Abstract 5137: Humoral factors of breast tumor environment may boost PD-1 and CTLA4 gene expression of T cells, which has already been upregulated by CD28 stimulation
Authors: Kawaguchi Shun , Suzuki Eiji , Kawaguchi Kosuke , Pu Fengling , Yamaguchi Ayane , Tsuda Moe , Toi Masakazu 
Published: July 2016

Abstract
Background:
It has been reported that PD-1 and CTLA4 expression of T cells is up-regulated by CD28 stimulation in vitro, however the influence of humoral factors on their up-regulation is still incompletely understood. Therefore, in the current study, we evaluated if humoral factors of breast tumor environment induce further enhancement of their expression in vitro.
Materials and Methods:
Conditioned medium from MDA-MB 231 (triple negative human breast cancer cell line)(M), THP-1 (human monocytic cell line) (T) and co-culture of MDA-MB 231 and THP-1 (MT) were prepared. Pan T cells were isolated from PBMC of a healthy volunteer by MACS Pan T cell isolation kit (Miltenyi Biotec) and activated with Dynabeads Human T-activator CD3/CD28 (Thermo Fisher scientific). Activated and non-activated T cells were cultured with each conditioned medium M, T, MT and original RPMI-1640 medium. After cultivation for 24 hours, we analyzed mRNA expression level of PD-1, CTLA4 and FOXP3 of activated and non-activated T cells stimulated with each conditioned medium by quantitive real time-PCR using TaqMan gene expression probes. We also measured cytokines released into each conditioned culture supernatants by using human Bio-Plex multiplex assay system (Bio-Rad).
Results:
As it has been reported, mRNA expression of PD-1 and CTLA4 was up-regulated by CD28 stimulation in normal medium. Interestingly, PD-1 and CTLA4 mRNA of activated T cells incubated with MT was further up-regulated by 2 times and 1.5 times as compared to that of activated T cells with normal medium (PD-1 and CTLA4, respectively) while there was no up-regulation of PD-1 and CTLA4 in non-activated T cells incubated with MT. We further found that abundance of MIP-1β, VEGF, IL-7 and IP-10 was significantly higher in supernatant of MT-treated T cells than that of activated T cells in normal medium (98135.37 +/- 22.50 vs 4957.47 +/- 281.00 pg/ml (MIP-1β), 670.18 +/- 5.00 vs 311.77 +/- 26.75 pg/ml (VEGF), 4.65 +/- 0.50 vs 2.69 +/- 0 pg/ml (IL-7), 1654.53 +/- 53.00 vs 1013.91+/- 16.50 pg/ml (IP-10)).
Conclusions:
These findings suggest that humoral factors may contribute to immune escape by further up-regulation of PD-1 and CTLA4 on T cells which has already been activated by CD28 stimulation (in addition to antigen-TCR signal activation). It is assumed that a certain humoral factor, which might not be detected in this experiment although the candidate factors may be IL-7 and VEGF according to our cytokine study, induces inhibition of T cells via immune checkpoints.
Citation Format: Shun Kawaguchi, Eiji Suzuki, Kosuke Kawaguchi, Fengling Pu, Ayane Yamaguchi, Moe Tsuda, Masakazu Toi. Humoral factors of breast tumor environment may boost PD-1 and CTLA4 gene expression of T cells, which has already been upregulated by CD28 stimulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5137.