Abstract
E3 ubiquitin ligase Casitas B lineage Lymphoma b (Cbl-b) is a negative regulator in peripheral T cell activation. Cbl-b deficient T cells are hyper-reactive due to CD28-independent activation. Here we studied the effect of Cbl-b deficiency on T cell homeostasis in hypercholesterolemia and on atherogenesis in 20 wk old Cbl-b-/-/ApoE-/- mice. Flow cytometric analysis of lymphoid tissues and aortic arches showed decreased CD4+:CD8+ T cell ratios in Cbl-b-/-/ApoE-/- mice. Cbl-b deficiency induced central memory CD8+ T cells expansion, whereas the proportion of naïve T cells decreased. We found that Cbl-b deficient CD8+ T cells are less apoptotic as indicated by decreased AnnexinV positivity and elevated expression of anti-apoptosis markers, such as Bcl-2. Pro-inflammatory, TNFa and IFNy, and cytotoxicity markers, granzyme B, are increased in Cbl-b deficient CD8+ T cells. As expected from the increase of CD8+ T-cells in the aortic arch, Cbl-b-/-/ApoE-/- mice showed significantly more plaque development in the aortic root. The plaques contained higher leukocyte and T cell counts, but contained surprisingly less macrophages. The latter is caused by decreased monocyte recruitment resulting from lower MCP-1 levels. Moreover, the excess of CD8+ T cells induced enhanced cell death of macrophages. In vitro co-culture of Cbl-b deficient and wildtype CD8+ T cells with bone marrow derived macrophages revealed enhanced macrophage apoptosis in increased CD8+:macrophage ratios, irrespective of the CD8+ T cell genotype. Expression of the M1 macrophage markers, CD115 and CD64 was upregulated in Cbl-b deficient aortic arches, whereas M2 markers, CD206 and Arg-1, were decreased. In conclusion, we show that Cbl-b deficiency decreases CD4+:CD8+ T cell ratio during hypercholesterolemia, through reduced apoptosis and possibly less susceptibility of CD8+ T cells to regulatory T cell suppression. This contributes to exacerbated atherosclerosis in Cbl-b deficient mice. Although plaques contained an excess of lymphocytes and T-cells, and macrophages where of an M1 phenotype, macrophage counts were decreased. This was caused by low MCP-1 levels due to CD8+ T cell induced macrophage apoptosis. These results reveal that Cbl-b balances immune reactions in atherosclerosis.
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