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

Enose-Akahata Y, Abrams A, Massoud R, Bialuk I, Johnson KR, Green PL, Maloney EM, Jacobson S - Retrovirology (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Viral Immunology Section, Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA.

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.

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Related in: MedlinePlus

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.
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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.

Mentions: Since HBZ is involved in both regulation of viral gene transcription and T-cell proliferation [18,20-23], we asked whether HBZ-specific antibody responses have potentially beneficial roles in suppressing immune activation in HAM/TSP patients. To confirm the inhibitory effect of HBZ-specific antibody on T cell activation, we further attempted to generate immortalized memory B cells producing HBZ-specific antibody from HAM/TSP patients and isolate the specific antibody from the B cell culture supernatants. Production of HBZ-specific antibodies was detected in 9.2-41.4% of immortalized memory B cell cultures from all three HAM/TSP patients with HBZ-specific antibody response (#1, #3 and #4; Figure 4A). As control, HTLV-I-specific antibodies to Gag, Env and Tax were also able to be detected in memory B cell pools of both HAM/TSP patients with and without HBZ-specific antibody response, but HBZ-specific antibody production was not detected in the memory B cell cultures of HAM/TSP patients without HBZ-specific antibody response (data not shown). To confirm the reactivity of HBZ-specific antibody produced from memory B cells, we examined HBZ protein detection using an anti-HBZ (+) B cell culture supernatant by western blot. The nuclear proteins were extracted from HTLV-1-uninfected (Jurkat and MOLT-3) and infected cell lines (MT-2 and HUT102) and HBZ/pRen2-untransfected and transfected 293 T cells. As shown in Figure 4 Bi, HBZ protein (25 kDa) and HBZ-Renilla luciferase (Ruc) fusion protein (61 kDa) was detected in nuclear protein extract of HTLV-1 infected cell lines (MT-2 and HUT102) and HBZ/pRen2 transfected 293T cells, respectively, using anti-HBZ (+) B cell culture supernatant. Rabbit anti-HBZ serum was used as a positive control and also reacted with the HBZ protein and HBZ-Ruc fusion protein similar to the anti-HBZ (+) B cell culture supernatant (Figure 4 Bi and ii). Furthermore, we isolated HBZ-specific IgG from supernatants of a memory B cell culture from HAM/TSP patient (#1), and examined the inhibitory effect on spontaneous lymphoproliferation in PBMCs of HAM/TSP patients without anti-HBZ response. The representative dot plots showed the inhibition of spontaneous proliferation by HBZ-specific IgG in PBMCs of a HAM/TSP patient without anti-HBZ response (Figure 4C). As shown in Figure 4 Di, HBZ-specific IgG significantly inhibited the spontaneous lymphoproliferation predominantly in CD8+ T cells of HAM/TSP patients without anti-HBZ response. Since CD4+ T cells of HAM/TSP patients showed significantly less proliferation than CD8+ T cells, it was difficult to observe an inhibitory effect of HBZ-specific IgG on CD4+ T cell proliferation in HAM/TSP patients without anti-HBZ response (Figure 4 Dii). Consistent with previous reports [33], the frequency of CD4+CD25+ T cells was higher in HAM/TSP patients without anti-HBZ antibody responses than those of ND, but HAM/TSP patients with anti-HBZ antibody responses showed significantly less CD4+CD25+ T cells compared to patients without anti-HBZ antibody responses (Figure 4 Ei). Also in CD8+ T cells, HAM/TSP patients without anti-HBZ antibody response showed significantly higher frequency of CD25+ cells than those of NDs (Figure 4 Eii). The frequency of CD8+CD25+ T cells was less in HAM/TSP patients with anti-HBZ response than those of patients without anti-HBZ response and approached significance (P = 0.0553; Figure 4 Eii). These results demonstrate that HBZ-specific antibody responses may have a role in suppressing T cell activation in HAM/TSP patients.


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

Enose-Akahata Y, Abrams A, Massoud R, Bialuk I, Johnson KR, Green PL, Maloney EM, Jacobson S - Retrovirology (2013)

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.
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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.
Mentions: Since HBZ is involved in both regulation of viral gene transcription and T-cell proliferation [18,20-23], we asked whether HBZ-specific antibody responses have potentially beneficial roles in suppressing immune activation in HAM/TSP patients. To confirm the inhibitory effect of HBZ-specific antibody on T cell activation, we further attempted to generate immortalized memory B cells producing HBZ-specific antibody from HAM/TSP patients and isolate the specific antibody from the B cell culture supernatants. Production of HBZ-specific antibodies was detected in 9.2-41.4% of immortalized memory B cell cultures from all three HAM/TSP patients with HBZ-specific antibody response (#1, #3 and #4; Figure 4A). As control, HTLV-I-specific antibodies to Gag, Env and Tax were also able to be detected in memory B cell pools of both HAM/TSP patients with and without HBZ-specific antibody response, but HBZ-specific antibody production was not detected in the memory B cell cultures of HAM/TSP patients without HBZ-specific antibody response (data not shown). To confirm the reactivity of HBZ-specific antibody produced from memory B cells, we examined HBZ protein detection using an anti-HBZ (+) B cell culture supernatant by western blot. The nuclear proteins were extracted from HTLV-1-uninfected (Jurkat and MOLT-3) and infected cell lines (MT-2 and HUT102) and HBZ/pRen2-untransfected and transfected 293 T cells. As shown in Figure 4 Bi, HBZ protein (25 kDa) and HBZ-Renilla luciferase (Ruc) fusion protein (61 kDa) was detected in nuclear protein extract of HTLV-1 infected cell lines (MT-2 and HUT102) and HBZ/pRen2 transfected 293T cells, respectively, using anti-HBZ (+) B cell culture supernatant. Rabbit anti-HBZ serum was used as a positive control and also reacted with the HBZ protein and HBZ-Ruc fusion protein similar to the anti-HBZ (+) B cell culture supernatant (Figure 4 Bi and ii). Furthermore, we isolated HBZ-specific IgG from supernatants of a memory B cell culture from HAM/TSP patient (#1), and examined the inhibitory effect on spontaneous lymphoproliferation in PBMCs of HAM/TSP patients without anti-HBZ response. The representative dot plots showed the inhibition of spontaneous proliferation by HBZ-specific IgG in PBMCs of a HAM/TSP patient without anti-HBZ response (Figure 4C). As shown in Figure 4 Di, HBZ-specific IgG significantly inhibited the spontaneous lymphoproliferation predominantly in CD8+ T cells of HAM/TSP patients without anti-HBZ response. Since CD4+ T cells of HAM/TSP patients showed significantly less proliferation than CD8+ T cells, it was difficult to observe an inhibitory effect of HBZ-specific IgG on CD4+ T cell proliferation in HAM/TSP patients without anti-HBZ response (Figure 4 Dii). Consistent with previous reports [33], the frequency of CD4+CD25+ T cells was higher in HAM/TSP patients without anti-HBZ antibody responses than those of ND, but HAM/TSP patients with anti-HBZ antibody responses showed significantly less CD4+CD25+ T cells compared to patients without anti-HBZ antibody responses (Figure 4 Ei). Also in CD8+ T cells, HAM/TSP patients without anti-HBZ antibody response showed significantly higher frequency of CD25+ cells than those of NDs (Figure 4 Eii). The frequency of CD8+CD25+ T cells was less in HAM/TSP patients with anti-HBZ response than those of patients without anti-HBZ response and approached significance (P = 0.0553; Figure 4 Eii). These results demonstrate that HBZ-specific antibody responses may have a role in suppressing T cell activation in HAM/TSP patients.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Viral Immunology Section, Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA.

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.

Show MeSH
Related in: MedlinePlus