UCLA

Solid organ transplantation is a lifesaving therapy for patients with end-stage organ failure. Although modern immunosuppressive strategies have allowed organ survival one-year post-transplants, chronic allograft rejection remains a main clinical challenge. Approximately 20% of recipients lose their graft within 5 years, and 50% within 10 years. Antibody-mediated rejection (AMR) described by the recipient production of donor-specific antibodies (DSA) remains the main risk factor contributing to late graft loss. DSA target human leukocyte antigen (HLA) class I and class II molecules present of the donor graft endothelium. DSA triggers strong alloimmune responses against allograft endothelial cells (ECs) leading to vascular injury and inflammation. Chronic inflammation induces vascular remodeling processes consisting of EC, smooth muscle cell (SMC), and myofibroblast proliferation leading to thickening of the intimal layer of vessels (neointima). This process ultimately leads to vessel occlusion, a condition termed transplant vasculopathy (TV) and specifically in heart transplant, cardiac allograft vasculopathy (CAV). Although macrophages remain a distinguishing feature of graft pathology in both AMR and CAV lesions, their precise phenotype and function in the context of HLA class I DSA remain poorly understood. Furthermore, there is a lack of research exploring the vascular characteristics of CAV-affected vessels in patients with DSA.In this dissertation, we utilized both in vitro and in vivo approaches to investigate the mechanisms by which HLA class I DSA activate EC signaling and the impact on monocyte-to-macrophage polarization and functions. Our findings revealed that crosslinking of anti-HLA I (IgG and F(ab')2) antibodies with HLA class I molecules (HLA I) forms a complex with TLR4. Signaling through TLR4 and the adaptor protein MyD88 triggers the release of Weibel-Palade bodies (WPbs) containing P-selectin. P-selectin surface expression mediates the capture of monocytes to ECs via interactions with monocyte P-selectin glycoprotein ligand-1 (PSGL-1). Second, we identified that anti-HLA I antibodies (IgG and F(ab')2) antibody-activated ECs induced the polarization of M2-like macrophages with distinct cytokine/chemokine secretion and transcriptomic expression. We further delineated that M2-macrophage polarization was enhanced via Fc-gamma receptor (FcR) interactions. For example, monocytes co-cultured with HLA I IgG-stimulated ECs differentiated into CD68+CD206+CD163+ macrophages, while monocytes co-cultured with HLA I F(ab’)2 only upregulated CD206. Thirdly, by inhibiting TLR4 signaling and PSGL-1-P-selectin interaction during monocyte transmigration across HLA I (IgG or F(ab’)2) antibody-activated ECs, we discovered that macrophage expression of CD206 and the secretion of matrix metalloproteinase-9 (MMP9) is regulated by P-selectin. We validated the expression of CD68+CD206+CD163+MMP9+ M2-like macrophages within CAV affected lesions of DSA+ rejected cardiac explants. Finally, using innovative spatial multi-omics techniques, we examined protein and transcriptomic expression in arterial regions of DSA+ CAV+ human rejected cardiac allografts. Our analysis revealed distinct profiles in regions with low and high neointima, encompassing differentially expressed proteins, transcripts, gene modules, and gene regulatory networks. Notably, low neointimal regions exhibited elevated inflammatory profiles, while high neointima regions demonstrated features associated with fibrotic and remodeling processes. These results suggested a speculative sequential time-frame of the arterial changes that may occur during CAV progression. This dissertation provides valuable insights into the immunological mechanisms driving AMR and CAV progression. The study highlights the role of HLA I DSA-activated ECs in regulating macrophage functions and uncovers molecular signatures of different arterial regions from DSA+ CAV+ rejected cardiac allografts. These findings offer a deeper understanding of AMR and CAV pathogenesis, suggesting potential therapeutic targets to prevent leukocyte infiltration and EC activation in rejecting grafts. Finally, this research contributes to the development of interventions for improved long-term graft survival in transplantation.