The human CD8 coreceptor effects cytotoxic T cell activation and antigen sensitivity primarily by mediating complete phosphorylation of the T cell receptor zeta chain.

Recognition of antigen by cytotoxic T lymphocytes (CTL) is determined by interaction of both the T cell receptor and its CD8 coreceptor with peptide-major histocompatibility complex (pMHC) class I molecules. We examine the relative roles of these receptors in the activation of human CTL using mutations in MHC class I designed to diminish or abrogate the CD8/pMHC interaction. We use surface plasmon resonance to determine that point mutation of the alpha3 loop of HLA A2 abrogates the CD8/pMHC interaction without affecting the affinity of the T cell receptor/pMHC interaction. Antigen-presenting cells expressing HLA A2 which does not bind to CD8 fail to activate CTL at any peptide concentration. Comparison of CTL activation by targets expressing HLA A2 with normal, abrogated, or diminished CD8/pMHC interaction show that the CD8/pMHC interaction enhances sensitivity to antigen. We determine that the biochemical basis for coreceptor dependence is the activation of the 23-kDa phosphoform of the CD3zeta chain. In addition, we produce mutant MHC class I multimers that specifically stain but do not activate CTL. These reagents may prove useful in circumventing undesirable activation-related perturbation of intracellular processes when pMHC multimers are used to phenotype antigen-specific CD8+ lymphocytes.     

Rational design of cytotoxic T-cell inhibitors

This study describes the use of the CD8/major histocompatibility complex (MHC) class I crystal structure as a template for the de novo design of low-molecular-weight surface mimetics. The analogs were designed from a local surface region on the CD8 alpha-chain directly adjacent to the bound MHC class I, to block the protein associations in the T-cell activation cluster that occur upon stimulation of the cytotoxic T lymphocytes (CTLs). One small conformationally restrained peptide showed dose-dependent inhibition of a primary allogeneic CTL assay while having no effect on the CD4-dependent mixed lymphocyte reaction (MLR). The analog's activity could be modulated through subtle changes in its side chain composition. Administration of the analog prevented CD8-dependent clearance of a murine retrovirus in BALB/c mice. In C57BL/6 mice challenged with the same retrovirus, the analog selectively inhibited the antiviral CTL responses without affecting the ability of the CTLs to generate robust allogeneic responses.     

Classical and nonclassical class I major histocompatibility complex molecules exhibit subtle conformational differences that affect binding to CD8alphaalpha

The cell surface molecules CD4 and CD8 greatly enhance the sensitivity of T-cell antigen recognition, acting as "co-receptors" by binding to the same major histocompatibility complex (MHC) molecules as the T-cell receptor (TCR). Here we use surface plasmon resonance to study the binding of CD8alphaalpha to class I MHC molecules. CD8alphaalpha bound the classical MHC molecules HLA-A*0201, -A*1101, -B*3501, and -C*0702 with dissociation constants (K(d)) of 90-220 microm, a range of affinities distinctly lower than that of TCR/peptide-MHC interaction. We suggest such affinities apply to most CD8alphaalpha/classical class I MHC interactions and may be optimal for T-cell recognition. In contrast, CD8alphaalpha bound both HLA-A*6801 and B*4801 with a significantly lower affinity (>/=1 mm), consistent with the finding that interactions with these alleles are unable to mediate cell-cell adhesion. Interestingly, CD8alphaalpha bound normally to the nonclassical MHC molecule HLA-G (K(d) approximately 150 microm), but only weakly to the natural killer cell receptor ligand HLA-E (K(d) >/= 1 mm). Site-directed mutagenesis experiments revealed that variation in CD8alphaalpha binding affinity can be explained by amino acid differences within the alpha3 domain. Taken together with crystallographic studies, these results indicate that subtle conformational changes in the solvent exposed alpha3 domain loop (residues 223-229) can account for the differential ability of both classical and nonclassical class I MHC molecules to bind CD8.     

Mapping the binding site on CD8 beta for MHC class I reveals mutants with enhanced binding

In an effective immune response, CD8+ T cell recognition of virally derived Ag, bound to MHC class I, results in killing of infected cells. The CD8alphabeta heterodimer acts as a coreceptor with the TCR, to enhance sensitivity of the T cells to peptide/MHC class I, and is two orders of magnitude more efficient as a coreceptor than the CD8alphaalpha. To understand the important interaction between CD8alphabeta and MHC class I, we created a panel of CD8beta mutants and identified mutations in the CDR1, CDR2, and CDR3 loops that decreased binding to MHC class I tetramers as well as mutations that enhanced binding. We tested the coreceptor function of a subset of reducing and enhancing mutants using a T cell hybridoma and found similar reducing and enhancing effects. CD8beta-enhancing mutants could be useful for immunotherapy by transduction into T cells to enhance T cell responses against weak Ags such as those expressed by tumors. We also addressed the question of the orientation of CD8alphabeta with MHC class I using CD8alpha mutants expressed as a heterodimer with wild-type CD8alpha or CD8beta. The partial rescuing of binding with wild-type CD8beta compared with wild-type CD8alpha is consistent with models in which either the topology of CD8alphaalpha and CD8alphabeta binding to MHC class I is different or CD8alphabeta is capable of binding in both the T cell membrane proximal and distal positions.     

CD8alphabeta has two distinct binding modes of interaction with peptide-major histocompatibility complex class I

Interaction of CD8 (CD8alphaalpha or CD8alphabeta) with the peptide-major histocompatibility complex (MHC) class I (pMHCI) is critical for the development and function of cytolytic T cells. Although the crystal structure of CD8alphaalpha.pMHCI complex revealed that two symmetric CD8alpha subunits interact with pMHCI asymmetrically, with one subunit engaged in more extensive interaction than the other, the details of the interaction between the CD8alphabeta heterodimer and pMHCI remained unknown. The Ig-like domains of mouse CD8alphabeta and CD8alphaalpha are similar in the size, shape, and surface electrostatic potential of their pMHCI-binding regions, suggesting that their interactions with pMHCI could be very similar. Indeed, we found that the CD8alpha variants CD8alpha(R8A) and CD8alpha(E27A), which were functionally inactive as homodimers, could form an active co-receptor with wild-type (WT) CD8beta as a CD8alpha(R8A)beta or CD8alpha(E27A)beta heterodimer. We also identified CD8beta variants that could form active receptors with WT CD8alpha but not with CD8alpha(R8A). This observation is consistent with the notion that the CD8beta subunit may replace either CD8alpha subunit in CD8alphaalpha.pMHCI complex. In addition, we showed that both anti-CD8alpha and anti-CD8beta antibodies were unable to completely block the co-receptor activity of WT CD8alphabeta. We propose that CD8alphabeta binds to pMHCI in at least two distinguishable orientations.     

The beta1 and beta3 integrins promote T cell receptor-mediated cytotoxic T lymphocyte activation

Recognition by CD8+ cytotoxic T lymphocytes (CTLs) of antigenic peptides bound to major histocompatibility class (MHC) I molecules on target cells leads to sustained calcium mobilization and CTL degranulation resulting in perforin-dependent killing. We report that beta1 and beta3 integrin-mediated adhesion to extracellular matrix proteins on target cells and/or surfaces dramatically promotes CTL degranulation. CTLs, when adhered to fibronectin but not CTL in suspension, efficiently degranulate upon exposure to soluble MHC.peptide complexes, even monomeric ones. This adhesion induces recruitment and activation of the focal adhesion kinase Pyk2, the cytoskeleton linker paxillin, and the Src kinases Lck and Fyn in the contact site. The T cell receptor, by association with Pyk2, becomes part of this adhesion-induced activation cluster, which greatly increases its signaling.     

Antigen presentation of a modified tumor-derived peptide by tumor infiltrating lymphocytes

CD8+ T-lymphocytes recognize peptides in the context of major histocompatibility complex (MHC) class I antigens. Upon activation, these cells differentiate into effector cytotoxic T lymphocytes (CTL) and no longer require formal antigen presentation by professional antigen presenting cells (APC). Subsequently, any cell expressing MHC class I/cognate peptide can stimulate CTL. Using TIL specific for a melanoma antigen-derived peptide, IMDQVPFSV (g209 2M), we sought to determine whether these CTL could present peptide to each other. Our findings demonstrate that peptide presentation of the g209 2M peptide epitope by TIL is comparable to conventional methods of using T2 cells as APC. We report here that CTL are capable of self-presentation of antigenic peptide to neighboring CTL resulting in IFN-gamma secretion, proliferation, and lysis of peptide-loaded CTL. These results demonstrate that human TIL possess both APC functions as well as cytotoxic functions and that this phenomenon could influence CTL activity elicited by immunotherapy.     

Macrophages escape inhibition of major histocompatibility complex class I-dependent antigen presentation by cytomegalovirus

The mouse cytomegalovirus (MCMV) m152- and m06-encoded glycoproteins gp40 and gp48, respectively, independently downregulate major histocompatibility complex (MHC) class I surface expression during the course of productive MCMV infection in fibroblasts. As a result, presentation of an immediate-early protein pp89-derived nonapeptide to H-2L(d)-restricted CD8(+) cytotoxic T cells is completely prevented in fibroblasts. Here we demonstrate that MCMV-infected primary bone marrow macrophages and the macrophage cell line J774 constitutively present pp89 peptides during permissive MCMV infection to cytotoxic T lymphocytes (CTL). In contrast to fibroblasts, expression of the m152 and m06 genes in macrophages does not affect surface expression of MHC class I. Assessment of pp89 synthesis and quantification of extracted peptide revealed a significantly higher efficiency of macrophages than of fibroblasts to process pp89 into finally trimmed peptide. The yield of pp89 peptide determined in MCMV-infected tissues of bone marrow chimeras confirmed that bone marrow-derived cells represent a prime source of pp89 processing in parenchymal organs. The finding that macrophages resist the viral control of MHC I-dependent antigen presentation reconciles the paradox of efficient induction of CMV-specific CD8(+) CTL in vivo despite extensive potential of CMVs to subvert MHC class I.     

The role of T8 in the cytotoxic activity of cloned cytotoxic T lymphocyte lines specific for class II and class I major histocompatibility complex antigens

It is reported here that most cytotoxic T lymphocytes (CTL), which recognize class I major histocompatibility complex (MHC) loci, express the T cell differentiation antigen T8. However, a minority of T8+ CTL clones was found to recognize class II MHC antigens. To test the hypothesis that T8 is involved only in T cell recognition of class I MHC antigens, we studied the role of T8 in the cytotoxic activity of class II MHC-specific CTL. Monoclonal antibodies specific for T8 blocked the activity of most class I MHC-specific CTL clones but did not affect the activity of class II MHC-specific CTL clones. Moreover, a mild trypsin treatment of the clones, which removed and T8 determinant, affected the activity of class I MHC but not that of class II MHC-specific CTL clones. These findings indicate that the class II-specific MHC CTL clones described here did not require T8 for their cytolytic activity. The activity of one T8+ class I MHC-specific (HLA-B27) CTL clone (HG-61) against the B cell line JY, which was used to raise this CTL clone, was not blocked by trypsin treatment of this clone. However, the activity of CTL clone HG-61 against target cells different from JY but carrying the appropriate HLA specificity was blocked by anti-T8 antibodies and trypsin treatment. The implications of these findings for the hypothesis that T8 is involved only in the activity of CTL with a relatively low avidity for class I MHC antigens are discussed.     

Construction of bioactive chimeric MHC class I tetramer by expression and purification of human-murine chimeric MHC heavy chain and beta(2)m as a fusion protein in Escherichia coli

Major histocompatibility (MHC) class I tetramers are used in the quantitative analysis of epitope peptide-specific CD8+ T-cells. An MHC class I tetramer was composed of 4 MHC class I complexes and a fluorescently labeled streptavidin (SA) molecule. Each MHC class I complex consists of an MHC heavy chain, a beta(2)-microglobulin (beta(2)m) molecule and a synthetic epitope peptide. In most previous studies, an MHC class I complex was formed in the refolding buffer with an expressed MHC heavy chain molecule and beta(2)m, respectively. This procedure inevitably resulted in the disadvantages of forming unwanted multimers and self-refolding products, and the purification of each kind of monomer was time-consuming. In the present study, the genes of a human/murine chimeric MHC heavy chain (HLA-A2 alpha1, HLA-A2 alpha2 and MHC-H2D alpha3) and beta(2)m were tandem-cloned into plasmid pET17b and expressed as a fusion protein. The recombinant fusion protein was refolded with each of the three HLA-A2 restricted peptides (HBc18-27 FLPSDFFPSI, HBx52-60 HLSLRGLPV, and HBx92-100 VLHKRTLGL) and thus three chimeric MHC class I complexes were obtained. Biotinylation was performed, and its level of efficiency was observed via a band-shift assay in non-reducing polyacrylamide gel electrophoresis (PAGE). Such chimeric MHC class I tetramers showed a sensitive binding activity in monitoring HLA/A2 restrictive cytotoxic T lymphocytes (CTLs) in immunized HLA/A*0201 transgenic mice.     

The complementarity-determining region-like loops of CD8 alpha interact differently with beta 2-microglobulin of the class I molecules H-2Kb and thymic leukemia antigen, while similarly with their alpha 3 domains

The murine CD8 glycoprotein interacts with both classical MHC class I molecules and some nonclassical molecules, including the thymic leukemia Ag (TL). TL binds preferentially to CD8alphaalpha homodimers with a 10-fold higher affinity than H-2K(b) class I molecules. To understand the molecular basis for this difference, we created a panel of CD8alpha mutants and tested the ability of the CD8alphaalpha homodimers to bind to H-2K(b) tetramers and TL tetramers. Mutations in three CD8 residues located on the complementarity-determining region-like loops contacting the negatively charged loop in the alpha3 domain of MHC class I greatly reduced binding to both tetramers. Because TL and H-2K(b) class I sequences are highly conserved in the alpha3 domain of MHC class I, this suggests that CD8 contacts the alpha3 domain of TL and H-2K(b) in a similar manner. In contrast, mutations in residues on the A and B beta strands of CD8 that are involved in contact with beta(2)-microglobulin affected interaction with the H-2K(b) tetramer, but not the TL tetramer. Therefore, the orientation of interaction of TL with CD8 appears to be different from that of H-2K(b). The unique high affinity binding of TL with CD8alphaalpha is most likely a result of amino acid differences in the alpha3 domain between TL and H-2K(b), particularly at positions 198 (K to D) and 228 (M to T), which are contact residues in the CD8alphaalpha-H-2K(b) cocrystal.     

Computational design and crystal structure of an enhanced affinity mutant human CD8 alphaalpha coreceptor

Human CD8 is a T cell coreceptor, which binds to pHLA I and plays a pivotal role in the activation of cytotoxic T lymphocytes. Soluble recombinant CD8 alphaalpha has been shown to antagonize T cell activation, both in vitro and in vivo. However, because of a very low affinity for pHLA I, high concentrations of soluble CD8 alphaalpha are required for efficient inhibition. Based upon our knowledge of the wild-type CD8/pHLA I structure, we have designed and produced a mutated form of soluble CD8 alphaalpha that binds to pHLA I with approximately fourfold higher affinity. We have characterized the binding of the high affinity CD8 mutant using surface plasmon resonance and determined its structure at 2.1 A resolution using X-ray crystallography. The analysis of this structure suggests that the higher affinity is achieved by providing a larger side chain that allows for an optimal contact to be made between the HLA alpha3 loop and the mutated CDR-like loops of CD8.     

CD8 Raft localization is induced by its assembly into CD8alpha beta heterodimers, Not CD8alpha alpha homodimers

The coreceptor CD8 is expressed as a CD8alphabeta heterodimer on major histocompatibility complex class I-restricted TCRalphabeta T cells, and as a CD8alphaalpha homodimer on subsets of memory T cells, intraepithelial lymphocytes, natural killer cells, and dendritic cells. Although the role of CD8alphaalpha is not well understood, it is increasingly clear that this protein is not a functional homologue of CD8alphabeta. On major histocompatibility complex class I-restricted T cells, CD8alphabeta is a more efficient TCR coreceptor than CD8alphaalpha. This property has for the mouse protein been attributed to the recruitment of CD8alphabeta into lipid rafts, which is dependent on CD8beta palmitoylation. Here, these divergent distributions of CD8alphabeta and CD8alphaalpha are demonstrated for the human CD8 proteins as well. However, although palmitoylation of both CD8alpha and CD8beta chains was detected, this modification did not contribute to raft localization. In contrast, arginines in the cytoplasmic domain are crucial for raft localization of CD8betabeta. Most strikingly, the assembly of a non-raft localized CD8beta chain with a non-raft localized CD8alpha chain resulted in raft-localized CD8alphabeta heterodimers. Using chimeric CD8 proteins, this property of the heterodimer was found to be determined by the assembly of CD8alpha and CD8beta extracellular regions. The presence of two CD8alpha extracellular regions, on the other hand, appears to preclude raft localization. Thus, heterodimer formation and raft association are intimately linked for CD8alphabeta. These results emphasize that lipid raft localization is a key feature of human CD8alphabeta that clearly distinguishes it from CD8alphaalpha.     

The CD8 T cell coreceptor exhibits disproportionate biological activity at extremely low binding affinities

T lymphocytes recognize peptides presented in the context of major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells. Recognition specificity is determined by the alphabeta T cell receptor (TCR). The T lymphocyte surface glycoproteins CD8 and CD4 enhance T cell antigen recognition by binding to MHC class I and class II molecules, respectively. Biophysical measurements have determined that equilibrium binding of the TCR with natural agonist peptide-MHC (pMHC) complexes occurs with KD values of 1-50 microm. The pMHCI/CD8 and pMHCII/CD4 interactions are significantly weaker than this (KD >100 microm), and the relative roles of TCR/pMHC and pMHC/coreceptor affinity in T cell activation remain controversial. Here, we engineer mutations in the MHCI heavy chain and beta2-microglobulin that further reduce or abolish the pMHCI/CD8 interaction to probe the significance of pMHC/coreceptor affinity in T cell activation. We demonstrate that the pMHCI/CD8 coreceptor interaction retains the vast majority of its biological activity at affinities that are reduced by over 15-fold (KD > 2 mm). In contrast to previous reports, we observe that the weak interaction between HLA A68 and CD8, which falls within this spectrum of reduced affinities, retains substantial functional activity. These findings are discussed in the context of current concepts of coreceptor dependence and the mechanism by which TCR coreceptors facilitate T cell activation.     

High affinity soluble ILT2 receptor: a potent inhibitor of CD8+ T cell activation

Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin superfamily receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t_1/2) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8⁺ cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with sub-nanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8⁺ CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.     

Development of the CD4 and CD8 lineage of T cells: instruction versus selection

T cells bearing the alpha beta T cell receptor (TCR) can be divided into CD4+8- and CD4-8+ subsets which develop in the thymus from CD4+8+ precursors. The commitment to the CD4 and CD8 lineage depends on the binding of the alpha beta TCR to thymic major histocompatibility complex (MHC) coded class II and class I molecules, respectively. In an instructive model of lineage commitment, the binding of the alpha beta TCR, for instance to class I MHC molecules, would generate a specific signal instructing the CD4+8+ precursors to switch off the expression of the CD4 gene. In a selective model, the initial commitment, i.e. switching off the expression of either the CD4 or the CD8 gene would be a stochastic event which is then followed by a selective step rescuing only CD4+ class II and CD8+ class I specific T cells while CD4+ class I and CD8+ class II specific cells would have a very short lifespan. The selective model predicts that a CD8 transgene which is expressed in all immature and mature T cells should rescue CD4+ class I MHC specific T cells from cell death. We have performed experiments in CD8 transgenic mice which fail to support a selective model and we present data which show that the binding of the alpha beta TCR to thymic class I MHC molecules results in up-regulation of the TCR in the CD4+8+ population. Therefore, these experiments are consistent with an instructive model of lineage commitment.     

Comparison of phosphorylation and internalization of the antigen receptor/CD3 complex, CD8, and class I MHC-encoded proteins on T cells. Role of intracytoplasmic domains analyzed with hybrid CD8/class I molecules

We analyzed the phosphorylation and the dynamics of TCR/CD3, CD8 and MHC class I molecules during the activation of a CD8+ cytotoxic T lymphocyte clone and of CD8- T helper hybridomas transfected with the gene coding for the native (J. Gabert, C. Langlet, R. Zamoyska, J.R. Parnes, A.M. Schmitt-Verhulst, and B. Malissen. 1987. Reconstitution of MHC class I specificity by transfer of the T cell receptor and Lyt-2 genes. Cell 50:545) or truncated CD8 alpha molecule. The CD3 components gamma and epsilon and the CD8 alpha subunit were phosphorylated after activation of the CTL clone with the protein kinase C activator PMA. Class I MHC molecules were phosphorylated irrespective of PMA activation. Constitutive phosphorylation of the MHC class I products was found to be intrinsic to the transmembrane/cytoplasmic portion of the molecules because it was transferred to the CD8 alpha hybrid molecules composed of extracellular CD8 and MHC class I transmembrane and intracytoplasmic domains (CD8-e/MHC-t-i). Measurements of the dynamics of these cell surface molecules by using radiolabeled mAb revealed distinct behaviors: TCR/CD3 complex ligand internalization was increased (around 50% after 40 to 60 min) after PMA activation, whereas the ligand of class I MHC molecules was internalized at constant rate irrespective of PMA activation. Ligand bound to native CD8 molecules was poorly internalized, irrespective of the activation of the T cells with PMA. The same ligand bound to the CD8-e/MHC-t-i hybrid molecule was internalized at the same rate as a class I MHC molecule ligand, indicating that the behavior of the hybrid molecule was characteristic of the transmembrane/cytoplasmic portion of MHC class I molecules.     

Kinetics and thermodynamics of beta 2-microglobulin binding to the alpha 3 domain of major histocompatibility complex class I heavy chain

The major histocompatibility complex (MHC) class I molecule plays a crucial role in cytotoxic lymphocyte function. Functional class I MHC exists as a heterotrimer consisting of the MHC class I heavy chain, an antigenic peptide fragment, and beta2-microglobulin (beta2m). beta2m has been previously shown to play an important role in the folding of the MHC heavy chain without continued beta2m association with the MHC complex. Therefore, beta2m is both a structural component of the MHC complex and a chaperone-like molecule for MHC folding. In this study we provide data supporting a model in which the chaperone-like role of beta2m is dependent on initial binding to only one of the two beta2m interfaces with class 1 heavy chain. beta2-Microglobulin binding to an isolated alpha3 domain of the class I MHC heavy chain accurately models the biochemistry and thermodynamics of beta2m-driven refolding. Our results explain a 1000-fold discrepancy between beta2m binding and refolding of MHC1. The biochemical study of the individual domains of complex molecules is an important strategy for understanding their dynamic structure and multiple functions.     

Expression of herpes simplex virus ICP47 and human cytomegalovirus US11 prevents recognition of transgene products by CD8(+) cytotoxic T lymphocytes

The in vivo persistence of gene-modified cells may be limited by the development of a host immune response to vector-encoded proteins. Herpesviruses evade cytotoxic T-lymphocyte (CTL) recognition by expressing genes which interfere selectively with presentation of viral antigens by class I major histocompatibility complex (MHC) molecules. Here, we studied the use of retroviral vectors encoding herpes simplex virus ICP47, human cytomegalovirus (HCMV) US3, or HCMV US11 to decrease presentation of viral proteins and transgene products to CD8(+) CTL. Human fibroblasts and T cells transduced to express the ICP47, US3, or US11 genes alone exhibited a decrease in cell surface class I MHC expression. The combination of ICP47 and US11 rendered fibroblasts negative for surface class I MHC and allowed a class I MHC-low population of T cells to be sorted by flow cytometry. Fibroblasts and T cells expressing both ICP47 and US11 were protected from CTL-mediated lysis and failed to stimulate specific memory T-cell responses to transgene products in vitro. Our findings suggest that expression of immunoregulatory viral gene products could be a potential strategy to prolong transgene expression in vivo.