Humoral immune response to HTLV-1 basic leucine zipper factor (HBZ) in HTLV-1-infected individuals

Abstract

Background: Human T cell lymphotropic virus type 1 (HTLV-1) infection can lead to development of adult T cell leukemia/lymphoma (ATL) or HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a subset of infected subjects. HTLV-1 basic leucine zipper factor (HBZ) gene has a critical role in HTLV-1 infectivity and the development of ATL and HAM/TSP. However, little is known about the immune response against HBZ in HTLV-1-infected individuals. In this study, we examined antibody responses against HBZ in serum/plasma samples from 436 subjects including HTLV-1 seronegative donors, asymptomatic carriers (AC), ATL, and HAM/TSP patients using the luciferase immunoprecipitation system.

Results: Immunoreactivity against HBZ was detected in subsets of all HTLV-1-infected individuals but the test did not discriminate between AC, ATL and HAM/TSP. However, the frequency of detection of HBZ-specific antibodies in the serum of ATL patients with the chronic subtype was higher than in ATL patients with the lymphomatous subtype. Antibody responses against HBZ were also detected in cerebrospinal fluid of HAM/TSP patients with anti-HBZ in serum. Antibody responses against HBZ did not correlate with proviral load and HBZ mRNA expression in HAM/TSP patients, but the presence of an HBZ-specific response was associated with reduced CD4+ T cell activation in HAM/TSP patients. Moreover, HBZ-specific antibody inhibited lymphoproliferation in the PBMC of HAM/TSP patients.

Conclusions: This is the first report demonstrating humoral immune response against HBZ associated with HTLV-I infection. Thus, a humoral immune response against HBZ might play a role in HTLV-1 infection.

Figure 1

Antibody responses against HBZ from serum/plasma of ND, AC, ATL patients and HAM/TSP patients. (A) Comparison of antibody responses against HBZ in serum/plasma of ND, AC, ATL and HAM/TSP patients using Mann–Whitney Test. The data were obtained from 98 ND, 145 AC, 89 ATL patients and 104 HAM/TSP patients. Antibody responses against HBZ were detected in HTLV-1-infected groups (AC, ATL and HAM/TSP). The horizontal line represents the mean. Dotted line represents cut-off value (6853LU). (B) Frequency of subjects with antibody response against HBZ in serum/plasma of ND, AC, ATL and HAM/TSP patients. The distribution of subjects with antibody response against HBZ among the groups was compared by Chi-Square Test. (C) Comparison of antibody responses against HBZ in serum of ATL patients by ATL subtypes using Mann–Whitney Test. The data were obtained from 30 acute, 29 chronic and 30 lymphoma ATL patients. The horizontal line represents the mean. Dotted line represents cut-off value (6853LU). (D) Frequency of subjects with antibody response against HBZ in serum of ATL patients. The distribution of subjects with antibody response against HBZ among the groups was compared by Chi-Square Test.

Figure 2

Comparison of antibody responses against HTLV-1, proviral loads and HBZ mRNA expression of HTLV-1-infected individuals. Comparison of antibody responses against Gag (A), Env (B) and Tax (C) in serum/plasma of HTLV-1-infected individuals with and without antibody response against HBZ by Mann–Whitney Test. The data were obtained from 338 HTLV-1-infected individuals; 145 AC, 89 ATL patients and 104 HAM/TSP patients. Anti-HBZ (−) group includes 130 AC, 78 ATL patients and 90 HAM/TSP patients. Anti-HBZ (+) group includes 15 AC, 11 ATL patients and 14 HAM/TSP patients. The horizontal line represents the mean. (D) Correlation of HTLV-1 proviral load with HBZ mRNA expression in 13 HAM/TSP patients including 6 patients with antibody response against HBZ (closed circles) and 7 patients without antibody response against HBZ (closed triangles) by Spearman’s correlation test. (E) Comparison of HTLV-1 proviral loads between HAM/TSP patients with and without antibody response against HBZ. HTLV-1 proviral loads were examined in PBMCs of HAM/TSP patients using Mann–Whitney Test. The horizontal line represents the mean. (F) Comparison of HBZ mRNA expression between HAM/TSP patients with and without antibody response against HBZ. The expression of HBZ mRNA was examined in PBMCs of HAM/TSP patients using Mann–Whitney Test. The horizontal line represents the mean. (G) Correlation of immunoreactivity against HBZ with HTLV-1 proviral loads (closed circles) and HBZ mRNA expression (opened circles) in 6 HAM/TSP patients with antibody response against HBZ by Spearman’s correlation test.

Figure 3

Detection of antibody responses against HTLV-1 in serum and CSF of HAM/TSP patients. The data were obtained from 5 HAM/TSP patients including 2 patients (#1 and #2) and 3 patients (#5, #6 and #7) with and without HBZ-specific antibody response, respectively. Antibody responses for Gag (A), Env (B), Tax (C) and HBZ (D) were examined in serum (opened bar) and in CSF (closed bar). (E) Ratio of immunoreactivities against HTLV-1 Gag, Env, Tax and HBZ in CSF to those in serum of two HAM/TSP patients with HBZ-specific antibody responses.

Figure 4

Inhibitory effects of HBZ-specific antibody on T cell activation of HAM/TSP patients. (A) Detection of immortalized memory B cells producing HBZ-specific antibodies in HAM/TSP patients. The immortalized B cells were generated from three HAM/TSP patients with anti-HBZ in serum, and production of HBZ-specific antibodies (closed bars) was detected using LIPS assay. (B) Detection of HBZ proteins using an anti-HBZ (+) B cell culture supernatant and rabbit anti-HBZ serum by western blot. The nuclear proteins were extracted from HTLV-1-uninfected (Jurkat and MOLT-3) and HTLV-I-infected (MT-2 and HUT102) cell lines and HBZ/pRen2-untransfected and transfected 293T cells. Detection of HBZ proteins was confirmed by western blot (i) representative image and (ii) the intensity of HBZ proteins. (C) Representative dot plots of CFSE staining in CD8⁺ T cells of a HAM/TSP patient with HBZ-specific antibody or control IgG after culture for 6 days. (D) Inhibitory effects of HBZ-specific antibody on spontaneous proliferation of CD8⁺ T cells (i) and CD4⁺ T cells (ii) in PBMCs of HAM/TSP patients after culture for 6 days by Wilcoxson matched-pairs signed rank test. The data were obtained from 7 HAM/TSP patients without anti-HBZ response (closed circles) and one ND (opened circle) as control. (E) Comparison of frequencies of CD4⁺CD25⁺ T cells (i) and CD8⁺CD25⁺ T cells (ii) of NDs, HAM/TSP patients with and without antibody responses for HBZ by Mann–Whitney test. The data were obtained from twelve NDs, and five and ten HAM/TSP patients with and without antibody responses for HBZ, respectively. The horizontal line represents the mean.