Abstract 4350116: β ₁ -adrenergic autoantibodies (β ₁ -AA) augment macropinocytosis in CD4 ⁺ T cells, leading to the expansion of CD4 ⁺ CD28 ⁻ T cell subsets in heart failure.

Background: β 1 -adrenergic autoantibodies (β 1 -AA), pathogenic autoantibodies targeting β 1 -AR, drive adverse cardiac remodeling and elevate rehospitalization risk in HF. It has been established that β 1 -AA induce both proportional imbalances and functional dysregulation of CD4 + T lymphocytes. However, the mechanisms underlying β 1 -AA-induced CD4 + T cell dysfunction and its pathophysiological relationship with HF progression remain unelucidated. Methods and Results: Peripheral blood was collected from heart failure (HF) patients and analyzed by flow cytometry, revealing a significant reduction in CD28 expression on CD4 + T cells along with increased granzyme secretion in HF patients. HF mouse model (passive transfer of heart failure patient derived β 1 -AA, H-β 1 -AA) exhibited enhanced cardiac infiltration of CD4 + T cells and immune dysregulation in peripheral blood. In vivo , H-β 1 -AA stimulated CD4 + T cells were adoptively transferred into nude mice, resulting in cardiac dysfunction accompanied by massive CD4 + T cell infiltration into the heart. Cell coculture confirmed that H-β 1 -AA stimulated CD4 + T cells induced myocardial injury in vitro . Moreover, CD28 downregulation was consistently observed in: splenic CD4 + T cells from β 1 -AA passively immunized mice, cardiac-infiltrating CD4 + T cells in nude mice, and in vitro -stimulated CD4 + T cells by H-β 1 -AA. Proteomic analysis of β 1 -AA-stimulated CD4 + T cells revealed enhanced CD28 endocytosis. Further experiments employing spinning-disk confocal microscopy and related techniques demonstrated that β 1 -AA stimulation induces CD28 internalization via macropinocytosis. Upon Ehd2 (a pivotal regulator of macropinocytosis) knockdown in CD4 + T cells, the heart failure phenotype induced by β 1 -AA-CD4 + T cells in nude mice was reversed. β 1 -AA-CD4 + T cells were then subjected to IP with anti-CD28 antibody, followed by mass spectrometry analysis, and validated, which identified increased GRP75 translocation to membrane and interaction with CD28 upon β 1 -AA stimulation. The GRP75 inhibitors MKT-077 and Withanone reversed β 1 -AA-induced macropinocytosis and CD28 loss in CD4 + T cells. Withanone also reversed the HF phenotype in nude mice receiving β 1 -AA-CD4 + T cells. Conclusion: Our study reveals for the first time that β 1 -AA induce GRP75 membrane translocation, which mediates CD28 molecule internalization via macropinocytosis in CD4 + T cells, ultimately contributing to heart failure pathogenesis.