Development of a cytotoxic T-cell assay in rabbits to evaluate early immune response to human T-lymphotropic virus type 1 infection

Abstract

Human T-lymphotropic virus type 1 (HTLV-1) infection causes adult T-cell lymphoma/leukemia (ATL) following a prolonged clinical incubation period, despite a robust adaptive immune response against the virus. Early immune responses that allow establishment of the infection are difficult to study without effective animal models. We have developed a cytotoxic T-lymphocyte (CTL) assay to monitor the early events of HTLV-1 infection in rabbits. Rabbit skin fibroblast cell lines were established by transformation with a plasmid expressing simian virus 40 (SV40) large T antigen and used as autochthonous targets (derived from same individual animal) to measure CTL activity against HTLV-1 infection in rabbits. Recombinant vaccinia virus (rVV) constructs expressing either HTLV-1 envelope surface unit (SU) glycoprotein 46 or Tax proteins were used to infect fibroblast targets in a (51)Cr-release CTL assay. Rabbits inoculated with Jurkat T cells or ACH.2 cells (expressing ACH HTLV-1 molecule clone) were monitored at 0, 2, 4, 6, 8, 13, 21, and 34 wk post-infection. ACH.2-inoculated rabbits were monitored serologically and for viral infected cells following ex vivo culture. Proviral load analysis indicated that rabbits with higher proviral loads had significant CTL activity against HTLV-1 SU as early as 2 wk post-infection, while both low- and high-proviral-load groups had minimal Tax-specific CTL activity throughout the study. This first development of a stringent assay to measure HTLV-1 SU and Tax-specific CTL assay in the rabbit model will enhance immunopathogenesis studies of HTLV-1 infection. Our data suggest that during the early weeks following infection, HTLV-1-specific CTL responses are primarily targeted against Env-SU.

FIG. 1.

Immortalized rabbit primary skin fibroblasts. (A) Photomicrograph of immortalized rabbit primary skin fibroblasts taken at 400 × magnification by an inverted microscope. (B) Immunohistochemistry: Photomicrograph of rabbit primary skin fibroblasts taken at 400 × magnification by an inverted microscope. The rabbit primary skin fibroblasts were positive for vimentin. (C) Immunofluorescence: Photomicrograph of rabbit primary skin fibroblasts taken at 400 × magnification by a fluorescence microscope. Rabbit primary skin fibroblasts were positive for large-T-antigen expression.

FIG. 2.

Expression of HTLV-1 Tax and SU in primary rabbit fibroblasts. (A) Western blot analysis depicting Tax expression from CMV-Tax-transfected and rVV-Tax-infected rabbit primary skin fibroblasts. The arrow indicates the Tax-specific band at 40 kDa. rVV-wt-infected rabbit primary skin fibroblasts did not express Tax. (B) Western blot analysis depicting Env-SU expression in HTLV-1-positive HuT102 cells and rVV-Env-SU-infected rabbit primary skin fibroblasts. rVV-wt did not express Env-SU.

FIG. 3.

Anti-HTLV-1 Western blot. Representative results from selected animals are shown. ROS1 was inoculated with HTLV-1-negative Jurkat T lymphocytes, and never seroconverted. ROS-2, -3, and -4 were inoculated with HTLV-1-positive ACH.2 cells. All ACH.2-inoculated rabbits seroconverted by week 4 (gp46I, glycoprotein 46 HTLV-1 Env surface unit; gp46II, glycoprotein 46 HTLV-2 Env surface unit; gp24, HTLV-1 capsid; p19, HTLV-1 matrix; p21, HTLV-1 Env transmembrane unit; asterisks denote serum loading control bands, indicating comparable concentrations of serum immunoglobulin levels among the samples).

FIG. 4.

Ex-vivo p19 production from cultured rabbit lymphocytes. Rabbit lymphocytes were isolated from PBMCs and cultured for 24 h. p19 MA, a measure of virus expression, was measured with an HTLV-1 ELISA. p19 expression was highest at 13 wk post-infection in all rabbits. At 34 wk post-infection ex-vivo p19 production was at a similar level in all rabbits.

FIG. 5.

Proviral load. Genomic DNA was isolated from PBMCs and used in a real-time PCR assay to detect provirus. The average proviral load was 84, 116, 89, and 166 proviral copies per 10,000 PBMCs at 8, 13, 21, and 34 wk post-infection, respectively. ROS1, the negative control, was below the level of detection for the entire length of the study.

FIG. 6.

Specific lysis of rVV-SU- and Tax-infected immortalized rabbit fibroblasts. Freshly isolated PBMCs were placed into culture with rVV-infected autochthonous immortalized rabbit primary skin fibroblasts expressing either SU gp46, Tax, or VV-wt proteins at a 40:1 E:T ratio. Specific lysis of rabbit fibroblasts detected above 3 standard deviations (3 SD) of the VV-wt-infected control was counted as true lysis, and appears to the left of the 3 SD in parentheses. Note detection of anti-SU CTL responses more often and earlier than anti-Tax CTL responses. Bold type indicates true specific lysis.