Macrophage PD-L1 Expression is Increased by Toll-Like Receptor Activation with Select Oral Bacteria

Presentation: S006
Topic: Cancer Biology
Type: Oral
Date: Sunday, July 9, 2023
Session: 10:45 AM - 12:15 PM Cancer Biology Session 1
Authors: Olivia L Ricks, RN, BSN1; Juhi J Patel2; Zoë E Evans3; Gabriela S Fonseca1; Sarah I Elimam3; Shilpa M Rao, MD2; Yolanda E Hartman2; Adia Ingram, MS2; Jason M Warram, PhD2; Carissa M Thomas, MD, PhD2
Institution(s): 1Heersink School of Medicine, University of Alabama at Birmingham, Birmingham, AL; 2Department of Otolaryngology - Head and Neck Surgery, University of Alabama at Birmingham, Birmingham, AL; 3Cancer Biology Program, University of Alabama at Birmingham, Birmingham, AL

Background: Programmed Death Ligand 1 (PD-L1) acts as an immune checkpoint inhibitor and is expressed by immune cells in the tumor microenvironment (TME) as well as cancer cells in oral squamous cell carcinoma (OSCC). PD-L1 overexpression enables immune detection evasion, however, increased PD-L1, as determined by the combined positive score (CPS), is also needed for response to immunotherapy. Activation of toll-like receptors (TLRs) on antigen presenting cells (APCs) such as macrophages, which recognize bacteria, may upregulate PD-L1 in the TME.

Objective: To determine if TLR activation with TLR2 and TLR4 agonists or select oral bacteria, Streptococcus sanguinis and Fusobacterium nucleatum, will increase PD-L1 expression on macrophages.

Materials/Methods: THP-1 cells (human monocytic cell line) were treated with phorbol-12-myristate-13-acetate (PMA) for 72 hours to induce differentiation into macrophages (pTHP-1). After a 24-hour recovery, macrophages were activated with lipopolysaccharides (LPS), a component of gram-negative bacteria that is recognized by TLR4, or Pam3CSK4 (PCK), a component of gram-positive bacteria that is recognized by TLR2. Macrophages were treated with LPS or PCK (100 ng/mL) for 6, 12, 18, 24, 36 and 48 hours. Bacteria of interest, S. sanguinis and F. nucleatum, were grown to stationary phase and filter sterilized to obtain bacteria conditioned media (BCM). Macrophages were treated with the BCM for 12, 24, and 48 hours. PD-L1 expression was determined through western blotting and fluorescent immunohistochemistry (IHC) staining. Flow cytometry was used to assess baseline PD-L1 expression on THP-1 cells. A231 cells were used as a positive control for PD-L1 expression.

Results: THP-1 cells (monocytes) do not express PD-L1 at baseline, however, pTHP-1 cells (macrophages) have low levels of PD-L1 expression. When pTHP-1 cells are activated with either LPS or PCK, PD-L1 expression increases compared to baseline with peak expression of PD-L1 at 36 hours and 18 hours, respectively, seen on both western blot and fluorescent IHC staining. Treatment with F. nucleatum BCM (secreted factors from a gram negative bacteria found in the oral microbiome of OSCC) also significantly increases PD-L1 expression. A similar effect was not seen with S. sanguinis BCM (secreted factors from a gram positive bacteria in the oral microbiome of OSCC).

Conclusion: TLR activation of macrophages leads to increased PD-L1 expression. Secreted factors from F. nucleatum also increased PD-L1 expression but not secreted factors from S. sanguinis. Select bacteria in the oral microbiome may influence expression of immune checkpoint inhibitor signaling in the TME of oral squamous cell carcinoma.