Preferential Localization of Effector Memory Cells in Nonlymphoid Tissue David Masopust, Vaiva Vezys, Amanda Marzo, Leo Lefrançois

Supplementary Material

Supplemental Figure 1. Virus-specific CD8 memory T cells in peripheral but not lymphoid tissues are constitutively cytolytic. At 111 days after VSV infection, cells were isolated from the indicated tissues and incubated for 5 hr with ⁵¹Cr-labeled untreated EL4 target cells (data not shown) or target cells pulsed with N52-59 peptide. Effector to target ratio was 300:1 for spleen and LP and 200:1 for lung. E:T shown in plots are corrected for the number of tetramer⁺ cells in each population. The percentage of tetramer⁺ cells among total lymphocytes was: spleen, 1.4%; LP, 1.6%; lung, 4.0%. Data shown is derived from a single spleen (similar results were obtained using spleens from 5 other mice) and pools of lung or LP cells from two mice (two additional equivalent pools gave similar results). Spleen cells were isolated using the same protocol as that for isolation of lung cells including collagenase treatment and Percoll gradient purification.

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Supplemental Figure 2. Size and phenotype of CD8 memory T cells in different tissues. (A) Tetramer⁺ cells from spleen, lung, liver and LP of mice infected with VSV 6 or 20 days previously or from mice 224 days after secondary VSV infection, were analyzed for forward light scatter (FSC) properties by flow cytometry. The red histogram represents naive CD8 splenocytes (tetramer^neg, CD11a^low). (B) At 81 days after VSV infection, cells were isolated and analyzed by fluorescence flow cytometry for the indicated cell-surface molecules. Plots shown are analyses of gated CD8⁺ tetramer⁺ lymphocytes from each tissue. LP memory cells expressed high levels of b7 integrins which was primarily due to high aEb7 expression. The 1B11 determinant is a carbohydrate epitope expressed on CD43 and CD45 molecules [D. A. Carlow, B. Ardman, H. J. Ziltener, J. Immunol. 163, 1441 (1999)] and has been reported to be upregulated after CD8 T cell activation followed by the loss of the determinant on splenic CD8 memory cells [L. E. Harrington, M. Galvan, L. G. Baum, J. D. Altman, R. Ahmed, J. Exp. Med. 191, 1241 (2000)]. However, our results did not agree with this finding since we observed two distinct populations of memory cells in spleen based on 1B11 expression and nearly all LP memory cells expressed high levels of 1B11. In addition, the lack of CD62L expression did not correlate with the ability to mediate direct ex vivo lytic activity, as has been suggested for LCMV-specific memory cells [S. Oehen, K. Brduscha-Riem, J. Immunol. 161, 5338 (1998)].

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