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. 1999 Jun;73(6):4575-81.
doi: 10.1128/JVI.73.6.4575-4581.1999.

The role of human T-lymphotropic virus type 1 (HTLV-1)-infected dendritic cells in the development of HTLV-1-associated myelopathy/tropical spastic paraparesis

M Makino  1 , S Shimokubo, S I Wakamatsu, S Izumo, M Baba
Affiliations

The role of human T-lymphotropic virus type 1 (HTLV-1)-infected dendritic cells in the development of HTLV-1-associated myelopathy/tropical spastic paraparesis

M Makino et al. J Virol. 1999 Jun.

Abstract

The development of human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is closely associated with the activation of T cells which are HTLV-1 specific but may cross-react with neural antigens (Ags). Immature dendritic cells (DCs), differentiated from normal donor monocytes by using recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleukin-4, were pulsed with HTLV-1 in vitro. The pulsed DCs contained HTLV-1 proviral DNA and expressed HTLV-1 Gag Ag on their surface 6 days after infection. The DCs matured by lipopolysaccharides stimulated autologous CD4(+) T cells and CD8(+) T cells in a viral dose-dependent manner. However, the proliferation level of CD4(+) T cells was five- to sixfold higher than that of CD8(+) T cells. In contrast to virus-infected DCs, DCs pulsed with heat-inactivated virions activated only CD4(+) T cells. To clarify the role of DCs in HAM/TSP development, monocytes from patients were cultured for 4 days in the presence of the cytokines. The expression of CD86 Ag on DCs was higher and that of CD1a Ag was more down-regulated than in DCs generated from normal monocytes. DCs from two of five patients expressed HTLV-1 Gag Ag. Furthermore, both CD4(+) and CD8(+) T cells from the patients were greatly stimulated by contact with autologous DCs pulsed with inactivated viral Ag as well as HTLV-1-infected DCs. These results suggest that DCs are susceptible to HTLV-1 infection and that their cognate interaction with T cells may contribute to the development of HAM/TSP.

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Figures

FIG. 1
FIG. 1
(a) Detection of HTLV-1 proviral DNA by nested PCR. Immature DCs were exposed to live HTLV-1 virions for 6 days in vitro. A genomic DNA was extracted from mock- or HTLV-1-infected DCs and amplified by PCR. Lane 1, mock-infected DCs; lanes 2 and 3, HTLV-1-infected DCs. (b) Expression of HTLV-1 Ags on virus-infected DCs. Immature DCs were pulsed with live HTLV-1 for 6 days and examined for HTLV-1 Gag and Env Ag expression by FACScan. –––, control MAb; ———, indicated MAb. The number in each panel represents percent positive cells.
FIG. 2
FIG. 2
Expression of various molecules on DCs pulsed with HTLV-1. Immature DCs were differentiated from normal healthy donor monocytes, pulsed with 20 CCID50 of live HTLV-1 or equivalent dose of heat-inactivated virions, and matured by LPS (10 ng/ml). –––, control MAb; ———, indicated MAb. The number in each panel represents the mean fluorescence intensity.
FIG. 3
FIG. 3
(a) Time-dependent T-cell proliferation stimulated by HTLV-1-infected DCs. Mature HTLV-1-infected DCs were generated by exposure of immature DCs to 20 CCID50 of HTLV-1 and cocultured with unseparated T cells (105/well) at a DC:T-cell ratio of 1:20 or 1:40 in triplicate. Mock-infected DCs were used as a negative control. (b) Ag dose-dependent proliferation of T cells. Immature DCs were pulsed with various doses of live HTLV-1, and unseparated or CD4+ T cells (5 × 104/well) were cocultured with HTLV-1- or mock-infected DCs at a DC:T-cell ratio of 1:20 in triplicate.
FIG. 4
FIG. 4
Inhibitory effect of various MAbs on DC-CD4 interaction. HTLV-1-infected mature DCs and CD4+ T cells (105/well) were cocultured for 6 days in the presence or absence of MAbs (10 μg/ml) at a DC:CD4+ T-cell ratio of 1:20. The proliferation of T cells in the absence of test MAb was 96,514 ± 1,783 cpm (mean ± standard deviation of triplicate assays). A mixture of normal mouse IgG subclasses (IgG1, Ig2a, Ig2b, and Ig3) was used as control antibody. A representative of three independent experiments is shown.
FIG. 5
FIG. 5
Comparison of T-cell-stimulating activities of HTLV-1-infected DCs and inactivated virion-pulsed DCs. (a) Immature DCs were either infected with various doses of HTLV-1 or pulsed with an equivalent dose of heat-inactivated virions, subsequently matured by LPS, and cocultured with autologous unseparated, CD4+ and CD8+ T cells (105/well) for 5 days at a DC:T-cell ratio of 1:20. (b) LPS-matured DCs were exposed to live or inactivated virus for 4 days and cocultured with various T cells (105/well) for 5 days at a DC:T-cell ratio of 1:20. A representative result of three independent experiments is shown. Each experiment was performed in triplicate.
FIG. 6
FIG. 6
Expression of various molecules on DCs generated from uninfected donors and HAM/TSP patients. DCs were generated by using rGM-CSF and rIL-4 from peripheral monocytes for 4 days. Monocytes were purified from PBMCs depleted of CD2+ T cells. The number in the panel for HTLV-1 gag staining of HAM/TSP patient DCs represents the percent positive cells. –––, control MAb; ———, indicated MAb.
FIG. 7
FIG. 7
Autologous T-cell proliferation by DCs from HAM/TSP patients. DCs were generated from peripheral monocytes obtained from an uninfected donor and HAM/TSP patients. Plastic-adherent monocytes were obtained from the population of CD2+ T cells depleted in advance by using beads. CD4+ or CD8+ T cells (5 × 104/well) were stimulated with 2.5 × 103 autologous HTLV-1-infected, inactivated Ag-pulsed or unpulsed DCs per well for 5 days in triplicate. Mature DCs from HAM/TSP patient 2 expressed HTLV-1 Ag on the surface without any exposure to HTLV-1 Ag in vitro and those from patient 1 did not.
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