Date of Award

6-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Medicinal Chemistry

Research Advisor

Ae-Kyung Yi, PhD

Committee

David Brand, PhD; Wei Li, PhD; Edward F. Rosloniec, Jr., PhD; Paul Thomas, PhD

Keywords

Collagen, Inhibitory Receptor, LAIR-1, Rheumatoid Arthritis, Src Homology Phosphatase, T cells

Abstract

Inflammation is a natural process in which the immune system concertedly responds to pathogens and abnormal cell growth to protect the host. For an efficient/effective immune response that circumvents tissue atrophy, a delicate balance between stimulatory and inhibitory mechanisms is essential for the proper functioning of the immune system. Immuno-receptor Tyrosine-based Inhibitory Motif (ITIM)-bearing receptors in immune cells, including T lymphocytes, play a major role in preventing autoimmune responses. Leukocyte-associated Ig-like Inhibitory Receptor 1 (LAIR-1) is one of ITIM-bearing receptors. The mechanism by which LAIR-1 attenuates T-cell response has yet to be completely understood. In this study, I investigated a part of the mechanisms by which LAIR-1 transduces inhibitory signal to attenuate T cell activation. I investigated whether the LAIR-1 signaling complex contains SH2 domain-containing phosphatase (SHP)-1, SHP-2, C-terminal src kinase (Csk), and/or the protein tyrosine phosphatase, non-receptor 22 (PTPN22), whether these phosphatases are activated upon LAIR-1 and its ligand interaction, and whether SHP1 plays a role in LAIR-1 signaling and T cell activation.

I validated previous findings that suggested LAIR-1 stimulation by its natural ligand collagen suppresses T cell antigen receptor (TCR)-mediated T-cell activation. To study proximal LAIR-1 signaling, transformed human T cells (Jurkat) that overexpress LAIR-1 were treated with the constituent chain of collagen type II [α1(II)] to stimulate LAIR-1. LAIR-1 activation by α1(II) induced phosphorylation of both SHP-1 and SHP-2, as well as phosphorylation of LAIR-1. In addition, LAIR-1 stimulation by α1(II) recruited the phosphorylated form of SHP-1, the phosphorylated form of SHP-2, and PTPN22 to LAIR-1 in T cells. However, Csk was constitutively bound to LAIR-1 in T cells. These results indicate that upon ligand binding LAIR-1 forms a signaling complex with SHP1, SHP-2, Csk, and PTPN22 and activates SHP-1 and SHP-2 in human T cells. To determine the role of SHP-1 in LAIR-1 signaling, the expression of SHP-1 in Jurkat-LAIR1 cells was knocked down by stably expressing SHP-1-specific short hairpin RNA interference (shRNA). I found that expression levels of SHP-1 correlated with expression levels of LAIR1 in Jurkat-LAIR-1 cells, indicating a possibility that SHP1 and LAIR-1 regulate expression of each other in T cells as a feedback mechanism. Phosphorylation of SHP-2 was not inhibited in SHP-1- knockdown cells, indicating that SHP-2 activation by LAIR-1 is not affected by SHP-1. To investigate whether SHP-1 mediates LAIR-1 inhibitory signaling to suppress TCR signal transduction, control and SHP-1-knockdown Jurkat-LAIR-1 cells were treated with α1(II) followed by TCR stimulation by anti-CD3.

SHP-1-knockdown in Jurkat-LAIR1 cells resulted in ablation of ZAP70 and ERK phosphorylation regardless of LAIR-1 signaling and TCR signaling, indicating that SHP-1 is required for activation of ZAP70 and ERKs in T cells. Thus, SHP-1 may play a dual function; an intermediate for LAIR-1 to regulate signaling phosphoproteins and as a positive regulator of signaling pathways like the TCR to promote cellular activation and effector function. These results are insightful because they show that inhibitory receptors like LAIR-1 can engage multiple SH2 domain-containing phosphatases, some of which have a dynamic function that can positively and negatively regulate intracellular biochemical activities to modulate cellular behavior and hyperactive T-cell response to avoid autoimmunity.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0009-0008-8051-4090

DOI

10.21007/etd.cghs.2023.0631

Additional Files
2023-018-Zacheaus-DOA.pdf (152 kB)
Declaration of Authorship
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