Key Points

Crosslinking of HLA I triggers complex formation between TLR4 and HLA I in ECs.

P-selectin externalization and monocyte binding induced by HLA I Ab depend on TLR4.

TLR4 functions as a coreceptor of HLA I in human ECs.

Abstract

Donor-specific HLA Abs contribute to Ab-mediated rejection (AMR) by binding to HLA molecules on endothelial cells (ECs) and triggering intracellular signaling, leading to EC activation and leukocyte recruitment. The molecular mechanisms involving donor-specific HLA Ab–mediated EC activation and leukocyte recruitment remain incompletely understood. In this study, we determined whether TLRs act as coreceptors for HLA class I (HLA I) in ECs. We found that human aortic ECs express TLR3, TLR4, TLR6, and TLR10, but only TLR4 was detected on the EC surface. Consequently, we performed coimmunoprecipitation experiments to examine complex formation between HLA I and TLR4. Stimulation of human ECs with HLA Ab increased the amount of complex formation between HLA I and TLR4. Reciprocal coimmunoprecipitation with a TLR4 Ab confirmed that the crosslinking of HLA I increased complex formation between TLR4 and HLA I. Knockdown of TLR4 or MyD88 with small interfering RNAs inhibited HLA I Ab–stimulated P-selectin expression, von Willebrand factor release, and monocyte recruitment on ECs. Our results show that TLR4 is a novel coreceptor for HLA I to stimulate monocyte recruitment on activated ECs. Taken together with our previous published results, we propose that HLA I molecules form two separate signaling complexes at the EC surface, that is, with TLR4 to upregulate P-selectin surface expression and capture of monocytes to human ECs and integrin β4 to induce mTOR-dependent firm monocyte adhesion via ICAM-1 clustering on ECs, two processes implicated in Ab-mediated rejection.

Footnotes

This work was supported by National Institute of Allergy and Infectious Diseases Grant R01AI135201 (to E.F.R., N.M.V., and E.R.). E.F.R. was also supported by National Institute of Allergy and Infectious Diseases Grants R21AI156592, U19AI128913, and P01AI120944 and National Institute of Allergy and Infectious Diseases Contract 75N93019C00052. E.R. was also supported by National Cancer Institute Grant P01CA236585, National Institute of Allergy and Infectious Diseases Grant R21AI156592, and by Department of Veterans Affairs Merit Award 1I01BX003801. In addition, this work was supported by the Norman E. Shumway Career Development Award from the International Society of Heart and Lung Transplantation and Enduring Hearts (to N.M.V., 2017–2019) and the UCLA Faculty Development Award (to N.M.V., 2019–2020). J.N.-M. was supported by the Ruth L. Kirschstein National Research Service Award T32HL069766.

The online version of this article contains supplemental material.

Abbreviations used in this article:

AMRAb-mediated rejectionECendothelial cellHLA IHLA class IHLA IIHLA class IIPFAparaformaldehydesiRNAsmall interfering RNATRIFToll/IL-1R domain–containing adaptor inducing IFN-βWPBWeibel–Palade bodyvWFvon Willebrand factorReceived April 18, 2022.Accepted July 22, 2022.Copyright © 2022 by The American Association of Immunologists, Inc.