Analysis of pulmonary T lymphocytes in ovine lentivirus infection

Abstract

Ovine lentivirus- (OvLV-) induced lymphoid interstitial pneumonia (LIP) is a model for a pulmonary disease associated with human immunodeficiency virus (HIV) infection. While the mechanisms of lentivirus-associated lymphoproliferative diseases are poorly understood. T cells are thought to play an important role in the initiation and perpetuation of the lesions. To further elucidate their involvement in LIP, T cells present in the lungs of lambs experimentally-infected with OvLV were characterized. Neonatal lambs were inoculated with OvLV and infection was confirmed through the detection of anti-viral antibodies, the isolation of virus, and histological assessment of pulmonary inflammation. Increased numbers of T cells were present in bronchoalveolar lavage (BAL) fluids of OvLV-infected lambs relative to BAL fluids of sham-infected, age-matched control lambs. Moreover, the normal ratio of CD4+ to CD8+T cells was inverted in the BAL fluid of infected animals, consistent with the selective expansion of virus-specific cytotoxic T lymphocytes (CTLs). To further examine the T cells involved in LIP, a semi-quantitative RT-PCR method for analyzing ovine T cell receptor β-chain variable region (TCRBV) gene expression was developed. The PCR method used TCRBV primers that were derived from an analysis of the ovine TCRBV gene repertoire in which 34 TCRBV gene rearrangements were isolated from an ovine spleen anchored PCR library. Eighteen unique TCRBV gene segments were represented among these cDNA clones, which were clustered into 13 families based on their similarity to human TCRBV genes. Semiquantitative RT-PCR analyses of TCRBV gene expression revealed increases in the relative expression of TCRBV 4S1 (P=0.05) and TCRBV 7S1 (P=0.01) by T cells in the lungs of OvLV-infected versus control animals. Analysis of TCRBV complimentary determining region 3 (CDR3) nucleotide sequences, suggests that TCRBV 4S1 T cells were oligoclonally expanded, while TCRBV 7S1 populations were polyclonally expanded. Additionally. RT-PCR analysis of cytokine gene expression by these lung T cells revealed a 35-fold increase in IFN-γ expression and a 3-fold increase in IL-2 expression in infected versus control lambs, while IL-10 and IL-4 were expressed at equivalent levels in both groups. Analysis of magnetically-selected lung T cell subsets revealed TCRBV 4S1 T cells to be primarily CD4+, while TCRBV 7S1 T cells were predominantly CD8+. Further analysis of T cell subsets correlated IFN-y expression predominantly with CD8+ T cells. Collectively, these findings suggest that the heterogeneity of virus-specific. CD8+ T cells in the lungs of OvLV-infected lambs is limited, and that in situ proliferation of these T cells contributes to the pathogenesis of lentivirus-associated LIP.