Saturday, April 20, 2024
4/20/2024
Abstract Globoid cell leukodystrophy (GLD) or Krabbe's disease is a fatal genetic demyelinating disease of the central nervous system affecting 1 in 100,000 live births with no cure or effective long-term treatment. GLD is caused by loss-of-function mutations in the galactosylceramidase (galc) gene, where loss of GALC enzymatic function results in toxic accumulation of its substrate, a lipid called galactosylsphingosine or `psychosine'. Psychosine cytotoxicity is considered the basis of several key pathologies in GLD. Neuropathology in GLD is marked by profound demyelination and inflammation. However, molecular details of these processes are limited, leaving few therapeutic options. Early histological evidence for CD8+ T cells within demyelinated lesions in both the twi mouse brain and human GLD brain had suggested a role for adaptive immunity in this disease. However, the function of CD8+ T cells in GLD has not been previously determined. To address this gap in our knowledge, we analyzed the timing of T cell population changes by flow cytometry in CNS tissues from the twitcher mouse model of GLD and compared with wt littermates. We identified a rapid and protracted elevation of T cells in the twi CNS that was coincident with the onset of clinical disease at postnatal day 21 (P21). These data were confirmed using single-cell RNA sequencing on twi and wt littermate brain tissues from which identified a 9-fold increase in CD8+ T cells in twi mouse brains at P21. Our transcriptomic data also defined the CD8+ population as cytotoxic T lymphocytes (CTLs). To test the function of CD8+ CTLs in GLD-like disease in twi mice, we depleted CD8+ T cells by administering anti-CD8 antibody to twi mice and found that this treatment effectively prevented disease onset, preserved wellness and completely prevented CNS demyelination while also attenuating pro-inflammatory cytokine levels in brain and blood. These novel and highly translational data portend a pathogenic role for CD8+ T cells in GLD and underlie the basis for our overall hypothesis that CD8+ T cells are pathogenic in GLD and directly contribute to disease. Accordingly, Aim 1 will characterize the spatial and temporal development of CD8+ T cells in twi mice and challenge the contribution of CD8+ T cells using knockout mouse lines to then assess clinical, biochemical, and pathological outcomes. Aim 2 will define the overall nature of adaptive immunity by evaluating contribution of CD4+ T cells to CD8+ T cell-mediated immunity in twi mice using targeted depletion and genetic strategies. Aim 3 will identify and profile the timing, and location T cells responding to authentic antigen, the clonality of the T cells using T cell ß chain VDJ phenotyping, and then identify the nature of the antigen presenting cell types using transgenic reporter mice. These multi-disciplinary studies will interrogate a previously unrecognized CD8+ T cell neuroinflammatory response in GLD. Outcomes of these studies are expected to fill an important gap in our understanding on the fundamental cellular pathological mechanisms underlying the development CD8+ T cell mediated neuropathology and disease in GLD.
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