Major histocompatibility complex-dependent cytotoxic T lymphocyte repertoire and functional avidity contribute to strain-specific disease susceptibility after murine respiratory syncytial virus infection

Abstract

Susceptibility to respiratory syncytial virus (RSV) infection in mice is genetically determined. While RSV causes little pathology in C57BL/6 mice, pulmonary inflammation and weight loss occur in BALB/c mice. Using major histocompatibility complex (MHC)-congenic mice, we observed that the H-2(d) allele can partially transfer disease susceptibility to C57BL/6 mice. This was not explained by altered viral elimination or differences in the magnitude of the overall virus-specific cytotoxic T lymphocyte (CTL) response. However, H-2(d) mice showed a more focused response, with 70% of virus-specific CTL representing Vβ8.2(+) CTL directed against the immunodominant epitope M2-1 82, while in H-2(b) mice only 20% of antiviral CTL were Vβ9(+) CTL specific for the immunodominant epitope M187. The immunodominant H-2(d)-restricted CTL lysed target cells less efficiently than the immunodominant H-2(b) CTL, probably contributing to prolonged CTL stimulation and cytokine-mediated immunopathology. Accordingly, reduction of dominance of the M2-1 82-specific CTL population by introduction of an M187 response in the F1 generation of a C57BL/6N × C57BL/6-H-2(d) mating (C57BL/6-H-2(dxb) mice) attenuated disease. Moreover, disease in H-2(d) mice was less pronounced after infection with an RSV mutant failing to activate M2-1 82-specific CTL or after depletion of Vβ8.2(+) cells. These data illustrate how the MHC-determined diversity and functional avidity of CTL responses contribute to disease susceptibility after viral infection.

Fig. 1.

Weight loss after RSV infection is more pronounced in mice carrying the H-2^d MHC haplotype, but disease is independent of peak virus titers and elimination. Mice were infected intranasally (i.n.) with 1 × 10⁶ PFU RSV. (A and B) Weight was monitored for 8 days. (A) The data show mean values and SD from mice in one experiment representative of 6 independent experiments. (B) Each box represents the weight on day six after infection in 12 independent experiments for BALB/c and C57BL/6 mice (n = 44). In six of those experiments, C57BL/6-H-2^d and C57BL/6-H-2^dxb mice were included (n = 18). *, P < 0.05; ***, P < 0.001; n.s. (not significant), P > 0.05. (C) Pulmonary virus load was determined at days four and six after infection. Data were pooled from two independent experiments with 3 to 4 mice per group and time point. The dashed line indicates the detection limit. ***, P < 0.001; n.s. (not significant), P > 0.05.

Fig. 2.

Different cytokine patterns in supernatants of BALF and of lung homogenates of BALB/c, C57BL/6, C57BL/6-H-2^d, and C57BL/6-H-2^dxb mice. Mice were infected with 1 × 10⁶ PFU RSV, and the indicated cytokines, IFN-γ (A and E), IL-6 (B and F), MCP-1 (C and G), and TNF-α (D and H), were determined in supernatants of BAL fluid on d7 (A to D) or of lung homogenates on d6 (E to H) following infection by cytometric bead array. The graphs show results of two independent experiments (n = 5 to 9). *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s. (not significant), P > 0.05.

Fig. 3.

Total antiviral CTL responses are comparable in all groups. Mice were infected i.n. with 1 × 10⁶ PFU RSV. Seven days later, BAL fluid CTL were analyzed for total numbers of CD8 T cells and IFN-γ production after restimulation with RSV-infected RAW macrophages. (A) Absolute numbers of CD8 T cells in BAL fluid were determined by a combination of microscopic cell counts and flow cytometry. (B) Representative fluorescence-activated cell sorter (FACS) plots gated on CD3⁺ T cells are shown. (C) Frequency of IFN-γ-producing CD8 T cells following restimulation with RAW cells. Pooled data from 6 independent experiments with 3 to 5 mice/group for BALB/c and C57BL/6 mice are shown. In two (A) or three (C) of those experiments, C57BL/6-H-2^d and C57BL/6-H-2^dxb mice were included. n.s. (not significant), P > 0.05.

Fig. 4.

TCR repertoire of BALB/c, C57BL/6-H-2^d, C57BL/6, and C57BL/6-H-2^dxb mice after RSV infection. Mice were infected i.n. with 1 × 10⁶ PFU RSV. Seven days later, BAL fluid cells were stained with antibodies against CD3, CD8, and the indicated Vβ chains and analyzed by flow cytometry. The mean percentages of the indicated Vβ⁺ CD8⁺ T cells from BAL fluid of RSV-infected BALB/c (A), C57BL/6 (C), or C57BL/6-H-2^dxb (D) mice (n = 4), normalized by subtraction of the mean values from spleens of naive control mice, are shown. (B) Vβ usage among BAL fluid CTL from BALB/c and C57BL/6-H-2^d mice is shown for 5 selected Vβ chains. The experiments were repeated with similar results.

Fig. 5.

CTL response against the immunodominant peptide epitopes of BALB/c and C57BL/6 mice. Seven days after intranasal inoculation with 1 × 10⁶ PFU RSV, epitope-specific BAL fluid CTL were quantified by flow cytometry. (A and D) Intracellular IFN-γ production was measured following restimulation with the indicated peptides: H-2K^d epitope M2-1 82-90 (A) or H-2D^b epitope M187-195 (D). (B) M2-1 82-specific CTL were quantified by tetramer staining. (C and E) Percentages of Vβ8.1/8.2⁺ cells among the M2-1 82-specific IFN-γ⁺ cells (C) or percentages of Vβ9⁺ cells among the M187-specific IFN-γ⁺ cells (E). (A to E) Pooled data from 3 to 5 independent experiments are shown, with 3 to 5 mice per group. (F) Estimated contribution of the dominant CTL population to the overall RSV-specific response. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s. (not significant), P > 0.05.

Fig. 6.

Reduced weight loss after infection of C57BL/6-H-2^d mice with an RSV strain carrying a mutation in the immunodominant epitope or after depletion of Vβ8.2⁺ CTL in BALB/c mice. (A) C57BL/6-H-2^d mice were infected i.n. with 1 × 10⁶ PFU of recombinant RSV (rRSV) wild type (wt) or rRSV 8A. Weight was monitored for 7 days. Data show mean values and SD for 6 mice per group, obtained in 2 independent experiments. (B) Specific lysis of target cells labeled with M2-1 82 peptide is shown for BAL fluid CTL of mice infected with rRSV 8A or rRSV wt. (C to E) Mice were treated with the hybridoma supernatant (αVβ8.2 [F23.2] d-1, d2, and d5) i.p. and infected i.n. with 1 × 10⁶ PFU of RSV. (C) Efficacy of depletion was monitored by flow cytometry of BAL fluid CTL at d7 after infection. (D) After in vitro restimulation with M2-1 82, the percentage of IFN-γ⁺ CD8⁺ T cells is shown. (E) Percent body weight is shown for depleted and PBS-treated mice (d6). Results shown are from one of two independent experiments with 4 mice per group. *, P < 0.05; ***, P < 0.001.

Fig. 7.

The highly focused M2-1 82-specific CTL population has a lower avidity than the M187-specific CTL population. The specific lysis of target cells loaded with a peptide concentration of 10⁻⁸ M (A) or 10⁻¹⁰ M (B) or loaded with peptides in decreasing concentrations (C) is shown for BAL fluid CTL of BALB/c and C57BL/6 mice 7 days following infection with RSV. (D) Dose-response curves of M2-182- and M187-specific CD8 T cells stimulated with decreasing peptide concentrations are shown for TNF-α⁺ (left panel) or IFN-γ⁺ (right panel) CTL. Values obtained after stimulation with a peptide concentration of 10⁻⁶ M represented the maximum response and were set as 100% response. Mean values and SD for 5 mice per group are shown.