Volume 21, Issue 1, Pages 42-48 (January 2013) Reducing Mitochondrial ROS Improves Disease-related Pathology in a Mouse Model of Ataxia-telangiectasia  Anthony D D'Souza, Ian A Parish, Diane S Krause, Susan M Kaech, Gerald S Shadel  Molecular Therapy  Volume 21, Issue 1, Pages 42-48 (January 2013) DOI: 10.1038/mt.2012.203 Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Mitochondrial catalase suppresses thymic lymphomagenesis in ATM−/− mice. (a) Total thymocytes from 12-week-old ATM+/+ (white-filled bar, n = 10), ATM+/+ mCAT+ (gray-filled bar, n = 10), normal ATM−/− (white filled with black diagonal lines, n = 6), lymphomagenic ATM−/− (white filled with black vertical lines, n = 6), normal ATM−/− mCAT+ (gray filled with black diagonal lines, n = 8), and lymphomagenic ATM−/− mCAT+ (gray filled with black vertical lines, n = 4) mice. Analyses of percent CD8+ CD4+ (double-positive), CD8− CD4− (double-negative), CD8+ (single-positive), and CD4+ (single-positive) cells from the mice in a. FACS analyses of (b) mitochondrial ROS (MitoSox), (c) cellular ROS/redox (H2DCFDA), and (d) TORC1 activity (phospho-S6) of CD8+ CD4+ (double-positive) cells from the mice in a. (e) L-lactate measured from 107 thymocytes from 12-week-old ATM+/+ (white-filled bar, n = 3), ATM+/+ mCAT+ (gray-filled bar, n = 3), ATM−/− (white filled with black diagonal lines, n = 3), and ATM−/− mCAT+ (gray filled with black diagonal lines, n = 3) mice. Statistical significance is denoted by *(<0.05), **(<0.005), ***(<0.0005) or ns (not significant). FACS, fluorescence-activated cell sorting; ROS, reactive oxygen species. Molecular Therapy 2013 21, 42-48DOI: (10.1038/mt.2012.203) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Survival Analysis. Kaplan–Meier survival curves for ATM+/+ (n = 9), ATM+/+ mCAT+ (n = 14), ATM−/− (n = 22), and ATM−/− mCAT+ (n = 21) mice. P value was <0.0001 on comparing ATM−/− and ATM−/− mCAT+ curves. The experiment was terminated at 300 days. Molecular Therapy 2013 21, 42-48DOI: (10.1038/mt.2012.203) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Bone marrow analyses of 12-week-old ATM−/− and ATM−/− mCAT+ mice. (a) FACS analyses of total bone marrow cells from femurs of the same mice analyzed in a, followed by (b) total number of hematopoietic stem cells (HSC, Lin− cKit+ Sca-1+), (c) granulocytes-monocytes (Gr-1+ Mac-1+), erythroid cells (Ter119+ CD71+), and lymphoid cells (B220+). (d) Total macrophages obtained upon differentiation in vitro of bone marrow cells from ATM+/+ (n = 4), ATM+/+ mCAT+ (n = 4), ATM−/− (n = 4), and ATM−/− mCAT+ (n = 4) mice. Statistical significance is denoted by *(<0.05), **(<0.005), ***(<0.0005) or ns (not significant). FACS, fluorescence-activated cell sorting. Molecular Therapy 2013 21, 42-48DOI: (10.1038/mt.2012.203) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Analysis of CD8+ T cell memory-differentiation phenotypes in vivo and in vitro. (a) Analyses of LCMV-specific Gp33+ splenic CD8+ T cells on day 30 post-LCMV infection of ATM+/+ (white-filled bar, n = 10), ATM+/+ mCAT+ (white filled with black stripes, n = 7), ATM−/− (gray-filled bar, n = 10), and ATM−/− mCAT+ (gray filled with black stripes, n = 9) mice. (b) Percent CD62Lhi, KLRG1+ (effector-memory TEM) and CD127+ (central memory, TCM) GP33+ CD8+ T cells from the mice analyzed in a. (c) Phospho-S6 MFI of splenic CD8+ T cells 72 hours post-TCR activation in vitro and (d) number of TCR-activated CD8+ T cells after additional 72 hours of IL-15 treatment from ATM+/+ (white-filled bar, n = 5), ATM+/+ mCAT+ (white filled with black stripes, n = 4), ATM−/− (gray-filled bar, n = 5), and ATM−/− mCAT+ (gray filled with black stripes, n = 5) mice. Statistical significance is denoted by *(<0.05), **(<0.005), ***(<0.0005) or ns (not significant). IL, interleukin; LCMV, lymphochoriomeningitis virus; MFI, median fluorescence intensity; TCR, T cell receptor. Molecular Therapy 2013 21, 42-48DOI: (10.1038/mt.2012.203) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions