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Natural OX40L expressed on human T cell leukemia virus type-I-immortalized T cell lines interferes with infection of activated peripheral blood mononuclear cells by CCR5-utilizing human immunodeficiency virus.

Kasahara D, Takara A, Takahashi Y, Kodama A, Tanaka R, Ansari AA, Tanaka Y - Virol. J. (2013)

Bottom Line: Results of our studies showed that HTLV-1+ T cell lines bound exogenous OX40 but not OX40L, indicating that HTLV-1+ T cell lines express an active form of OX40L but an inactive form of OX40.Anti-OX40 non-blocking monoclonal antibody (mAb), but not blocking mAb, stained HTLV-1+ T cell lines, suggesting that the OX40 might be saturated with endogenous OX40L.Altogether, these results demonstrated that autologous T cell lines immortalized by HTLV-1 can be utilized as a conventional source of physiologically functional OX40L.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan. yuetsu@s4.dion.ne.jp.

ABSTRACT

Background: OX40 ligand (OX40L) co-stimulates and differentiates T cells via ligation of OX40 that is transiently induced on T cells upon activation, resulting in prolonged T cell survival and enhanced cytokine production by T cells. This view has led to the targeting of OX40 as a strategy to boost antigen specific T cells in the context of vaccination. In addition, the ligation of OX40 has also been shown to inhibit infection by CCR5-utilizing (R5) but not CXCR4-utilizing (X4) human immunodeficiency virus type-1 (HIV-1) via enhancement of production of CCR5-binding β-chemokines. It was reasoned that human T cell leukemia virus type-I (HTLV-1) immortalized T cell lines that express high levels of OX40L could serve as an unique source of physiologically functional OX40L. The fact that HTLV-1+ T cell lines simultaneously also express high levels of OX40 suggested a potential limitation.

Results: Results of our studies showed that HTLV-1+ T cell lines bound exogenous OX40 but not OX40L, indicating that HTLV-1+ T cell lines express an active form of OX40L but an inactive form of OX40. Anti-OX40 non-blocking monoclonal antibody (mAb), but not blocking mAb, stained HTLV-1+ T cell lines, suggesting that the OX40 might be saturated with endogenous OX40L. Functionality of the OX40L was confirmed by the fact that a paraformaldehyde (PFA)-fixed HTLV-1+ T cell lines inhibited the infection of autologous activated peripheral blood mononuclear cells (PBMCs) with R5 HIV-1 which was reversed by either anti-OX40L blocking mAb or a mixture of neutralizing mAbs against CCR5-binding β-chemokines.

Conclusions: Altogether, these results demonstrated that autologous T cell lines immortalized by HTLV-1 can be utilized as a conventional source of physiologically functional OX40L.

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Western blot analysis of OX40. OX40-expressing CEM cells (CEM/OX40), in vitro activated PBMCs and MT-2 cells were cell-surface labeled with biotin, lysed and immunoprecipitated with anti-OX40 (B-7B5). The precipitates were subjected to 10% PAGE and blotted onto nitrocellulose sheets. The sheets were then probed with HRP-labeled anti-HIV-1 p24 (as a control), anti-OX40 (B-7B5) or streptavidin. Mol. Wt. markers are shown on the right. Data shown are representative of 3 independent experiments.
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Figure 2: Western blot analysis of OX40. OX40-expressing CEM cells (CEM/OX40), in vitro activated PBMCs and MT-2 cells were cell-surface labeled with biotin, lysed and immunoprecipitated with anti-OX40 (B-7B5). The precipitates were subjected to 10% PAGE and blotted onto nitrocellulose sheets. The sheets were then probed with HRP-labeled anti-HIV-1 p24 (as a control), anti-OX40 (B-7B5) or streptavidin. Mol. Wt. markers are shown on the right. Data shown are representative of 3 independent experiments.

Mentions: A series of studies were subsequently conducted in efforts to identify the potential reason(s) for the failure of HTLV-1+ T cell lines to bind rec-OX40L. Western Blot analysis of OX40 expressed by HTLV-1+ T cell line was first carried out to determine whether the OX40 expressed by these cells was truncated. Cell lysates prepared from surface biotinylated in vitro activated PBMCs and the OX40 transfected CEM cell line (CEM/OX40) were analyzed in parallel with the HTLV-1+ T cell line MT-2 using standard Western Blot techniques. Results of these studies displayed in Figure 2 showed that there were no detectable differences in the molecular weight of the glycosylated authentic OX40 (50 kDa) among these three samples. The 35 kDa band corresponding to the non-glycosylated form of OX40 was apparent in CEM/OX40 cells and activated PBMCs, but it was faint in MT-2 cells. These data indicated that there was no detectable deletion or modification in the glycosylated OX40 molecules expressed by the HTLV-1+ T cell lines.


Natural OX40L expressed on human T cell leukemia virus type-I-immortalized T cell lines interferes with infection of activated peripheral blood mononuclear cells by CCR5-utilizing human immunodeficiency virus.

Kasahara D, Takara A, Takahashi Y, Kodama A, Tanaka R, Ansari AA, Tanaka Y - Virol. J. (2013)

Western blot analysis of OX40. OX40-expressing CEM cells (CEM/OX40), in vitro activated PBMCs and MT-2 cells were cell-surface labeled with biotin, lysed and immunoprecipitated with anti-OX40 (B-7B5). The precipitates were subjected to 10% PAGE and blotted onto nitrocellulose sheets. The sheets were then probed with HRP-labeled anti-HIV-1 p24 (as a control), anti-OX40 (B-7B5) or streptavidin. Mol. Wt. markers are shown on the right. Data shown are representative of 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225675&req=5

Figure 2: Western blot analysis of OX40. OX40-expressing CEM cells (CEM/OX40), in vitro activated PBMCs and MT-2 cells were cell-surface labeled with biotin, lysed and immunoprecipitated with anti-OX40 (B-7B5). The precipitates were subjected to 10% PAGE and blotted onto nitrocellulose sheets. The sheets were then probed with HRP-labeled anti-HIV-1 p24 (as a control), anti-OX40 (B-7B5) or streptavidin. Mol. Wt. markers are shown on the right. Data shown are representative of 3 independent experiments.
Mentions: A series of studies were subsequently conducted in efforts to identify the potential reason(s) for the failure of HTLV-1+ T cell lines to bind rec-OX40L. Western Blot analysis of OX40 expressed by HTLV-1+ T cell line was first carried out to determine whether the OX40 expressed by these cells was truncated. Cell lysates prepared from surface biotinylated in vitro activated PBMCs and the OX40 transfected CEM cell line (CEM/OX40) were analyzed in parallel with the HTLV-1+ T cell line MT-2 using standard Western Blot techniques. Results of these studies displayed in Figure 2 showed that there were no detectable differences in the molecular weight of the glycosylated authentic OX40 (50 kDa) among these three samples. The 35 kDa band corresponding to the non-glycosylated form of OX40 was apparent in CEM/OX40 cells and activated PBMCs, but it was faint in MT-2 cells. These data indicated that there was no detectable deletion or modification in the glycosylated OX40 molecules expressed by the HTLV-1+ T cell lines.

Bottom Line: Results of our studies showed that HTLV-1+ T cell lines bound exogenous OX40 but not OX40L, indicating that HTLV-1+ T cell lines express an active form of OX40L but an inactive form of OX40.Anti-OX40 non-blocking monoclonal antibody (mAb), but not blocking mAb, stained HTLV-1+ T cell lines, suggesting that the OX40 might be saturated with endogenous OX40L.Altogether, these results demonstrated that autologous T cell lines immortalized by HTLV-1 can be utilized as a conventional source of physiologically functional OX40L.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan. yuetsu@s4.dion.ne.jp.

ABSTRACT

Background: OX40 ligand (OX40L) co-stimulates and differentiates T cells via ligation of OX40 that is transiently induced on T cells upon activation, resulting in prolonged T cell survival and enhanced cytokine production by T cells. This view has led to the targeting of OX40 as a strategy to boost antigen specific T cells in the context of vaccination. In addition, the ligation of OX40 has also been shown to inhibit infection by CCR5-utilizing (R5) but not CXCR4-utilizing (X4) human immunodeficiency virus type-1 (HIV-1) via enhancement of production of CCR5-binding β-chemokines. It was reasoned that human T cell leukemia virus type-I (HTLV-1) immortalized T cell lines that express high levels of OX40L could serve as an unique source of physiologically functional OX40L. The fact that HTLV-1+ T cell lines simultaneously also express high levels of OX40 suggested a potential limitation.

Results: Results of our studies showed that HTLV-1+ T cell lines bound exogenous OX40 but not OX40L, indicating that HTLV-1+ T cell lines express an active form of OX40L but an inactive form of OX40. Anti-OX40 non-blocking monoclonal antibody (mAb), but not blocking mAb, stained HTLV-1+ T cell lines, suggesting that the OX40 might be saturated with endogenous OX40L. Functionality of the OX40L was confirmed by the fact that a paraformaldehyde (PFA)-fixed HTLV-1+ T cell lines inhibited the infection of autologous activated peripheral blood mononuclear cells (PBMCs) with R5 HIV-1 which was reversed by either anti-OX40L blocking mAb or a mixture of neutralizing mAbs against CCR5-binding β-chemokines.

Conclusions: Altogether, these results demonstrated that autologous T cell lines immortalized by HTLV-1 can be utilized as a conventional source of physiologically functional OX40L.

Show MeSH
Related in: MedlinePlus