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HLA I Antibody-Activated Endothelium Polarized M2-Like Macrophages with Increased Efferocytic and Phagocytic Capacity

J. Nevarez-Mejia1, X. Wei1, N. M. Valenzuela1, M. Rossetti1, H. Pickering1, G. A. Fishbein1, A. Mulder2, E. F. Reed1

1Pathology and Laboratory Medicine, UCLA, Los Angeles, CA, 2Immunohematology and Blood Transfusion, LUMC, Leiden, Netherlands

Meeting: 2021 American Transplant Congress

Abstract number: 542

Keywords: Apoptosis, Endothelial cells, HLA antibodies, Immunogenicity

Topic: Basic Science » Endothelial Cell Biology

Session Information

Session Name: Endothelial Cell Biology

Session Type: Poster Abstract

Session Date & Time: None. Available on demand.

Location: Virtual

*Purpose: The role of intragraft macrophages (Mϕs) during antibody-mediated rejection (AMR) remains unclear. Anti-HLA Abs during AMR drives vascular inflammation as seen by monocyte recruitment and endothelial cell (EC) injury. Prior studies in our lab have indicated that monocyte and HLA I Ab-activated EC interactions skew monocytes to resolving M2-like phenotypes (CD68+CD206+CD163+). In this study, we validated the capacity of HLA I Ab-activated EC polarized Mϕs to a) efferocytose apoptotic ECs and b) phagocytose E.coli particles as in the setting of graft injury or infection, respectively. Moreover, we identified immune responses post-engulfment.

*Methods: Using a transwell in vitro co-culture system, human primary aortic ECs were stimulated with allele specific HLA I hIgG or HLA I F(ab’)2 Ab portion, anti-CD105 Ab, TNF-a, hIgG isotype control, or left untreated. Third-party human monocytes added above activated ECs transmigrate to the bottom chamber and differentiate into Mϕs (5-days). Cytokine-polarized Mϕs (M1/M2) were generated as controls. EC apoptosis was induced using TNF-a and cycloheximide (16-hours) and was validated via caspase-3 activation. Apoptotic ECs and E. coli particles were labeled with pHRodo (pH sensitive dye; measured by FITC). Mϕs were co-cultured with labeled apoptotic ECs or E.coli particles for 24-hours. The phenotype of FITC Pos Mϕs was measured by flow cytometry. Cytokine secretion of culture supernatants from Mϕs treated with target cells and untreated Mϕs was measured via Luminex.

*Results: All transwell polarized Mϕs exhibited significantly higher levels of apoptotic EC efferocytosis (~20-40% FITCPos) and E.coli phagocytosis (~60-80% FITCPos) compared to cytokine-polarized Mϕs. Immune profiling results show that FITCPos Mϕs post-apoptotic EC efferocytosis increased expression of CD40, CR1, and CD71 compared to FITCNeg Mϕs. Precisely, TNF-a activated EC polarized Mϕs significantly increased CD163 (*p=0.03) compared to untreated EC polarized Mϕs. Moreover, all transwell conditions exhibited an added increased in adhesive molecules (ICAM-1, CCR2, and PSGL-1) post-E.coli phagocytosis. Specifically, HLA I IgG Ab-activated EC polarized Mϕs significantly increased CR1 (*p=0.03) compared to untreated EC polarized Mϕs. Mϕ culture supernatants following apoptotic EC efferocytosis significantly increased FGF-2 while culture supernatants post-E.coli phagocytosis increased inflammatory cytokines (e.g. IL-6, IL-8, and IL-1b).

*Conclusions: Our studies indicate that endothelium polarized M2-like Mϕs exhibit high efferocytic and phagocytic capacities, and activate immune responses which are enhanced by TNF-a and HLA I Abs. This provides insight into Mϕ functions which may help mediate vascular repair, and clearance of pathogens following transplantation.

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To cite this abstract in AMA style:

Nevarez-Mejia J, Wei X, Valenzuela NM, Rossetti M, Pickering H, Fishbein GA, Mulder A, Reed EF. HLA I Antibody-Activated Endothelium Polarized M2-Like Macrophages with Increased Efferocytic and Phagocytic Capacity [abstract]. Am J Transplant. 2021; 21 (suppl 3). https://atcmeetingabstracts.com/abstract/hla-i-antibody-activated-endothelium-polarized-m2-like-macrophages-with-increased-efferocytic-and-phagocytic-capacity/. Accessed May 9, 2025.

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