Author ORCID Identifier

https://orcid.org/0000-0003-3470-0921

Date of Graduation

8-2022

Document Type

Thesis (MS)

Program Affiliation

Experimental Therapeutics

Degree Name

Masters of Science (MS)

Advisor/Committee Chair

Matthew Gubin

Committee Member

Stephanie Watowich

Committee Member

Michael Curran

Committee Member

Powel Brown

Abstract

Advancements in immunotherapy treatments have become essential in the treatment of various cancers, such as those with high tumor burden like melanoma(Brahmer et al., 2012; Hodi et al., 2010). Immunotherapy, specifically immune checkpoint therapy (ICT) provides antagonist effect on specific immune checkpoints to control effector T cell functions(Pardoll, 2012). This therapy allows for the modulation of anti-tumor responses through the use of monoclonal antibodies, such as anti-CTLA-4 or anti-PD-1 that block the receptors from binding with their ligands, thus allowing for CD4+ and CD8+ T cell priming and activation(Wei et al., 2017). However, the required epigenetic and transcriptional features that T cells must possess to efficaciously respond to ICT remains unclear. We show the transcription factor BHLHE40 is required in T cells for effective ICT response in mice bearing immune-edited tumors. Furthermore, we show BHLHE40 is upregulated in tumor antigen-specific CD8+ and CD4+ T cells upon ICT treatment with anti-CTLA-4 or anti-PD-1. Using single-cell RNA sequencing (scRNAseq), we revealed ICT-induced immune compartment remodeling in intratumoral immune cells taken from BHLHE40-deficent mice.

These BHLHE40-dependent gene expression changes indicated dysregulated metabolism, NF-kB signaling, and IFNg response within certain subpopulations of CD4+ and CD8+ T cells. Intratumoral CD4+ and CD8+ T cells from BHLHE40-deficient mice also exhibited higher expression of the inhibitory receptor gene Tigit and displayed alterations in chemokine/chemokine receptor gene expression with altered gene expression of granzyme family members. Mice lacking BHLHE40 had reduced ICT-driven IFNg production by CD4+ and CD8+ T cells and defects in ICT-induced remodeling of macrophages from a CX3CR1+ CD206+ subpopulation to an iNOS+ subpopulation that is typically observed during effective ICT. Although both anti-PD-1 and anti-CTLA-4 ICT in BHLHE40-deficient mice lead to 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.

Keywords

BHLHE40, ICT, anti-CTLA-4, anti-PD-1, scRNAseq, biomarker

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