AUTHOR=Muller Yannick D. , Nguyen Duy P. , Ferreira Leonardo M. R. , Ho Patrick , Raffin Caroline , Valencia Roxxana Valeria Beltran , Congrave-Wilson Zion , Roth Theodore L. , Eyquem Justin , Van Gool Frederic , Marson Alexander , Perez Laurent , Wells James A. , Bluestone Jeffrey A. , Tang Qizhi 

TITLE=The CD28-Transmembrane Domain Mediates Chimeric Antigen Receptor Heterodimerization With CD28

JOURNAL=Frontiers in Immunology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2021.639818

DOI=10.3389/fimmu.2021.639818

ISSN=1664-3224

ABSTRACT=<p>Anti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge domain (HD) and the transmembrane domain (TMD) between the extracellular antigen-targeting CARs and the intracellular signaling modalities of CARs has not been systemically studied. In this study, a series of 19-CARs differing only by their HD (CD8, CD28, or IgG<sub>4</sub>) and TMD (CD8 or CD28) was generated. CARs containing a CD28-TMD, but not a CD8-TMD, formed heterodimers with the endogenous CD28 in human T cells, as shown by co-immunoprecipitation and CAR-dependent proliferation of anti-CD28 stimulation. This dimerization was dependent on polar amino acids in the CD28-TMD and was more efficient with CARs containing CD28 or CD8 HD than IgG<sub>4</sub>-HD. The CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but had a significantly reduced CD28 cell-surface expression. These data unveiled a fundamental difference between CD28-TMD and CD8-TMD and indicated that CD28-TMD can modulate CAR T-cell activities by engaging endogenous partners.</p>