Generation of Potent Tumor-Specific CTLs from High-Dose IL-2 Activated naïve CD8 T Cells with Overcoming the Tumor-Derived Immune Suppression
Authors: Nguyen Hong-Hanh , Kim Dong-hwan , Lee Hyun-Ju , Jung Sung-Hoon , Kim Hyeoung-Joon , Lee Je-Jung , Yang Deok-Hwan 
Published: October 2019

Abstract
(Background) Adoptive T cell therapy using tumor-infiltrating lymphocytes (TILs) is a promising treatment for cancer patients unresponsive to conventional therapies. However, clonal expansion of T cells easily faced with T cell exhaustion or terminal differentiation which related to suppression of the tumor-killing function in tumor microenvironment. Naïve CD8+T cells have an astounding capacity to react to antigens by massive expansion and differentiation into cytotoxic effector cells. However, it is the main key factor that how can increase the limited number of tumor-specific naïve CD8 T cells due to the negative selection during T cell thymic development.
(Methods) In human, naïve, memory T cells and TILs were activated with CD3/CD28 dynabeads and culture on CD2-coated plate to generate various effector T cell populations (Teff N, Teff M and activated TILs). Those different effectors were analyzed the expression of surface exhaustion phenotypes, intracellular transcription factors (T-bet/Eomes) and granzyme/perforin secretions using FACS and western blot assay. Telomere length of three kinds of effector cells was analyzed. High-dose IL-2 (1ug/mL) activated naïve CD8 T cells were stimulated with tumor antigen-loaded dendritic cells (DCs) and then, ELISPOT/cytokine ELISA assay was performed to evaluate tumor-specific (TA) CTL function. In murine model, we also checked the functional difference of each effector cells as like the same methods. In addition, tumor challenging test using EG7-EL4 cell line (OVAp expression) was performed in C57BL/6 mice which adoptively transferred with OT-I thy1.1 Teff N, Teff M and activated TILs.
(Results) In vitro expansion of all human naïve, memory and TIL CD8+ T cells was induced successfully. After 3-5 days of expansion, effectors from different progenitors were assessed for the several activation markers CD44, OX40 and CD27 and were considered as the CD62LlowCD44highOX40highCD27high populations. Population frequency of Teff N was significantly higher than Teff M (p < 0.05) or activated TILs (p < 0.005). Telomere length, which correlates with replicative capacity, was greatest in TeffN, shorter in TeffM and shortest in aTILs. When compared the T cell exhaustion phenotypes in three different effector cells, TeffN showed the very low expression of inhibitory markers including PD-1, CTLA-4, and KLRG-1.aTILs expressed most high exhaustion phenotypes with shorter telomere length. Moreover, the secretion of cytotoxic granules such as granzyme B and perforin gradually increased in all effector cells but, among the fully effector cells status, TeffN possess the highest expression level compared to TeffM and aTILs wheras similar level of IFN-r. To further confirm expression profile of T-box transcription factors, the expression of both T-bet and Eomes in TeffN increased at relatively early time point (D+3) and sustained high expression levels during effector status (D+5 and D+8). High expression of T-bet in TeffN promoted the full effector generation and Eomes expression linked to formation of long-term tumor-specific memory population. In CTL function, high-dose IL2-activated naïve T cells were successfully increased and generated the tumor-specific CTLs co-cultured with TA DCs, which inducing the outstanding CTL function compared to those from memory CD8 T cells or aTILs.
(Discussion) High-dose IL-2 could increase the tumor-specific naïve CD8 T cells without resulting in clonal exhaustion and could generate the potent tumor-specific CTLs with overcoming the tumor-derived immune suppression compared with CD8+ TILs or secondary effectors from memory T cells.

Disclosures
No relevant conflicts of interest to declare.