Altered function in CD8+ T cells following paramyxovirus infection of the respiratory tract

For many respiratory pathogens, CD8+ T cells have been shown to play a critical role in clearance. However, there are still many unanswered questions with regard to the factors that promote the most efficacious immune response and the potential for immunoregulation of effector cells at the local site of infection. We have used infection of the respiratory tract with the model paramyxovirus simian virus 5 (SV5) to study CD8+ T-cell responses in the lung. For the present study, we report that over time a population of nonresponsive, virus-specific CD8+ T cells emerged in the lung, culminating in a lack of function in approximately 85% of cells specific for the immunodominant epitope from the viral matrix (M) protein by day 40 postinfection. Concurrent with the induction of nonresponsiveness, virus-specific cells that retained function at later times postinfection exhibited an increased requirement for CD8 engagement. This change was coupled with a nearly complete loss of functional phosphoprotein-specific cells, a response previously shown to be almost exclusively CD8 independent. These studies add to the growing evidence for immune dysregulation following viral infection of the respiratory tract.     

A quantitative analysis of the variables affecting the repertoire of T cell specificities recognized after vaccinia virus infection

Many components contribute to immunodominance in the response to a complex virus, but their relative importance is unclear. This was addressed using vaccinia virus and HLA-A*0201 as the model system. A comprehensive analysis of 18 viral proteins recognized by CD8(+) T cell responses demonstrated that approximately one-fortieth of all possible 9- to 10-mer peptides were high-affinity HLA-A*0201 binders. Peptide immunization and T cell recognition data generated from 90 peptides indicated that about one-half of the binders were capable of eliciting T cell responses, and that one-seventh of immunogenic peptides are generated by natural processing. Based on these results, we estimate that vaccinia virus encodes approximately 150 dominant and subdominant epitopes restricted in by HLA-A*0201. However, of all these potential epitopes, only 15 are immunodominant and actually recognized in vivo during vaccinia virus infection of HLA-A*0201 transgenic mice. Neither peptide-binding affinity, nor complex stability, nor TCR avidity, nor amount of processed epitope appeared to strictly correlate with immunodominance status. Additional experiments suggested that vaccinia infection impairs the development of responses directed against subdominant epitopes. This suggested that additional factors, including immunoregulatory mechanisms, restrict the repertoire of T cell specificities after vaccinia infection by a factor of at least 10.     

A novel CD8-independent high-avidity cytotoxic T-lymphocyte response directed against an epitope in the phosphoprotein of the paramyxovirus simian virus 5

Adoptive transfer studies have shown that cytotoxic T lymphocytes (CTL) of high avidity, capable of recognizing low levels of peptide-MHC I molecules, are more efficient at reducing viral titers than are low-avidity CTL, thus establishing CTL avidity as a critical parameter for the ability of a CTL to clear virus in vivo. It has been well documented that CTL of high avidity are relatively CD8 independent, whereas low-avidity CTL require CD8 engagement in order to become activated. In this study we have analyzed the antiviral CTL response elicited following infection with the paramyxovirus simian virus 5 (SV5). We have identified the immunodominant and subdominant CTL responses and subsequently assessed the avidity of these responses by their CD8 dependence. This is the first study in which the relationship between immunodominance and CTL avidity has been investigated. The immunodominant response was directed against an epitope present in the viral M protein, and subdominant responses were directed against epitopes present in the P, F, and HN proteins. Similarly to other CTL responses we have analyzed, the immunodominant response and the subdominant F and HN responses were comprised of both high- and low-avidity CTL. However, the subdominant response directed against the epitope present in the P protein is novel, as it is exclusively high avidity. This high-avidity response is independent of both the route of infection and expression by recombinant SV5. A further understanding of the inherent properties of P that elicit only high-avidity CTL may allow for the design of more efficacious vaccine vectors that preferentially elicit high-avidity CTL in vivo.     

Modulation of CD8+ T cell avidity by increasing the turnover of viral antigen during infection

The increased potency of high avidity CD8+ T cells for the clearance of viral infections has been well documented. We have previously reported the novel finding that intranasal infection with the paramyxovirus SV5 induces a CD8+ T cell response to the SV5 P protein that is almost exclusively of high avidity. Based on our results that the level of peptide presentation is a critical factor in the selective expansion of high versus low avidity cells in vitro, we hypothesized that the avidity of the anti-viral response generated in vivo could be altered by increasing the turnover of the P protein during viral infection through linkage to ubiquitin (UbP). Infection with a virus expressing UbP (VV-UbP) elicited a significant increase in low avidity cells in both BALB/c and C3H mice compared to the almost exclusively high avidity response elicited by VV-P. Our results are the first demonstration of the control of avidity during the antiviral response through an engineered change to a viral antigen. The implications of our findings for vaccine development are discussed.     

In vivo expansion of the residual tumor antigen-specific CD8+ T lymphocytes that survive negative selection in simian virus 40 T-antigen-transgenic mice

Mice that express the viral oncoprotein simian virus 40 (SV40) large T antigen (T-Ag) as a transgene provide useful models for the assessment of the state of the host immune response in the face of spontaneous tumor progression. Line SV11 (H2(b)) mice develop rapidly progressing choroid plexus tumors due to expression of full-length T-Ag from the SV40 promoter. In addition, T-Ag expression in the thymus of SV11 mice results in the deletion of CD8(+) T cells specific for the three H2(b)-restricted immunodominant epitopes of T-Ag. Whether CD8(+) T cells specific for the immunorecessive H2-D(b)-restricted epitope V of T-Ag survive negative selection in SV11 mice has not been determined. Immunization of SV11 mice with rVV-ES-V, a recombinant vaccinia virus expressing epitope V as a minigene, resulted in the induction of weak, but reproducible, epitope V-specific cytotoxic T-lymphocyte (CTL) responses. This weak lytic response corresponded with a decreased frequency of epitope V-specific CTL that could be recruited in SV11 mice. In addition, CTL lines derived from rVV-ES-V-immunized SV11 mice had reduced avidities compared to that seen with CTL derived from healthy mice. Despite this initial weak response, significant numbers of epitope V-specific CD8(+) T cells were detected in SV11 mice ex vivo following a priming-boosting approach and these cells demonstrated high avidity for epitope V. The results suggest that low numbers of tumor-reactive CD8(+) T cells with high avidity for epitope V survive negative selection in SV11 mice but can be expanded by specific boosting approaches in the tumor bearing host.     

CD8 T cells are essential for recovery from a respiratory vaccinia virus infection

The precise immune components required for protection against a respiratory Orthopoxvirus infection, such as human smallpox or monkeypox, remain to be fully identified. In this study, we used the virulent Western Reserve strain of vaccinia virus (VACV-WR) to model a primary respiratory Orthopoxvirus infection. Naive mice infected with VACV-WR mounted an early CD8 T cell response directed against dominant and subdominant VACV-WR Ags, followed by a CD4 T cell and Ig response. In contrast to other VACV-WR infection models that highlight the critical requirement for CD4 T cells and Ig, we found that only mice deficient in CD8 T cells presented with severe cachexia, pulmonary inflammation, viral dissemination, and 100% mortality. Depletion of CD8 T cells at specified times throughout infection highlighted that they perform their critical function between days 4 and 6 postinfection and that their protective requirement is critically dictated by initial viral load and virulence. Finally, the ability of adoptively transferred naive CD8 T cells to protect RAG(-/-) mice against a lethal VACV-WR infection demonstrated that they are both necessary and sufficient in protecting against a primary VACV-WR infection of the respiratory tract.     

Stability and function of secondary Th1 memory cells are dependent on the nature of the secondary stimulus

Following acute infection in some mouse models, CD4(+) memory T cells steadily decline over time. Conversely, in humans, CD4(+) memory T cells can be maintained for many years at levels similar to CD8(+) T cells. Because we previously observed that the longevity of Th1 memory cell survival corresponded to their functional avidity, we hypothesized that secondary challenge, which enriches for high functional avidity Th1 responders, would result in more stable Th1 memory populations. We found that following a heterologous secondary challenge, Th1 memory cells were maintained at stable levels compared with primary Th1 memory cells, showing little to no decline after day 75 postinfection. The improved stability of secondary Th1 memory T cells corresponded to enhanced homeostatic turnover; enhanced trafficking of effector memory Th1 cells to tissue sites of infection, such as the liver; and acquisition or maintenance of high functional avidity following secondary challenge. Conversely, a weaker homologous rechallenge failed to induce a stable secondary Th1 memory population. Additionally, homologous secondary challenge resulted in a transient loss of functional avidity by Th1 memory cells recruited into the secondary response. Our findings suggest that the longevity of Th1 memory T cells is dependent, at least in part, on the combined effects of primary and secondary Ag-driven differentiation. Furthermore, they demonstrate that the quality of the secondary challenge can have profound effects on the longevity and function of the ensuing secondary Th1 memory population.     

Mycobacterium tuberculosis directs immunofocusing of CD8+ T cell responses despite vaccination

Vaccines that elicit T cell responses try to mimic protective memory T cell immunity after infection by increasing the frequency of Ag-specific T cells in the immune repertoire. However, the factors that determine immunodominance during infection and after vaccination and the relation between immunodominance and protection are incompletely understood. We previously identified TB10.4(20-28) as an immunodominant epitope recognized by H2-K(d)-restricted CD8(+) T cells after M. tuberculosis infection. Here we report a second epitope, EspA(150-158), that is recognized by a substantial number of pulmonary CD8(+) T cells. The relative abundance of these T cells in the naive repertoire only partially predicts their relative frequency after M. tuberculosis infection. Furthermore, although vaccination with recombinant vaccinia virus expressing these epitopes changes their relative immunodominance in the preinfection T cell repertoire, this change is transient after challenge with M. tuberculosis. We speculate that factors intrinsic to the chronic nature of M. tuberculosis infection establishes the hierarchy of immunodominance and may explain the failure of some vaccines to provide protection.     

CD127+CCR5+CD38+++ CD4+ Th1 effector cells are an early component of the primary immune response to vaccinia virus and precede development of interleukin-2+ memory CD4+ T cells

The stages of development of human antigen-specific CD4+ T cells responding to viral infection and their differentiation into long-term memory cells are not well understood. The inoculation of healthy adults with vaccinia virus presents an opportunity to study these events intensively. Between days 11 and 14 postinoculation, there was a peak of proliferating CCR5+CD38+++ CD4+ effector cells which contained the cytotoxic granule marker T-cell intracellular antigen 1 and included gamma interferon (IFN-gamma)-producing vaccinia virus-specific CD4+ T cells. The majority of these initial vaccinia virus-specific CD4+ T cells were CD127+ and produced interleukin-2 (IL-2) but not CTLA-4 in response to restimulation in vitro. Between days 14 and 21, there was a switch from IFN-gamma and IL-2 coexpression to IL-2 production only, coinciding with a resting phenotype and an increased in vitro proliferation response. The early CCR5+CD38+++ vaccinia virus-specific CD4+ T cells were similar to our previous observations of human immunodeficiency virus (HIV)-specific CD4+ T cells in primary HIV type 1 (HIV-1) infection, but the vaccinia virus-specific cells expressed much more CD127 and IL-2 than we previously found in their HIV-specific counterparts. The current study provides important information on the differentiation of IL-2+ vaccinia virus-specific memory cells, allowing further study of antiviral effector CD4+ T cells in healthy adults and their dysfunction in HIV-1 infection.     

Novel features of the respiratory tract T-cell response to influenza virus infection: lung T cells increase expression of gamma interferon mRNA in vivo and maintain high levels of mRNA expression for interleukin-5 (IL-5) and IL-10.

Analysis of the respiratory tract before and after primary influenza virus infection revealed a virus-induced preferential accumulation of a CD8+ T-cell population that coexpresses mRNA for interleukin-5 (IL-5) and IL-10 with virus dose-dependent high levels of gamma interferon. However, cytokine production in lung tissues was not restricted to the T-cell population, since CD3- cells were found to express mRNA for various cytokines, including IL-4 and particularly IL-6 and granulocyte-macrophage colony-stimulating factor. These data provide in vivo evidence for a local respiratory tract immune response to influenza virus infection dominated by cytokine-producing CD8+ T cells.     

Expression of intracellular IFN-gamma in HSV-1-specific CD8+ T cells identifies distinct responding subpopulations during the primary response to infection

Cutaneous infection in the footpads of C57BL/6 mice with HSV-1 results in an accumulation of activated (CD44high CD25+) CD8+ T cells within the draining popliteal lymph node (PLN). These studies were undertaken to evaluate the frequency and phenotype of the CD8+ T cell population within the PLN, recognizing the single immunodominant HSV-1 epitope derived from the viral envelope glycoprotein, glycoprotein B (gB), using an intracellular IFN-gamma-staining assay. It revealed that approximately 6% of the CD8+ T cells were specific for the gB epitope. Phenotypic analysis of the IFN-gamma-producing gB-specific CD8+ T cells generated in the PLN during the course of the acute infection expressed the CD44high CD25+ phenotype on days 3-5 postinfection. Surprisingly, IFN-gamma-producing CD8+ T cells expressed the CD44high CD25- phenotype on days 5-8 postinfection, in contrast to expectations for a CD8+ effector T cell. IFN-gamma-producing CD25- CD8+ T cells were detected in the PLN on day 21 postinfection, long after infectious virus had been cleared. Throughout the response, the spleen was found to be the major reservoir of gB-specific CD8+ T cells, even during the peak of the response. In contrast to the gB-specific CD8+ T cell population within the PLN, the entire gB-specific CD8+ T cell population within the spleen was CD25-. Collectively, these results suggest the generation of subpopulations of virus-specific CD8+ T cells, distinguished by the expression of CD25, during the acute phase of the primary response to a localized viral infection.     

Differential requirements for CD80/86-CD28 costimulation in primary and memory CD4 T cell responses to vaccinia virus

Vaccinia virus infection can confer immunity to smallpox by inducing potent T cell and antibody responses. While the CD8 T cell response to vaccinia virus has been well characterized, less is known about factors required for priming and memory for the CD4 T cells. Focusing on two recently described epitopes, we show that after intranasal infection, both I1L and L4R epitopes are co-dominant during the acute response, but the I1L epitope dominates during memory. CD4 T cell priming was intact in the absence of CD80/86, however secondary responses were reduced. This contrasts with our previous data showing CD80/86–CD28 interaction is required for optimal primary and memory CD8 T cell responses. The absence of CD80/86 also changed the immunodominance hierarchy during memory, with the I1L and L4R responses becoming co-dominant in knockout mice. These data highlight different costimulatory requirements for primary CD4 and CD8 T cell responses to vaccinia virus.     

Type I IFN substitutes for T cell help during viral infections

Certain virus infections depend on the presence of T cell help for the generation of primary CD8(+) T cell responses. However, the mechanisms that render these particular viral infections T cell help dependent is largely unknown. In this study, we compared CD8(+) T cell responses elicited by lymphocytic choriomeningitis virus infection, as prototype of a T cell help independent infection, with T cell help dependent CD8(+) T cell responses induced by vaccinia virus infection. In this paper, we show that a key parameter decisive for T cell help independence is the ability of an infectious agent to stimulate early and robust production of type I IFN. Experimental provision of type I IFN during VV infection rendered the ensuing CD8(+) T cell response completely T cell help independent. Our results support a model in which type I IFN has to be present during the first 3 d of Ag encounter and has to act directly on the responding CD8(+) T cells to promote their survival and effector differentiation. We show that type I IFN signaling on responding CD8(+) T cells induces profound upregulation of CD25 and increased IL-2 expression; however, neither this nor IL-15 accounts for the type I IFN effects on responding CD8(+) T cells. Thus, type I IFN can effectively replace the requirement of T cell help by directly promoting CD8(+) T cell survival and differentiation independent of the type I IFN-induced cytokines IL-2 and IL-15.     

Selective delivery of augmented IL-2 receptor signals to responding CD8+ T cells increases the size of the acute antiviral response and of the resulting memory T cell pool

CD8(+) T cells respond to IL-2 produced both endogenously and by CD4(+) Th during an antiviral response. However, IL-2R signals can potentially promote CD8(+) T cell death as well as proliferation, making it unclear whether IL-2R signals provide a predominantly positive or negative effect upon CD8(+) T cell responses to viral infection. To more precisely define the direct role of IL-2R signaling on CD8(+) T cells during the response to a virus, we examined the effect of delivering augmented IL-2R signals selectively to CD8(+) T cells responding to lymphocytic choriomeningitis virus infection. Although naive CD8(+) T cells are competent to produce IL-2, CD8(+) T cells lose this capacity upon differentiation into effector CD8(+) T cells. However, effector CD8(+) T cells do retain the capacity to produce GM-CSF upon Ag stimulation. Thus, to deliver enhanced autocrine IL-2R signals to CD8(+) T cells, we established a transgenic mouse strain expressing a chimeric GM-CSF/IL-2R (GMIL2R). As GM-CSF production is Ag dependent, the GMIL2R delivers an augmented IL-2R signal exclusively to CD8(+) T cells responding to Ag. Following lymphocytic choriomeningitis virus infection, GMIL2R transgenic mice exhibited an increase in both the peak CD8(+) T cell response achieved and the size of the resulting memory pool established. Upon secondary viral challenge, the GMIL2R also enhanced the proliferative response of memory CD8(+) T cells. Thus, our findings indicate that IL-2 delivery to responding CD8(+) T cells is a limiting factor in both the acute and memory antiviral responses.     

CD8 marks a subpopulation of lung-derived dendritic cells with differential responsiveness to viral infection and toll-like receptor stimulation

An increasing number of studies suggest that individual subsets of dendritic cells (DC) exhibit distinct capabilities with regard to the generation of the adaptive immune response. In this study, we evaluated the properties of a relatively unexplored DC subset present in the lung-draining mediastinal lymph node. This subset expresses the airway dendritic cell marker CD103 together with CD8. These DC were of interest given that our previous studies using a model of respiratory infection with vaccinia virus revealed a distinct difference in the ability of CD103(+) DC to prime T cells that correlated inversely with the expression of CD8, suggesting a differential role of these DC in the context of respiratory virus infection. To expand our understanding of the role of this DC population, we performed analyses to elucidate the phenotype, migratory capacity, responsiveness to innate stimuli, and priming capacity of CD8(+) CD103(+) DC. We found that expression of surface markers on these DC was similar to that of CD8(-) CD103(+) DC, supporting their close relationship. Further, the two DC types were similar with regard to antigen uptake. However, although both CD103(+) subsets originated from the lung, CD8-bearing CD103(+) DC appeared in the lymph node with delayed kinetics following virus infection. While this subset exhibited increased responsiveness to a number of Toll-like receptor (TLR) agonists, their response to infection was virus specific, demonstrating poor responsiveness to vaccinia virus infection but robust maturation following infection with parainfluenza virus 5 or influenza virus. These findings show that CD8 marks a population of lung airway-derived DC with distinct migratory and maturation responses that likely contribute differentially to the immune response depending on the infecting pathogen.     

Naive precursor frequencies and MHC binding rather than the degree of epitope diversity shape CD8+ T cell immunodominance

The primary CD8(+) T cell response of C57BL/6J mice against the 28 known epitopes of lymphocytic choriomeningitis virus (LCMV) is associated with a clear immunodominance hierarchy whose mechanism has yet to be defined. To evaluate the role of epitope competition in immunodominance, we manipulated the number of CD8(+) T cell epitopes that could be recognized during LCMV infection. Decreasing epitope numbers, using a viral variant lacking dominant epitopes or C57BL/6J mice lacking H-2K(b), resulted in minor response increases for the remaining epitopes and no new epitopes being recognized. Increasing epitope numbers by using F(1) hybrid mice, delivery by recombinant vaccinia virus, or epitope delivery as a pool in IFA maintained the overall response pattern; however, changes in the hierarchy did become apparent. MHC binding affinity of these epitopes was measured and was found to not strictly predict the hierarchy since in several cases similarly high binding affinities were associated with differences in immunodominance. In these instances the naive CD8(+) T cell precursor frequency, directly measured by tetramer staining, correlated with the response hierarchy seen after LCMV infection. Finally, we investigated an escape mutant of the dominant GP33-41 epitope that elicited a weak response following LCMV variant virus infection. Strikingly, dominance loss likely reflects a substantial reduction in frequencies of naive precursors specific for this epitope. Thus, our results indicate that an intrinsic property of the epitope (MHC binding affinity) and an intrinsic property of the host (naive precursor frequency) jointly dictate the immunodominance hierarchy of CD8(+) T cell responses.     

Mice deficient in perforin,CD4+ T cells,or CD28-mediated signaling maintain the typical immunodominance hierarchies of CD8+ T-cell responses to influenza virus

CD8 T-cell (T(CD8+)) responses elicited by viral infection demonstrate the phenomenon of immunodominance: the numbers of T(CD8+) responding to different viral peptides vary over a wide range in a reproducible manner for individuals with the same major histocompatibility complex class I alleles. To better understand immunodominance, we examined T(CD8+) responses to multiple defined viral peptides following infection of mice with influenza virus. The immunodominance hierarchy of influenza virus-specific T(CD8+) was not greatly perturbed by the absence of either perforin or T-helper cells or by interference with B7 (CD80)-mediated signaling. These findings indicate that costimulation by antigen-presenting cells (APCs) or killing of APCs by T(CD8+) plays only a minor role in establishing the immunodominance hierarchy of antiviral T(CD8+) in this system. This points to intrinsic features of the T(CD8+) repertoire as major contributors to immunodominance.     

Gamma interferon and perforin control the strength, but not the hierarchy, of immunodominance of an antiviral CD8+ T cell response

The two major antiviral effector mechanisms of CD8(+) T cells are thought to be perforin (Prf)-mediated cell lysis and gamma interferon (IFN-γ)-mediated induction of an antiviral state. By affecting the expression of proteins involved in antigen presentation, IFN-γ is also thought to shape the magnitude and specificity of the CD8(+) T cell response. Here we studied the roles of Prf and IFN-γ in shaping the effector and memory CD8(+) T cell responses to vaccinia virus (VACV). IFN-γ deficiency resulted in increased numbers of anti-VACV effector and memory CD8(+) T cells, which were partly dependent on increased virus loads. On the other hand, Prf-deficient mice showed an increase in the number of VACV-specific CD8(+) T cells only in the memory phase. Treatment of the mice with the antiviral drug cidofovir reduced the numbers of effector and memory cells closer to wild-type levels in IFN-γ-deficient mice and reduced the numbers of memory CD8(+) T cells to wild-type levels in Prf-deficient mice. These data suggest that virus loads are the main reason for the increased strength of the CD8 response in IFN-γ- and Prf-deficient mice. Neither Prf deficiency nor IFN-γ deficiency had an effect on the immunodominance hierarchy of five K(b)-restricted CD8(+) T cell determinants either during acute infection or after recovery. Thus, our work shows that CD8(+) T cell immunodominance during VACV infection is not affected by the effects of IFN-γ on the antigen presentation machinery.     

Altered immunodominance hierarchies of CD8+ T cells in the spleen after infection at different sites is contingent on high virus inoculum

Activated epitope-specific CD8+ T cells after virus infection can be organized into hierarchies (immunodominance), based on their ability to focus the response on few viral determinants. The mechanisms responsible for immunodominance can be multifactorial, with CD8+ T cells precursor frequencies recently highlighted as a key regulator. Employing the LCMV infection model, we demonstrate that the hierarchies were altered when comparing different sites of infection but only at high viral doses. These findings have significant implications when investigating immunity to viruses with different replication abilities that may override the influence of T cell precursor frequencies.     

Genome-wide analysis reveals a highly diverse CD8 T cell response to murine cytomegalovirus

Human CMV establishes a lifelong latent infection in the majority of people worldwide. Although most infections are asymptomatic, immunocompetent hosts devote an extraordinary amount of immune resources to virus control. To increase our understanding of CMV immunobiology in an animal model, we used a genomic approach to comprehensively map the C57BL/6 CD8 T cell response to murine CMV (MCMV). Responses to 27 viral proteins were detectable directly ex vivo, the most diverse CD8 T cell response yet described within an individual animal. Twenty-four peptide epitopes were mapped from 18 Ags, which together account for most of the MCMV-specific response. Most Ags were from genes expressed at early times, after viral genes that interfere with Ag presentation are expressed, consistent with the hypothesis that the CD8 T cell response to MCMV is largely driven by cross-presented Ag. Titration of peptide epitopes in a direct ex vivo intracellular cytokine staining assay revealed a wide range of functional avidities, with no obvious correlation between functional avidity and the strength of the response. The immunodominance hierarchy varied only slightly between mice and between experiments. However, H-2(b)-expressing mice with different genetic backgrounds responded preferentially to different epitopes, indicating that non-MHC-encoded factors contribute to immunodominance in the CD8 T cell response to MCMV.