Herpes simplex virus-induced stromal keratitis: role of T-lymphocyte subsets in immunopathology.

Herpetic stromal keratitis (SK), a frequent cause of visual impairment, is considered to represent an immune-mediated inflammatory response to persistent herpes simplex virus virions or subcomponents within the corneal stroma. The experimental disease in mice involves the essential participation of T lymphocytes, but the role of T-lymphocyte subsets in either mediating or controlling the disease is uncertain. In this report, rat monoclonal antibodies were used to selectively deplete mice in vivo of CD4+ (helper-inducer) and CD8+ (cytotoxic-suppressor) T-cell populations and the effect on herpetic SK was evaluated. As measured by flow cytometry, mice treated with anti-CD4 monoclonal antibody (GK 1.5) were greater than 95% depleted of CD4+ T lymphocytes and mice treated with anti-CD8 monoclonal antibody (2.43) were 90% depleted of CD8+ T lymphocytes. Depleted and nonspecific mouse ascites-treated control mice were infected topically on the corneas with herpes simplex virus type 1, and the induction of various immune parameters during the acute infection was evaluated. CD4+-depleted mice failed to produce either a significant antiviral antibody or delayed-type hypersensitivity response but were capable of producing normal cytotoxic T-lymphocyte responses. In contrast, CD8+-depleted mice produced antiviral antibody and delayed-type hypersensitivity responses comparable with those in control animals, but cytotoxic T-lymphocyte responses were markedly reduced. Clinical observations of the corneas revealed that SK in CD4+-depleted mice was significantly reduced, whereas in CD8+-depleted mice SK developed more rapidly, was more severe, and involved a greater percentage of mice. These observations implicate the CD4+ T-lymphocyte subset as the principal mediators of SK and CD8+ T lymphocytes as possible regulators that control the severity of SK.     

Complement component C3 promotes T-cell priming and lung migration to control acute influenza virus infection

The complement cascade defines an important link between the innate and the specific immune system. Here we show that mice deficient for the third component of complement (C3-/- mice) are highly susceptible to primary infection with influenza virus. C3-/- mice showed delayed viral clearance and increased viral titers in lung, whereas mice deficient for complement receptors CR1 and CR2 (Cr2-/- mice) cleared the infection normally. Priming of T-helper cells and cytotoxic T cells (CTLs) in lung-draining lymph nodes was reduced, and the recruitment into the lung of virus-specific CD4+ and CD8+ effector T cells producing interferon-gamma was severely impaired in C3-/- but not in Cr2-/- mice. Consequently, T-helper cell-dependent IgG responses were reduced in C3-/- mice but remained intact in Cr2-/- mice. These results demonstrate that complement induces specific immunity by promoting T-cell responses.     

Selection of CD8+ T cells with highly focused specificity during viral persistence in the central nervous system

The relationships between T cell populations during primary viral infection and persistence are poorly understood. Mice infected with the neurotropic JHMV strain of mouse hepatitis virus mount potent regional CTL responses that effectively reduce infectious virus; nevertheless, viral RNA persists in the central nervous system (CNS). To evaluate whether persistence influences Ag-specific CD8+ T cells, functional TCR diversity was studied in spleen and CNS-derived CTL populations based on differential recognition of variant peptides for the dominant nucleocapsid epitope. Increased specificity of peripheral CTL from persistently infected mice for the index epitope compared with immunized mice suggested T cell selection during persistence. This was confirmed with CD8+ T cell clones derived from the CNS of either acutely (CTLac) or persistently (CTLper) infected mice. Whereas CTLac clones recognized a broad diversity of amino acid substitutions, CTLper clones exhibited exquisite specificity for the wild-type sequence. Highly focused specificity was CD8 independent but correlated with longer complementarity-determining regions 3 characteristic of CTLper clonotypes despite limited TCR alpha/beta-chain heterogeneity. Direct ex vivo analysis of CNS-derived mononuclear cells by IFN-gamma enzyme-linked immunospot assay confirmed the selection of T cells with narrow Ag specificity during persistence at the population level. These data suggest that broadly reactive CTL during primary infection are capable of controlling potentially emerging mutations. By contrast, the predominance of CD8+ T cells with dramatically focused specificity during persistence at the site of infection and in the periphery supports selective pressure driven by persisting Ag.     

CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection.

In this study, we have examined the relative contributions of CD4+ and CD8+ T cells in controlling an acute or chronic lymphocytic choriomeningitis virus (LCMV) infection. To study acute infection, we used the LCMV Armstrong strain, which is cleared by adult mice in 8 to 10 days, and to analyze chronic infection, we used a panel of lymphocyte-tropic and macrophage-tropic variants of LCMV that persist in adult mice for several months. We show that CD4+ T cells are not necessary for resolving an acute LCMV infection. CD4+ T-cell-depleted mice were capable of generating an LCMV-specific CD8+ cytotoxic T-lymphocyte (CTL) response and eliminated virus with kinetics similar to those for control mice. The CD8+ CTL response was critical for resolving this infection, since beta 2-microglobulin knockout (CD8-deficient) mice were unable to control the LCMV Armstrong infection and became persistently infected. In striking contrast to the acute infection, even a transient depletion of CD4+ T cells profoundly affected the outcome of infection with the macrophage- and lymphocyte-tropic LCMV variants. Adult mice given a single injection of anti-CD4 monoclonal antibody (GK1.5) at the time of virus challenge became lifelong carriers with high levels of virus in most tissues. Unmanipulated adult mice infected with the different LCMV variants contained virus for prolonged periods (> 3 months) but eventually eliminated infection from most tissues, and all of these mice had LCMV-specific CD8+ CTL responses. Although the level of CTL activity was quite low, it was consistently present in all of the chronically infected mice that eventually resolved the infection. These results clearly show that even in the presence of an overwhelming viral infection of the immune system, CD8+ CTL can remain active for long periods and eventually resolve and/or keep the virus infection in check. In contrast, LCMV-specific CTL responses were completely lost in chronically infected CD4-depleted mice. Taken together, these results show that CD4+ T cells are dispensable for short-term acute infection in which CD8+ CTL activity does not need to be sustained for more than 2 weeks. However, under conditions of chronic infection, in which CD8+ CTLs take several months or longer to clear the infection, CD4+ T-cell function is critical. Thus, CD4+ T cells play an important role in sustaining virus-specific CD8+ CTL during chronic LCMV infection. These findings have implications for chronic viral infections in general and may provide a possible explanation for the loss of human immunodeficiency virus-specific CD8+ CTL activity that is seen during the late stages of AIDS, when CD4+ T cells become limiting.     

Viral infection of the thymus.

We have examined infection of the thymus during congenitally acquired chronic lymphocytic choriomeningitis virus (LCMV) infection of mice, a classic model of antigen-specific T-cell tolerance. Our results show that (i) infection starts at the fetal stage and is maintained throughout adulthood, and (ii) this chronic infection of the thymus can be eliminated by transfer of virus-specific cytotoxic T lymphocytes (CTL) that infiltrate the thymus and clear all viral products from both medullary and cortical regions. Elimination of virus from the thymus results in abrogation of tolerance. During the fetal stage, the predominant cell type infected is the earliest precursor of T cells with a surface phenotype of Thy1+ CD4- CD8- J11d+. In the adult thymus, infection is confined primarily to the cortisone-resistant thymocytes present in the medullary region. The infected cells are CD4+ and J11d+. The presence of J11d, a marker usually associated with immature thymocytes, on infected single positive CD4+ "mature" thymocytes is intriguing and suggests that infection by this noncytolytic virus may affect development of T cells. There is minimal infection of the CD8+ medullary thymocytes or of the double positive (CD4+ CD8+) cells present in the cortex. Infection within the cortex is confined to the stromal cells. Interestingly, there is infection of the double negative (CD4- CD8-) thymocytes in the adult thymus, showing that even during adulthood the newly developing T cells are susceptible to infection by LCMV. Virus can be eliminated from the thymuses of these carrier mice by adoptive transfer of medullary region first and then from the thymic cortex. This result clearly shows the need to reevaluate the widely held notion that mature T cells are unable to reenter the thymus. In fact, in our experiments the donor T cells made up to 20 to 30% of the total cells in the thymus at 5 to 7 days after the transfer. The number of donor T cells declined as virus was eliminated from the thymus, and at 1 month posttransfer, the donor T cells were hardly detectable. The results of this study examining the dynamics of viral infection and clearance from the thymus, the primary site of T-cell development, have implications for understanding tolerance induction in chronic viral infections.     

Epitope-specific CD8+ T lymphocytes cross-recognize mutant simian immunodeficiency virus (SIV) sequences but fail to contain very early evolution and eventual fixation of epitope escape mutations during SIV infection

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) evade containment by CD8(+) T lymphocytes through focused epitope mutations. However, because of limitations in the numbers of viral sequences that can be sampled, traditional sequencing technologies have not provided a true representation of the plasticity of these viruses or the intensity of CD8(+) T lymphocyte-mediated selection pressure. Moreover, the strategy by which CD8(+) T lymphocytes contain evolving viral quasispecies has not been characterized fully. In the present study we have employed ultradeep 454 pyrosequencing of virus and simultaneous staining of CD8(+) T lymphocytes with multiple tetramers in the SIV/rhesus monkey model to explore the coevolution of virus and the cellular immune response during primary infection. We demonstrated that cytotoxic T lymphocyte (CTL)-mediated selection pressure on the infecting virus was manifested by epitope mutations as early as 21 days following infection. We also showed that CD8(+) T lymphocytes cross-recognized wild-type and mutant epitopes and that these cross-reactive cell populations were present at a time when mutant forms of virus were present at frequencies of as low as 1 in 22,000 sequenced clones. Surprisingly, these cross-reactive cells became enriched in the epitope-specific CD8(+) T lymphocyte population as viruses with mutant epitope sequences largely replaced those with epitope sequences of the transmitted virus. These studies demonstrate that mutant epitope-specific CD8(+) T lymphocytes that are present at a time when viral mutant epitope sequences are detected at extremely low frequencies fail to contain the later accumulation and fixation of the mutant epitope sequences in the viral quasispecies.     

Dengue virus-specific cross-reactive CD8+ human cytotoxic T lymphocytes

Stimulation with live dengue virus of peripheral blood mononuclear cells from a dengue virus type 4-immune donor generated virus-specific, serotype-cross-reactive, CD8+, class I-restricted cytotoxic T lymphocytes (CTL) capable of lysing dengue virus-infected cells and cells pulsed with dengue virus antigens of all four serotypes. These CTL lysed autologous fibroblasts infected with vaccinia virus-dengue virus recombinant viruses containing the E gene or several nonstructural dengue virus type 4 genes. These results demonstrate that both dengue virus structural and nonstructural proteins are targets for the cytotoxic T-cell-mediated immune response to dengue virus and suggest that serotype-cross-reactive CD8+ CTL may be important mediators of viral clearance and of virus-induced immunopathology during secondary dengue virus infections.     

Expression of CD154 by a simian immunodeficiency virus vector induces only transitory changes in rhesus macaques

Human immunodeficiency virus infection is characterized by dysregulation of antigen-presenting cell function and defects in cell-mediated immunity. Recent evidence suggests that impaired ability of CD4+ T cells to upregulate the costimulatory molecule CD154 is at the core of this dysregulation. To test the hypothesis that increased expression of CD154 on infected CD4+ T cells could modulate immune function, we constructed a replication-competent simian immunodeficiency virus (SIV) vector that expressed CD154. We found that this recombinant vector directed the expression of CD154 on the surface of infected CD4+ T cells and that expression of CD154 resulted in activation of B cells present in the same cultures. Experimental infection of rhesus macaques resulted in very low viral loads for the CD154-expressing virus and the control virus, indicating that expression of CD154 did not result in increased viral replication. Analyses of the anti-SIV immune responses and the phenotype of lymphocytes in blood and lymphoid tissues showed changes that occurred during the acute phase of infection only in animals infected with the CD154-expressing SIV, but that became indistinguishable from those seen in animals infected with the control virus at later time points. We conclude that the level of expression of CD154 in itself is not responsible for affecting the immune response to an attenuated virus. Considering that the CD154-expressing SIV vector and the virus control did not carry an active nef gene, our results suggest that, in CD4+ T cells infected with wild-type virus, Nef is the viral factor that interferes with the immune mechanisms that regulate expression of CD154.     

CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy.

The outcome of antigen recognition by naive CD8+ cytotoxic T lymphocytes (CTLs) in the periphery is orchestrated by CD4+ T-helper cells, and can either lead to priming or tolerization. The presence of T-helper cells favors the induction of CTL immunity, whereas the absence of T-helper cells can result in CTL tolerance. The action of T helper cells in CTL priming is mediated by CD40-CD40 ligand interactions. We demonstrate here that triggering of CD40 in vivo can considerably enhance the efficacy of peptide-based anti-tumor vaccines. The combination of a tolerogenic peptide vaccine containing a minimal essential CTL epitope with an activating antibody against CD40 converts tolerization into strong CTL priming. Moreover, CD40 ligation can provide an already protective tumor-specific peptide vaccine with the capacity to induce therapeutic CTL immunity in tumor-bearing mice. These findings indicate that the CD40-CD40 ligand pair can act as a 'switch', determining whether naive peripheral CTLs are primed or tolerized, and support the clinical use of CD40-stimulating agents as components of anti-cancer vaccines.     

Role of CD4+ and CD8+ T cells in murine resistance to street rabies virus.

Mice of the SJL/J and BALB/cByJ inbred strains are naturally resistant to street rabies virus (SRV) injected via the intraperitoneal route. To determine the cellular mechanism of resistance, monoclonal antibodies specific for CD4+ or CD8+ subsets of T cells were used to deplete the respective cell population in SRV-infected animals. Elimination of CD4+ T-helper cells abrogated the production of immunoglobulin G (IgG) neutralizing antibodies in response to rabies virus infection and reversed the resistant status of SJL/J and BALB/cByJ mice. In contrast, in vivo depletion of CD8+ cytotoxic T cells had no measurable effect on host resistance to SRV. These results indicate that serum neutralizing antibodies of the IgG class are a primary immunological mechanism of defense against rabies virus infection in this murine model of disease. CD8+ cytotoxic T lymphocytes, which have been shown to transfer protection in other rabies virus systems, appear to have no role in protecting mice against intraperitoneally injected SRV.     

Identification of a protective CD4+ T-cell epitope in p15gag of Friend murine leukemia virus and role of the MA protein targeting the plasma membrane in immunogenicity

Recent studies have demonstrated an essential role of Gag-specific CD4+ T-cell responses for viral control in individuals infected with human immunodeficiency virus type 1. However, little is known about epitope specificities and functional roles of the Gag-specific helper T-cell responses in terms of vaccine-induced protection against a pathogenic retroviral challenge. We have previously demonstrated that immunization with Friend murine leukemia virus (F-MuLV) Gag proteins protects mice against the fatal Friend retrovirus (FV) infection. We report here the structure of a protective T helper cell (Th) epitope, (I)VTWEAIAVDPPP, identified in the p15 (MA) region of F-MuLV Gag. In mice immunized with the Th epitope-harboring peptide or a vaccinia virus-expressed native full-length MA protein, FV-induced early splenomegaly regressed rapidly. In these mice, FV-infected cells were eliminated within 4 weeks and the production of virus-neutralizing antibodies was induced rapidly after FV challenge, resulting in strong protection against the virus infection. Interestingly, mice immunized with the whole MA mounted strong CD4+ T-cell responses to the identified Th epitope, whereas mice immunized with mutant MA proteins that were not bound to the plasma membrane failed to mount efficient CD4+ T-cell responses, despite the presence of the Th epitope. These mutant MA proteins also failed to induce strong protection against FV challenge. These data indicate the importance of the properly processible MA molecule for CD4+ T-cell priming and for the resultant induction of an effective immune response against retrovirus infections.     

Molecular basis for cytolytic T-lymphocyte recognition of the murine cytomegalovirus immediate-early protein pp89

The murine cytomegalovirus protein pp89, which is encoded by gene ieI, is a nonstructural regulatory protein expressed in the immediate-early phase of the viral replication cycle and located mainly in the nucleus of infected cells. Protection of BALB/c (H-2d) mice against a lethal murine cytomegalovirus challenge infection is achieved by vaccination with a recombinant vaccinia virus, MCMV-ieI-VAC, expressing pp89 as the only murine cytomegalovirus gene product. The protection is entirely mediated by T lymphocytes of the CD8+ subset. In the present report, we analyzed the molecular basis of the recognition of pp89 by BALB/c CD8+ cytolytic T lymphocytes. A series of internal and terminal deletion mutants of gene ieI was constructed and cloned in vaccinia virus, and the antigenicity and immunogenicity of the fragments of pp89 expressed by the recombinants were studied. A region of only one-sixth of the protein, from amino acids 154 to 249 and encoded by the fourth exon of gene ieI, was sufficient for both the recognition in vitro of the protein by pp89-specific cytotoxic T lymphocytes and the induction in vivo of pp89-specific cytotoxic T lymphocytes. By using synthetic peptides, the sequence between residues 161 and 179, which is located within the defined domain, was identified as an epitope presented to BALB/C cytotoxic T lymphocytes by the class I major histocompatibility antigen Ld.     

Virus-lymphocyte interactions. II. Expression of viral sequences during the course of persistent lymphocytic choriomeningitis virus infection and their localization to the L3T4 lymphocyte subset

Viruses that cause in vivo persistent infections need to selectively compromise the host's immunologic surveillance machinery in order to survive. To understand the molecular basis of how this is accomplished we have analyzed persistent virus infection by lymphocytic choriomeningitis in its normal host, the mouse. Earlier we noted by infectious center analysis that five in 10(4) lymphocytes carried by persistently infected mice contained infectious materials throughout the course of infection. A previous publication extended these results, in BALB mice by showing that the L3T4+ lymphocyte subset in lymph nodes and spleens was predominantly involved. Using cDNA labeled probes to the viral genome and in situ hybridization we report that 1 to 2% of circulating lymphocytes from several mouse strains contain viral RNA sequences for the three viral structural genes. By FACS analysis, the Thy-1.2+, L3T4+ subset primarily harbors virus while viral sequences are usually not detected in the Lyt-2+ subset as early as 6 days after initiating infection in newborns and throughout the course of the persistence. These findings suggest that incomplete, presumably defective, virus is generated in a subset of Th lymphocytes during persistent infection and that during this time infection of cytotoxic T cell subsets is minimal.     

The role of CD4+ cells in sustaining lymphocyte proliferation during lymphocytic choriomeningitis virus infection.

The murine immune response to lymphocytic choriomeningitis virus (LCMV) infection involves the activation of CD8+, class I MHC-restricted and virus-specific CTL. At times coinciding with CTL activation, high levels of IL-2 gene expression and production occur, the IL-2R is expressed, and T cell blastogenesis and proliferation are induced. We have previously found that, although both CD4+ and CD8+ T cell subsets transcribe IL-2, the CD4+ subset appears to be the major producer of IL-2 whereas the CD8+ subset appears to be the major proliferating population when the subsets are separated after activation in vivo. The studies presented here were undertaken to examine the contribution made by the CD4+ subset to lymphocyte proliferation in vivo. Responses to LCMV infection were examined in intact mice and in mice depleted of CD4+ or CD8+ subsets by antibody treatments in vivo. Protocols were such that in vivo treatments with anti-CD4 or anti-CD8 depleted the respective subset by greater than 90%. In situ hybridizations demonstrated that the IL-2 gene was expressed in non-B lymphocytes isolated from either CD4+ cell-depleted or CD8+ cell-depleted mice on day 7 post-infection with LCMV. When placed in culture, however, cells from CD8+ cell-depleted mice produced significantly higher levels of detectable IL-2 than did cells isolated from CD4+ cell-depleted mice on day 7 post-infection. IL-2 was apparently produced in vivo in mice depleted of either CD4+ or CD8+ cells, as expression of the gene for the p55 chain of the IL-2R, IL-2 responsiveness, and lymphocyte proliferation were observed with cells isolated from both sets of mice. Lymphocyte proliferation was shown to be sustained in mice depleted of CD4+ cells in vivo by three criteria: 1) non-B lymphocytes isolated from infected mice depleted of CD4+ cells underwent more DNA synthesis than did those isolated from uninfected mice or from infected mice depleted of CD8+ cells; 2) leukocyte yields were expanded during infection of CD4+ cell-depleted mice; and 3) CD8+ cell numbers were increased during infection of CD4+ cell-depleted mice. The majority of non-B lymphocytes having the characteristics of blast lymphocytes was recovered in the CD8+ populations isolated from infected CD4+ cell-depleted mice. These findings suggest that the requirement for the CD4+ subset to sustain CD8+ lymphocyte proliferation in vivo is limited, and that CD4+ and CD8+ cell types can function independently in many aspects of their responses to viral infections.     

Characterization of primary and memory CD8 T-cell responses against ranavirus (FV3) in Xenopus laevis

In mammals, resistance to primary and secondary viral infections critically involves major histocompatibility complex class I-restricted cytotoxic CD8+ T lymphocytes (CTLs). Although many gene homologues involved in CTL function have been identified in all vertebrate classes, antiviral CTL responses have been poorly characterized for ectothermic vertebrates. Because of the threat of emerging wildlife viral diseases to global biodiversity, fundamental research on comparative viral immunity has become crucial. Ranaviruses (family Iridoviridae) are double-stranded DNA viruses possibly implicated in the worldwide decline of amphibian populations. We used the frog Xenopus laevis as a model to evaluate adaptive immune responses to the ranavirus frog virus 3 (FV3). FV3 infects the kidneys of adults but is cleared within 4 weeks, with faster clearance upon secondary infections. In vivo depletion of CD8+ T cells markedly decreases the survival of adults after viral infection. To further investigate the involvement of anti-FV3 CD8+ T-cell effectors in host resistance in vivo, we determined the proliferation kinetics of CD8+ T cells in the spleen by bromodeoxyuridine incorporation and their infiltration of kidneys by immunohistology. Upon primary infection, CD8+ T cells significantly proliferate in the spleen and accumulate in infected kidneys from day 6 onward, in parallel with virus clearance. Earlier proliferation and infiltration associated with faster viral clearance were observed during a secondary infection. These results provide in vivo evidence of protective antigen-dependent CD8+ T-cell proliferation, recognition, and memory in fighting a natural pathogen in Xenopus.     

CD8 is needed for development of cytotoxic T cells but not helper T cells.

A mutant mouse strain without CD8 (Lyt-2 and Lyt-3) expression on the cell surface has been generated by disrupting the Lyt-2 gene using embryonic stem cell technology. In these mice, CD8+ T lymphocytes are not present in peripheral lymphoid organs, but the CD4+ T lymphocyte population seems to be unaltered. Cytotoxic response of T lymphocytes from these mice against alloantigens and viral antigens is dramatically decreased. Proliferative response against alloantigens and in vivo help to B lymphocytes, however, are not affected. These data suggest that CD8 is necessary for the maturation and positive selection of class I MHC restricted cytotoxic T lymphocytes but is not required on any of the intermediate thymocyte populations (CD8+CD4-TcR- or CD4+CD8+TcRlow) during the development of functional class II MHC restricted helper T cells.     

Contact of human immunodeficiency virus type 1-infected and uninfected CD4+ T lymphocytes is highly cytolytic for both cells.

Individuals infected with the human immunodeficiency virus (HIV) experience a marked loss of CD4+ T lymphocytes, leading to fatal immunodeficiency. The mechanisms causing the depletion of these cells are not yet understood. In this study, we observed that CD4+ T lymphocytes from HIV type 1 (HIV-1)-infected and uninfected individuals rapidly lysed B lymphoblasts expressing the HIV-1 envelope glycoprotein on the cell surface and Jurkat cells expressing the complete virus. Contact of uninfected CD4+ T cells with envelope glycoprotein-expressing cells also resulted in the lysis of the uninfected CD4+ T cells. Cytolysis did not require priming or in vitro stimulation of the CD4+ T cells and was not restricted by major histocompatibility complex molecules. Cytotoxicity was inhibited by soluble CD4 and anti-CD4 monoclonal antibodies that block binding of CD4 to gp120. In addition, neutralizing anti-CD4 and anti-gp120 monoclonal antibodies which block postbinding membrane fusion events and syncytium formation also inhibited cell lysis, suggesting that identical mechanisms in HIV-infected cultures underlie cell-cell fusion and the cytolysis observed. However, cytotoxicity was not always accompanied by the formation of visible syncytia. Rapid cell lysis after contact of uninfected and HIV-1-infected CD4+ T cells may explain CD4+ T-cell depletion in the absence of detectable syncytia in infected individuals. Moreover, because of its vigor, lysis of envelope-expressing targets by contact with unprimed CD4+ T lymphocytes may at first glance resemble antigen-specific immune responses and should be excluded when cytotoxic T-lymphocyte responses in infected individuals and vaccinees are evaluated.     

H-2 Kd-restricted hepatitis B virus-derived epitope whose specific CD8+ T lymphocytes can produce gamma interferon without cytotoxicity

It is necessary to evaluate the cytokine secretion status of CD8+ T lymphocytes and elucidate the factors influencing cytokine secretion, because the secretion of cytokines is also an important feature of CD8+ T lymphocytes, and the cytokines usually play critical roles in the outcome of diseases. We showed here that peptide AYRPPNAPI, derived from the core antigen of hepatitis B virus (HBV), could bind to H-2 Kd and induce primed splenocytes from HBcAg expression plasmid-immunized mice to produce gamma interferon (IFN-gamma) in H-2 Kd- and CD8-dependent manners instead of in a CD4-dependent manner. The induced cells were mainly CD3 and CD8 positive but had no cytotoxic effect on the corresponding target cells. When administered into HBV transgenic mice, these cells can decrease the serum HBV load without causing liver damage. These results suggest that this peptide is a special kind of CD8+ T-cell epitope, for which specific CD8+ T cells can produce IFN-gamma when antigenic stimulation is encountered but which have no cytotoxic effect on the corresponding target cells both in vitro and in HBV transgenic mice. This phenomenon indicates initially that the functional mechanisms of CD8+ T cells can be determined by their epitope specificity, which may be associated with the development of epitope-based immunotherapeutic approaches for infectious diseases and tumors.     

Gamma interferon is a major mediator of antiviral defense in experimental measles virus-induced encephalitis.

Measles virus infection of the central nervous system in the murine model of experimental measles virus-induced encephalitis is successfully controlled by virus-specific T-helper lymphocytes. T cells from BALB/c mice that are resistant to measles virus encephalitis proliferate well against measles virus in vitro, and bulk cultures recognize viral nucleocapsid and hemagglutinin as well as fusion proteins. The measles virus-specific T cells secrete large amounts of interleukin 2 (IL-2), gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) but no IL-4, IL-6, or IL-10, and hence the cytokine pattern is consistent with that of subtype 1 T-helper lymphocytes. In contrast, cells obtained from measles virus-infected susceptible C3H mice recognize measles virus proteins only weakly and secrete little IFN-gamma and TNF-alpha. Treatment of infected mice with anti-TNF-alpha antibodies has no effect on survival or virus clearance from the brain. Upon neutralization of IFN-gamma in vivo, the phenotype of measles virus-specific T-helper cells isolatable from BALB/c mice is reversed from subtype 1 to subtype 2-like. Anti-IFN-gamma antibody-treated BALB/c mice are susceptible to measles virus encephalitis, and viral clearance from the central nervous system is impaired. These results indicate that IFN-gamma plays a significant role in the control of measles virus infection of the central nervous system.     

Virus-specific CD8+ T-cell responses in mice transgenic for a T-cell receptor beta chain selected at random.

The consequences of severely limiting the T-cell receptor (TCR) repertoire available for the response to intranasal infection with an influenza A virus or with Sendai virus have been analyzed by using H-2k mice (TG8.1) transgenic for a TCR beta-chain gene (V beta 8.1D beta 2J beta 2.3C beta 2). Analyzing the prevalence of V beta 8.1+ CD8+ T cells in lymph node cultures from nontransgenic (non-TG) H-2k controls primed with either virus and then stimulated in vitro with the homologous virus or with anti-CD3 epsilon showed that this TCR is not normally selected from the CD8+ T-cell repertoire during these infections. However, the TG8.1 mice cleared both viruses and generated virus-specific effector cytotoxic T lymphocytes (CTL) and memory CTL precursors, though the responses were delayed compared with the non-TG controls. Depletion of the CD4+ T-cell subset had little effect on the course of influenza virus infection but substantially slowed the development of the Sendai virus-specific CTL response and virus elimination in both the TG8.1 and non-TG mice, indicating that CD4+ helpers are promoting the CD8+ T-cell response in the Sendai virus model. Even so, restricting the available T-cell repertoire to lymphocytes expressing a single TCR beta chain still allows sufficient TCR diversity for CD8+ T cells (acting in the presence or absence of the CD4+ subset) to limit infection with an influenza A virus and a parainfluenza type 1 virus.