Breast cancer is one of the most common malignancies among woman. Decades of scientific study have linked the
overexpression of ERBB2 antigen to aggressive tumors. To target aggressive breast cancer, chimeric antigen receptor
(CAR) technology can be utilized. For this, human T-cells are transduced with a gene sequence encoding a CAR that is
specific for tumor-associated antigens (TAAs). These genetically-engineered CAR transduced T-cells (CAR-T cells) are
able to target the tumor antigen without the need for major histocompatibility complex (MHC) recognition, rendering
it a potentially universal immunotherapeutic option. However, efficient transduction of therapeutic gene into human
T-cells and further cell expansion are challenging. In this study, we reported a successful optimization of a transduction
protocol using spinoculation on CD3+ T-cells with different concentrations of lentiviral plasmid encoding the CAR gene.
CD3+T-cells were isolated from the peripheral blood mononuclear cells (PBMCs). The constructed CAR gene was inserted
into a lentiviral plasmid containing the green fluorescent protein (GFP) tag and lentiviral particles were produced. These
lentiviral particles were used to transduce activated T-cells by spinoculation. T-cells were activated using Dynabeadconjugated
CD3/CD28 human T-cell activator and interleukin-2 (IL-2) before transduction. CD3+ T-cells were selected
and GFP expression, which indicated transduction, was observed. Future studies will focus on in vitro and in vivo models
to determine the efficiency of CAR-T cells in specifically targeting ERBB2-expressing cells.
T cells undergo a series of complex phenotypic changes before achieving maturation. Discrete stages of T-cell differentiation are simplified to four stages (pro-, pre-, cortical and mature-T cell) and used in the classification of T-cell leukaemia. HLA-DR has been reported to be expressed in immature T-cell acute lymphoblastic leukemia (ALL) and also confer a poorer treatment outcome. Simultaneously, the genotype goes through distinct pattern changes due to rearrangement of T-cell receptor (TCR) genes. TCR gene rearrangement is important in the diagnosis of clonality and used as markers to detect minimal residual disease in lymphoproliferative disorders. We identified a subset within Pro-T and Pre-T cell cases distinguished by the expression of HLA-DR. These subgroups appeared to be more immature as rearrangement of the TCR-gamma gene was either at germline or involved only the first constant region (C1) unlike a more rearranged pattern in the HLA-DR-subgroups. We also observed a higher incidence of mediastinal mass (67%) in the HLA-DR-subgroup in the Pre-T stage. These characteristics may be useful as markers to further refine staging of T-cell ALL and determine prognosis.