Antigen-specific CD8(+) T cells persist in the upper respiratory tract following influenza virus infection.

Because little is known about lymphocyte responses in the nasal mucosa, lymphocyte accumulation in the nasal mucosa, nasal-associated lymphoid tissue (NALT), and cervical lymph nodes (CLN) were determined after primary and heterosubtypic intranasal influenza challenge of mice. T cell accumulation peaked in the nasal mucosa on day 7, but peaked slightly earlier in the CLN (day 5) and later (day 10) in the NALT. Tetrameric staining of nasal mucosal cells revealed a peak accumulation of CD8 T cells specific for either the H-2D(b) influenza nucleoprotein epitope 366-374 (D(b)NP(366)) or the H-2D(b) polymerase 2 protein epitope 224-233 (D(b)PA(224)) at 7 days. By day 13, D(b)PA(224)-specific CD8 T cells were undetectable in the mucosa, whereas D(b)NP(366)-specific CD8 T cells persisted for at least 35 days in the mucosa and spleen. After heterosubtypic virus challenge, the accumulation of CD8 T cells in the nasal mucosa was quicker, more intense, and predominantly D(b)NP(366) specific relative to the primary inoculation. The kinetics and specificity of the CD8 T cell response were similar to those in the CLN, but the responses in the NALT and spleen were again slower and more protracted. These results indicate that similar to what was reported in the lung, D(b)NP(366)-specific CD8 T cells persist in the nasal mucosa after primary influenza infection and predominate in an intensified nasal mucosal response to heterosubtypic challenge. In addition, differences in the kinetics of the CD8 T cell responses in the CLN, NALT, and spleen suggest different roles of these lymphoid tissues in the mucosal response.

Vaccination against murine gamma-herpesvirus infection.

The gamma-herpesviruses establish life-long latency in the host and are important human pathogens. T cells play a major role in controlling the initial acute infection and subsequently maintaining the virus in a quiescent state. However, the nature of the T-cell response to gamma-herpesvirus infection and the requirements for effective vaccination are poorly understood. The recent development of a murine gamma-herpesvirus (murine herpesvirus-68 [MHV-68]) has made it possible to analyze T-cell responses and test vaccination strategies in a small animal model. Intranasal infection with MHV-68 induces an acute infection in the lung and the subsequent establishment of long-term latency, which is associated with splenomegaly and an infectious mononucleosis-like syndrome. Here we review the T-cell response to different phases of the infection and the impact of vaccination against either lytic-cycle, or latency-associated T-cell epitopes.

Identification of quantitative trait loci controlling activation of TRBV4 CD8+ T cells during murine gamma-herpesvirus-induced infectious mononucleosis.

The murine gamma-herpesvirus, MHV-68, shares important biological and genetic features with the human gamma-herpesvirus, Epstein-Barr virus. Following intranasal infection, mice develop an infectious mononucleosis-like syndrome accompanied by increased numbers of activated CD8+ T cells in the blood. A consistent feature of the CD8+ T-cell activation is a marked increase in the frequency of cells expressing a TRBV4+ T-cell receptor. Previous studies suggested that the magnitude of TRBV4 expansion varied significantly among mouse strains, and was influenced by both MHC and non-MHC genes. Detailed analysis of strains with high (C57BL/6) or low (DBA/2) TRBV4 CD8+ T-cell expansion showed that differences in the degree of expansion were not a consequence of variation in genetic susceptibility to the viral infection. Rather, the magnitude of the TRBV4 CD8+ T-cell expansion correlated with differences in expression of the unidentified stimulatory ligand on activated, latently infected B cells. In the present study, analysis of TRBV4 expansion in C57BL/6, DBA/2, B6D2 F1 mice, BXD recombinant inbred strains, and the progeny of C57BL/6xDBA/2 F1 hybrids backcrossed to C57BL/6 demonstrated strong cumulative dominance of the low DBA/2 trait and moderately high heritability (h2 approximately 0.5). Two quantitative trait loci (QTLs) strongly associated with variance in TRBV4 expansion were identified using simple and composite mapping procedures. The first QTL is located on Chromosome (Chr) 17, near or proximal to H2. The second QTL is located on Chr 6 in a region spanning the Tcrb and Cd8a loci.

Cellular immunity and memory to respiratory virus infections.

Respiratory virus infections, such as those caused by influenza and parainfluenza viruses, are a major cause of morbidity and mortality worldwide. Current vaccines against these pathogens rely on the induction of humoral immune responses that target viral coat proteins. Although this type of immunity provides solid protection against homologous virus strains, it is ineffective against heterologous virus strains that express serologically distinct coat proteins. In contrast, cellular immune responses can target internal antigens that are shared between heterologous viral strains. This form of immunity, sometimes referred to as heterosubtypic immunity, can mediate a substantial degree of protection. Thus, vaccines that emphasize cellular immune responses would be a valuable complement to available humoral vaccines. However, we only have a rudimentary understanding of which T cell subsets mediate protective immunity, how T cell memory is established and maintained, how that memory is recalled in a secondary infection, and why cellular immunity wanes rapidly with time. Here we review the role of CD4+ and CD8+ T cells in the recall response to influenza and parainfluenza viruses. In particular we focus on the recent observation that substantial numbers of memory T cells are established in the lung tissues and discuss the potential role of these cells in mediating a recall response. A thorough understanding of the cellular immune response to infection in the lungs is essential for future vaccine development.

Latent antigen vaccination in a model gammaherpesvirus infection.

Vaccines that can reduce the load of latent gammaherpesvirus infections are eagerly sought. One attractive strategy is vaccination against latency-associated proteins, which may increase the efficiency with which T cells recognize and eliminate latently infected cells. However, due to the lack of tractable animal model systems, the effect of latent-antigen vaccination on gammaherpesvirus latency is not known. Here we use the murine gammaherpesvirus model to investigate the impact of vaccination with the latency-associated M2 antigen. As expected, vaccination had no effect on the acute lung infection. However, there was a significant reduction in the load of latently infected cells in the initial stages of the latent infection, when M2 is expressed. These data show for the first time that latent-antigen vaccination can reduce the level of latency in vivo and suggest that vaccination strategies involving other latent antigens may ultimately be successfully used to reduce the long-term latent infection.

Antibody-independent antiviral function of memory CD4+ T cells in vivo requires regulatory signals from CD8+ effector T cells.

Previous studies have shown that vaccine-primed CD4(+) T cells can mediate accelerated clearance of respiratory virus infection. However, the relative contributions of Ab and CD8(+) T cells, and the mechanism of viral clearance, are poorly understood. Here we show that control of a Sendai virus infection by primed CD4(+) T cells is mediated through the production of IFN-gamma and does not depend on Ab. This effect is critically dependent on CD8(+) cells for the expansion of CD4(+) T cells in the lymph nodes and the recruitment of memory CD4(+) T cells to the lungs. Passive transfer of a CD8(+) T cell supernatant into CD8(+) T cell-depleted, hemagglutinin-neuraminidase (HN)(421-436)-immune muMT mice substantially restored the virus-specific memory CD4(+) response and enhanced viral control in the lung. Together, the data demonstrate for the first time that in vivo primed CD4(+) T cells have the capacity to control a respiratory virus infection in the lung by an Ab-independent mechanism, provided that CD8(+) T cell "help" in the form of soluble factor(s) is available during the virus infection. These studies highlight the importance of synergistic interactions between CD4(+) and CD8(+) T cell subsets in the generation of optimal antiviral immunity.

Analysis of virus-specific CD4(+) t cells during long-term gammaherpesvirus infection.

Major histocompatibility complex class II-mediated antigen presentation after intranasal infection with murine gammaherpesvirus 68 differs in mediastinal lymph nodes and spleen. Evidence that virus-specific CD4(+) T cells were being stimulated was found as late as 6 to 8 months after infection, and cells specific for the viral gp150(67-83) and ORF11(168-180) peptides were maintained as a fairly stable proportion of the total response.

Identification of MHC class II-associated peptides that promote the presentation of toxic shock syndrome toxin-1 to T cells.

Previous studies have shown that the DM-deficient cell line, T2-I-A(b), is very inefficient at presenting toxic shock syndrome toxin 1 (TSST-1) to T cells, suggesting that I-A(b)-associated peptides play an essential role in the presentation of this superantigen. Consistent with this, the loading of an I-A(b)-binding peptide, staphylococcal enterotoxin B 121-136, onto T2-I-A(b) cells enhanced TSST-1 presentation >1000-fold. However, despite extensive screening, no other peptides have been identified that significantly promote TSST-1 presentation. In addition, the peptide effect on TSST-1 presentation has been demonstrated only in the context of the tumor cell line T2-I-A(b). Here we show that peptides that do not promote TSST-1 presentation can be converted into "promoting" peptides by the progressive truncation of C-terminal residues. These studies result in the identification of two peptides derived from IgGV heavy chain and I-Ealpha proteins that are extremely strong promoters of TSST-1 presentation (47,500- and 12,000-fold, respectively). We have also developed a system to examine the role of MHC class II-associated peptides in superantigen presentation using splenic APC taken directly ex vivo. The data confirmed that the length of the MHC class II-bound peptide plays a critical role in the presentation of TSST-1 by splenic APC and showed that different subpopulations of APC are equally peptide dependent in TSST-1 presentation. Finally, we demonstrated that the presentation of staphylococcal enterotoxin A, like TSST-1, is peptide dependent, whereas staphylococcal enterotoxin B presentation is peptide independent.

Protection from respiratory virus infections can be mediated by antigen-specific CD4(+) T cells that persist in the lungs.

Although CD4(+) T cells have been shown to mediate protective cellular immunity against respiratory virus infections, the underlying mechanisms are poorly understood. For example, although phenotypically distinct populations of memory CD4(+) T cells have been identified in different secondary lymphoid tissues, it is not known which subpopulations mediate protective cellular immunity. In this report, we demonstrate that virus-specific CD4(+) T cells persist in the lung tissues and airways for several months after Sendai virus infection of C57BL/6 mice. A large proportion of these cells possess a highly activated phenotype (CD44(hi), CD62L(lo), CD43(hi), and CD25(hi)) and express immediate effector function as indicated by the production of interferon gamma after a 5-h restimulation in vitro. Furthermore, intratracheal adoptive transfer of lung memory cells into beta2m-deficient mice demonstrated that lung-resident virus-specific CD4(+) T cells mediated a substantial degree of protection against secondary virus infection. Taken together, these data demonstrate that activated memory CD4(+) T cells persisting at mucosal sites play a critical role in mediating protective cellular immunity.

Activated antigen-specific CD8+ T cells persist in the lungs following recovery from respiratory virus infections.

The poor correlation between cellular immunity to respiratory virus infections and the numbers of memory CD8(+) T cells in the secondary lymphoid organs suggests that there may be additional reservoirs of T cell memory to this class of infection. Here we identify a substantial population of Ag-specific T cells in the lung that persist for several months after recovery from an influenza or Sendai virus infection. These cells are present in high numbers in both the airways and lung parenchyma and can be distinguished from memory cell populations in the spleen and peripheral lymph nodes in terms of the relative frequencies among CD8(+) T cells, activation status, and kinetics of persistence. In addition, these cells are functional in terms of their ability to proliferate, express cytolytic activity, and secrete cytokines, although they do not express constitutive cytolytic activity. Adoptive transfer experiments demonstrated that the long-term establishment of activated T cells in the lung did not require infection in the lung by a pathogen carrying the inducing Ag. The kinetics of persistence of Ag-specific CD8(+) T cells in the lung suggests that they play a key role in protective cellular immunity to respiratory virus infections.

Antigen expression during murine gamma-herpesvirus infection.

Gammaherpesviruses (gammaHV) establish a life-long latency in the host and are associated with a number of malignant human diseases. It is generally believed that T cells play a major role in controlling the initial acute infection and subsequently maintaining the virus in a quiescent state. However, the nature of the T cell response to gamma-herpesvirus infections is poorly understood. In the current report we took advantage of a mouse model of gammaHV infection (murine herpesvirus-68, MHV-68) to investigate the T cell response to different phases of the infection. Intranasal infection with MHV-68 induces an acute infection in lung epithelial cells and long-term latency in B cells. The kinetics of the CD8+ T cell response to different lytic cycle and latency-associated antigens was highly complex and distinct patterns of response could be identified. These responses were regulated by multiple factors including differences in temporal expression of the relevant antigens, differences in the presentation of antigen in different organs, and differential expression of antigen in different types of antigen presenting cells. For example, some antigens were expressed at distinct phases of the infection and in specific organs or subsets of antigen presenting cells. In addition, recent data suggest that in addition to B cells, both macrophages and dendritic cells harbor latent MHV-68 infection, adding further complexity to their role in controlling the T cell response to this infection.

Factors controlling levels of CD8+ T-cell lymphocytosis associated with murine gamma-herpesvirus infection.

Intranasal infection of mice with murine gamma-herpesvirus 68 (MHV-68) elicits a striking CD8+ T-cell lymphocytosis following the establishment of latency, which includes a marked increased frequency of Vbeta4+ CD8+ T cells. The Vbeta4+ CD8+ T cells do not recognize a conventional viral peptide, but are stimulated by an uncharacterized ligand expressed on latently infected, activated B cells. The selective expansion of Vbeta4+ CD8+ T cells after MHV-68 infection is observed in all mouse strains examined, although the fold-increase varies widely, ranging from less than twofold to greater than 10-fold. The factors controlling the variation are currently undefined. In the current study, CD8+ T cell activation and Vbeta4+ CD8+ T-cell frequencies were analyzed in 18 inbred strains of mice. The data show that the magnitude of the Vbeta4+ CD8+ T-cell response correlates with the degree of CD8+ T cell-activation, and that both major histocompatibility complex (MHC) and non-MHC genes contribute to the magnitude of the activation. Furthermore, the magnitude of the response does not reflect major differences in susceptibility to viral infection and/or corresponding differences in the acute response. Rather the degree of Vbeta4+ CD8+ T cell activation may be determined by differences in levels of expression of the stimulatory ligand at the peak of latency.

Control of gammaherpesvirus latency by latent antigen-specific CD8(+) T cells.

The contribution of the latent antigen-specific CD8(+) T cell response to the control of gammaherpesvirus latency is currently obscure. Some latent antigens induce potent T cell responses, but little is known about their induction or the role they play during the establishment of latency. Here we used the murine gammaherpesvirus system to examine the expression of the latency-associated M2 gene during latency and the induction of the CD8(+) T cell response to this protein. M2, in contrast to the M3 latency-associated antigen, was expressed at day 14 after infection but was undetectable during long-term latency. The induction of the M2(91-99)/K(d) CD8(+) T cell response was B cell dependent, transient, and apparently induced by the rapid increase in latently infected cells around day 14 after intranasal infection. These kinetics were consistent with a role in controlling the initial "burst" of latently infected cells. In support of this hypothesis, adoptive transfer of an M2-specific CD8(+) T cell line reduced the initial load of latently infected cells, although not the long-term load. These data represent the first description of a latent antigen-specific immune response in this model, and suggest that vaccination with latent antigens such as M2 may be capable of modulating latent gammaherpesvirus infection.

Murine gamma-herpesvirus infection causes V(beta)4-specific CDR3-restricted clonal expansions within CD8(+) peripheral blood T lymphocytes.

Infection of mice with the gamma-herpesvirus MHV-68 results in lytic infection in the lung cleared by CD8(+) cells and establishment of lifelong latency. An Epstein-Barr virus (EBV)-like infectious mononucleosis (IM) syndrome emerges approximately 3 weeks after infection. In human IM, the majority of T cells in the peripheral blood are monoclonal or oligoclonal and are frequently specific for lytic or latent viral epitopes. However, a unique feature of MHV-68-induced IM is a prominent MHC haplotype-independent expansion of CD8(+) T cells, the majority of which utilize V(beta)4 chains in their alphabetaTCR. The ligand driving the V(beta)4 expansion is unknown, but the V(beta) bias and MHC haplotype independence raised the possibility that these cells were responding to a virally encoded or a virally induced endogenous superantigen (sAg). The aim of this study was to determine whether this rapidly proliferating subset is composed of polyclonally or clonally expanded T cells. Complementarity-determining region (CDR)-3 size analysis of V(beta)4(+)CD8(+) cells in infected mice demonstrated CDR3-restricted expansions in the V(beta)4 family as a whole. More refined analysis demonstrated major distortions in every J(beta) subfamily. V-D-J junctional region sequencing indicated that these CDR3 size-restricted expansions were composed of clonal or oligoclonal populations. The sequences were largely unique in individual mice, although evidence for 'public' or highly conserved T cell expansions was also seen between different mice. Taken together with previous studies showing an apparent MHC independence, the data suggest that a novel ligand, distinct from conventional sAg and peptide-MHC, drives proliferation of V(beta)4(+)CD8(+) T cells.

Latent murine gamma-herpesvirus infection is established in activated B cells, dendritic cells, and macrophages.

Intranasal infection of mice with the murine gamma-herpesvirus MHV-68 results in an acute lytic infection in the lung, followed by the establishment of lifelong latency. Development of an infectious mononucleosis-like syndrome correlates with the establishment of latency and is characterized by splenomegaly and the appearance of activated CD8+ T cells in the peripheral blood. Interestingly, a large population of activated CD8+ T cells in the peripheral blood expresses the V beta 4+ element in their TCR. In this report we show that MHV-68 latency in the spleen after intranasal infection is harbored in three APC types: B cells, macrophages, and dendritic cells. Surprisingly, since latency has not previously been described in dendritic cells, these cells harbored the highest frequency of latent virus. Among B cells, latency was preferentially associated with activated B cells expressing the phenotype of germinal center B cells, thus formally linking the previously reported association of latency gene expression and germinal centers to germinal center B cells. Germinal center formation, however, was not required for the establishment of latency. Significantly, although three cell types were latently infected, the ability to stimulate V beta 4+CD8+ T cell hybridomas was limited to latently infected, activated B cells.

CD4+ T cell priming accelerates the clearance of Sendai virus in mice, but has a negative effect on CD8+ T cell memory.

Current vaccines designed to promote humoral immunity to respiratory virus infections also induce potent CD4+ T cell memory. However, little is known about the impact of primed CD4+ T cells on the immune response to heterologous viruses that are serologically distinct, but that share CD4+ T cell epitopes. In addition, the protective capacity of primed CD4+ T cells has not been fully evaluated. In the present study, we addressed these two issues using a murine Sendai virus model. Mice were primed with an HN421-436 peptide that represents the dominant CD4+ T cell epitope on the hemagglutinin-neuraminidase (HN) of Sendai virus. This vaccination strategy induced strong CD4+ T cell memory to the peptide, but did not induce Abs specific for the Sendai virus virion. Subsequent Sendai virus infection of primed mice resulted in 1) a substantially accelerated virus-specific CD4+ T cell response in the pneumonic lung; 2) enhanced primary antiviral Ab-forming cell response in the mediastinal lymph nodes; and 3) accelerated viral clearance. Interestingly, the virus-specific CD8+ T cell response in the lung and the development of long-term memory CD8+ T cells in the spleen were significantly reduced. Taken together, our data demonstrate that primed CD4+ T cells, in the absence of pre-existing Ab, can have a significant effect on the subsequent immune responses to a respiratory virus infection.

DNA vaccination as a tool to identify subdominant CD8 T cell epitopes.

DNA vaccination is highly efficient at inducing CD8(+) T cell responses in animal models. Here we investigated whether DNA vaccine technology could be exploited to identify subdominant cytotoxic T lymphocytes (CTL) epitopes. Previous studies have shown that the Sendai virus HN protein does not induce a CD8(+) T cell response in C57BL/6 mice. Thus, we vaccinated C57BL/6 mice with a DNA vaccine encoding Sendai virus hemagglutinin neuraminidase (HN) protein. The data show that this strategy elicited a potent D(b)-restricted CD8(+) CTL response against at least one subdominant HN-derived epitope. These CTL were able to lyse Sendai virus-infected target cells, demonstrating that the epitope was appropriately processed and present at sufficient levels for T cell recognition. However, these cells did not confer protection against lethal challenge with Sendai virus. These data demonstrate the capacity of DNA vaccine to raise CTL responses to subdominant epitopes, but show that such responses may be limited in their efficacy against non-persistent viruses.

T-cell vaccination alters the course of murine herpesvirus 68 infection and the establishment of viral latency in mice.

Diseases caused by gammaherpesviruses such as Epstein-Barr virus are a major health concern, and there is significant interest in developing vaccines against this class of viral infections. However, the requirements for effective control of gammaherpesvirus infection are only poorly understood. The recent development of the murine herpesvirus MHV-68 model provides an experimental tool to dissect the immune response to gammaherpesvirus infections. In this study, we investigated the impact of priming T cells specific for class I- and class II-restricted epitopes on the acute phase of the infection and the subsequent establishment of latency and infectious mononucleosis. The data show that vaccination with either major histocompatibility complex class I- or class II-restricted T-cell epitopes derived from lytic cycle proteins significantly reduced lung viral titers during the acute infection. Moreover, the peak level of latently infected spleen cells was significantly reduced following vaccination with immunodominant CD8(+) T-cell epitopes. However, this vaccination approach did not prevent the long-term establishment of latency or the development of the infectious mononucleosis-like syndrome in infected mice. Thus, the virus is able to establish latency efficiently despite strong immunological control of the lytic infection.

Apoptotic cells are generated at every division of in vitro cultured T cell lines.

Long- and short-term T cell lines form the backbone of many assays for T cell function and also represent important tools for use in human immunotherapy. Despite much study concerning the requirements for T cell activation and growth in culture there is relatively little information about the kinetics of proliferation and cell death in such cultures. Here we studied these parameters in a long-term CD8(+) T cell line using a tetrameric MHC reagent and the fluorescent dye CFSE. We observed proliferation of the T cells within 24 h of restimulation with antigen and IL-2 and the cells continued to divide once every 12 h on average. Interestingly, a proportion of cells entered apoptosis with each cell division, showing that a degree of programmed cell death occurred constantly in vitro, not merely at the end of the culture period when antigen or the necessary growth factors became limiting. This information should assist in the design of more efficient protocols for generating large numbers of specific T cells for clinical use.