Pembrolizumab Interferes with the Differentiation of Human FOXP3+–Induced T Regulatory Cells, but Not with FOXP3 Stability, through Activation of mTOR

Abstract

Programmed cell death 1 (PD-1) is critical for T regulatory cells (Tregs) to maintain peripheral tolerance to self-antigens. In the tumor microenvironment, interaction between PD-1 and its ligands supports tumor immune evasion. Pembrolizumab blocks interactions of PD-1 with its ligands, enhancing antitumor and clinical responses. We and others have reported that pembrolizumab does not affect function or phenotype of thymic-derived Tregs; however, little is known about its effect on extrathymic differentiation of peripheral Tregs. In this study, we investigated the effect of pembrolizumab on in vitro–induced Tregs (iTregs). Our work showed that PD-1 blockade interferes with iTreg differentiation and has no potential effect on the stability of FOXP3 after differentiation. Additionally, we found that both nontreated and pembrolizumab-treated iTregs were suppressive. However, pembrolizumab-treated iTregs were relatively less suppressive in higher Treg ratios and failed to produce IL-10 compared with their nontreated counterparts. Different methods including transcriptomic analyses confirmed that the downregulation of FOXP3 was mediated by activating mTOR and STAT1 and inhibiting MAPK pathways, shifting the iTreg polarization in favor of Th1 and Th17 subsets. To confirm the role of mTOR activation, we found that rapamycin diminished the effect of pembrolizumab-mediated downregulation of FOXP3. Ingenuity pathway analysis revealed that pembrolizumab-treated iTregs showed upregulation of genes promoting DNA repair and immune cell trafficking, in addition to downregulation of genes supporting cellular assembly and organization. To our knowledge, this is the first study to show that pembrolizumab interferes with differentiation of human FOXP3+ iTregs and to disclose some of the molecular pathways involved.

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

“…Therefore, we investigated the effects of different ICIs on tumor-infiltrating FoxP3 + Tregs in breast cancer patients. We have previously reported that anti-PD-1 (pembrolizumab) does not affect the phenotype or function of Tregs, as it instead affects the differentiation of FoxP3 + -induced Tregs [17,18]. In the present study, we found that the levels of FoxP3 + Helios + Treg remained unaffected upon the treatment with anti-PD-1.…”

“…Unlike anti-TIM-3 mAb, pembrolizumab treatment resulted in a complete blockade of the PD-1 surface expression on T cell subsets; this complete blockade was most likely due to a "masking effect" blocking the epitope of PD-1 by pembrolizumab, so the fluorescence-activated cell sorting (FACS) detection antibody would not be able to bind the same epitope of PD-1 showing no expression. This justification was supported and confirmed by qPCR and Western plot data indicating that pembrolizumab does not alter the expression of PD-1 at the mRNA and protein levels, as it only blocks its epitope [17].…”

“…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.…”

“…Quality-trimmed pair end reads were aligned to the hg19 human reference genome in CLC Genomics Workbench-12 (Qiagen) [17,24]. The abundance of the expression of transcripts was determined as the score of TPM (transcripts per million) mapped reads.…”

Hyperprogression Under Immune Checkpoint-Based Immunotherapy—Current Understanding, The Role of PD-1/PD-L1 Tumour-Intrinsic Signalling, Future Directions and a Potential Large Animal Model

“…The results were analogous when PD-1 was blocked with a mAb. On the other hand, Nair et al described inhibition of peripheral FoxP3 T-reg cells differentiation and a FoxP3 down-regulation through mTOR pathway under pembrolizumab-mediated PD-1 blockade [52]. The treated T-cells were less suppressive, but the study has not been validated in vivo.…”