Generation of virus-specific cytotoxic T cells in vitro. I. Induction conditions of primary and secondary Sendai virus-specific cytotoxic T cells.

H-2-restricted cytotoxic T cells specific for Sendai virus were generated in vitro in a primary response from normal mouse lymphocytes cultured in the presence of infective as well as inactivated Sendai virus. Antigen-presenting cells of different origin, including T cells, were found to be effective stimulators. Antibodies to Sendai virus were shown to inhibit the activation of specific precursor killer cells when added to cultures before, but not after, the addition of viral antigen. Data obtained by Lyt phenotyping, revealed that precursor killer cells specific for Sendai virus reside in the Lyt-2,3+ T cell population and that Lyt-1,2,3+ T cells are not required for the generation of cytotoxic lymphocytes. Different activation kinetics were demonstrated for primary and secondary antiviral cytotoxic responses, and the analysis of the proliferation and stimulation requirements suggests qualitative differences.     

Shared determinants between virus-infected and trinitrophenyl-conjugated H-2-identical target cells detected in cell-mediated lympholysis.

Infection of H-2-identical mice with either lymphocytic choriomeningitis (LCM) virus, vaccinia virus, or paramyxo (Sendai) virus resulted in the generation of specifically sensitized cytotoxic T lymphocytes (CTL). CTL generated in vitro against 2,4,6-trinitrophenyl (TNP)-conjugated syngeneic stimulator cells were specifically cytotoxic for TNP-conjugated H-2K (D) region identical targets. Both LCM and vaccinia-induced CTL, however, were found to be strongly cytotoxic towards TNP-conjugated, H-2K(D) region-identical target cells. In contrast, Sendai virus-induced CTL did not lyse TNP-conjugated, syngeneic target cells. Inhibition experiments using cold targets suggested that shared antigenic determinants can be detected on either LCM virus-infected and TNP-conjugated targets, which are not present on the cell surface of normal target cells.     

The proteasome regulator PA28alpha/beta can enhance antigen presentation without affecting 20S proteasome subunit composition

PA28alpha/beta is a regulatory complex of the 20S proteasome which consists of two IFN-gamma inducible subunits. Both subunits, alpha and beta, contribute equally to the formation of hexa- or heptameric rings which can associate with the 20S proteasome. Previously, we have shown that overexpression of the PA28alpha subunit enhanced the MHC class I-restricted presentation of two viral epitopes and that purified PA28alpha/beta accelerated T cell epitope generation by the 20S proteasome in vitro, indicating a role for PA28alpha/beta in antigen presentation. This conclusion was recently confirmed in PA28beta gene targeted mice which were severely deficient in MHC class I-restricted antigen presentation. These mice displayed a defect in the assembly of immunoproteasomes, suggesting that a lack of the proteasome subunits LMP2, LMP7, and MECL-1 may account for the deficiency in antigen presentation. In this study we investigated whether the effect of PA28alpha/beta on antigen presentation is dependent on a change of proteasome subunit composition. We have analyzed the assembly and subunit composition of proteasomes in fibroblast transfectants overexpressing both, alpha and beta subunits of PA28. In these transfectants we found a marked enhancement in the presentation of the immunodominant H-2Ld-restricted pp89 epitope of murine cytomegalovirus, although the 20S proteasome composition was the same as in recipient cells. We, therefore, conclude that PA28alpha/beta can enhance antigen processing independently of changes in 20S proteasome subunit composition or assembly.     

Transgenic mice expressing a soluble foreign H-2 class I antigen are tolerant to allogeneic fragments presented by self class I but not to the whole membrane-bound alloantigen.

The properties of transmembrane and soluble transplantation antigens were compared with respect to the induction of tolerance and the selection of the T-cell repertoire. For this purpose, transgenic (H-2b x H-2d)F1 mice were constructed that carry integrated copies of a modified H-2Kk gene resulting in the secretion from various cell types including thymocytes of soluble H-2Kk molecules. Despite the presence of H-2Kk antigen, these mice were still able to generate an H-2Kk-specific T-cell response. This response was comparable to that produced by normal littermates when stimulated with cells expressing membrane H-2Kk in a mixed lymphocyte reaction. In contrast, only transgenic mice failed to generate a cytolytic T-cell response to soluble H-2Kk antigen expressed by recombinant vaccinia virus and presented by the H-2Db molecule. These data imply the presence of two populations of alloreactive cytolytic T cells. A small fraction of T cells recognizes alloantigen as antigenic peptide(s) presented by other major histocompatibility complex class I molecules and tolerance can be induced in this population by soluble alloantigen. The majority of T cells, however, require the whole cell membrane-expressed class I molecule for recognition. This population is not affected by tolerance induction to the soluble major histocompatibility complex class I molecule.     

MCMV glycoprotein gp40 confers virus resistance to CD8+ T cells and NK cells in vivo

The susceptibility of certain inbred mouse strains to murine cytomegalovirus (MCMV) is related to their inability to generate a strong natural killer (NK) cell response. We addressed here whether the MCMV susceptibility of the BALB/c strain is due to viral functions that control NK cell activation in a strain-specific manner. MCMV expresses two proteins, gp48 and gp40, that are encoded by the genes m06 and m152, respectively; they down-regulate major histocompatibility complex (MHC) class I expression at the plasma membrane. Using MCMV deletion mutants and revertants, we found that gp40 but not gp48 controls NK cell activation. Absence of gp40 improved antiviral NK cell control in BALB/c, but not C57BL/6, mice. Down-regulation of H-60, the high-affinity ligand for the NKG2D receptor, was the mechanism by which gp40 modulates NK cell activation. Thus, a single herpesvirus protein has a dual function in inhibiting both the adaptive as well as the innate immune response.     

Site-restricted persistent cytomegalovirus infection after selective long-term depletion of CD4+ T lymphocytes

We have established a murine model system for exploring the ability of a CD4 subset-deficient host to cope with cytomegalovirus infection, and reported three findings. First, an antiviral response of the CD8 subset of T lymphocytes could be not only initiated but also maintained for a long period of time despite a continued absence of the CD4 subset, whereas the production of antiviral antibody proved strictly dependent upon help provided by the CD4 subset. Second, no function in the defense against infection could be ascribed as yet to CD4-CD8- T lymphocytes, which were seen to accumulate to a new subset as a result of depletion of the CD4 subset. This newly arising subset did not substitute for CD4+ T lymphocytes in providing help to B lymphocytes, and was also not effective in controlling the spread of virus in host tissues. As long as a function of these cells in the generation and maintenance of a CD8 subset-mediated response is not disproved, caution is indicated with concern to an autonomy of the CD8 subset. Third, even though with delay, the CD8+ effector cells raised in the CD4 subset-deficient host were able of clear vital tissues from productive infection and to restrict asymptomatic, persistent infection to acinar glandular epithelial cells in salivary gland tissue.     

Inactivation of a Defined Active Site in the Mouse 20S Proteasome Complex Enhances Major Histocompatibility Complex Class I Antigen Presentation of a Murine Cytomegalovirus Protein

Proteasomes generate peptides bound by major histocompatibility complex (MHC) class I molecules. Avoiding proteasome inhibitors, which in most cases do not distinguish between individual active sites within the cell, we used a molecular genetic approach that allowed for the first time the in vivo analysis of defined proteasomal active sites with regard to their significance for antigen processing. Functional elimination of the δ/low molecular weight protein (LMP) 2 sites by substitution with a mutated inactive LMP2 T1A subunit results in reduced cell surface expression of the MHC class I H-2L^d and H-2D^d molecules. Surface levels of H-2L^d and H-2D^d molecules were restored by external loading with peptides. However, as a result of the active site mutation, MHC class I presentation of a 9-mer peptide derived from a protein of murine cytomegalovirus was enhanced about three- to fivefold. Our experiments provide evidence that the δ/LMP2 active site elimination limits the processing and presentation of several peptides, but may be, nonetheless, beneficial for the generation and presentation of others.     

Dendritic cells under influence of mouse cytomegalovirus have a physiologic dual role: to initiate and to restrict T cell activation

The aim of this study is to analyze the dynamics of the mouse cytomegalovirus (MCMV)-dendritic cell (DC) interaction. Immature and mature DCs derived from the mouse stem cell line factor-dependent cell Paterson mixed potential were infected with a recombinant MCMV expressing green fluorescent protein. Infection of immature DCs resulted in DC activation and virus production, both of which may contribute to viral dissemination. The infection of mature DCs was nonproductive and was restricted to immediate-early and early viral protein expression. During early stages of MCMV infection, mature DCs up-regulated major histocompatibility complex (MHC) and costimulatory molecules and activated autologous, but not allogeneic, naive T cells. At later times of MCMV infection, DCs prevented T cell activation by down-regulation of MHC and costimulatory molecules. Thus, DCs under the influence of MCMV have a physiologic dual role: to initiate and to restrict T cell activation. The lack of immunostimulation in allogeneic settings may explain the increased risk of MCMV morbidity after allogeneic transplantation.     

Redistribution of critical major histocompatibility complex and T cell receptor-binding functions of residues in an antigenic sequence after biterminal substitution

Residues critical for establishing a trimolecular interaction with a major histocompatibility complex (MHC)-encoded receptor and a T cell antigen receptor (TcR) were determined for an antigenic nonapeptide. The N-terminal residue proved to be involved in binding of the peptide to both receptors and the C-terminal residue was essential for MHC binding. While substitution of either of these critical terminal residues by alanine resulted in an almost complete loss of peptide antigenicity, simultaneous substitution of both created a new functional ligand for the same MHC molecule and the same TcR. Notably, in the biterminally substituted peptide, the core residues took on new roles in the trimolecular interaction in that a residue critical in the authentic nonapeptide for TcR binding became critical for MHC binding and former spacer residues became essential to various degrees for the interaction with either receptor or both. Thus, apparently, the loss of the terminal residues' contribution was at least partially compensated by a redistribution of the roles among the remaining residues. These results reflect a cooperative contribution of all residues of an antigenic peptide to its binding to both receptors and thus challenge a static definition of agretope and epitope as MHC and TcR binding sites.