Functional Impairment of Co-Inhibitory Receptors Promotes T-Cell Proliferation in HTLV-1 Associated Adult T-Cell Leukemia Cells
Authors: Kinosada Haruka , Yasunaga Jun-ichirou , Shimura Kazuya , Matsuoka Masao 
Published: October 2019

Abstract
Introduction
Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1). It has been reported that co-inhibitory receptors such as programmed cell death 1 (PD-1) and T cell immunoglobulin and ITIM domain (TIGIT) are highly expressed on ATL cells and HTLV-1 infected cells. However, ATL cells and HTLV-1 infected cells proliferate regardless of their high expression. Although it has been known that HTLV-1 bZIP factor (HBZ), which is constitutively expressed in ATL cells and HTLV-1 infected cells, promotes T cell proliferation, the detailed mechanisms remain unclear. In this study, we found that HBZ promotes T-cell proliferation by interfering the suppressive function of co-inhibitory receptors.
Methods
We analyzed T-cell proliferation of HBZ transgenic (HBZ-Tg) mice that specifically express HBZ in CD4+ T cells, and expression of co-inhibitory and co-stimulatory molecules on ATL cells and CD4+ T cells of HBZ-Tg mice. Furthermore, the function of TIGIT and PD-1 was studied using HBZ-transduced murine CD4+T cells. The co-localization of SHP-2 and PD-1 in the presence of HBZ was analyzed by immunoprecipitation and confocal microscope. The immunoprecipitation and confocal microscope were also used to analyze interaction between HBZ and THEMIS and HBZ localization in the presence of THEMIS.
Results
Although HBZ promotes T-cell proliferation, we found that some co-inhibitory receptors, TIGIT and PD-1, were highly expressed on CD4+ T cells of HBZ-Tg mice and ATL cells. As mechanisms, HBZ upregulated transcriptions of these genes. Based on these observations, we hypothesized that HBZ impairs suppressive functions of TIGIT and PD-1 while it increases expression of these co-inhibitory receptors. To address this question, we analyzed the suppressive activity of TIGIT and PD-1 in the presence of HBZ. We transduced HBZ by the retrovirus vector into primary murine T cells and evaluated the proliferation after stimulated TIGIT or PD-1 with anti-CD3 and its ligand. As a result, TIGIT and PD-1 did not inhibit T-cell proliferation in the presence of HBZ, indicating that HBZ impairs the suppressive function of TIGIT and PD-1. Both TIGIT and PD-1 possess SHP-2, a tyrosine phosphatase, binding domains in its cytoplasmic tail, ITIM or ITSM motif. Therefore, we next studied whether HBZ influences the interaction between PD-1 and SHP-2. Tyrosine phosphorylation of PD-1 was induced with pervanadate and then SHP-2 recruitment and PD-1/SHP-2 co-localization were investigated. HBZ inhibited recruitment of SHP-2 to the ITSM motif of PD-1. Indeed, phosphorylation of SHP-2 was decreased in CD4+T cells of HBZ-Tg mice and HBZ-transduced murine primary T cells. Furthermore, function of SHP-2 to dephosphorylate ZAP-70 and CD3-zeta was suppressed in the presence of HBZ. These data showed that HBZ inhibited recruitment of SHP-2 to ITSM motif of PD-1 and suppressed its inhibitory function. Next, we examined how HBZ inhibits the interaction between PD-1 and SHP-2. HBZ did this by interacting with THEMIS, which forms a complex with Grb2 and SHP-2. Moreover, HBZ hindered the interaction between THEMIS and Grb2. In general, THEMIS is localized in the cytoplasm, whereas it has been reported that HBZ is localized in the nucleus. When we expressed THEMIS or HBZ, THEMIS existed in the cytoplasm (50 of 50 cells: 100%) whereas HBZ was mainly localized in the nucleus (67 of 74 cells: 90.5%). Interestingly, when both proteins were expressed in the same cells, HBZ changed its localization to cytoplasm (28 of 79 cells: 35.4%) and co-localized with THEMIS. These findings suggest that THEMIS changes the localization of HBZ from nucleus to cytoplasm. Thus, HBZ functions not only in the nucleus but also in the cytoplasm by interacting with host factors. Since THEMIS is expressed only in T-lineage cells, inhibition of the suppressive effects of co-inhibitory receptors by HBZ accounts for how HTLV-1 induced proliferation only T cells in vivo.
Conclusions
Our findings demonstrated that HBZ promotes T-cell proliferation upon TCR stimulation by impairing the suppressive signal of co-inhibitory receptors. This study presents the first evidence of mechanisms how HBZ attenuates the inhibitory signals and promotes T-cell proliferation.

Disclosures
No relevant conflicts of interest to declare.