Dual cytoplasmic and nuclear localization of HTLV-1-encoded HBZ protein is a unique feature of adult T-cell leukemia

Abstract

Adult T-cell leukemia-lymphoma (ATL), is a highly malignant T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1), characterized by a poor prognosis. Two viral proteins, Tax-1 and HBZ play important roles in the pathogenesis of ATL. While Tax-1 can be found in both cytoplasm and nucleus of HTLV-1 infected patients, HBZ is exclusively localized in the cytoplasm of HTLV-1 asymptomatic carriers and patients with chronic neurologic disease HAM/TSP, and only in the nucleus of ATL cell lines, suggesting that the nuclear localization of HBZ can be a hallmark of neoplastic transformation. To clarify this crucial point, here we investigated in detail the pattern of HBZ expression in ATL patients. We made use of our monoclonal antibody 4D4-F3, that at present is a uniquely reported reagent, among the few described, able to detect endogenous HBZ by immunofluorescence and confocal microscopy in cells from asymptomatic carriers, HAM/TSP and ATL patients. We found that HBZ localizes both in the cytoplasm and in the nucleus of cells of ATL patients irrespective of their clinical status, with a strong preference for the cytoplasmic localization. Also Tax-1 localized in both compartments. As HBZ is exclusively localized in the cytoplasm in asymptomatic carriers and in non-neoplastic pathologies, this finding shows that neoplastic transformation consequent to HTLV-1 infection is accompanied and associated with the capacity of HBZ to translocate to the nucleus, which suggests a role of cytoplasmic-to-nuclear translocation in HTLV-1-mediated oncogenesis.

Figure 1.

HBZ subcellular localization in peripheral blood mononuclear cells of patients with acute adult T-cell leukemia-lymphoma. (A) Peripheral blood mononuclear cells (PBMC) from PH131213, PH1401263, PH160822 and PH1612N07 patients were stained with the 4D4-F3 anti-HBZ monoclonal antibody (mAb) followed by Alexa Fluor 546-conjugated goat anti-mouse IgG1 antibody (red) and analyzed by confocal microscopy; DRAQ5 was used to detect the nucleus. (B). Specific counterstaining of cytoplasmic compartment in PH1612N07 patient’s PBMC was performed by using anti-vimentin rabbit polyclonal antibody followed by goat anti-rabbit IgG conjugated to Alexa Fluor 488 (green). DIC represents the differential interference contrast image. At least 200 cells were analyzed. A representative image of HBZ staining is shown of each patient. All scale bars are 5mm.

Figure 2.

Tax-1 subcellular localization in peripheral blood mononuclear cells of patients with acute adult T-cell leukemia-lymphoma. (A) Peripheral blood mononuclear cells (PBMC) of representative acute adult T-cell leukemia-lymphoma patients PH40126 (A) and PH1612N07 (B) were stained with the A51-2 anti-Tax-1 monoclonal antibody (mAb) followed by Alexa Fluor 488-conjugated goat-anti-mouse IgG2a antibody (green) and analyzed by confocal microscopy. Counterstaining of the nuclear or cytoplasmic compartments was performed by using DRAQ5 fluorescence probe to detect the nucleus (blue) and anti-vimentin rabbit polyclonal antibody followed by goat anti-rabbit IgG conjugated to Alexa Fluor 546 (B, red) to stain the cytoplasmic compartment. (C) PBMC of representative acute ATL patient PH160822 were costained with the 4D4-F3 anti-HBZ mAb followed by Alexa Fluor 546-conjugated goat anti-mouse IgG1 antibody (red) and with the A51-2 anti-Tax- 1 mAb followed by Alexa Fluor 488-conjugated goat-anti-mouse IgG2a antibody (green) and analyzed by confocal microscopy. DRAQ5 fluorescence probe was used to detect the nucleus (blue). DIC represents the differential interference contrast image. At least 300 cells were analyzed. All scale bars are 5mm.

Figure 3.

HBZ subcellular localization in peripheral blood mononuclear cells of patients with chronic adult T-cell leukemia-lymphoma. (A) Peripheral blood mononuclear cells (PBMC) of representative chromic leukemia patients PH150610, PH170706, PH170918 and PH171206 were stained with the 4D4-F3 anti-HBZ monoclonal antibody (mAb) followed by Alexa Fluor 546-conjugated goat anti-mouse IgG1 antibody (red) and analyzed by confocal microscopy. DRAQ5 fluorescence probe was used to detect the nucleus. (B) PBMC of representative chronic leukemia patient PH170706 was stained with the A51-2 anti-Tax-1 mAb followed by Alexa Fluor 488-conjugated goat-anti-mouse IgG2a antibody (green) and analyzed by confocal microscopy. Nucleus was stained with DRAQ5. DIC represents the differential interference contrast image. At least 300 cells were analyzed. One representative image of HBZ staining derived from PBMC samples of each patient is shown. At least 300 cells were analyzed. All scale bars are 5mm.

Figure 4.

HBZ expression in T-cell subpopulations of chronic adult T-cell leukemia-lymphoma. Confocal microscopy analysis of peripheral blood mononuclear cells (PBMC) from representative chronic (CH) leukemic PH170918. (A) Upper panels: PBMC of patient CH PH170918 were costained with the 4D4-F3 anti-HBZ monoclonal anitbody (mAb) followed by Alexa Fluor 546-conjugated goat anti-mouse IgG1 antibody (red) and with the anti-CD4 rabbit mAb (RabmAb) followed by Alexa Fluor 488-conjugated goat-anti-rabbit IgG antibody (green). Nucleus was stained with DRAQ5. (B) PBMC of CH patient PH170918 were costained with the 4D4-F3 anti-HBZ mAb followed by Alexa Fluor 546-conjugated goat anti-mouse IgG1 antibody (red) and with the anti-CD8 RabmAb directly conjugated to Alexa Fluor 647 (blue). At least 200 cells were analyzed; DIC represents the differential interference contrast image. representative images derived from each sample are shown. All scale bars are 5mm.

Figure 5.

Cytoplasmic HBZ is retained in this compartment and does not shuttle to the nucleus. (A) Peripheral blood mononuclear cells (PBMC) of representative acute PH140126 and chronic PH170918, were either treated (+LMB, bottom panels) or not treated (-LMB, top panels) with Leptomycin B (LMB), an inhibitor CRM1/exportin-mediated nuclear export, before fixing and stained with the anti-HBZ 4D4-F3 monoclonal antibody (mAb) followed by Alexa Fluor 546-conjugated goat anti-mouse IgG1 antibody (red) and analyzed by confocal microscopy. (B) As control of inhibition of nuclear export by LMB, treated (+LMB) and untreated (-LMB) cells were fixed and stained with antibodies against p65/RelA followed by Alexa Fluor 546-conjugated goat-anti-rabbit secondary antibody (red) and analyzed by confocal microscopy. One representative image on the field and an enlargement of this are shown. Nucleus was stained with DRAQ5 (blue). DIC represents the differential interference contrast image. One representative image is shown for each sample. At least 300 cells were analyzed. All scale bars are 5mm.

Figure 6.

Subcellular localization of HTLV-1 HBZ protein during adult T-cell leukemia-lymphoma progression. Primary infection of T cells by HTLV-1 is characterized by the expression and localization of HBZ protein exclusively in the cytoplasm, at variance with Tax-1 that can be found both in the cytoplasm and nucleus. This feature is conserved during the progression to chronic neurologic inflammatory HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) disease possibly due to retention by a cytoplasmic factor present in both asymptomatic carriers and HAM/TSP patients. Leukemogenesis, instead, is marked by the progressive translocation of HBZ from the cytoplasm to the nucleus, both in chronic and in acute adult T-cell leukemia-lymphoma (ATL). On the basis of our results presented here, it is likely that HBZ cytoplasmic localization in ATL is mediated by the same retention factor/mechanism present in asymptomatic carriers and HAM/TSP, that is gradually lost during neoplastic transformation allowing HBZ protein to dislocate into the nucleus.