Supplemental Figure 1. Activated splenocytes upregulate Serpina3g and Serpina3f expression.
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1 Relative Serpin expression Serpina3f Serpina3g C57BL/6 DBA/2 Supplemental Figure 1. Activated splenocytes upregulate Serpina3g and Serpina3f expression. Splenocytes from C57BL/6 or DBA/2 mice were cultured for the indicated periods in 1 μg / ml concanavalin A. Real time PCR using primers specific for Serpina3g or Serpina3f was performed on cdna. The serpin expression levels were normalized against β2microglobulin expression levels. Data show mean ± SEM from n = 2 mice.
2 Spleen ns ILN ns Thy1.2 + donor gp33 + CD8 + cells Supplemental Figure 2. Equivalent expansion of wild type and Spi2A-deficient memory gp33 + CD8 + cells Gp33 Tetramer + CD8 + splenocytes were isolated from and mice that had been previously infected with LCMV for >9 days and purified by FACS to >98% purity. 5 memory gp33 + CD8 + cells were transferred into naive Thy1.1 + congenic recipients, which were subsequently infected with LCMV. Eight days post infection, these mice were dissected and analyzed by flow cytometry. The number of Thy1.2 + donor gp33 + CD8 + cells in the spleen and both inguinal lymph nodes (ILN) is shown. No significant difference (ns) was found in the expansion of and Spi2A-deficient memory gp33 + CD8 + cells. Data show mean ± sem of n = 4 mice.
3 A Bcl-2 family Mean Fluorescence Intensity CD44 Bcl-2 Bcl-X L Mcl-1 Bim B % Annexin V + C % YoPro n. s. CD8 + CD n. s. 1 5 gp33 + CD8 + gp33 + CD8 + Supplemental Figure 3. Memory-phenotype CD8 + T cells possess similar levels of Bcl-2 family members and both memory-phenotype and LCMV-specific memory CD8 + cells undergo similar levels of cell death in wild type and mice. (A) Upper row: Representative intracellular flow cytometry staining of indicated Bcl-2 family members gated on wild type CD8 + bone marrow cells. Lower row: Mean fluorescence intensity of the intracellular Bcl-2 family staining in the CD8 + and CD8 + populations. Dotted line indicates MFI of isotype control staining. (B) Proportion of Annexin V + cells within the bone marrow CD8 + T cell population. (C) Wild type and mice were infected with LCMV Armstrong. After > 13 days, bone marrow leukocytes were stained and analyzed by flow cytometry. The DNA dye YoPro-1 was used to detect apoptosis among CD8 + gp33 Tetramer + cells. Data show mean ± SEM of n = 5 mice. Significance values were calculated by Studentʼs two-tailed t-test (n.s., P >.5) Results represent two independent experiments.
4 Byrne et al., Cathepsin B and memory T cell self-renewal Cell Number (x 1 3 cells / μl) Leukocytes 4.55 ± ±.15 Erythrocytes 41 ± 2 43 ± 2 Neutrophils.29 ±.6.51 ±.16 Lymphocytes 3.97 ± ±.32 Monocytes.18 ±.3.16 ±.5 Eosinophils.9 ±.2.39 ±.21 Supplemental Table 1. mice possess normal blood counts. Blood samples from 16-week-old wild type and mice were analyzed by a Sysmex F82 hematology analyzer. Blood smears were also stained with a modified Wright-Giemsa stain. Four part differential counts on 2 cells per slide were performed following standard morphological criteria. Each sample was assessed blind and in triplicate. Data list mean cell counts ± SEM of n = 5 mice, and are representative of three independent experiments. No statistically significant reproducible differences were found in any of the subsets. 1
5 Byrne et al., Cathepsin B and memory T cell self-renewal PBL: % CD ± ± 1.1 % CD ± ± 1.5 % B ± ± 4.5 Frequency of lymphocytes (%) Lymph node: % CD ± ±.8 % CD ± ±.7 % B ± ± 1.5 Bone marrow: % CD ±.1.7 ±.1 % CD ± ±.2 % B ± ±.1 Liver: % CD ± ±.2 % CD ± ± 1.1 % B ± ± 2. Spleen: % CD ± ±.8 % CD ± ± 1. % B ± ± 1.3 Lung: % CD ± ±.8 % CD ± ± 1.1 % B ± ±.4 Supplemental Table 2. mice possess normal proportions of lymphocyte subsets. Frequency of lymphocyte subsets in various organs of 16-week-old wild type and mice. Data show mean percentage of total lymphocytes ± SEM of at least n = 4 mice, and are representative of three independent experiments. No statistically significant and reproducible differences were found. 2
6 Byrne et al., Cathepsin B and memory T cell self-renewal Spleen Number of cells (x 1 3 ) gp33 + CD8 + CD62L hi gp33 + CD ± ± ± 32.8* ± 23.1* x Cath B KO ± ± 41.9 Cath B KO ± ± 56. Inguinal Lymph Nodes (both) Number of cells (x 1 3 ) gp33 + CD8 + CD62L hi gp33 + CD ± ± ± 1.34* 6.6 ±.97* x Cath B KO ± ± 1.49 Cath B KO ± ± 2.99 Bone marrow (one femur) Number of cells (x 1 3 ) gp33 + CD8 + CD62L + gp33 + CD ± ± ± ±.3* x Cath B KO ± ± 1.37 Cath B KO 2.69 ± ± 1.43 Intrahepatic Lymphocytes Number of cells (x 1 3 ) gp33 + CD8 + CD62L hi gp33 + CD ± ± ±.9.9 ±.2** x Cath B KO 5.9 ± ±.7 Cath B KO 9.5 ± ±.5 Supplemental Table 3. Spi2A-deficient mice develop fewer central memory CD8 + T cells after LCMV infection. Wild type,, x Cath B KO, and Cath B KO mice were infected with LCMV Armstrong and dissected 13 days post infection. Cells were counted and analyzed by flow cytometry as in Figure 2. Cell counts are expressed as mean ± SEM of at least n = 4 mice. Significance values were calculated between wild type and mice by Student s twotailed t test (*, P <.5; **, P <.1). 3
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