Functionally heterogeneous CD8(+) T-cell memory is induced by Sendai virus infection of mice.

It has recently been established that memory CD8(+) T cells induced by viral infection are maintained at unexpectedly high frequencies in the spleen. While it has been established that these memory cells are phenotypically heterogeneous, relatively little is known about the functional status of these cells. Here we investigated the proliferative potential of CD8(+) memory T cells induced by Sendai virus infection. High frequencies of CD8(+) T cells specific for both dominant and subdominant Sendai virus epitopes persisted for many weeks after primary infection, and these cells were heterogeneous with respect to CD62L expression (approximately 20% CD62L(hi) and 80% CD62L(lo)). Reactivation of these cells with the antigenic peptide in vitro induced strong proliferation of antigen-specific CD8(+) T cells. However, approximately 20% of the cells failed to proliferate in vitro in response to a cognate peptide but nevertheless differentiated into effector cells and acquired full cytotoxic potential. These cells also expressed high levels of CD62L (in marked contrast to the CD62L(lo) status of the proliferating cells in the culture). Direct isolation of CD62L(hi) and CD62L(lo) CD8(+) T cells from memory mice confirmed the correlation of this marker with proliferative potential. Taken together, these data demonstrate that Sendai virus infection induces high frequencies of memory CD8(+) T cells that are highly heterogeneous in terms of both their phenotype and their proliferative potential.

Long-term CD8+ T cell memory to Sendai virus elicited by DNA vaccination.

The capacity of DNA vaccines to prime CD8+ T cells makes them excellent candidates for vaccines that are designed to emphasize cellular immunity. However, the long-term stability of CD8+ T cell memory induced by DNA vaccination is poorly characterized. Here, the quality of CD8+ T cell recall responses in mice was investigated more than 1 year after DNA vaccination with the Sendai virus nucleoprotein gene. Cytotoxic T lymphocyte (CTL) activity specific for both dominant and subdominant epitopes could be recalled readily 1 year after vaccination and the frequencies of CTL precursors specific for both of these epitopes were relatively high. These CTL responded strongly to subsequent Sendai virus infection in terms of their ability to migrate to the lung and to differentiate into effector cells. In addition, the recall response to virus infection, as determined by CTL activity in the lungs and IFN-gamma responses in the spleen, was both faster and greater in magnitude than that in control-immunized mice. Significantly, virus titres were reduced at least 100-fold in the lungs of mice that were immunized more than 1 year before infection, as compared with control mice. These data demonstrate that CD8+ T cell memory elicited by DNA vaccination is functionally relevant and persists for at least 1 year.

Enumeration of antigen-presenting cells in mice infected with Sendai virus.

Substantial progress has been made in understanding Ag presentation to T cells; however, relatively little is known about the location and frequency of cells presenting viral Ags during a viral infection. Here, we took advantage of a highly sensitive system using lacZ-inducible T cell hybridomas to enumerate APCs during the course of respiratory Sendai virus infection in mice. Using lacZ-inducible T cell hybridomas specific for the immunodominant hemagglutinin-neuraminidase HN421-436/I-Ab and nucleoprotein NP324-332/Kb epitopes, we detected APCs in draining mediastinal lymph nodes (MLNs), in cervical lymph nodes, and also in the spleen. HN421-436/I-Ab- and NP324-332/Kb-presenting cells were readily detectable between days 3 and 9 postinfection, with more APCs present in the MLN than in the cervical lymph nodes. Interestingly, no infectious virus was detected in lymphoid tissue beyond day 6, suggesting that a depot of noninfectious viral Ag survives, in some form, for 2-3 days after viral clearance. Fractionation of the MLN demonstrated that APC frequency was enriched in dendritic cells and macrophages but depleted in the B cell population, suggesting that B cells do not form a large population of APCs during the primary response to this virus.

Efficient priming of CD8+ memory T cells specific for a subdominant epitope following Sendai virus infection.

The relationship between the primary effector CTL response to viral infection and the subsequent pool of memory CTL precursors (CTLp) is poorly understood. Here, we have analyzed the induction of both effector CTL and memory CTLp to dominant and subdominant epitopes following Sendai virus infection of C57BL/6 mice. A single peptide derived from the Sendai virus nucleoprotein (NP(324-332)) binds to both H-2 Kb and Db MHC class I molecules, generating both immunodominant (NP(324-332)/Kb) and subdominant (NP(324-332)/Db) epitopes. Following intranasal Sendai virus infection, NP(324-332)/Kb-specific CTL dominated the primary effector CTL response in the lung and were present at high frequency in the memory CTLp pool. In contrast, NP(324-332)/Db-specific CTL were not a detectable component of the effector response to primary Sendai virus infection. However, memory CTLp specific for this subdominant epitope were induced at frequencies approaching those of CTLp specific for the immunodominant epitope. These data indicate that memory CTLp specific for subdominant epitopes can be primed by Sendai virus infection in the absence of a detectable effector response. To determine whether CTLp memory to subdominant epitopes is functional in the context of Sendai virus infection, memory CTLp specific for a subdominant epitope were selectively primed by vaccination. These cells dominated the subsequent effector CTL response to Sendai virus infection, demonstrating that memory CTLp primed against subdominant epitopes can participate in an immune response and effectively compete with T cells specific for immunodominant epitopes. These data have implications for the development of vaccines designed to emphasize cellular immunity.

Carboxy-terminal residues of major histocompatibility complex class II-associated peptides control the presentation of the bacterial superantigen toxic shock syndrome toxin-1 to T cells.

Previous studies have shown that the presentation of some bacterial superantigens by major histocompatibility complex (MHC) class II molecules is strongly influenced by class II-associated peptides. For example, presentation of the toxic shock syndrome toxin-1 (TSST-1) superantigen by antigen-processing-defective T2-I-Ab cells (which expresses I-Ab that is either empty or associated with invariant chain-derived peptides) can be strongly enhanced by some, but not other, I-Ab-binding peptides. Here we investigate the contribution of I-Ab-associated peptides in the presentation of TSST-1 to T cells. The data show that overlapping peptides expressing the same core I-Ab-restricted epitope, but with various N and C termini, can differ profoundly in their ability to promote TSST-1 presentation to T cells. Analysis of altered and truncated peptides indicates that residues at the C-terminal end of the peptide have a dramatic effect on TSST-1 presentation. This effect does not involve a cognate interaction between the peptide and the TSST-1 molecule, but appears to depend on the length of the C-terminal region. These data are consistent with crystallographic studies suggesting that TSST-1 may interact with the C-terminal residues of MHC class II-associated peptides. We also examined the capacity of naturally processed peptides to promote TSST-1 binding using a superantigen blocking assay. The data demonstrated that a naturally processed epitope is dominated by peptides that do not promote strong TSST-1 binding to I-Ab. Taken together, these data suggest that TSST-1 binding to MHC class II molecules is controlled by the C-terminal residues of the associated peptide, and that many naturally processed peptide/class II complexes do not present TSST-1 to T cells. Thus, the peptide dependence of TSST-1 binding to class II molecules may significantly reduce the capacity of TSST-1 to stimulate T cells.

Binding motifs predict major histocompatibility complex class II-restricted epitopes in the Sendai virus M protein.

Major histocompatibility complex (MHC) class I ligand motifs have been defined for a number of class I molecules and have been successfully used to identify class I-restricted cytotoxic T-cell epitopes. In contrast, the relative degeneracy of sequence motifs in naturally processed MHC class II ligands has suggested that they may be of more limited use. Here, we use a predicted I-Ab ligand motif to identify antigenic peptides in the Sendai virus Enders strain matrix (M) protein. The entire coding sequence of the M protein was derived, and seven peptide sequences that contained the predicted I-Ab motif were identified. Analysis of I-Ab-restricted M-specific T-cell hybridomas for reactivity to these synthetic peptides identified two distinct epitopes. These data demonstrate that MHC class II motifs can be valuable in predicting T-cell epitopes.

T cell recognition of the immunodominant Sendai virus NP324-332/Kb epitope is focused on the center of the peptide.

The CTL response to Sendai virus in C57BL/6 mice is directed almost exclusively to a single H-2Kb-restricted epitope derived from the virus nucleoprotein, NP324-332 (FAPGNYPAL). We have previously shown that the repertoire of T cells elicited by this epitope following primary Sendai virus infection is very diverse. The current experiments were undertaken to determine how a diverse array of TCR are able to interact with a single class I epitope. Crystallographic analysis of NP324-332 bound to Kb has shown that the side chains of peptide residues F1, G4, N5, and A8 protrude toward the solvent and are potentially available for recognition by the TCR. Notably, the N5 residue protrudes prominently from the peptide-binding site due to its localization on a bulge in the center of NP324-332. To determine the importance of these residues for T cell recognition, we analyzed the response of a large panel of hybridomas to NP324-332 analogues substituted at these four positions. The data suggested that there is dominant recognition of the central G4 and N5 residues at the center of the peptide. However, individual hybridomas exhibited distinct patterns of fine specificity for residues F1 and A8, in that they were dependent on one, both, or neither of these residues for recognition of NP324-332. These data are consistent with a critical role for the G4 and N5 residues in governing NP324-332/Kb recognition by T cells and may have implications for T cell recognition of class-I restricted epitopes in general.

Analysis of the primary T-cell response to Sendai virus infection in C57BL/6 mice: CD4+ T-cell recognition is directed predominantly to the hemagglutinin-neuraminidase glycoprotein.

Sendai virus infection of C57BL/6 mice elicits a strong CD4+ and CD8+ T-cell response in the respiratory tract. To investigate the specificity of the CD4+ T-cell response, a panel of hybridomas was generated from cells recovered from the respiratory tracts of infected mice. Using vaccinia virus recombinants expressing individual Sendai virus proteins, we found that the majority of these hybridomas (34 of 37) were specific for the hemagglutinin-neuraminidase (HN) glycoprotein. The hybridomas were then analyzed for reactivity to a set of overlapping peptides spanning the entire length of the hemagglutinin-neuraminidase glycoprotein. At least five H-2 I-Ab-restricted epitopes were defined in HN. The strong bias toward recognition of class II epitopes derived from a single viral protein contrasts with T-cell recognition of epitopes of several proteins in influenza A virus as found previously by others.

The MHC class I-restricted T cell response to Sendai virus infection in C57BL/6 mice: a single immunodominant epitope elicits an extremely diverse repertoire of T cells.

We have used Sendai virus infection of C57BL/6 mice as a model with which to study the T cell response to a single MHC class I epitope. Cells taken from the bronchoalveolar lavage or restimulated in vitro from the mediastinal lymph nodes of virus-infected mice were strongly cytotoxic for a single nucleoprotein epitope, NP324-332/Kb. To correlate TCR usage with specificity for the immunodominant epitope, we generated T cell hybridomas from the bronchoalveolar lavage and mediastinal lymph node cells of C57BL/6 mice at the peak of infection. Altogether, 20 hybridomas were identified that specifically secreted IL-2 in response to NP324-332-pulsed L929-Kb cells. TCR usage in this panel of hybridomas was extremely diverse. Over half of the available J beta and V beta elements present in the C57BL/6 strain of mouse were represented in the hybridomas. Similarly, V alpha usage was also diverse and all 12 of the alpha chains sequenced used distinct J alpha elements. The only relatively conserved feature of the TCR in these hybridomas was the presence of an arginine residue in the junctions of 70% of the beta chains. These data demonstrate that a diverse repertoire of TCR is able to recognize a single MHC class I epitope. Moreover, the data demonstrate that mice make use of this potential diversity in the primary response to a natural viral infection.