Tfeb regulates CD8+ T-cell proliferation In Vivo

Abstract CD8+ T-cells are critical components of anti-viral and anti-tumor immunity. Our accumulating understanding of their function has allowed for T-cell based immunotherapies that have widespread clinical utility in various malignancies albeit without universal benefit. Further understanding the molecular mechanisms underlying CD8+ T-cell function can improve T-cell based immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy and bispecific antibodies. Transcription factor EB (Tfeb) is a regulator of autophagy and lysosome biogenesis that has a major impact on the metabolic state of several tissue types. Tfeb regulates CD4+ regulatory T cell differentiation through the regulation of mitochondrial integrity and metabolism. However, it remains unknown whether any role for Tfeb exists in CD8+ T-cells. Using a T-cell conditional Tfeb knockout mouse (Tfebfl/flCD4Cre; TfebKO), we have determined that Tfeb is dispensable for baseline quantity of CD4+and CD8+ T-cells in the thymus, spleen and lymph nodes (LN). Baseline phenotypes of CD8+ T-cells were similar to controls at baseline in these tissues. To test whether Tfeb impacts the fate of activated CD8+ T-cells, we utilized the murine model of acute viral infection (lymphocytic choriomeningitis virus, LCMV) and co-transferred control and TfebKO TCR-transgenic CD8+ T-cell specific for LCMV in a 1:1 ratio into congenic wildtype recipients. Cells from both donors persisted in vivo post-transfer. Several days following LCMV infection, transferred T-cells were analyzed by flow cytometry. At 8 days post-LCMV, a time point typically corresponding with peak virus-specific CD8+ T-cell expansion, a marked predominance of T-cells from control donors versus TfebKO donors was observed in the peripheral blood of recipients (median 99.1% control donorvs 0.1% TfebKO donor; p<0.001). T-cell distribution in lymph node and spleen at this time point was similar to the peripheral blood, arguing against a primary trafficking defect. Thus, we looked at earlier timepoints. At 3 days post-LCMV, we noted early signs of a predominance of control versus TfebKO donor T-cells in both the lymph nodes (median 61.8% vs 38.2%; p<0.0001) and spleen (median 67.4% vs 32.1%; p<0.0001). This difference grew with time – with control donor T-cells outcompeting TfebKO T-cells in peripheral blood (median 92.7% vs 7.1%; p <0.01), spleen (median 74.9% vs 24.1%; p<0.01), and lymph nodes (median 97.8% vs 2.2%; p<0.01) at day 5 post-LCMV. Importantly, uninfected control mice harbored equivalent ratios of control to TfebKO donor cells akin to the original transferred 1:1 ratio. Together, these data suggest that Tfeb is required in a cell-intrinsic manner for antigen-specific CD8+ T-cell expansion following LCMV infection. Given the defect in expansion, we asked if Tfeb was required for the survival or proliferation of activated LCMV-specific CD8+ T -cells in vivo. There was no statistically significant difference in the viability of TfebKO vs control donor cells . However, Tfeb-deficient T-cells were found to have a proliferative disadvantage in comparison to their control counterparts in both the LNs (undivided cells 47% in control vs 71.3% in TfebKO; p= 0.036) and spleen (17.2% in control vs 48.9% in TfebKO; p=0.024). These data suggest that Tfeb plays a critical role in T cell proliferation after antigen activation. In assessing degrees of activation post-LCMV, we find that TfebKO cells expressed decreased levels of CD25 compared to control cells at early time points (day 3 lymph node CD25 MFI 180 vs 4.7; p=0.036; day 3 spleen CD25 MFI 1286 vs 43.1; p=0.0089) as well as later time points (day 8 peripheral blood CD25 MFI 76.6 vs 16.7; p=0.0286). Other markers of activation such as CD71 and CD44 were similarly decreased in TfebKO cells compared to controls. Together, these data demonstrate that Tfeb plays a crucial cell-intrinsic role in CD8+ T cell activation and subsequent T-cell proliferation in vivo. Mechanisms by which Tfeb impacts CD8+ T-cell activation and proliferation are being actively investigated. RNA-sequencing of control and TfebKO T-cells from this in vivo model is underway and will be presented at the annual meeting.