Recognition of bovine respiratory syncytial virus proteins by bovine CD8+ T lymphocytes

Abstract

CD8+ T lymphocytes play a major role in the clearance of bovine respiratory syncytial virus (BRSV), an important respiratory pathogen of young calves that shares many of the epidemiological and pathological features of human respiratory syncytial virus (HRSV) in infants. Recombinant vaccinia virus (rVV) and recombinant fowlpox virus (rFPV), expressing individual BRSV proteins, were used to demonstrate that the F, N and M2 proteins were the major antigens recognized by bovine CD8+ T cells in major histocompatibility complex (MHC)-defined cattle. BRSV protein recognition by CD8+ T cells was analysed using cytotoxic T lymphocyte (CTL) assays or by the production of interferon-gamma (IFN-gamma) following restimulation with BRSV proteins. Strong recognition of the G protein by CD8+ T cells was observed in cattle that had been vaccinated with rVV expressing this protein and subsequently challenged with BRSV. Although there is variation in the number of expressed MHC genes in cattle with different class I haplotypes, this did not appear to influence BRSV protein recognition by CD8+ T cells. Knowledge of the antigenic specificity of BRSV-specific CD8+ T cells will facilitate the qualitative and quantitative analysis of BRSV-specific CD8+ T-cell memory in cattle and help to ensure that potential vaccines induce a qualitatively appropriate CD8+ T-cell response.

Figure 4

Two-colour flow cytometric analysis of intracellular interferon-γ (IFN-γ) production by peripheral blood CD8⁺ T cells taken from an A20 homozygous calf (animal 4) which had been experimentally infected with bovine respiratory syncytial virus (BRSV) ≈ 12 months previously. Quadrants were set to include only CD8 high T cells by (a) single staining for CD8 and (b) by double staining for CD8 and IFN-γ, following stimulation in the presence of phorbol 12-myristate 13-acetate (PMA) and ionomycin (IO). PBMC were stimulated in vitro for 22 hr in the presence of: (c) medium alone; (d) control wild-type fowlpox virus (FPV); (e) recombinant (r)FPV-F; (f)rFPV-N; (g) rFPV-M2; or (h) BRSV (Snook). The values shown in the upper right quadrants represent the percentage of CD8⁺ high cells that are IFN-γ⁺.

Figure 1

Presentation of bovine respiratory syncytial virus (BRSV) by class I alleles expressed on haplotypes A18, A31 and A14. (a) Lysis of A18/A18 homozygous (▴) or A20/A11 (▪) BRSV-infected cells by peripheral blood mononuclear cells (PBMC) from calf no. 3397 (A18/A31) infected 10 days previously with BRSV. (b) Lysis of A18/A18 homozygous (▴) or A10/w8 (▪) BRSV-infected cells by PBMC from calf no. 7292 (A18/A31) infected 10 days previously with BRSV. (c) Lysis of A31/A31 homozygous (▴) or untyped (▪) BRSV-infected cells by PBMC from calf no. 3796 (A19/A31) infected 9 days previously with BRSV. (d) Lysis of A31/A31 homozygous (▴) or A14/A11 (▪) BRSV-infected cells by lung lymphocytes from calf no. 3395 (A31/A31), infected 10 days previously and restimulated in vitro with BRSV-infected autologous fibroblasts. (e) Lysis of A14/A31 (▴), A14/A11 (▪) or A20/A11 (•) BRSV-infected cells by lung lymphocytes from calf no. 6130 (A14/A31) infected 10 days previously with BRSV. E : T, effector : target ratio.

Figure 2

Lysis of bovine respiratory syncytial virus (BRSV)-infected HD6 transfected mouse L-cells by BRSV-specific cytotoxic T lymphocytes (CTL). The percentage specific lysis of BRSV-infected HD6 transfected mouse L-cells (▪), uninfected HD6 transfected cells (□), BRSV-infected A18/A18 fibroblasts (▴) and uninfected A18/A18 fibroblasts (▵) by lung lymphocytes from calf no. 4795 (A18/A10), infected 11 days previously with BRSV and restimulated in vitro with BRSV-infected autologous fibroblasts. E : T, effector : target ratio.

Figure 3

Recognition of the F, N and M2 proteins by major histocompatibility complex (MHC) class I A14- and A18-restricted bovine respiratory syncytial virus (BRSV)-specific cytotoxic T lymphocytes (CTL). (a) Lysis of BRSV-infected, MHC-matched A18/A14 (white bars), partially MHC-matched A14/A11 (black bars) or MHC-mismatched A11/A20 (hatched bars) target cells by lung lymphocytes from an A18/A14 calf, infected 10 days previously with BRSV and restimulated in vitro with glutaraldehdye-fixed BRSV-infected NM7 fibroblasts, recombinant vaccinia virus (rVV)-F-infected concanavalin A (Con A) blasts or rVV-M2-infected Con A blasts [effector : target (E : T) ratio of 100 : 1] (b) Lysis of A18/A18 targets infected with rVV expressing individual BRSV proteins, or with BRSV-infected, MHC-matched (A18/A31) partially matched (A18/A18) and mismatched (A10/W8) targets, by peripheral blood mononuclear cells (PBMC) from an A18/A31 calf, infected 7 days previously with BRSV (E : T ratio of 100 : 1).

Figure 5

Recognition of bovine respiratory syncytial virus (BRSV) proteins by CD8⁺ memory T cells. The percentages of CD8⁺ T cells positive for intracellular interferon-γ (IFN-γ) production are shown. Peripheral blood mononuclear cells (PBMC) from each of the calves within the five groups (Table 1) were stimulated with: (a) BRSV (b) recombinant fowlpox virus (rFPV)-F; (c) rFPV-N; and (d) rFPV-M2. The broken bars represent values that exceed the maximum value shown. The actual values for these are as follows: calf no. 4 response to N = 1·74; calf no. 8 response to F = 2·55 and to BRSV = 1·86; calf no. 12 response to F = 1·09, to N = 2·09 and to BRSV = 4·85. *Values that are considered significant.