A Rare Population of Stem Cell Memory T Cells is an Apex of the Hierarchy of Adult T-Cell Leukemia
Authors: Nagai Yuya , Kawahara Masahiro , Hishizawa Masakatsu , Sugino Noriko , Shimazu Yayoi , Takaori-Kondo Akifumi 
Published: October 2019

Abstract
Adult T-cell leukemia (ATL) is a mature T cell neoplasm causatively associated with HTLV-1. It takes a long period, around 50 years, to develop ATL after the transmission of HTLV-1 in infancy through breastfeeding, suggesting the pool for the generation of ATL clone may exist somewhere. Recent reports showed human CD34+ bone marrow cells infected by HTLV-1 ex vivo can generate CD4+ T-cell lymphomas in immunodeficiency mice or that CD117+ early hematopoietic progenitors can develop lymphoma in transgenic mice model of Tax which is an oncogenic protein of HTLV-1. These results suggest the model of leukemia initiating cells, that leukemia is organized hierarchically and sustained by a small subset of stem/progenitors at the apex, might be acceptable in ATL. A more recent report demonstrated that a novel compartment of normal T cells, termed stem cell memory T cells (TSCM), possesses stem cell-like capacity including relatively enhanced self-renewal capacity and multipotent ability to generate all memory and effector T cell subsets. This concept prompts us to hypothesize that TSCM subset might contribute to the development of ATL.
First, to investigate whether hematopoietic stem/progenitor cells in primary samples of ATL patients are infected with HTLV-1, we sorted lineage(CD3, CD4, CD8, CD11b, CD14, CD19, CD20, CD56, CD235)-negative CD34+ cells from bone marrow samples of ATL patients by FACS AriaII, purified genomic DNA, and then performed PCR of pX region which is a virus specific gene. No amplification of pX was detected in all samples we studied.
Next, to focus on TSCM population, we established the eight-color staining method with lineage of NK/Monocyte/DC (CD11b, CD14 and CD56), CD4, CD45RA, CD45RO, CCR7, CD95, CD122 and CD25. We successfully separated CD4+ T cells from ATL patients into four subsets; naïve T (TN: CD4+CD45RA+CD45RO-CCR7+CD95-CD122-), stem cell memory T (TSCM: CD4+CD45RA+CD45RO-CCR7+CD95+CD122dim), central memory T (TCM: CD4+CD45RA-CD45RO+CCR7+), and effector memory T (TEM: CD4+CD45RA-CD45RO+CCR7-). Although ATL cells are generally CD45RO positive, so that TCM and TEM were extremely dominant, CD45RA positive populations did exist in all ATL samples we studied.
To assess the frequency of infected cells, we performed quantitative PCR with the sensitivity of 5 copies detectable at the minimum and calculated by dividing copy number of pX in HTLV-1 provirus with that of RNase P in human genome in genomic DNA from 1,000 cells of each subset. Almost all cells in TCM and TEM and more than 10% of cells in TSCM were infected in most of cases while none of infected cells were detected in TN in our experiment design. Since ATL is caused by monoclonal expansion from plenty of HTLV-1 infected clones, we focused on the existence of ATL original clone in each subset. We determined the ATL original clone by inverse PCR and the clone-specific provirus integration site by sequencing of the inverse PCR product in each case, and then constructed PCR primers being capable of detecting each ATL original clone by regular PCR. Interestingly we found each ATL clone was detected in TSCM, TCM, and TEM but not in TN subset in all studied cases, indicating TSCM could be an origin of ATL cells.
To investigate whether there is a hierarchy in ATL cells, we next performed in vitro culture with IL-7 and examined the status of CD45RA and CCR7 after 2 weeks. Intriguingly TSCM generated TCM and TEM while TCM and TEM did not generate TSCM, suggesting TSCM is an apex in HTLV-1 infected T cells.
To investigate tumorigenicity of TSCM, we adopted xenogeneic transplantation assay. When we inoculated 3x104 cells of each subset from one ATL patient into immune deficient mice (NOG mice) intraperitoneally, we observed high number of cells in peripheral blood and spleen of recipient mice transplanted with TSCM compared to TCM and TEM. Phenotypically TCM and TEM were generated in mice transplanted with TSCM as well as TCM and TEM while TSCM was only generated from TSCM but not TCM and TEM. Furthermore we analyzed the clonality of engrafted cells by ATL clone-specific PCR and inverse PCR, and found that the patient original clone becomes dominant in recipient mice inoculated with TSCM but not in those with TCM and TEM.
Taken together, our findings provide new insights that TSCM could be an apex of the hierarchy of ATL and play an important role in development and maintenance of ATL clone.

Disclosures:
No relevant conflicts of interest to declare.