Unexpected Monoclonal T Cell Line Derived From the Bone Marrow of a Multiple Myeloma Patient.
Authors: Levy Debora , Brocardo Graciela Aparecida , de Godoy Carla Rosa Teixeira , Linardi Camila da Cruz Gouveia , Ruiz Jorge Maria , Martinez Gracia , Kleber Adilson Ferreira , Maria Durvanei Augusto , Pereira Juliana , Bydlowski Sergio Paulo 
Published: October 2019

Abstract
Abstract 4905

Background
Multiple Myeloma (MM) is a mature B-cell lymphoproliferative disorder in which the microenvironment of the bone marrow (BM) is important in its pathophysiology. BM niche is composed of osteoblasts, endothelial cells, stromal cells, adipocytes, extracellular matrix proteins and lymphocytes. The interaction between MM cells and BM triggers multiple proliferative and antiapoptotic signaling pathways. Moreover, an adequate microenvironment could increase survival and chemoresistance of MM cells to current therapies. However, our knowledge in this field is still poor and many details are unknown. Therefore, there is a strong need to further study the MM BM niche and understand how it influences MM cell growth, survival and development of resistance to chemotherapy.

Methods
BM (with 31.6% of plasma cells) was obtained from a 67-yr old woman (EC DS IIIA, ISS II) before treatment. Whole BM was cultivated in RPMI with10% FBS. After 36 days several colonies were observed and the cells had a fibroblast-like appearance. They were transferred again to another flask and after 30 days, small rounded floating cells were seen in the media or attached to the adherent fibroblast-like cells. The floating cells were transferred to another flask. They were named MOX cells. Immunophenotyping was done by flow cytometry before and after 9 days of culture and in the MOX cells. PCR was performed to detect clonality in gamma T cell receptor (TCR). In other experiment, seven Nude/SCID mice were injected IV or subcutaneously (SC) with MOX cells (4×106/100mL PBS) to test for tumorigenicity. After 15 and 30 days blood was drawn and examined for the presence of these cells. Animals were sacrificed at the 30th day when immunophenotyping was performed in blood and bone marrow.

Results
Cells prior to culture were CD45-, CD19-, CD38+++ and CD56+++ compatible with plasma cell phenotype. MOX cells had a doubling time of 20 h, DNA index of 1,19 (hyperdiploid) with 22.5% of cells in S, 13.88% in G2-M and 63.62% in G0-G1 phase. Unexpectedly, flow cytometry results of MOX cells showed a phenotype of mature T cell (CD45+++/CD38+CD3+/CD4+/CD8-/CD7+/CD5+/CD2-/TCRab+). The analysis of V(D)J rearrangement in T cell receptor demonstrated that the MOX cells were monoclonal biallelic to TCR gamma gene. We demonstrated the malignant feature of the MOX cells in Nude/SCID mice. MOX cells were observed in peripheral blood from all animals 15 and 30 days after IV or SC injection of MOX cells. After 30 days MOX cells were found in the BM in a considerable amount (10%).

Conclusions
1) Monoclonal malignant T cells were unexpectedly obtained from the BM of a patient with MM, a neoplasia of the B-cell linage. 2) This new cell line is potentially useful for future studies in this field.

Disclosures
No relevant conflicts of interest to declare.