Proliferation response to interleukin-2 and Jak/Stat activation of T cells immortalized by human T-cell lymphotropic virus type 1 is independent of open reading frame I expression

Abstract

Human T-cell lymphotropic virus type 1 (HTLV-1), a complex retrovirus, encodes a hydrophobic 12-kD protein from pX open reading frame (ORF) I that localizes to cellular endomembranes and contains four minimal SH3 binding motifs (PXXP). We have demonstrated the importance of ORF I expression in the establishment of infection and hypothesize that p12(I) has a role in T-cell activation. In this study, we tested interleukin-2 (IL-2) receptor expression, IL-2-mediated proliferation, and Jak/Stat activation in T-cell lines immortalized with either wild-type or ORF I mutant clones of HTLV-1. All cell lines exhibited typical patterns of T-cell markers and maintained mutation fidelity. No significant differences between cell lines were observed in IL-2 receptor chain (alpha, beta, or gamma(c)) expression, in IL-2-mediated proliferation, or in IL-2-induced phosphorylated forms of Stat3, Stat5, Jak1, or Jak3. The expression of ORF I is more likely to play a role in early HTLV-1 infection, such as in the activation of quiescent T cells in vivo.

FIG. 1

Conservation of the ΔPstI mutation and absence of ORF I expression in PBMC immortalized with ACH.p12^I. (A) PCR amplification of genomic DNA from ACH- and ACH.p12^I-immortalized PBMC, HTLV-1-transformed MT-2, and naive PBMC. The native (−) or PstI-digested (+) 938-bp HTLV-1 ORF I/II-specific product is present in HTLV-1-positive cells and lacks the PstI site corresponding to the ORF I exon 3 splice acceptor site in ACH.p12^I-immortalized cells. (B) Reverse transcription-PCR of total RNA from ACH- and ACH.p12^I-immortalized PBMC, MT-2 cells, and naive PBMC. The 232-bp fragment specific for the pX-Rex-ORF I message is indicated and present in ACH-immortalized cells and MT-2 but is absent in ACH.p12^I-immortalized cells and HTLV-1-negative PBMC. The constitutive 183-bp abl transcript is indicated and present in all samples.

FIG. 2

Surface IL-2R chain expression on HTLV-1-immortalized cells as measured by flow cytometric detection of fluorescence intensity following labeling with PE-conjugated antibodies. The receptor chain detected by direct (IL-2β and -γ_c) or indirect (IL-2α) labeling with monoclonal antibodies is indicated along the top. For each IL-2R-chain-specific antibody (intensity histogram depicted as a solid line), fluorescence intensity with the isotype-matched control antibody is concurrently shown (intensity histogram depicted as a dotted line). Group mean channel fluorescence intensity of ACH- versus ACH.p12-immortalized PBMC were not different for IL-2R α, β, or γ_c chains (P > 0.05).

FIG. 3

Basal and IL-2-responsive proliferation of HTLV-1-immortalized cells. Results are expressed as the relative increase in colorimetric conversion of MTS by viable cells, measured by absorbance before and after culturing for 72 h in the presence of varying amounts of IL-2. Data are the means of duplicate wells plated in a 96-well microtiter plate and are representative of three independent experiments. Group mean proliferations for ACH- versus ACH.p12-immortalized PBMC were not statistically different at any concentration of IL-2 (P > 0.05).

FIG. 4

Constitutive and IL-2-responsive Stat3 (A) and Stat5 (B) phosphorylation in HTLV-1-immortalized PBMC, as measured by immunoblot assay. Cells used are indicated at the top of each panel. Cells were treated with (+) or without (−) 2 nM IL-2. Native or phospho-specific Stat antibody types used for immunoblotting are indicated to the left of each panel. Molecular mass is indicated to the right (MW). The identity of the 85-kD band recognized by the phospho-specific Stat5 antibody is unknown.

FIG. 5

Constitutive and IL-2-responsive Jak and Stat phosphorylation in HTLV-1-immortalized PBMC, as measured by immunoprecipitation. Cells used are indicated at the top of each panel. Cells were treated with (+) or without (−) 2 nM IL-2. Immunoprecipitations were performed with anti-Stat3 (A), -Stat5 (B), -Jak1 (C), or -Jak3 (D) antibodies. Immunoblotting was performed with antiphosphotyrosine (4G10) or anti-Jak/Stat antibodies, as indicated to the left of each panel. Panel B sample ACH.p12.1 (prestimulated Jak-1) failed to load in the gel. Molecular mass is indicated to the right (MW).