Breast Cancer Cells and PD-1/PD-L1 Blockade Upregulate the Expression of PD-1, CTLA-4, TIM-3 and LAG-3 Immune Checkpoints in CD4+ T Cells

Abstract

: Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype, and it exhibits resistance to common breast cancer therapies. Immune checkpoint inhibitors (ICIs) targeting programmed cell death 1 (PD-1) and its ligand, PD-L1, have been approved to treat various cancers. However, the therapeutic efficacy of targeting PD-1/PD-L1 axis in breast cancer is under clinical investigation. In addition, the mechanisms of action of drugs targeting PD-1 and PD-L1 have not been fully elucidated. In this study, we investigated the effect of human TNBC cell lines, MDA-MB-231 and MDA-MB-468, and the non-TNBC cell line, MCF-7, on the expression of immune checkpoints (ICs) on CD4+ T cell subsets, including regulatory T cells (Tregs), using a co-culture system. We also examined the effect of blocking PD-1 or PD-L1 separately and in combination on IC expression by CD4+ T cell subsets. We found that breast cancer cells upregulate the expression of ICs including PD-1, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) and lymphocyte activation gene-3 (LAG-3) in CD4+ T cell subsets. We also found that the co-blockade of PD-1 and PD-L1 further upregulates the co-expression of TIM-3 and LAG-3 on CD4+CD25+ T cells and CD4+CD25+FoxP3+Helios+ Tregs in the presence of TNBC cells, but not in non-TNBC cells. Our results indicate the emergence of compensatory inhibitory mechanisms, most likely mediated by Tregs and activated non-Tregs, which could lead to the development of TNBC resistance against PD-1/PD-L1 blockade.

Blockade of PD-1, PD-L1, and TIM-3 Altered Distinct Immune- and Cancer-Related Signaling Pathways in the Transcriptome of Human Breast Cancer Explants

“…Together, these data suggested that blocking one IC or IC ligand can result in the upregulation of alternative ICs, which potentially leads to T cell exhaustion [56] and gives rise to compensatory mechanisms for tumor cell survival and immune response evasion leading to acquired resistance [20,41]. In parallel with this, we previously investigated the effect of blocking PD-1 and PD-L1 on CD4 + T cells, including Tregs, using a co-culture model of the MDA231 human TNBC cell line [20]. We found that the single or dual blockade of PD-1 and PD-L1 resulted in the upregulation of other ICs on CD4 + T cells, such as TIM-3, CTLA-4 and LAG-3, indicating the emergence of compensatory mechanisms that potentially lead to resistance to ICIs [20].…”

“…In parallel with this, we previously investigated the effect of blocking PD-1 and PD-L1 on CD4 + T cells, including Tregs, using a co-culture model of the MDA231 human TNBC cell line [20]. We found that the single or dual blockade of PD-1 and PD-L1 resulted in the upregulation of other ICs on CD4 + T cells, such as TIM-3, CTLA-4 and LAG-3, indicating the emergence of compensatory mechanisms that potentially lead to resistance to ICIs [20]. Other studies have also shown that resistance-mediated mechanisms in response to PD-1 or PD-L1 blockades are associated with the overexpression of alternative ICs in murine and human studies [57][58][59][60].…”

“…Tissue pieces were then cultured in a 6-well tissue culture-treated plate in complete media (RPMI-1640 media (Life Technologies) supplemented with 10% FCS (Hyclone) and 1% penicillin/streptomycin (Hyclone)) and 600 international units (IU) of interleukin-2 (IL-2, PeproTech, Hamburg, Germany) in the presence or absence of different immune checkpoint inhibitors, including 2 µg/mL of anti-PD-1 mAb (pembrolizumab; Merck, Branchburg, NJ, USA), 0.5 µg/mL of anti-PD-L1 mAb (atezolizumab, BioVision Inc., Milpitas, CA, USA), or 10 µg/mL of anti-TIM-3 (functional grade, Biolegend, San Diego, CA, USA). The dosage used of mAbs was based on titration that was previously performed [ 17 , 18 ], as previously published [ 19 , 20 , 21 , 22 ]. The treatment with ICIs was done on day 0.…”

“…Multiple ICs are expressed on activated T cells, but excessive stimulation during in vitro expansion may lead to T cell exhaustion, which is characterized by the overexpression of inhibitory ICs [28]. Previously, we showed that the co-blockade of PD-1 and PD-L1 upregulated the surface expression of CTLA-4, TIM-3, and LAG-3 on CD4 + T cell subsets by using a co-culture system with human breast cancer cell lines [20].…”

CD4 T-cell immune stimulation of HER2+ breast cancer cells alters response to trastuzumab in vitro

“…Culturing of cancer cells with immune cells, or onco-immune co-culturing, has been used to study immune interactions between cancer cells and tumor associated macrophages (TAMs) [21][22][23]. Castellaro et al co-cultured MCF-7 breast cancer cells with TAMs and found TAMs promoted cell proliferation and metastasis.…”