The mechanistic basis of anti-CD6 as a novel form for the treatment of autoimmune diseases and cancer – UROP Summer Symposium 2021

The mechanistic basis of anti-CD6 as a novel form for the treatment of autoimmune diseases and cancer

Sarah Ory

Sarah Ory

Pronouns: She/Her/Hers

UROP Fellowship: Biomedical and Life Sciences
Research Mentor(s): Mikel Gurrea Rubio, PhD
Research Mentor Institution/Department: Michigan Medicine, Department of Internal Medicine – Division of Rheumatology

Presentation Date: Wednesday, August 4th
Session: Session 1 (3pm-3:50pm EDT)
Breakout Room: Room 2
Presenter: 5

Event Link

Abstract

Background: The use of Immune Checkpoint Inhibitors (ICIs) is limited by the induction of immune-related adverse events. CD6 is expressed by most T lymphocytes and a subset of natural killer (NK) cells, and engages the ligands CD166/ALCAM and CD318. Interrupting CD6 interaction with its ligands using UMCD6 (anti-CD6) reverses autoimmunity in mouse models of rheumatoid arthritis, multiple sclerosis and uveitis, due to suppression of differentiation of effector Th1 and Th17 cells. Recently, we have demonstrated that UMCD6 directly activates CD8+T and NK cells, enhancing these cells to kill breast, lung, and prostate cancer lines, even more robustly than ICIs directed to the PD-1/PD-1L pathway. We now explore the mechanisms by which UMCD6 activates NK cells while controlling the differentiation of CD4 cells.

Methods: RNAseq was used to study the molecular changes occurring during NK activation by CD6 blockade. Data analysis was conducted using DESeq2 software, and genes showing adjustment ≤0.05 were considered significantly differentially expressed. RNAseq data was confirmed by RT-PCR and Western Blot. Isolation of total RNA was carried out by TRIZOL treatment (Invitrogen, Carlsbad, CA, USA) followed by RNeasy mini kit (Qiagen, Hilden, Germany). Equal amounts of protein (20µg per lane) were separated by Tris-Glycine SDS-PAGE and electro-blotted onto nitrocellulose membranes. Chemotaxis of NK cells was assessed by the number of isolated human NK cells using an IncuCyte® Chemotaxis System assay. One-way ANOVA was used to compare groups. Significance was defined as p< 0.05. Results: Our preliminary RNAseq data from UMCD6-treated NK cells demonstrate extensive changes in gene expression induced by UMCD6. Expression of 180 genes was altered significantly, with 94 upregulated and 86 downregulated. These genes included: i) activating NK receptors (e.g. Klrk1 [NKG2D], Hcst [DAP10], and CD244 [2B4]), ii) genes whose protein products are shown to be important in the immunoregulatory signaling pathways PI3K, AKT and mTOR, all shown to be involved in the activation of several NK receptors by enhancing calcium flux, and iii) activation of genes that encode chemokines (e.g. CCL3 and CCL5, both found in activated NK cells). NK cells migrate in response to sCD318 with a peak response at 200 pg/ml (p< 0.01). This concentration of sCD318 is detectable in supernatants of cancer cell lines that we have used as targets for UMCD6-stimulated lymphocytes. NK cells also migrate toward stromal cell-derived factor (SDF)-1α (CXCL12) at a concentration of 25 ng/ml (p< 0.01), consistent with studies of NK chemotaxis to CXCL12 into sites of inflammatory responses or malignancies. Conclusion: The profound changes in gene expression induced by UMCD6 (anti-CD6) are consistent with our previous studies showing UMCD6 to concurrently control autoimmunity through effects on CD4+ lymphocyte differentiation while enhancing killing of cancer cells through activation of CD8+ and NK cells. The chemotactic properties of sCD318, a ligand of CD6, toward NK cells also demonstrates the importance of CD6 in migration of cytolytic lymphocytes into the tumor microenvironment. Altogether, these data point to a potential new approach to cancer immunotherapy (anti-CD6) that would suppress rather than instigate autoimmunity. Authors: Sarah Ory, Mikel Gurrea-Rubio, Qi Wu, Pei-Suen Tsou, Phillip L. Campbell, and David A. Fox
Research Method: Laboratory Research

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