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Tissue-specific regulation of CD8+ T-lymphocyte immunodominance in respiratory syncytial virus infection - PubMed

Tissue-specific regulation of CD8+ T-lymphocyte immunodominance in respiratory syncytial virus infection

Sujin Lee et al. J Virol. 2007 Mar.

Abstract

Cytotoxic T lymphocytes (CTLs) are critical for control of respiratory syncytial virus (RSV) infection in humans and mice. To investigate cellular immune responses to infection, it is important to identify major histocompatibility complex (MHC) class I-restricted CTL epitopes. In this study, we identified a new RSV-specific, H-2K(d)-restricted subdominant epitope in the M2 protein, M2(127-135) (amino acids 127 to 135). This finding allowed us to study the frequency of T lymphocytes responding to two H-2K(d)-presented epitopes in the same protein following RSV infection by enzyme-linked immunospot (ELISPOT) and intracellular cytokine assays for both lymphoid and nonlymphoid tissues. For the subdominant epitope, we identified an optimal nine-amino-acid peptide, VYNTVISYI, which contained an H-2K(d) consensus sequence with Y at position 2 and I at position 9. In addition, an MHC class I stabilization assay using TAP-2-deficient RMA-S cells transfected with K(d) or L(d) indicated that the epitope was presented by K(d). The ratios of T lymphocytes during the peak CTL response to RSV infection that were specific for M2(82-90) (dominant) to T lymphocytes specific for M2(127-135) (subdominant) were approximately 3:1 in the spleen and 10:1 in the lung. These ratios were observed consistently in primary or secondary infection by the ELISPOT assay and in secondary infection by MHC/peptide tetramer staining. The number of antigen-specific T lymphocytes dropped in the 6 weeks after infection; however, the proportions of T lymphocytes specific for the immunodominant and subdominant epitopes were maintained to a remarkable degree in a tissue-specific manner. These studies will facilitate investigation of the regulation of immunodominance of RSV-specific CTL epitopes.

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Figures

FIG. 1.
FIG. 1.

Frequencies of RSV M2-1-specific IFN-γ-secreting mouse splenocytes after RSV immunization. A panel of peptides spanning the RSV M2 protein was tested for the ability of individual peptides to elicit IFN-γ production. At day 8 (acute) (A) or day 48 (memory) (B) after the second infection, mice were sacrificed and splenocytes were stimulated with 46 peptides individually. Data represent the results of a single experiment, which was one of three with similar results. A threshold of twice the average background value was used to identify peptides containing putative epitopes.

FIG. 2.
FIG. 2.

Analysis of peptide-reactive CD8+ T lymphocytes 48 days after a second infection. Splenocytes were incubated in the presence or absence of the peptides (1 μg/sample) in an intracellular cytokine staining assay for IFN-γ. The dot plots represent the production of IFN-γ from one of three representative experiments with similar results. Panels A to C show results for negative control stimulations. Flu NP147-155, influenza virus protein NP147-155. Panel D shows results for a positive control for maximal stimulation. Panels E and F show results for stimulations with M282-90. Panels G and H show results for stimulations with M2127-135.

FIG. 3.
FIG. 3.

Identification of the optimal epitope. IFN-γ responses were measured using truncated or mutated peptides. At day 5 (filled squares) or day 12 (open squares) after the second infection, animals were sacrificed and splenocytes were stimulated with the peptides indicated in Table 2. Data represent the results of an average of eight replicates. Group A, negative control medium or peptide; group B, truncated or mutated peptides for the subdominant epitope; group C, immunodominant epitope M282-90.

FIG. 4.
FIG. 4.

Increased cell surface expression levels of H-2Kd MHC class I protein caused by M2127-135. RMA-S cells expressing Ld (A) or Kd (B) molecules maintained at 26°C for 18 to 24 h were pulsed with increasing concentrations of the indicated peptides for 45 min at 26°C, washed, and incubated at 37°C for an additional 3 h. The RSV M282-90 SYIGSINNI peptide and RSV F85-93 KYKNAVTEL peptide were used as positive controls, and murine cytomegalovirus (CMV) pp89 YPHFMPTNL peptide was used as a negative control for Kd binding. For Ld binding, murine CMV pp89 peptide was used as the positive control and RSV M282-90 and RSV F85-93 were used as negative controls. Data represent the results from a single experiment, which was one of three with similar results.

FIG. 5.
FIG. 5.

Epitope hierarchy in the lung and spleen after the second RSV infection. Splenocytes (A) or lung lymphocytes (B) obtained at acute, intermediate, or memory phase after the second infection were restimulated in vitro with peptides and IFN-γ production was measured by using an ELISPOT assay. Data represent the averages from three independent experiments.

FIG. 6.
FIG. 6.

Epitope hierarchy in the lung and spleen after RSV primary infection. Lung lymphocytes isolated at day 10 (A) or day 20 (B) or splenocytes isolated at day 10 (C) or day 20 (D) after primary infection were restimulated with a panel of peptides and IFN-γ production was measured using the ELISPOT assay. The average number of spots is indicated at the peak of each bar for the dominant and subdominant epitopes. The dominant/subdominant (D:S) ratio is the ratio of the mean number of spots of peptides 20 and 21 to that of peptides 31 and 32.

FIG. 7.
FIG. 7.

Direct enumeration of M282-90 or M2127-135 epitope-specific CD8+ T lymphocytes. Groups of BALB/c mice were infected twice with RSV wild-type strain A2. Splenocytes (D, E, and F) were harvested 8 days following the second infection and tested by flow cytometric analysis after staining with the M282-90 tetramer (B and E) or M2127-135 tetramer (C and F). Splenocytes from naïve mice were used as negative controls (A, B, and C). Dot plots illustrate results for total CD3+ lymphocytes as well as 50,000 CD8+ T lymphocytes. Numbers shown in the rectangular gate correspond to the percentage of CD8+ lymphocytes stained by the respective tetramer (tet). Data represent the results of a single experiment, which was one of five with similar results.

FIG. 8.
FIG. 8.

Direct enumeration of M282-90 or M2127-135 epitope-specific CD8+ T lymphocytes. Groups of BALB/c mice were infected twice with RSV wild-type strain A2. Lung lymphocytes (A, B, and C) were harvested 8 days following the second infection and tested by flow cytometric analysis after staining with no tetramer (A), M282-90 tetramer (B), or M2127-135 tetramer (C). Dot plots illustrate results for total CD3+ lymphocytes as well as 20,000 CD8+ T lymphocytes. Numbers shown in the rectangular gate correspond to the percentages of CD8+ lymphocytes stained by the respective tetramers (tet). Data shown are representative of five experiments.

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