RT Journal Article
SR Electronic
T1 A high-throughput two-cell assay for interrogating inhibitory signaling pathways in T cells
JF Life Science Alliance
JO Life Sci. Alliance
FD Life Science Alliance LLC
SP e202302359
DO 10.26508/lsa.202302359
VO 7
IS 3
A1 Sharma, Sumana
A1 Whitehead, Toby
A1 Kotowski, Mateusz
A1 Ng, Emily Zhi Qing
A1 Clarke, Joseph
A1 Leitner, Judith
A1 Chen, Yi-Ling
A1 Santos, Ana Mafalda
A1 Steinberger, Peter
A1 Davis, Simon J
YR 2024
UL http://www.life-science-alliance.org/content/7/3/e202302359.abstract
AB The recent success of immunotherapies relying on manipulation of T-cell activation highlights the value of characterising the mediators of immune checkpoint signaling. CRISPR/Cas9 is a popular approach for interrogating signaling pathways; however, the lack of appropriate assays for studying inhibitory signaling in T cells is limiting the use of large-scale perturbation-based approaches. Here, we adapted an existing Jurkat cell-based transcriptional reporter assay to study both activatory and inhibitory (PD-1-mediated) T-cell signaling using CRISPR-based genome screening in arrayed and pooled formats. We targeted 64 SH2 domain-containing proteins expressed by Jurkat T cells in an arrayed screen, in which individual targets could be assessed independently, showing that arrays can be used to study mediators of both activatory and inhibitory signaling. Pooled screens succeeded in simultaneously identifying many of the known mediators of proximal activating and inhibitory T-cell signaling, including SHP2 and PD-1, confirming the utility of the method. Altogether, the data suggested that SHP2 is the major PD-1-specific, SH2 family mediator of inhibitory signaling. These approaches should allow the systematic analysis of signaling pathways in T cells.