Virus-specific CD8+ T lymphocytes downregulate T helper cell type 2 cytokine secretion and pulmonary eosinophilia during experimental murine respiratory syncytial virus infection

Abstract

T lymphocytes play a pivotal role in the immune response during viral infections. In a murine model of experimental respiratory syncytial virus (RSV) infection, mice sensitized to either of the two major glycoproteins of RSV develop distinct patterns of cytokine secretion and lung inflammation upon subsequent RSV infection. Mice sensitized to RSV-G (attachment) glycoprotein exhibit a strong interleukin (IL)-4 and IL-5 response and develop pulmonary eosinophilia, whereas mice sensitized to RSV-F (fusion) glycoprotein develop a predominantly T helper cell (Th)1 response and pulmonary inflammation characterized by mononuclear cell infiltration. In this study, we examined the potential role of virus-specific CD8+ T cytolytic T cells on the differentiation and activation of functionally distinct CD4+ T cells specific to these viral glycoproteins. Mice primed with recombinant vaccinia virus expressing RSV-F glycoprotein mounted a strong RSV-specific, MHC class I-restricted cytolytic response, whereas priming with recombinant vaccinia virus expressing RSV-G glycoprotein failed to elicit any detectable cytolytic response. Priming for a RSV-specific CD8+ T cell response, either with a recombinant vaccinia virus expressing RSV-G glycoprotein in which a strong CD8+ T cell epitope from RSV-M2 (matrix) protein has been inserted or with a combination of vaccinia virus expressing the matrix protein and the RSV-G glycoprotein, suppressed the eosinophil recruitment into the lungs of these mice upon subsequent challenge with RSV. This reduction in pulmonary eosinophilia correlated with the suppression of Th2 type cytokine production. The importance of CD8+ T cells in this process was further supported by the results in CD8+ T cell deficient, beta 2 microglobulin KO mice. In these mice, priming to RSV-F glycoprotein (which in normal mice primed for a strong cytolytic response and a pulmonary infiltrate consisting primarily of mononuclear cells on RSV challenge) resulted in the development of marked pulmonary eosinophilia that was not seen in mice with an intact CD8+ T cell compartment. These results indicate that CD8+ T cells may play an important role in the regulation of the differentiation and activation of Th2 CD4+ T cells as well as the recruitment of eosinophils into the lungs during RSV infection.

Figure 1

Construction of cDNA encoding RSV-G glycoprotein containing a CTL epitope of RSV-M2(22K) protein. The indicated oligonucleotide heteroduplex (bold) was introduced into the NdeI site at base 535 of the RSV-G cDNA. The synthetic oligonucleotide encodes the nine residues SYIGSINNI from amino acids 82–90 of the RSV-M2(22K) matrix protein.

Figure 2

RSV-specific (A) and vaccinia virus–specific (B) cytolytic responses in mice primed with recombinant vaccinia virus expressing RSV-F (VF) or RSV-G (VG) glycoprotein. Splenocytes from mice previously primed with VF or VG were stimulated in vitro with RSV-infected naive spleen cells (A) or spleen cells infected with recombinant vaccinia virus not expressing RSV antigen (B). RSV-specific and vaccinia-specific cytolytic activity of these bulk cultures against target cells uninfected or infected with indicated recombinant vaccinia virus were analyzed in a standard ⁵¹Cr assay.

Figure 3

Target cells expressing G/22K protein are recognized by 22K-specific CTL. Target cells infected with indicated recombinant vaccinia virus were tested in a standard ⁵¹Cr assay for recognition by 22K-specific CTL generated from splenocytes of mice previously primed with V22K. Note that target cells infected with recombinant vaccinia virus expressing the native RSV-G glycoprotein were not recognized by 22K-specific CTL.

Figure 4

Splenocytes from mice primed with VG22K, but not VG or VSC11 exhibit 22K-specific cytolytic activity. Splenocytes from mice primed with VSC11, VG, or VG22K were stimulated in vitro with RSV-infected spleen cells. Cytolytic activity against target cells infected with V22K (gray bar) or control vaccinia virus VSC11 (black bar) was analyzed in a standard ⁵¹Cr assay.

Figure 5

Perivascular eosinophils in the lungs of mice sensitized to RSV-F, RSV-G, or RSV-G/22K glycoproteins. Mice were primed with the indicated recombinant vaccinia virus 3 wk before intranasal challenge with RSV. Mice were killed 5 d later and lung sections were stained for eosinophils. Eosinophils around the blood vessels were counted and the lengths of the vessels were measured by micrometer. Each data point represents cell counts from individual animals.

Figure 6

Cytokines produced by cells isolated from lungs of mice primed with recombinant vaccinia virus and challenged with RSV. Mice were primed with indicated recombinant vaccinia virus and challenged with live RSV intranasally 3 wk after priming. Mononuclear cells were isolated from lungs of these mice 5 d after challenge and stimulated with RSV. Supernatants were collected 48 h later and analyzed for cytokines by ELISA.

Figure 7

VG22K effectively primes for RSV-specific memory T lymphocyte response. Splenocytes from mice previously primed with VG or VG22K were stimulated in vitro with RSV-infected naive spleen cells. Supernatants were collected 48 h later and analyzed for IL-2, IL-4, IL-5, and IFN-γ. IL-4 and IL-5 were not detected in culture supernatants from either group of animals (data not shown).

Figure 8

Priming with V22K results in decreased pulmonary eosinophilia in mice sensitized to RSV-G glycoprotein. Mice were primed with a single or a mixture of two recombinant vaccinia viruses as indicated and subsequently challenged with RSV intranasally. Mice were killed 5 d later, and lung sections were stained for eosinophils. Eosinophils around the blood vessels were counted and the lengths of the vessels were measured by micrometer. Each data point represents cell counts from individual animals.

Figure 9

Lung histopathology of β2mKO mice sensitized to RSV glycoproteins. Mice were primed with VG (A), VF (B), or VSC11 (C) and challenged with live RSV intranasally 3 wk after priming. Mice were killed 5 d after challenge, and lung sections were stained for eosinophils with Leinert-Giemsa stain. Original magnification: 200.

Figure 9

Lung histopathology of β2mKO mice sensitized to RSV glycoproteins. Mice were primed with VG (A), VF (B), or VSC11 (C) and challenged with live RSV intranasally 3 wk after priming. Mice were killed 5 d after challenge, and lung sections were stained for eosinophils with Leinert-Giemsa stain. Original magnification: 200.

Figure 10

Perivascular eosinophils in the lungs of β2mKO mice sensitized to RSV glycoproteins. Mice were primed with the indicated recombinant vaccinia virus 3 wk before intranasal challenge with RSV. Mice were killed 5 d later and lung sections were stained for eosinophils. Eosinophils around the blood vessels were counted and the lengths of the vessels were measured by micrometer. Note that the magnitude of lung eosinophilia is four to five times higher in β2mKO mice than BALB/c mice.