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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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Blocking (clone W4-54) versus non-blocking (clone B-7B5) mAb against 2 distinct epitopes of OX40 distinguish between OX40L bound and unbound OX40. (A) OX40-expressing CEM and activated PBMCs were stained with the two mAbs in the absence (mock) or presence of 1 μg/ml of recombinant OX40L (rec-OX40L). (B) Various HTLV-1+ T cell lines were stained with B-7B5 andW4-54 labeled with FITC and Cy5, respectively. Data shown are representative of 3 independent experiments.
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Figure 3: Blocking (clone W4-54) versus non-blocking (clone B-7B5) mAb against 2 distinct epitopes of OX40 distinguish between OX40L bound and unbound OX40. (A) OX40-expressing CEM and activated PBMCs were stained with the two mAbs in the absence (mock) or presence of 1 μg/ml of recombinant OX40L (rec-OX40L). (B) Various HTLV-1+ T cell lines were stained with B-7B5 andW4-54 labeled with FITC and Cy5, respectively. Data shown are representative of 3 independent experiments.

Mentions: To further probe for the molecular basis for the inability of the OX40 expressed by the HTLV-1+ T cell lines to bind rec-OX40L, we utilized an additional anti-OX40 specific mAb (W4-54 mAb) along with B-7B5 mAb. While the clone W4-54 anti-OX40 mAb inhibited the binding of OX40 and OX40L, the clone B-7B5 failed to show any detectable inhibition (Additional file 1: Figure S1). These two mAbs are reasoned to react against conformational epitopes since they failed to bind any overlapping 15-mer peptides spanning the entire OX40 protein (data not shown). As shown in Figure 3(A), control mock treated CEM/OX40 and activated PBMCs, as expected, both stained dual-positive with the B-7B5 mAb and W4-54 mAbs. These data show that the comparative staining with B-7B5 and W4-54 mAbs can be potentially utilized to distinguish between non-ligated versus OX40L ligated forms of OX40. Figure 3(B) shows that although B-7B5 mAb stained HTLV-1+ T cell lines at high levels, little or no staining was noted with the use of the W4-54 mAb. In contrast, results of a WB analysis showed that the W4-54 mAb readily reacts to the p50 of the OX40 molecule in lysates of the HTLV-1+ T cell line, YT/cM1 (Additional file 2: Figure S2). These results suggest that the OX40L binding site of OX40 expressed by the HTLV-1+ T cell lines was altered, most probably due to pre-occupation with endogenous OX40L. To confirm this possibility, we explored the presence of OX40-OX40L complexes expressed by HTLV-1+ T cell lines using our in-house ELISA. Cell lysates of the ATL-derived HTLV-1+ T cell line (ILT-H2) were first captured with the use of immobilized anti-OX40L (clone HD-1) or anti-OX40 (clone B-7B5) mAb, respectively. The levels of captured antigens were assayed with the use of HRP-labeled anti-OX40 mAb or anti-OX40L mAb. Although it is reasonable to assume that the natural interaction between OX40 and OX40L on the living cell surface may be dissociated by the detergent treatment, as shown in Figure 4, low but significant levels of OX40-OX40L complex were still detectable in the cell lysates.


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)

Blocking (clone W4-54) versus non-blocking (clone B-7B5) mAb against 2 distinct epitopes of OX40 distinguish between OX40L bound and unbound OX40. (A) OX40-expressing CEM and activated PBMCs were stained with the two mAbs in the absence (mock) or presence of 1 μg/ml of recombinant OX40L (rec-OX40L). (B) Various HTLV-1+ T cell lines were stained with B-7B5 andW4-54 labeled with FITC and Cy5, respectively. Data shown are representative of 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Blocking (clone W4-54) versus non-blocking (clone B-7B5) mAb against 2 distinct epitopes of OX40 distinguish between OX40L bound and unbound OX40. (A) OX40-expressing CEM and activated PBMCs were stained with the two mAbs in the absence (mock) or presence of 1 μg/ml of recombinant OX40L (rec-OX40L). (B) Various HTLV-1+ T cell lines were stained with B-7B5 andW4-54 labeled with FITC and Cy5, respectively. Data shown are representative of 3 independent experiments.
Mentions: To further probe for the molecular basis for the inability of the OX40 expressed by the HTLV-1+ T cell lines to bind rec-OX40L, we utilized an additional anti-OX40 specific mAb (W4-54 mAb) along with B-7B5 mAb. While the clone W4-54 anti-OX40 mAb inhibited the binding of OX40 and OX40L, the clone B-7B5 failed to show any detectable inhibition (Additional file 1: Figure S1). These two mAbs are reasoned to react against conformational epitopes since they failed to bind any overlapping 15-mer peptides spanning the entire OX40 protein (data not shown). As shown in Figure 3(A), control mock treated CEM/OX40 and activated PBMCs, as expected, both stained dual-positive with the B-7B5 mAb and W4-54 mAbs. These data show that the comparative staining with B-7B5 and W4-54 mAbs can be potentially utilized to distinguish between non-ligated versus OX40L ligated forms of OX40. Figure 3(B) shows that although B-7B5 mAb stained HTLV-1+ T cell lines at high levels, little or no staining was noted with the use of the W4-54 mAb. In contrast, results of a WB analysis showed that the W4-54 mAb readily reacts to the p50 of the OX40 molecule in lysates of the HTLV-1+ T cell line, YT/cM1 (Additional file 2: Figure S2). These results suggest that the OX40L binding site of OX40 expressed by the HTLV-1+ T cell lines was altered, most probably due to pre-occupation with endogenous OX40L. To confirm this possibility, we explored the presence of OX40-OX40L complexes expressed by HTLV-1+ T cell lines using our in-house ELISA. Cell lysates of the ATL-derived HTLV-1+ T cell line (ILT-H2) were first captured with the use of immobilized anti-OX40L (clone HD-1) or anti-OX40 (clone B-7B5) mAb, respectively. The levels of captured antigens were assayed with the use of HRP-labeled anti-OX40 mAb or anti-OX40L mAb. Although it is reasonable to assume that the natural interaction between OX40 and OX40L on the living cell surface may be dissociated by the detergent treatment, as shown in Figure 4, low but significant levels of OX40-OX40L complex were still detectable in the cell lysates.

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