Supplementary Figure from BHLHE40 Regulates the T-Cell Effector Function Required for Tumor Microenvironment Remodeling and Immune Checkpoint Therapy Efficacy
<div>Abstract<p>Immune checkpoint therapy (ICT) using antibody blockade of programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) can provoke T cell–dependent antitumor activity that generates durable clinical responses in some patients. The epigenetic and transcriptional features that T cells require for efficacious ICT remain to be fully elucidated. Herein, we report that anti–PD-1 and anti–CTLA-4 ICT induce upregulation of the transcription factor BHLHE40 in tumor antigen–specific CD8<sup>+</sup> and CD4<sup>+</sup> T cells and that T cells require BHLHE40 for effective ICT in mice bearing immune-edited tumors. Single-cell RNA sequencing of intratumoral immune cells in BHLHE40-deficient mice revealed differential ICT-induced immune cell remodeling. The BHLHE40-dependent gene expression changes indicated dysregulated metabolism, NF-κB signaling, and IFNγ response within certain subpopulations of CD4<sup>+</sup> and CD8<sup>+</sup> T cells. Intratumoral CD4<sup>+</sup> and CD8<sup>+</sup> T cells from BHLHE40-deficient mice exhibited higher expression of the inhibitory receptor gene <i>Tigit</i> and displayed alterations in expression of genes encoding chemokines/chemokine receptors and granzyme family members. Mice lacking BHLHE40 had reduced ICT-driven IFNγ production by CD4<sup>+</sup> and CD8<sup>+</sup> T cells and defects in ICT-induced remodeling of macrophages from a CX3CR1<sup>+</sup>CD206<sup>+</sup> subpopulation to an iNOS<sup>+</sup> subpopulation that is typically observed during effective ICT. Although both anti–PD-1 and anti–CTLA-4 ICT in BHLHE40-deficient mice led to the same outcome—tumor outgrowth—several BHLHE40-dependent alterations were specific to the ICT that was used. Our results reveal a crucial role for BHLHE40 in effective ICT and suggest that BHLHE40 may be a predictive or prognostic biomarker for ICT efficacy and a potential therapeutic target.</p></div>
We previously developed a protocol for generating murine migratory dendritic cells type 1 (DC1s) from bone marrow and demonstrated polyI:C (pI:C)-activated DC1s loaded with tumor antigens (Ag) display efficacy against primary and metastatic mouse tumors, including B16-OVA melanoma. B16-OVA melanomas express OVA protein containing MHC class I (MHC-I) and MHC-II Ags, modeling expression of tumor-specific neoantigens (NeoAg). Intratumoral administration of OVA-loaded DC1 vaccines induced tumor regression in both treated and untreated B16-OVA tumors, indicating systemic anti-tumor immunity. The DC1 vaccine also displayed superior therapeutic efficacy compared to monocyte-derived DCs with the latter exclusively used in prior DC cancer vaccine clinical trials. Whereas DC1s specialize in cross-presenting Ags to CD8 T cells, in a non-vaccine-setting, DC1s also prime helper CD4 T cells by presenting MHC-II tumor Ags, with CD4 T cells then licensing DC1s in a CD40-dependent manner. This licensing enhances DC1 survival and expression of MHC and T cell costimulatory molecules (e.g., CD80/86, CD70) to support CD8 T cell priming. In a DC1 vaccine setting, how presentation of MHC-I and MHC-II NeoAgs shape the T cell response and therapeutic efficacy is unknown.
Methods
Using B16-OVA tumor bearing wild type (WT) mice, we compared vaccine efficacy and phenotype of DC1s using in vitro pI:C-activated, OVA-loaded WT DC1s or MHC-I-deficient or MHC-II-deficient DC1s generated from WT, H-2Kb-/- H-2Db-/- (MHC-I-/-), or I-Ab-/- (MHC-II-/-) mice.
Results
OVA-loaded pI:C-activated DC1 vaccines lacking either MHC-I or MHC-II expression or composed of a mixture of both were less effective than WT DC1 vaccines in B16-OVA tumor bearing WT mice, suggesting expression of both MHC-I and MHC-II on the same DC1 is necessary for efficacy. We then focused on mechanisms behind why OVA-loaded and pI:C activated DC1 vaccines require MHC-II expression and hypothesized they must present MHC-II NeoAgs to receive a CD40-dependent licensing signal from CD4 T cells. In vitro stimulation of DC1s with pI:C induced DC1 activation, but pI:C stimulation and induction of CD40 signaling via an agonist CD40 antibody induced expression of CD70, 4-1BBL, and the anti-apoptotic transcript Bcl2l1.
Conclusions
Our results reveal MHC-I and MHC-II expression on the same DC1 is critical for DC1 vaccine efficacy. Our data also indicate CD40 signaling, which likely requires MHC-II tumor Ag presentation by DC1s and interaction with CD4 T cells, may be critical for induction of CD70, 4-1BBL, and survival factors in DC1s to induce more potent antitumor responses. These data provide critical insights into mechanisms associated with DC1 vaccine efficacy.
Abstract Due to the poor correlation between steady state mRNA levels and protein products, traditional microarray analysis may miss many genes which are regulated primarily at the level of mRNA stability and translation. Posttranscriptional gene regulation mediated by microRNAs and RNA binding proteins (RBPs) is being recognized as an important form of gene regulation. The elav (embryonic lethal abnormal vision) family of RBPs, are paraneoplastic antigens, over-expressed in a variety of malignancies, including breast cancer. Antibodies against elav family members are believed to be cancer-protective. The elav family binds to the AU-rich elements (AREs) found in the 3’ untranslated regions (UTRs) of many early-response genes, including proto-oncogenes and cell cycle regulators. HuR, the ubiquitously expressed family member, has been described to play a role in cancer progression by stabilizing and translationally up regulating expression of its target mRNAs. Elevated levels of cytoplasmic HuR directly correlate with increased invasiveness of malignancy and poor prognosis for many cancers, including those of the breast. HuR has been described to positively control the expression of multiple genes in the acquired capabilities model, such as VEGF and HIF1α. Hence, it has been suggested that HuR may serve as a tumor maintenance gene which allows for cancers to proliferate. Therefore, it is of interest to discover in vivo HuR targets, as these genes may play vital roles in transformed cells. We have developed methods called RNA immunoprecipitations applied to microarrays, RIP-Chip. We used RIP-Chip to identify distinct subsets of HuR associated mRNAs in MDA-MB-231 and MCF-7 breast cancer cell lines and validated several novel targets. To further investigate the role of HuR in triple negative breast cancer, we over expressed HuR in MDA-MB-231 cells, which results in accelerated growth and alterations in cell cycle kinetics. Surprisingly, when employed in orthotopic mouse models of cancer, HuR over expression significantly inhibited growth of triple negative tumors by 90%. Putative mechanisms appear to be anti-angiogenic, as HuR over expression increases anti-angiogenic factors, but surprisingly, also down regulates pro-angiogenic factors such as VEGF. These results are highly significant because they implicate HuR as a master regulator of angiogenesis in triple negative breast cancer tumor formation and suggest potentially novel treatment methods for this aggressive form of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3271.
Supplementary Figure from BHLHE40 Regulates the T-Cell Effector Function Required for Tumor Microenvironment Remodeling and Immune Checkpoint Therapy Efficacy
