stimulating factor suggested that increased latency was associated with the presence of lymphoid-related (CD11c⁺ CD8α⁺) DCs, while reduced latency was associated with myeloid-related (CD11c⁺ CD8α⁻) DCs. Modulation of DC numbers by Flt3L DNA immunization or depletion did not alter acute virus replication in the eye or TG or eye disease in ocularly infected mice. Our results suggest that CD11c⁺ CD8α⁺ DCs directly or indirectly increase the amount of HSV-1 latency in mouse TGs.     

Memory CD4+ T-cell-mediated protection from lethal coronavirus encephalomyelitis

The antiviral role of CD4⁺ T cells in virus-induced pathologies of the central nervous system (CNS) has not been explored extensively. Control of neurotropic mouse hepatitis virus (JHMV) requires the collaboration of CD4⁺ and CD8⁺ T cells, with CD8⁺ T cells providing direct perforin and gamma interferon (IFN-γ)-mediated antiviral activity. To distinguish bystander from direct antiviral contributions of CD4⁺ T cells in virus clearance and pathology, memory CD4⁺ T cells purified from wild type (wt), perforin-deficient (PKO), and IFN-γ-deficient (GKO) immune donors were transferred to immunodeficient SCID mice prior to CNS challenge. All three donor CD4⁺ T-cell populations controlled CNS virus replication at 8 days postinfection, indicating IFN-γ- and perforin-independent antiviral function. Recipients of GKO CD4⁺ T cells succumbed more rapidly to fatal disease than untreated control infected mice. In contrast, wt and PKO donor CD4⁺ T cells cleared infectious virus to undetectable levels and protected from fatal disease. Recipients of all CD4⁺ T-cell populations exhibited demyelination. However, it was more severe in wt CD4⁺ T-cell recipients. These data support a role of CD4⁺ T cells in virus clearance and demyelination. Despite substantial IFN-γ-independent antiviral activity, IFN-γ was crucial in providing protection from death. IFN-γ reduced neutrophil accumulation and directed macrophages to white matter but did not ameliorate myelin loss.     

In Situ Patrolling of Regulatory T Cells Is Essential for Protecting Autoimmune Exocrinopathy

Background

Migration of T cells, including regulatory T (Treg) cells, into the secondary lymph organs is critically controlled by chemokines and adhesion molecules. However, the mechanisms by which Treg cells regulate organ-specific autoimmunity via these molecules remain unclear. Although we previously reported autoimmune exocrinopathy resembling Sjögren''s syndrome (SS) in the lacrimal and salivary glands from C-C chemokine receptor 7 (CCR7)-deficient mice, it is still unclear whether CCR7 signaling might specifically affect the dynamics and functions of Treg cells in vivo. We therefore investigated the cellular mechanism for suppressive function of Treg cells via CCR7 in autoimmunity using mouse models and human samples.

Methods and Findings

Patrolling Treg cells were detected in the exocrine organs such as lacrimal and salivary glands from normal mice that tend to be targets for autoimmunity while the Treg cells were almost undetectable in the exocrine glands of CCR7 ^−/− mice. In addition, we found the significantly increased retention of CD4⁺CD25⁺Foxp3⁺ Treg cells in the lymph nodes of CCR7 ^−/− mice with aging. Although Treg cell egress requires sphingosine 1-phosphate (S1P), chemotactic function to S1P of CCR7−/− Treg cells was impaired compared with that of WT Treg cells. Moreover, the in vivo suppression activity was remarkably diminished in CCR7 ^−/− Treg cells in the model where Treg cells were co-transferred with CCR7 ^−/− CD25^-CD4⁺ T cells into Rag2 ^−/− mice. Finally, confocal analysis showed that CCR7⁺Treg cells were detectable in normal salivary glands while the number of CCR7⁺Treg cells was extremely decreased in the tissues from patients with Sjögren''s syndrome.

Conclusions

These results indicate that CCR7 essentially governs the patrolling functions of Treg cells by controlling the traffic to the exocrine organs for protecting autoimmunity. Characterization of this cellular mechanism could have clinical implications by supporting development of new diagnosis or treatments for the organ-specific autoimmune diseases such as Sjögren''s syndrome and clarifying how the local immune system regulates autoimmunity.     

Impaired Innate Immunity in Tlr4 ?/? Mice but Preserved CD8+ T Cell Responses against Trypanosoma cruzi in Tlr4-, Tlr2-, Tlr9- or Myd88-Deficient Mice

The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8⁺ T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-γ secreting CD8⁺ T cells specific for H-2K^b-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2^−/−, Tlr4^−/−, Tlr9^{−/ −} or Myd88^−/− mice generated both specific cytotoxic responses and IFN-γ secreting CD8⁺ T cells at levels comparable to WT mice, although the frequency of IFN-γ⁺CD4⁺ cells was diminished in infected Myd88^−/− mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-γ, TNF-α and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4^−/− mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi.     

CD8+ T Cells Cause Disability and Axon Loss in a Mouse Model of Multiple Sclerosis

Background

The objective of this study was to test the hypothesis that CD8⁺ T cells directly mediate motor disability and axon injury in the demyelinated central nervous system. We have previously observed that genetic deletion of the CD8⁺ T cell effector molecule perforin leads to preservation of motor function and preservation of spinal axons in chronically demyelinated mice.

Methodology/Principal Findings

To determine if CD8⁺ T cells are necessary and sufficient to directly injure demyelinated axons, we adoptively transferred purified perforin-competent CD8⁺ spinal cord-infiltrating T cells into profoundly demyelinated but functionally preserved perforin-deficient host mice. Transfer of CD8⁺ spinal cord-infiltrating T cells rapidly and irreversibly impaired motor function, disrupted spinal cord motor conduction, and reduced the number of medium- and large-caliber spinal axons. Likewise, immunodepletion of CD8⁺ T cells from chronically demyelinated wildtype mice preserved motor function and limited axon loss without altering other disease parameters.

Conclusions/Significance

In multiple sclerosis patients, CD8⁺ T cells outnumber CD4⁺ T cells in active lesions and the number of CD8⁺ T cells correlates with the extent of ongoing axon injury and functional disability. Our findings suggest that CD8⁺ T cells may directly injure demyelinated axons and are therefore a viable therapeutic target to protect axons and motor function in patients with multiple sclerosis.     

Apoptotic Regulation of T Cells and Absence of Immune Deficiency in Virus-Infected Gamma Interferon Receptor Knockout Mice

Acute viral infections often induce a transient period of immune deficiency in which the host’s T cells fail to proliferate in response to T-cell mitogens and fail to make an antigen-specific memory recall response. This has been associated with the enhanced sensitivity of these highly activated T cells to undergo apoptosis, or activation-induced cell death (AICD), upon T-cell receptor ligation. Here we show that gamma interferon receptor-deficient (IFN-γ R^−/−) mice mount a T-cell response to lymphocytic choriomeningitis virus (LCMV) infection but fail to undergo the transient immune deficiency. Instead, their T cells were hyperproliferative and relatively, but not completely, resistant to AICD. The immune response returned to homeostasis, but with delayed kinetics, in parallel with delayed clearance of the virus. Wild-type mice receiving high doses of disseminating LCMV Clone 13 are known to undergo clonal exhaustion of their virus-specific cytotoxic T lymphocytes (CTL). To determine whether this process was mediated by AICD associated with IFN-γ or with Fas-Fas ligand interactions, LCMV-specific precursor CTL frequencies were examined in LCMV Clone 13-infected IFN-γ R^−/− or lpr (Fas-deficient) mice. In both instances, viral persistence was established and CTL precursors were greatly eliminated. This finding indicates that clonal exhaustion of CTL does not require IFN-γ or Fas, even though both molecules influence AICD and the transient immune deficiency seen in the LCMV infection.     

CD40 Ligand-Mediated Interactions Are Involved in the Generation of Memory CD8+ Cytotoxic T Lymphocytes (CTL) but Are Not Required for the Maintenance of CTL Memory following Virus Infection

CD8⁺ cytotoxic T lymphocytes (CTL) play a key role in the control of many virus infections, and the need for vaccines to elicit strong CD8⁺ T-cell responses in order to provide optimal protection in such infections is increasingly apparent. However, the mechanisms involved in the induction and maintenance of CD8⁺ CTL memory are currently poorly understood. In this study, we investigated the involvement of CD40 ligand (CD40L)-mediated interactions in these processes by analyzing the memory CTL response of CD40L-deficient mice following infection with lymphocytic choriomeningitis virus (LCMV). The maintenance of memory CD8⁺ CTL precursors (CTLp) at stable frequencies over time was not impaired in CD40L-deficient mice. By contrast, the initial generation of memory CTLp was affected. CD40L-deficient mice produced lower levels of CD8⁺ CTLp during the primary immune response to LCMV than did wild-type controls, despite the fact that the LCMV-specific effector CTL response of CD40L-deficient mice was indistinguishable from that of control animals. The differentiation of naïve CD8⁺ T cells into effector and memory CTL thus involves pathways that can be discriminated from each other by their requirement for CD40L-mediated interactions. Expression of CD40L by CTLp themselves was not an essential step during their expansion and differentiation from naïve CD8⁺ cells into memory CTLp; instead, the reduction in memory CTLp generation in CD40L-deficient mice was likely a consequence of defects in the CD4⁺ T-cell response mounted by these animals. These results thus suggest a previously unappreciated role for CD40L in the generation of CD8⁺ memory CTLp, the probable nature of which is discussed.