Reduction of human T-cell leukemia virus type 1 (HTLV-1) proviral loads in rats orally infected with HTLV-1 by reimmunization with HTLV-1-infected cells

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) persistently infects humans, and the proviral loads that persist in vivo vary widely among individuals. Elevation in the proviral load is associated with serious HTLV-1-mediated diseases, such as adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, it remains controversial whether HTLV-1-specific T-cell immunity can control HTLV-1 in vivo. We previously reported that orally HTLV-1-infected rats showed insufficient HTLV-1-specific T-cell immunity that coincided with elevated levels of the HTLV-1 proviral load. In the present study, we found that individual HTLV-1 proviral loads established in low-responding hosts could be reduced by the restoration of HTLV-1-specific T-cell responses. Despite the T-cell unresponsiveness for HTLV-1 in orally infected rats, an allogeneic mixed lymphocyte reaction in the splenocytes and a contact hypersensitivity response in the skin of these rats were comparable with those of naive rats. HTLV-1-specific T-cell response in orally HTLV-1-infected rats could be restored by subcutaneous reimmunization with mitomycin C (MMC)-treated syngeneic HTLV-1-transformed cells. The reimmunized rats exhibited lower proviral loads than untreated orally infected rats. We also confirmed that the proviral loads in orally infected rats decreased after reimmunization in the same hosts. Similar T-cell immune conversion could be reproduced in orally HTLV-1-infected rats by subcutaneous inoculation with MMC-treated primary T cells from syngeneic orally HTLV-1-infected rats. The present results indicate that, although HTLV-1-specific T-cell unresponsiveness is an underlying risk factor for the propagation of HTLV-1-infected cells in vivo, the risk may potentially be reduced by reimmunization, for which autologous HTLV-1-infected cells are a candidate immunogen.

FIG. 1.

HTLV-1 specificity of the T-cell unresponsiveness in orally HTLV-1-infected rats. (A) IFN-γ production in spleen T cells isolated from uninfected rats (naive) and orally (P.O.) or intraperitoneally (I.P.) HTLV-1-infected rats at 20 to 21 weeks after infection were examined by ELISA after 3 days of coculture without (░⃞) or with formalin-treated various syngeneic T-cell line cells, including G14 cells (□) negative for Tax, Tax-G14 cells (▪) expressing Tax, and FPM1 cells (▨) infected with HTLV-1. The results represent the mean ± the standard deviation (SD). Similar results were obtained in three other sets of orally or intraperitoneally HTLV-1-infected rats. Asterisks denote statistical significance compared to values without stimulator cells: ✽, P < 0.01; ✽✽, P < 0.05. (B) The alloreactivities of spleen T cells (2 × 10⁵/well) from uninfected (Naive) and orally HTLV-1-infected (P.O.) rats at 17 weeks after infection were examined by MLRs after culture without (□) or with 5 × 10⁴/well (░⃞) or 1 × 10⁵/well (▪) of MMC-treated ACI rat splenocytes in a 96-well plate for 5 days and evaluated by measuring [³H]thymidine incorporation during the last 16 h of culture. (C) The contact hypersensitivity responses in the skin of uninfected (Naive) and orally (P.O.) or intraperitoneally (I.P.) HTLV-1-infected rats were evaluated at 5 weeks after infection by ear swelling for 24 h after DNFB challenge, following sensitization with DNFB in their backs 1 week previously. The ear swelling was calculated as described in Materials and Methods. The results represent the mean ± the SD for three rats in each group.

FIG. 2.

HTLV-1-specific T-cell responses and proviral loads in orally infected and reimmunized rats. (A) A total of eight rats were orally infected with HTLV-1. At 7 weeks after the infection, four of the rats were left untreated (P.O. rats), while the other four rats were subcutaneously administered 2 × 10⁷ MMC-treated HTLV-1-infected syngeneic rat FPM1 cells (Reimmunized P.O. rats). At 4 to 5 weeks after the reimmunization, T-cell-enriched spleen cells from the P.O. or reimmunized P.O. rats were subjected to proliferation assays. The proliferation index of [³H]thymidine incorporation against Tax-G14 cells was calculated as described in Materials and Methods. (B) The HTLV-1 provirus loads in the spleens of the rats in panel A were measured by real-time PCR. The results represent the provirus copy numbers/10⁵ copies of GAPDH.

FIG. 3.

HTLV-1-specific T-cell responses and proviral loads in orally infected rats before and after reimmunization. A half-splenectomy was performed in two orally HTLV-1-infected rats (5Z-9 and 5Z-10) under anesthesia at 47 weeks after infection. The T-cell-enriched fractions of the excised spleen tissues were stored at −80°C. At 1 week after the surgery, 2 × 10⁷ MMC-treated FPM1 cells were administered subcutaneously. The residual spleens were harvested at 4 weeks after the reimmunization. (A and B) The cryopreserved spleen T-cell-enriched fractions before and after the FPM1 cell inoculation were examined for their proliferative (A) and IFN-γ production (B) responses to medium only (░⃞), formalin-treated G14 cells (□), or Tax-G14 cells (▪) by determining the [³H]thymidine incorporation and by ELISA, respectively. The results represent the mean ± the SD of triplicate wells. (C) Comparison of the amounts of HTLV-1 provirus in 5Z-9 and 5Z-10 rats before and after FPM1 cell inoculation. The spleen DNA samples were amplified by 35 cycles of PCR with Tax- and GAPDH-specific primers and visualized with ethidium bromide staining. (D) Quantification of HTLV-1 provirus loads by real-time PCR in the spleens of 5Z-9 (▪), 5Z-10 (⧫), and two additional orally HTLV-1-infected rats, 8H-7 (•) and 8H-9 (▴), that received a half-splenectomy at 20 weeks after infection and were immunized with MMC-treated FPM1 cells similarly to animals 5Z-9 and 5Z10.

FIG. 4.

Recovery of Tax-specific T-cell responses in orally HTLV-1-infected rats by subcutaneous (s.c.) inoculation with HTLV-1-infected primary T cells. Rats orally infected with HTLV-1 15 weeks previously were subcutaneously inoculated without (P.O. rat 1) or with (P.O. rats 2 to 5) primary spleen T cells (2 × 10⁷ cells), which had been isolated from other syngeneic orally HTLV-1-infected rats and treated with MMC before the inoculation, either immediately (uncultured T) or after 2 days of culture in RPMI 1640 medium containing 10% FCS (cultured T), as indicated. After 5 weeks, the spleen T cells were harvested from these rats and subjected to proliferation assays against medium only (░⃞), formalin-treated G14 cells (□), or Tax-G14 cells (▪). The results represent the mean [³H]thymidine incorporation ± the SD in triplicate samples.