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The functional avidity of a cytotoxic T lymphocyte (CTL) is known to be a critical determinant of the efficacy with which it clears pathogens. High avidity cells, which are by definition highly sensitive to peptide antigen, are superior for elimination of viruses and tumours. Our studies have established the ability of T cells to undergo avidity modulation as a result of antigen encounter. High and low avidity cells established in this manner exhibit significant differences in the amount of peptide required to elicit effector function. However, how signalling is regulated in these cells as it relates to the control of peptide sensitivity remains to be defined. To address this question, we compared T-cell receptor (TCR) signal transduction events in high and low avidity CTL generated from OT-I(rag2-) TCR transgenic mice. Our data suggest that divergent signalling is initiated at the TCR-associated CD3ζ, with low avidity CTL requiring higher amounts of pMHC to achieve threshold levels of phosphorylated CD3ζ compared with high avidity CTL. Further, this difference is transduced further downstream to mitogen-activated protein kinase and Ca(2+) signalling pathways. These results suggest that regulated control of the initiation of TCR signalling in high versus low avidity cells determines the amount of peptide required for T-cell activation.

A previously unknown mechanism for the active negative regulation of function in effector T cells is described. CD8+ T cells play a critical role in the clearance of respiratory pathogens. Thus, it is surprising that functional inactivation of lung effectors has been observed in many models of viral infection. Currently, the molecular defect responsible for the shut-off of function in these cells is unknown. In the present study, we addressed this question using a model of respiratory infection with the paramyxovirus SV5. Nonfunctional cells were found to exhibit decreases in SOCE, resulting in reduced NFAT1 activation. Notably, function could be restored by the provision of increased levels of extracellular calcium. The reduced ability to mobilize calcium was associated with reduced expression of ORAI1, the CRAC channel subunit. These findings reveal a previously unknown mechanism for the negative regulation of function in effector T cells.

The paramyxovirus simian virus 5 (SV5) is a poor activator of human dendritic cell (DC) maturation pathways in vitro, and infected DC do not upregulate cell surface costimulatory proteins or secretion of immunomodulatory cytokines. We evaluated the hypothesis that activation of SV5-infected DC would be enhanced by engineering SV5 to express a Toll-like-receptor (TLR) ligand. To test this hypothesis, a novel virus was engineered such that the gene encoding an intracellular form of the TLR5 ligand flagellin was expressed from the genome of wild-type (WT) SV5 (SV5-flagellin). Cells infected in vitro with the flagellin-expressing virus released low levels of biologically active flagellin, which was capable of stimulating TLR5 signaling. Infection of human peripheral blood mononuclear cell-derived immature DC with SV5-flagellin resulted in enhanced levels of interleukin-6 (IL-6) and IL-12 compared to infection with DC with the parental virus, WT SV5. In contrast to cytokine induction, the flagellin-expressing virus did not appreciably increase DC surface expression of the costimulatory molecule CD80 or CD86 above the level seen with WT SV5 alone. In mixed-culture assays, DC infected with the flagellin-expressing virus were more effective at activating gamma interferon secretion from both CD8+and CD4+allogeneic T cells than DC infected with WT SV5. Our results with SV5-directed intracellular expression of flagellin may be applicable to other vectors or pathogenic viruses where overcoming impairment of DC activation could contribute to the development of safer and more effective vaccines.

Recently there has been renewed interest in poxvirus pathogenesis, especially with regard to infection via the respiratory route. Members of this family are known to produce a number of proteins that have the potential to negatively regulate the immune response. Vaccinia virus (VACV) has been used for a number of years as a model for the study of poxvirus infection. We have previously reported a dose-dependent decrease in virus-specific CD8 + T cells following respiratory infection with VACV. In this study we have evaluated whether more generalized immunosuppressive effects are also observed following infection with a high dose of VACV. We have found that mice infected intranasally with a high, but non-lethal, dose of VACV exhibited significant weight loss as well as decreased thymocyte number. Although these mice mounted an immune response, there was a significant increase observed in bystander T and B cell apoptosis. While increased death was apparent in both naive and activated/memory T cells populations, naive T cells appeared more sensitive to this effect. These findings are important for our understanding of poxvirus regulation of the immune response and extends our previous understanding of VACV-mediated immunosuppression to include generalized apoptosis in the naive and activated/memory repertoires.

DC maturation is known to be a necessary step in the generation of an effective immune response. We have used vaccinia virus (VACV) as a model to investigate the regulation of DC subsets in vivo following infection. While a number of in vitro studies have shown that DC infected with VACV fail to undergo maturation, the effect of VACV infection on the maturation of and cytokine production by DC subsets in vivo remains less defined. We have found that following systemic infection with vaccinia virus, both CD8 + and CD8 − dendritic cells are infected. The number of infected DC peaked at 6h and was highly decreased by 24h post-infection. In both subsets, there was evidence of generalized upregulation of costimulatory molecules. Surprisingly, this included vaccinia infected DC, suggesting the regulation of DC maturation in vivo is much more complex andlikely influenced by DC extrinsic signals. However, while we observed generalized upregulation of costimulatory molecules, IL-12 production was restricted to a subset of non-infected cells in both the CD8 + and CD8 − DC populations. Importantly, the control of IL-12 production was differentially dependent on MyD88 signaling. IL-12 production was ablated in the absence of MyD88 in CD8 − DC, while it was unchanged in CD8 + DC. These findings provide new insights into the control of DC maturation in vivo and demonstrate that the regulation of maturation in vivo following virus infection can be differentially controlled in distinct types of DC.

CTL that possess a high functional avidity are known to be optimal for the clearance of pathogens in vivo. We have shown that the amount of peptide encountered by a CD8+ CTL determines its functional avidity. Notably, in these studies nonprofessional APC were used. However, it is mature dendritic cells (DC) that are predominantly responsible for the activation of naive T cells in vivo. Whether DC also direct dose dependent-differences in avidity is unknown. In this work we examined the ability of mature DC presenting a high vs low level of peptide to generate CTL of distinct avidities. In contrast to what was observed with nonprofessional APC, CTL generated by stimulation with mature DC were of high avidity regardless of the amount of peptide presented. This DC property may promote generation of highly effective CTL that retain plasticity, which would allow the tuning of avidity in the periphery to promote optimal pathogen recognition and clearance.

Infection of primary cultures of human immature monocyte-derived dendritic cells (moDC) with the paramyxovirus Simian Virus 5 (SV5) results in extensive cytopathic effect (CPE) and induction of apoptosis, but DC maturation pathways are not activated. In this study, we investigated the relationship between SV5-induced apoptosis and the lack of DC maturation. Reducing CPE and apoptosis in SV5-infected immature DC by the addition of a pancaspase inhibitor resulted in only low level expression of maturation markers CD40, CD80 and CD86, suggesting that SV5 infection either actively blocked maturation pathways or failed to provide sufficient signals to activate maturation. To distinguish between these hypotheses, SV5-infected immature DC were challenged with agonists that stimulate toll-like receptors (TLRs). Treatment with the TLR-4 agonist LPS or TLR-6 agonist FSL1 enhanced cell surface expression of CD40, CD80 and CD86 on SV5-infected cells to levels approaching that of mock-infected TLR-treated moDC, but treatment with agonists for TLR-2, -3, -5 or -8 had little effect. Addition of TLR-4 or -6 agonists to SV5-infected DC also dramatically reduced CPE and apoptosis, but the levels of viral protein and virus yield were not affected. Similarly, SV5-infected immature moDC were matured by treatment with IL-1β, and these mature infected cells also showed reduced CPE and apoptosis. In the presence of NFkB inhibitors, TLR-4 and -6 agonists did not promote maturation or reduce apoptosis of SV5-infected DC, indicating that maturation and cell survival were both dependent on signaling through NFkB-dependent pathways. Our results suggest a model whereby SV5 replication induces apoptosis in immature DC but fails to provide strong maturation signals, while activation of NFkB-dependent pathways by exogenous ligands can lead to moDC maturation and override SV5-induced cell death.

Professional antigen-presenting cells are critical components of both the innate and adaptive immune responses. Although dendritic cells (DCs) are generally thought to be the primary activators of naive T cells, macrophages have also been shown to fulfill this role. As with DCs, the capacity to induce optimal activation of T cells requires that macrophages undergo a process that results in the increased expression of costimulatory molecules, such as CD40, CD80, and CD86, and the production of cytokines. In this study we analyzed the effect of infection of macrophages generated from BALB/c mice with the paramyxovirus simian virus 5 (SV5). Here we have shown that bone marrow-derived macrophages (BMMs) are not productively infected at any multiplicity of infection tested. Analysis of activation markers revealed that SV5-infected BMMs robustly upregulated CD40 and modestly upregulated CD86, but did not upregulate the expression of CD80. Further, SV5-infected BMMs secreted low levels of interferon-beta and interleukin (IL)-12p40, but high levels of tumor necrosis factor-alpha and IL-6. Intriguingly, upregulation of these molecules on BMMs, unlike our previous results using bone marrow-derived dendritic cells, was not dependent on live virus. These findings provide evidence that different professional antigen-presenting cells can detect and respond to virus via distinct mechanisms.

Influenza virus can cause life-threatening infections in neonates and young infants. Although vaccination is a major countermeasure against influenza, current vaccines are not approved for use in infants less than 6 months of age, in part due to the weak immune response following vaccination. Thus, there is a strong need to develop new vaccines with improved efficacy for this vulnerable population. To address this issue, we established a neonatal African green monkey (AGM) nonhuman primate model that could be used to identify effective influenza vaccine approaches for use in young infants. We assessed the ability of flagellin, a Toll-like receptor 5 (TLR5) agonist, to serve as an effective adjuvant in this at-risk population. Four- to 6-day-old AGMs were primed and boosted with inactivated PR8 influenza virus (IPR8) adjuvanted with either wild-type flagellin or inactive flagellin with a mutation at position 229 (m229), the latter of which is incapable of signaling through TLR5. Increased IgG responses were observed following a boost, as well as at early times after challenge, in infants vaccinated with flagellin-adjuvanted IPR8. Inclusion of flagellin during vaccination also resulted in a significantly increased number of influenza virus-specific T cells following challenge compared to the number in infants vaccinated with the m229 adjuvant. Finally, following challenge infants vaccinated with IPR8 plus flagellin exhibited a reduced pathology in the lungs compared to that in infants that received IPR8 plus m229. This study provides the first evidence of flagellin-mediated enhancement of vaccine responses in nonhuman primate neonates. IMPORTANCE Young infants are particularly susceptible to severe disease as a result of influenza virus infection. Compounding this is the lack of effective vaccines for use in this vulnerable population. Here we describe a vaccine approach that results in improved immune responses and protection in young infants. Incorporation of flagellin during vaccination resulted in increased antibody and T cell responses together with reduced disease following virus infection. These results suggest that flagellin may serve as an effective adjuvant for vaccines targeted to this vulnerable population.

The paramyxovirus Simian virus 5 (SV5) is largely non-cytopathic in human epithelial and fibroblast cells. WF-PIV has been described previously as a naturally occurring SV5 variant that encodes P and V proteins differing from the wild-type (WT) SV5 proteins in eight and five amino acid positions, respectively. In this study, it is shown that WF-PIV is like WT SV5 by being largely non-cytopathic in A549 lung epithelial cells. However, substitution of the WF-PIV P/V gene into the background of WT SV5 resulted in a hybrid virus (P/V-WF) that induced apoptotic cell death not seen with either of the parental viruses. The kinetics of HeLa cell killing and induction of apoptosis by the P/V-WF chimera differed from those of the previously described P/V-CPI− chimera by being slower and less extensive. HeLa cell killing by the P/V-WF chimera was effectively reduced by inhibitors of caspase-9, but not of caspase-8. These results demonstrate that an exchange of P/V genes from two non-cytopathic SV5 variants can produce apoptosis-inducing chimeras, and that the role of the SV5 P/V gene products in limiting apoptosis can be dependent on expression in the context of a native viral genome.

CD80 and CD86 have been shown to play a critical role in the optimal activation of T cells. Although these two molecules bind the same ligand, CD28, the question of whether CD80 and CD86 provide unique signals or serve redundant roles remains controversial. Previous studies have suggested that CD80 binding to CD28 may be superior to CD86 for the activation of naive CD8+ T cells. This study provides a potential mechanism to explain these observations. Our study demonstrates a previously unappreciated role for CD80, its superiority over CD86 in promoting CD25 expression, increasing both the number of cells that express CD25 and the level expressed on a per cell basis. These findings provide new insights into the role of CD80 vs CD86 and have important implications for the design of vaccines and immunotherapeutics aimed at the generation of a robust CD8+ T cell response in vivo.

Human epithelial cells infected with the parainfluenza virus simian virus 5 (SV5) show minimal activation of host cell interferon (IFN), cytokine, and cell death pathways. In contrast, a recombinant SV5 P/V gene mutant (rSV5-P/V-CPI−) overexpresses viral gene products and is a potent inducer of IFN, proinflammatory cytokines, and apoptosis in these cells. In this study, we have compared the outcomes of wild-type (WT) SV5 and rSV5-P/V-CPI−infections of primary human dendritic cells (DC), important antigen-presenting cells for initiating adaptive immune responses. We have tested the hypothesis that a P/V mutant which activates host antiviral responses will be a more potent inducer of DC maturation and function than WT rSV5, which suppresses host cell responses. Infection of peripheral blood mononuclear cell-derived immature DC with WT rSV5 resulted in high levels of viral protein and progeny virus but very little increase in cell surface costimulatory molecules or secretion of IFN and proinflammatory cytokines. In contrast, immature DC infected with the rSV5-P/V-CPI−mutant produced only low levels of viral protein and progeny virus, but these infected cells were induced to secrete IFN-α and other cytokines and showed elevated levels of maturation markers. Unexpectedly, DC infected with WT rSV5 showed extensive cytopathic effects and increased levels of active caspase-3, while infection of DC with the P/V mutant was largely noncytopathic. In mixed-culture assays, WT rSV5-infected DC were impaired in the ability to stimulate proliferation of autologous CD4+T cells, whereas DC infected with the P/V mutant were very effective at activating T-cell proliferation. The addition of a pancaspase inhibitor to DC infected with WT rSV5 reduced cytopathic effects and resulted in higher surface expression levels of maturation markers. Our finding that the SV5 P/V mutant has both a reduced cytopathic effect in human DC compared to WT SV5 and an enhanced ability to induce DC function has implications for the rational design of novel recombinant paramyxovirus vectors based on engineered mutations in the viral P/V gene.

The first six months of life reflect a time of high susceptibility to severe disease following respiratory virus infection. Although this could be improved significantly by immunization, current vaccines are not approved for use in these very young individuals. This is the result of the combined effects of poor immune responsiveness and safety concerns regarding the use of live attenuated vaccines or potent adjuvants in this population. Vaccines to effectively combat respiratory viral infection ideally would result in robust CD4+ and CD8+ T cell responses, as well as high-affinity Ab. Inclusion of TLR agonists or single-cycle viruses is an attractive approach for provision of signals that can act as potent stimulators of dendritic cell maturation, as well as direct activators of T and/or B cells. In this article, I discuss the challenges associated with generation of a robust immune response in neonates and the potential for adjuvants to overcome these obstacles.

Dendritic cells are the most potent antigen-presenting cell for priming naive T cells. Optimal activation of T cells requires that dendritic cells undergo a process of maturation resulting in the increased expression of costimulatory molecules, such as CD40, CD86, and CD80, and the production of cytokines. In this study we analyzed the effect of infection of dendritic cells obtained from two strains of mice, BALB/c and C57BL/6, with the paramyxovirus simian virus 5 (SV5). Our results show that C57BL/6 bone marrow-derived dendritic cells (BMDC) are much more permissive to infection with SV5 at a multiplicity of infection (MOI) of 10 PFU/cell compared to BALB/c BMDC, as determined by the production of viral proteins and progeny. However, infection of BALB/c BMDC with a higher MOI of 50 PFU/cell resulted in a productive infection with the production of significant amounts of viral proteins and progeny. Regardless of the permissivity to infection, both BALB/c and C57BL/6 BMDC efficiently upregulated CD40 and CD86. However, CD80 upregulation correlated with the level of expression of viral proteins and the production of viral progeny. While secreted alpha/beta interferon was required for increased expression of all three molecules, optimal CD80 expression was dependent on an additional signal provided by a productive viral infection. These findings provide evidence that the signals controlling the expression of costimulatory molecules following viral infection are distinct. Further, they suggest that the amount of virus encountered and/or the permissivity of a dendritic cell to infection can alter the resulting maturation phenotype and functional capacity of the infected dendritic cell.

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 ∼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.

The primary goal of vaccination is the establishment of protective immunity. Thus there has been significant effort put toward the identification of attributes of the immune response that are associated with optimal protection. Although the number of virus-specific cells elicited is unquestionably important, recent studies have identified an additional parameter, functional avidity, as critical in determining the efficiency of viral clearance. T-cell avidity is a measure of the sensitivity of a cell to peptide antigen. High-avidity cells are those that can recognize antigen-presenting cells (APC) bearing very low levels of peptide antigen, whereas low-avidity cells require much higher numbers of peptide major histocompatibility complex (MHC) complexes in order to become activated or exert effector function. We are only now beginning to gain insights into the molecular control of avidity and the signals required for the optimal activation, expansion, and retention of high-avidity cells in vivo. This review summarizes the current knowledge regarding CD8+ T-cell avidity and explores some of the important issues that are, as of yet, unresolved.

CD28 has been shown to play an important role in T cell activation. Among the downstream events associated with CD28 engagement is the reorganization of the cytoskeleton resulting in lipid raft aggregation. In our previous studies we investigated the involvement of lipid rafts in the activation of high avidity CD8+ T lymphocytes, which recognize cells bearing very low levels of peptide antigen, versus low avidity cells, which require high levels of peptide antigen. In these studies we found that high avidity cells were much more sensitive to lipid raft disruption compared to low avidity cells. Given the important role for CD28 in lipid raft reorganization and our previous finding that high avidity cells are extremely dependent on lipid raft integrity, we hypothesized that high avidity cells could not be generated in the absence of CD28. Surprisingly, we have found that the absence of CD28 does not alter the ability to generate high or low avidity CD8+ T cells. In fact high and low avidity lines generated in parallel from CD28-deficient and WT mice exhibited very similar requirements for peptide antigen. We next compared the effect of lipid raft disruption on the activation of high versus low avidity cells from CD28-deficient and WT mice. While high avidity cells generated from WT mice exhibited the expected dependence on lipid raft integrity, high avidity cells from CD28-deficient mice were not affected. These data suggest that the lines generated from the CD28-deficient mice have developed alternative strategies to promote high sensitivity to peptide antigen.

The ability of CD8+ cytotoxic T lymphocytes (CTL) to clear viral infections may be limited when high avidity CTL encounter supra-optimal antigen density on antigen-presenting cells (APC) and undergo antigen-dependent apoptosis of CTL (ADAC). Previously, we have shown ADAC in CD8+ populations to be Fas independent, TNF-alpha receptor 2 (TNFR2) mediated, caspase dependent, and accompanied by a decrease in Bcl-2. We now employ flow cytometry to follow ADAC within individual CD8+ cells to demonstrate that the intense TCR signal induced in high avidity CTL by supra-optimal antigen density results 8 - 16 h later in a caspase-independent TNFR2 down-modulation that is directly related to the stimulating APC antigen density and concludes in a rapid onset of apoptosis by 18 - 24 h. Individual CTL undergoing apoptosis exhibit a dramatic and concurrent: (1) positive staining with Annexin V and propidium iodide; (2) transformation to a smaller cell size characteristic of apoptosis; and (3) a nearly complete loss of Bcl-2, c-IAP1, and TRAF2. We conclude that the antigen-dependent apoptosis of CD8+ CTL occurs when a tandem TCR/TNFR2 signal initiates an abrupt and concordant onset of multiple apoptotic events.

Previously, we observed that high-avidity CTL are much more effective in vivo than low-avidity CTL in elimination of infected cells, but the mechanisms behind their superior activity remained unclear. In this study, we identify two complementary mechanisms: 1) high-avidity CTL lyse infected cells earlier in the course of a viral infection by recognizing lower Ag densities than those distinguished by low-avidity CTL and 2) they initiate lysis of target cells more rapidly at any given Ag density. Alternative mechanisms were excluded, including: 1) the possibility that low-avidity CTL might control virus given more time (virus levels remained as high at 6 days following transfer as at 3 days) and 2) that differences in efficacy might be correlated with homing ability. Furthermore, adoptive transfer of high- and low-avidity CTL into SCID mice demonstrated that transfer of a 10-fold greater amount of low-avidity CTL could only partially compensate for their decreased ability to eliminate infected cells. Thus, we conclude that high-avidity CTL exploit two complementary mechanisms that combine to prevent the spread of virus within the animal: earlier recognition of infected cells when little viral protein has been made and more rapid lysis of infected cells.

Cytotoxic T lymphocytes (CTLs) are primary mediators of viral clearance, but high viral burden can result in deletion of antigen-specific CTLs. We previously reported a potential mechanism for this deletion: tumor necrosis factor (TNF)-α–mediated apoptosis resulting from stimulation with supraoptimal peptide–major histocompatibility complex. Here, we show that although death is mediated by TNF-α and its receptor (TNF-RII), surprisingly neither the antigen dose dependence of TNF-α production nor that of TNF-RII expression can account for the dose dependence of apoptosis. Rather, a previously unrecognized effect of supraoptimal antigen in markedly decreasing levels of the antiapoptotic protein Bcl-2 was discovered and is likely to account for the gain in susceptibility or competence to sustain the death signal through TNF-RII. This decrease requires a signal through the TCR, not just through TNF-RII. Although death mediated by TNF-RII is not as widely studied as that mediated by TNF-RI, we show here that it is also dependent on proteolytic cleavage by caspases and triggered by a brief initial encounter with antigen. These results suggest that determinant density can regulate the immune response by altering the sensitivity of CTLs to the apoptotic effects of TNF-α by decreasing Bcl-2 levels.

Activation of ICE/Ced-3 family proteases (caspases) has been proposed to mediate both the granule exocytosis and Fas–Fas ligand pathways of rapid target cell death by cytotoxic T lymphocytes. In agreement with this model, two peptide fluoromethyl ketone caspase inhibitors and baculovirus p35 blocked apoptotic nuclear damage and target cell lysis by the CTL-mediated Fas–Fas ligand pathway. The peptide caspase inhibitors also blocked drug-induced apoptotic cell death in tumor cells. In contrast, the caspase inhibitors blocked CTL granule exocytosis–induced target apoptotic nuclear damage, but did not inhibit target lysis. These results are consistent with recent demonstrations that granzyme B can activate caspases leading to apoptotic nuclear damage, but show that target cell lysis by CTL granule exocytosis occurs by a caspase-independent pathway.

Experimental data suggest that negative selection of thymocytes can occur as a result of supraoptimal antigenic stimulation. It is unknown, however, whether such mechanisms are at work in mature CD8+ T lymphocytes. Here, we show that CD8+ effector cytotoxic T lymphocytes (CTL) are susceptible to proliferative inhibition by high dose peptide antigen, leading to apoptotic death mediated by TNF-alpha release. Such inhibition is not reflected in the cytolytic potential of the CTL, since concentrations of antigen that are inhibitory for proliferation promote efficient lysis of target cells. Thus, although CTL have committed to the apoptotic pathway, the kinetics of this process are such that CTL function can occur before death of the CTL. The concentration of antigen required for inhibition is a function of the CTL avidity, in that concentrations of antigen capable of completely inhibiting high avidity CTL maximally stimulate low avidity CTL. Importantly, the inhibition can be detected in both activated and resting CTL. Blocking studies demonstrate that the CD8 molecule contributes significantly to the inhibitory signal as the addition of anti-CD8 antibody restores the proliferative response. Thus, our data support the model that mature CD8+ CTL can accommodate an activation signal of restricted intensity, which, if surpassed, results in deletion of that cell.

The expression of the HLA class I molecules on the cell surface was investigated in hepatocellular carcinoma (HCC) cell lines using complement-mediated cytotoxicity (CMC) and flow cytometric analysis. Although HLA-A antigens were detected by CMC in all cell lines tested, HLA-B and -C antigens were not detectable in six of seven HCC cell lines. These results were also confirmed by flow cytometric analysis focusing on HLA-Bw4 and Bw6 public antigens. Furthermore, complementary DNA (cDNA) from each cell line was tested for the expression of HLA-A, -B, -C and the transporter associated with antigen processing genes (TAP1 and TAP2). Two cell lines showed a reduced level of one or both of the TAP messenger RNAs (mRNAs), and one of these showed a reduction of HLA-B and -C gene expression as well, but the others had detectable mRNA levels. These results demonstrate that hepatocellular carcinoma cell lines tested in the current study lose or decrease the expression of HLA-B and -C alleles on the cell surface, even though mRNA encoding these alleles is present, suggesting that the loss of the HLA molecules might be caused by posttranscriptional events or failure to transport and load peptides necessary for HLA expression. The selective loss of HLA-B and -C, but not -A, molecules (which also excludes a beta 2-microglobulin defect) is intriguing, and may be attributable to the ability of some of the HLA-A molecules to load signal peptides not requiring TAP transport, or to natural selection of HLA-B or -C locus-specific immune surveillance.

P18IIIB is a highly immunogenic peptide from the V3 loop of the HIV-1 gp160 envelope protein that is presented promiscuously by multiple class I MHC molecules. Understanding the molecular basis for promiscuous presentation may have many practical applications. As the highly prevalent HLAA2.1 class I molecule is known to present P18lilB for recognition by cytotoxic T lymphocytes (CTL) found in peripheral blood mononuclear cells of HIV + donors, a P18MIB-specific CTL line was generated from an HLA-A2 +, HIV~ donor in order to define the molecular basis for, and ultimately improve upon the binding of, this peptide to HLA-A2.1. The minimal epitope recognized by the line was a decamer, 110, with the sequence RGPGRAFVTI. Interestingly, this decamer is identical to the minimal epitope from P18IIIB seen by murine CTL restricted by H-2Dd. A panel of Ala-substitut ed peptides was employed in MHC-binding and T cell response studies to identify MHC- and TCRbinding residues. Notably, many of the agretopic and epitopic residues identified were identical to those involved in the corresponding interactions of 110 with the H-2Dd MHC molecule and murine 110-specific CTL. The 110 peptide does not contain the described HLA-A2.1 binding motif. Instead a Pro at P3, a Phe at P7 and an Me at P10 are utilized for MHC binding. Agretopic residue similarities with the hepatitis B nucleocapsid decamer suggest that these residues may comprise an alternative motif of anchors utilized by decamers for binding to HLA-A2.1.

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.

Enveloped viruses can incorporate host cell membrane proteins during the budding process. Here we demonstrate that mumps virus (MuV) and vesicular stomatitis virus (VSV) assemble to include CD46 and CD55, two host cell regulators which inhibit propagation of complement pathways through distinct mechanisms. Using viruses which incorporated CD46 alone, CD55 alone, or both CD46 and CD55, we have tested the relative contribution of these regulators in resistance to complement-mediated neutralization. Virion-associated CD46 and CD55 were biologically active, with VSV showing higher levels of activity of both cofactors, which promoted factor I-mediated cleavage of C3b into iC3b as well as decay-accelerating factor (DAF) activity against the C3 convertase, than MuV. Time courses of in vitro neutralization with normal human serum (NHS) showed that both regulators could delay neutralization, but viruses containing CD46 alone were neutralized faster and more completely than viruses containing CD55 alone. A dominant inhibitory role for CD55 was most evident for VSV, where virus containing CD55 alone was not substantially different in neutralization kinetics from virus harboring both regulators. Electron microscopy showed that VSV neutralization proceeded through virion aggregation followed by lysis, with virion-associated CD55 providing a delay in both aggregation and lysis more substantial than that conferred by CD46. Our results demonstrate the functional significance of incorporation of host cell factors during virion envelope assembly. They also define pathways of virus complement-mediated neutralization and suggest the design of more effective viral vectors.

By the peak of the CD8+ T cell response, the effector cell pool consists of a heterogeneous population of cells that includes both those with an increased propensity to become long-lived memory cells (memory precursor effector cells; MPEC) and those that are terminally differentiated cells (short-lived effector cells; SLEC). Numerous studies have established the critical role that functional avidity plays in determining the in vivo efficacy of CD8+ effector cells. Currently, how functional avidity differs in MPEC versus SLEC and the evolution of this property within these two populations during the expansion and contraction of the response are unknown. The data presented in this study show that at the peak of the effector response generated after poxvirus infection, SLEC were of higher functional avidity than their MPEC counterpart. Over time, however, SLEC exhibited a decrease in peptide sensitivity. This is in contrast to MPEC, which showed a modest increase in peptide sensitivity as the response reached equilibrium. The decrease in functional avidity in SLEC was independent of CD8 modulation or the amount of Ag receptor expressed by the T cell. Instead, the loss in sensitivity was correlated with decreased expression and activation of ZAP70 and Lck, critical components of TCR membrane proximal signaling. These results highlight the potential contribution of avidity in the differentiation and evolution of the T cell effector response after viral infection.

A large number of viral infections are contracted via the respiratory route. Thus, an effective immune response at this site is of vital importance. Past studies in murine models analyzing a number of viruses have reported that CD8(+) effector T cells entering the lung after respiratory infection exhibit significant functional inactivation. The impaired function in these cells has been proposed to be the result of infection-induced changes in the lung; however, we have found that loss of function can occur in effector CD8(+) T cells present in the lung, even in the absence of infection. This functional inactivation takes place within 48 hours of entry into the lung, and is seen only in effector cells residing in the lung parenchyma, and not the airway. In this study, we have extended our findings to show that functional impairment of these effector cells is not initiated by bone marrow-derived cells, and is independent of proliferation in the lung tissue. Of critical importance, we have also determined that the susceptibility to functional inactivation is a common property shared by most effector cells. Finally, we show that the susceptibility to loss of function is actively regulated throughout differentiation. Although naive CD8(+) T cells, like effector cells, are negatively regulated as a result of residence in the lung, memory cells exhibit profound resistance to functional inactivation. The selective resistance of CD8(+) memory cells may allow the host to limit damage during the effector phase while retaining a protective response that can effectively limit subsequent infection.

A large number of dendritic cell (DC) subsets have now been identified based on the expression of a distinct array of surface markers as well as differences in functional capabilities. More recently, the concept of unique subsets has been extended to the lung, although the functional capabilities of these subsets are only beginning to be explored. Of particular interest are respiratory DCs that express CD103. These cells line the airway and act as sentinels for pathogens that enter the lung, migrating to the draining lymph node, where they add to the already complex array of DC subsets present at this site. Here we assessed the contributions of these individual populations to the generation of a CD8+T-cell response following respiratory infection with poxvirus. We found that CD103+DCs were the most effective antigen-presenting cells (APC) for naive CD8+T-cell activation. Surprisingly, we found no evidence that lymph node-resident or parenchymal DCs could prime virus-specific cells. The increased efficacy of CD103+DCs was associated with the increased presence of viral antigen as well as high levels of maturation markers. Within the CD103+DCs, we observed a population that expressed CD8α. Interestingly, cells bearing CD8α were less competent for T-cell activation than their CD8α−counterparts. These data show that lung-migrating CD103+DCs are the major contributors to CD8+T-cell activation following poxvirus infection. However, the functional capabilities of cells within this population differ with the expression of CD8, suggesting that CD103+cells may be divided further into distinct subsets.

Recently, several studies, including those with respiratory syncytial virus, mouse pneumovirus, and simian virus 5, have reported that virus-specific CD8+ effector cells entering the lung as a result of respiratory infection undergo significant loss of function. The impaired function in these cells has been proposed to be the result of infection-induced changes in the lung. Although virus-specific effects may contribute to regulation of T cells in the lung, the findings from this study provide evidence that the basal lung environment is sufficient to promote loss of function in effector cells. Loss of function occurs within 48 h of entry into the lung and is most evident in cells residing in the lung parenchyma. These findings suggest an additional paradigm for the immunoregulation of effector cells that enter the lung as a result of virus infection.

The generation of an optimal CD8(+) cytotoxic T lymphocyte (CTL) response is critical for the clearance of many intracellular pathogens. Previous studies suggest that one contributor to an optimal immune response is the presence of CD8(+) cells exhibiting high functional avidity. In this regard, CD8 expression has been shown to contribute to peptide sensitivity. Here, we investigated the ability of naive splenocytes to modulate CD8 expression according to the concentration of stimulatory peptide antigen. Our results showed that the level of CD8 expressed was inversely correlated with the amount of peptide used for the primary stimulation, with higher concentrations of antigen resulting in lower expression of both CD8alpha and CD8beta. Importantly the ensuing CD8(low) and CD8(high) CTL populations were not the result of the selective outgrowth of naive CD8(+) T-cell subpopulations expressing distinct levels of CD8. Subsequent encounter with peptide antigen resulted in continued modulation of both the absolute level and the isoform of CD8 expressed and in the functional avidity of the responding cells. We propose that CD8 cell surface expression is not a static property, but can be modulated to 'fine tune' the sensitivity of responding CTL to a defined concentration of antigen.

The property of functional avidity is recognized to be of critical importance in determining pathogen clearance. An unresolved question with regard to this property is whether distinct naive subsets exist that display inherent differences in their peptide sensitivity requirements for activation, i.e., functional avidity, or whether differences in peptide sensitivity are induced following peptide encounter. In this study, we demonstrate that naive populations that can give rise to both high- and low-avidity cells do not contain subsets that exhibit differences in the amount of peptide required for activation. Furthermore, we show that an individual T cell clone can generate both high- and low-avidity effectors. The work presented here provides the first formal demonstration that an individual cell can give rise to both high- and low-avidity progeny, suggesting that avidity modulation at the level of an individual cell may play an important role in the CD8+ T cell response generated in vivo.

Spontaneous regression/complete resistance (SR/CR) mice resist very high doses of cancer cells that are lethal to WT mice even at low doses. In this study, we show that this resistance is mediated by rapid infiltration of leukocytes, mostly of innate immunity, in both primary and repeated challenges. Formation of rosettes with infiltrating natural killer cells, neutrophils, and macrophages was required for the subsequent destruction of cancer cells through rapid cytolysis. Highly purified natural killer cells, macrophages, and neutrophils from the SR/CR mice independently killed cancer cellsin vitro. The independent killing activity by each subset of effector cells is consistent with the observation that the resistance was abolished by depleting total infiltrating leukocytes but not by depleting only one or two subsets of leukocytes. The resistance was completely transferable to WT recipient mice through SR/CR splenocytes, bone marrow cells, or enriched peritoneal macrophages, either for prevention against subsequent cancer challenges or eradication of established malignancy at distant sites.

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.

CD8 CTLs are a major effector for protection against cancer as well as many infectious diseases, including HIV/AIDS. CD8 CTL recognize antigenic peptides in the context of class I MHC. CTL functional avidity has been shown to be an important determinant of in vivo efficacy. CTL that can recognize peptide/MHC only at high antigen density are termed low avidity CTL, while those that can recognize their cognate antigen at low densities are termed high avidity CTL. Recent studies have demonstrated that high avidity CTLs are essential for the effective clearance of viral infections and for the elimination of tumor cells. At this time, approaches that can target high avidity cells for expansion in vivo are not well defined; however, new insights are beginning to emerge. A recent study has shown that prime-boost immunization may be an effective method to generate high avidity CTLs that recognize HIV antigens. In addition, we recently found that high levels of costimulation (signal 2) can skew the CTL response toward higher avidity cells. Thus, vectors expressing a triad of costimulatory molecules (TRICOM) or dendritic cells expressing higher levels of costimulatory molecules, can be used to induce high avidity CTL. Finally a critical role for CD4+ T cell help in the generation of high avidity cells has recently been identified (Palmer, manuscript submitted). While high avidity CTLs are superior for viral and tumor clearance, they also have a greater sensitivity to antigen induced cell death. In some types of chronic infections, such as HIV and HCV, as well as in cancer, the host may lose, by clonal exhaustion or other apoptotic mechanisms, the effector cells that are most critical to viral or tumor clearance. In this review, we examine the current knowledge concerning CTL avidity. We discuss the factors that may distinguish high avidity CTLs from low avidity CTLs and describe some of the mechanisms these cells use to clear viral infections. In addition, we study possible immunization strategies that may be used to elicit higher avidity CTLs and describe what is known about the factors that render these cells more susceptible to apoptosis than low avidity CTLs. Finally, we will incorporate these various elements into a general discussion of possible approaches for induction and maintenance of an effective immune response that can result in clearance of tumors or chronic viral infections and the relevance to vaccine development.

We have established and studied a colony of mice with a unique trait of host resistance to both ascites and solid cancers induced by transplantable cells. One dramatic manifestation of this trait is age-dependent spontaneous regression of advanced cancers. This powerful resistance segregates as a single-locus dominant trait, is independent of tumor burden, and is effective against cell lines from multiple types of cancer. During spontaneous regression or immediately after exposure, cancer cells provoke a massive infiltration of host leukocytes, which form aggregates and rosettes with tumor cells. The cytolytic destruction of cancer cells by innate leukocytes is rapid and specific without apparent damage to normal cells. The mice are healthy and cancer-free and have a normal life span. These observations suggest a previously unrecognized mechanism of immune surveillance, which may have potential for therapy or prevention of cancer.

Following intranasal administration, the model paramyxovirus simian virus 5 (SV5) establishes an infection in the respiratory tract of mice, which is subsequently cleared by CD8+ T cells. In this study, we sought to understand the maturation of the antiviral immune response over time by assessing the functional avidity of the responding T cells and the expansion of immunodominant populations. Surprisingly, we determined that the initial response to Ag at day 3 (d3) in the mediastinal lymph node was exclusively high avidity. However, by d5 postinfection, low avidity cells were ∼50% of the responding T cell population. Following secondary exposure to SV5, high avidity CD8+ T cells again are the exclusive cell type present at early times postinfection (d2). Similarly, high avidity cells were preferentially elicited at d3 following infection with the unrelated vaccinia virus. We also made the observation that the immunodominance profile has not been established at d3 postinfection with SV5. However, by d5 a clear immunodominance pattern arises and is permanently maintained. These data indicate that high avidity cells are the predominant population responding at early times postinfection following respiratory infection with SV5 or vaccinia virus. However, as the response progresses, low avidity cells are activated/expanded to a greater extent compared with high avidity cells.

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.

The importance of high avidity CTL for the effective clearance of viral infections is now well established. Thus one would predict that the preferential activation and expansion of high avidity CTL following viral challenge and retention of these cells in the memory pool would be optimal for the immune response. However, whether this actually occurs during the immune response to viral infection is unknown. In this report I have analyzed the avidity of the CTL specific for the OVA 257–264 peptide during acute infection with a recombinant vaccinia expressing ovalbumin and in the memory population. I have found that the relative ratio of high and low avidity CTL varies over the course of an immune response. Thus CTL avidity is an important factor in the expansion and survival of CTL in vivo.

During an immune response, CTL clones are present that exhibit a wide spectrum of peptide sensitivities. While the property of functional avidity has been well documented, the range of mechanism(s) by which this T cell property can be regulated is unknown. For example, an unresolved question is whether distinct naïve T cell subsets exist that display “inherent” differences in their peptide requirements for activation, i.e. functional avidity. In this scenario distinct naïve T cell clones would selectively respond upon encountering a high vs. low antigen concentration. Alternatively, differences in peptide sensitivity could be an “induced” property within the responding CTL population following encounter with an APC. Although the latter model is consistent with data from previous studies, formal demonstration that a single clone can give rise to both high and low avidity daughter cells is lacking. Using a TCR transgenic model in which both high and low avidity CTL can be generated, we now demonstrate that naïve T cells do not exhibit differences in the amount of peptide required for conjugate formation and subsequent proliferation/activation. Furthermore, we show that individual T cell clones are capable of differentiating into either a high or low avidity CTL. These results provide the first formal demonstration that an individual cell can give rise to both high and low avidity progeny, suggesting that avidity modulation at the level of an individual cell may be an important contributor to the functional avidity of the CD8+ T cell response generated in vivo. This work was supported by National Institutes of Health grant R01 AI43591 to M.A.A-M.

Cytotoxic T lymphocytes (CTL) form an important immune surveillance system against intracellular pathogens. Here we describe a simple, visual assay for identifying peptides specifically recognized by CTL, based on the discovery that CTL develop increased adhesive properties upon TCR triggering. Several CTL lines were shown to pellet to the bottom of a round bottom 96-well plate in the absence of peptide. In contrast, these same CTL lines incubated with their cognate peptide, allowing them to present peptide to each other, adhered to the sides of the well and were readily distinguished by macroscopic visual examination of the plate after 4-5 h or overnight incubation. This CTL adherence assay (CAA) demonstrated peptide specificity and MHC restriction, and was titratable with peptide concentration. With this technique, a minimal-sized, malaria CTL epitope was correctly identified from a panel of overlapping nonamers, although the adherence pattern of two mono-substituted, variant peptides was less predictive of lytic activity. Also, substitutions in an HIV-1 envelope CTL epitope that reduced lytic activity were correctly predicted. Inhibitors of RNA and protein synthesis, upon preincubation, abrogated the adherence, indicating, at minimum, a need for live cells. Wortmannin, a PI-3 kinase inhibitor, inhibited the peptide specific adherence, consistent with a role for TCR or integrin signal transduction in CAA. Other cytoskeletal and metabolic inhibitors had no effect. Adherence of the T cells may involve low affinity, nonspecific interactions since wells coated with FCS, BSA or milk powder all produced an effective CAA in the presence of peptide under serum free conditions. Consequently, CAA may represent a rapid, simple method for screening large numbers of peptides to find cytolytic epitopes for a given CTL line and may identify additional epitopes causing T cell activation and adherence but not cytolytic activity.

The conventional approach to cytotoxic T-lymphocyte (CTL) induction uses maximal antigen concentration with the intent of eliciting more CTL. However, the efficacy of this approach has not been systematically explored with regard to the quality of the CTLs elicited or their in vivo functionality. Here, we show that a diametrically opposite approach elicits CTLs that are much more effective at clearing virus. CTLs specific for a defined peptide epitope were selectively expanded with various concentrations of peptide antigen. CTLs generated with exceedingly low-dose peptide lysed targets sensitized with > 100-fold less peptide than CTLs generated with high-dose peptide. Differences in expression of T-cell antigen receptors or a number of other accessory molecules did not account for the functional differences. Further, high-avidity CTLs adoptively transferred into severe combined immunodeficient mice were 100- to 1000-fold more effective at viral clearance than the low-avidity CTLs, despite the fact that all CTL lines lysed virus-infected targets in vitro. Thus, the quality of CTLs is as important as the quantity of CTLs for adoptive immunotherapy, and the ability to kill virally infected targets in vitro is not predictive of in vivo efficacy, whereas the determinant density requirement described here is predictive. Application of these principles may be critical in developing effective adoptive cellular immunotherapy for viral infections and cancer.

CTLs that display high functional avidity are known to be optimal for pathogen clearance. However, the molecular mechanism that allows high avidity CTLs to respond at very low levels of peptide antigen remains to be elucidated. To investigate this question, we compared TCR signal transduction events in high and low avidity CD8+ T-cells generated from OT-I TCR transgenic mice. Upon stimulation with APCs preloaded with a range of Ova257-264 peptide concentrations (10^-6, 10^-9 and 10^-12 M), we found that high avidity CD8+ T-cells induced higher levels of MAPK ERK-1/2 phosphorylation as well as higher levels of the dephosphorylated form of NFAT at each peptide concentration used for stimulation. We next determined whether the observed divergence in NFAT activation was reflected in differences in calcium mobilization. We found efficient mobilization of Ca2+ in high avidity cells even when the lowest concentration of peptide was used for stimulation, a level of peptide at which no increase in calcium was observed in low avidity cells. The decreased peptide requirement for calcium mobilization in high avidity cells correlated with a decreased peptide requirement for ZAP-70 activation and CD3ζ phosphorylation, the latter of which was higher and prolonged in high versus low avidity cells. Thus the functional avidity of the CD8+ effector cells studies here is controlled by differential regulation of the most membrane proximal event, phosphorylation of CD3ζ. Studies are currently underway to determine the mechanism by which high avidity cells acheive CD3ζ phosphorylation at limiting peptide levels.

Recently there has been renewed interest in poxvirus pathogenesis. Vaccinia virus (VACV) serves as a useful model for the study of poxvirus infection in vivo. Understanding how vaccinia virus generates immunity necessitates an appreciation for how this virus interacts with dendritic cells (DC), which are the most potent activators of naïve T cells. A number of in vitro studies have shown that DC infected with VACV fail to undergo maturation. However, the effect of VACV infection on DC in vivo remains less defined. We have found that following intravenous administration of vaccinia virus, both CD8+ and CD8− dendritic cells are infected. The number of infected DC peaks at 6 hours and is highly decreased by 24h postinfection. Dendritic cells infected with vaccinia virus have an immature phenotype. However, non-infected dendritic cells, both CD8+ and CD8− subsets, show increased expression of CD80, CD86, and CD40, suggesting that there is a generalized maturation of non-infected DC. In contrast to the generalized upregulation of costimulatory molecules, cytokine analysis suggests that only a subset of these mature DC may be relevant for activation of T cells, as IL-12 production is restricted to a small percentage of cells. These findings provide new insights into the control of DC maturation in vivo following vaccinia virus infection. This work was supported by NIH grant P01 AI60642.

Understanding how vaccinia virus (VV) generates immunity necessitates an appreciation for how this virus interacts with dendritic cells (DC), which are the most potent activators of naive CD8(+) T cells. In order to optimally activate naive CD8(+) T cells, DC must undergo maturation, during which costimulatory molecules are upregulated and cytokines are produced. In this report, we show that VV infection of immature murine bone marrow-derived DC (BMDC) failed to induce maturation. Similar results were obtained when CD8(+) DC were analyzed, a subset shown previously to be important in vivo in the generation of a vaccinia-specific response. The finding that VV infection of DC resulted in APC that were incapable of initiating T-cell activation was surprising given the previously reported role for direct presentation in the generation of anti-VV CD8(+) T-cell responses in mice. To address the potential mechanism responsible for direct presentation, we tested the hypothesis that previously matured DC were susceptible to vaccinia virus infection and could present newly synthesized VV-derived epitopes for CD8(+) T-cell activation. Our results show, that during VV infection of mature DC, threshold levels of viral protein are produced that promote T-cell activation. These results suggest that, even though VV cannot mature DC, previously matured DC exposed to VV can generate a VV-specific CD8(+) T-cell response providing a potential mechanism by which direct infection results in T-cell activation in vivo.