Volume 25, Issue 4, Pages 643-654 (October 2006) Persistence and Responsiveness of Immunologic Memory in the Absence of Secondary Lymphoid Organs  Juan E. Moyron-Quiroz, Javier Rangel-Moreno, Louise Hartson, Kim Kusser, Michael P. Tighe, Kimberly D. Klonowski, Leo Lefrançois, Linda S. Cauley, Allen G. Harmsen, Frances E. Lund, Troy D. Randall  Immunity  Volume 25, Issue 4, Pages 643-654 (October 2006) DOI: 10.1016/j.immuni.2006.08.022 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 The Generation and Maintenance of Memory CD8 T Cells Occurs Independently of SLOs Recipient C57BL/6 or Lta−/− mice were irradiated and reconstituted with BM from C57BL/6 or Lta−/− mice. C57BL/6 mice receiving C57BL/6 BM are referred to as WT mice. Splenectomized Lta−/− mice receiving C57BL/6 BM are referred to as SLP mice. Lta−/− mice receiving Lta−/− BM are referred to as LTKO mice. (A–E) Chimeric mice were infected with 100 EIU of PR8 on day 0, and cells from the lungs of WT and SLP mice were analyzed by flow cytometry at the indicated times. (A) Expression of CD62L on NP-specific CD8 T cells. The plots shown are gated on CD8 T cells. (B) Expression of CD127 on NP-specific CD8 T cells. The plots shown are gated on CD8 T cells. (C) The total number of NP-specific CD8 T cells in the lungs was calculated as defined by the boxed areas in (A). (D) The total number of NP-specific memory T cells was calculated as defined by the boxed areas in (B). (E) The percentage of NP-specific CD8 T cells in the lung that expressed the memory marker, CD127, was calculated based on the boxed areas in (B). The data in (C)–(E) represent the mean and standard deviation from 3–4 mice per group at each time point. The data shown are representative of two independent experiments. (F and G) Groups of chimeric mice were infected with 100 EIU of PR8, provided BrdU in the drinking water starting on week 4, and analyzed 1, 2, and 3 weeks after the start of BrdU treatment. (F) Representative BrdU incorporation in NP-specific CD8 T cells 3 weeks after the provision of BrdU. The plots shown are gated on CD8 T cells. (G) Rate of BrdU incorporation into NP-specific memory CD8 T cells in lungs and BM of WT and SLP mice. The data represent the mean and standard deviation from three mice per group at each time point. The data shown are representative of three independent experiments. Immunity 2006 25, 643-654DOI: (10.1016/j.immuni.2006.08.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Influenza-Specific CD8 Memory T Cells Are Maintained Independently of SLOs and Rapidly Accumulate in the Lung after Heterotypic Challenge (A and B) Groups of chimeric mice were infected with 100 EIU PR8 (top and middle) or mock infected (bottom) on day 0. The chimeric mice were then challenged with 5000 EIU X31 on day 132 (middle and bottom) or left unchallenged (top). Cells from all groups were analyzed 6 days later on day 138. (A) Dot plots show representative expression of CD62L and H-2DbNP366-374 tetramer binding on CD8+ lymphocytes isolated from the lung on day 138 (day 6 postchallenge). (B) The mean percentage of CD8 cells that are antigen specific (tetramer binding) in each group of chimeras is shown. (C) The viral titers in the lungs of WT (square), SLP (diamond), and LTKO (circle) mice were determined on day 138 (day 6 postchallenge). Dashed line indicates limit of detection. (D) The mean change in weight of each group from day 132 to day 138 is shown. The data represent the mean and standard deviation from four mice per group. This data shown are representative of two independent experiments. Immunity 2006 25, 643-654DOI: (10.1016/j.immuni.2006.08.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Memory CD8 T Cells Proliferate Locally in the Lung after Challenge Infection WT and SLP mice were infected with PR8, and memory T cells were allowed to develop for 11 weeks. Mice were then challenged with X31 and treated with FTY720 5 days later. After 12 hr, they were pulsed with BrdU for 2 hr and analyzed. (A) Total live cells were enumerated in the BM, lung, and blood of treated mice. (B) CD8 T cells were enumerated in the BM, lung, and blood of treated mice by flow cytometry. (C) Live thick sections were probed with antibodies to BrdU (green) and CD8 (red) as well as tetramers (blue) to identify NP-specific CD8 T cells that had divided during the BrdU pulse. Red arrows pointing to the left indicate CD8+ cells, while violet arrows pointing to the right indicate CD8+tetramer+ cells. Overexposed autofluorescence is pseudocolored in white to identify structural elements in the lung. (D) Selected cells from the images in (C) were enlarged to more clearly show the difference between CD8+tetramer− cells (red) and CD8+tetramer+ cells (violet). (E) The frequency of BrdU incorporation in NP-specific CD8 cells from the BM and lung was determined by flow cytometry. Images are representative of multiple sections from 2–3 mice and flow cytometric data was obtained from 4–5 mice per group. The data shown are representative of two independent experiments. Immunity 2006 25, 643-654DOI: (10.1016/j.immuni.2006.08.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Neither Central Memory nor Effector Memory CD8 Cells Require SLOs to Respond to Challenge Infection B6.CD45.1 mice were infected with 100 EIU of PR8 and allowed to develop memory CD8 cells for 30 days, when CD8 T cells were positively selected from spleen and lymph nodes via magnetic beads. The positively selected CD8 cells were sorted into CD8+CD44hiCD62Llo effector memory cells or CD8+CD44hiCD62Lhi central memory T cells. 7 × 104 total cells from these populations (not normalized for the number of influenza-specific cells) were then injected into WT and SLP mice (both CD45.2), which were challenged with 100 EIU PR8 the next day. (A) CD8 T cells from the lung tissue were analyzed by flow cytometry for NP tetramer binding and for the expression of the CD45.1 donor marker on day 10 after challenge. The plots shown are gated on CD8 T cells. (B) The number of donor-derived (CD45.1+) NP-specific CD8 T cells was enumerated in the lung tissue. (C) CD8 T cells from the BAL were analyzed by flow cytometry for NP tetramer binding and for the expression of the CD45.1 donor marker on day 10 after challenge. The plots shown are gated on CD8 T cells. (D) The number of donor-derived (CD45.1+) NP-specific CD8 T cells was enumerated in the BAL. The data in (B) and (D) represent the mean and standard deviation from five mice per group. Statistical differences were determined by unpaired t test. The data shown are representative of three independent experiments. Immunity 2006 25, 643-654DOI: (10.1016/j.immuni.2006.08.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Influenza-Specific Memory CD8 T Cells Recirculate between Nonlymphoid Tissues Independently of SLOs WT and SLP chimeric mice were infected with 300 EIU of X31 and allowed to develop memory CD8 T cells for 30 days. The chimeric mice (both CD45.2) were then parabiotically joined with naive age-matched CD45.1 partners for 10–15 days. (A and B) Parabiotic partners were separated on day 10, and cells from the lungs of each partner were analyzed for NP-specific CD8 T cells of CD45.1 and CD45.2 genotypes. The top row of plots is gated on lymphocytes and the bottom row of plots is gated on CD8 T cells. (C and D) Donor-derived (CD45.2+) NP-specific and total CD8 T cells were enumerated in the lungs and spleens of WT/partner and SLP/partner pairs of mice on day 10 after parabiosis. The data in (C) and (D) represent the mean and standard deviation from four WT and four SLP parabiotic pairs. Statistical differences were determined by paired t test. (E and F) Donor-derived, NP-specific CD8 T cells were analyzed in the lungs, livers, pLNs, and spleens of WT/partner and SLP/partner pairs of mice on day 15 after parabiosis. The plots shown are gated on CD8+CD45.2+ cells. Three WT and three SLP parabiotic pairs were used for this analysis and the cells from each tissue/partner were pooled. Immunity 2006 25, 643-654DOI: (10.1016/j.immuni.2006.08.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Influenza-Specific, Virus-Neutralizing Antibody Is Generated and Maintained Independently of SLOs Groups of chimeric mice were either infected with 100 EIU of PR8 or mock infected on day 0. Mice were bled for the analysis of serum antibodies at days 0, 28, and 120 after primary infection. Mice were then challenged on day 132 with 1000 EIU of PR8 and analyzed on day 138 (day 6 postchallenge). (A) The titers of influenza-specific IgM and IgG in the serum of WT (square), SLP (diamond), and LTKO (circle) mice were determined by influenza-specific ELISA. (B) Viral titers in the lungs of WT (square), SLP (diamond), and LTKO (circle) mice were determined on day 138 (day 6 postchallenge). Dashed line indicates limit of detection. (C) The mean change and standard deviation in weight of each group from day 132 to day 138 is shown. (D) Dot plots show representative expression of CD62L and H-2DbNP366-374 tetramer binding on CD8+ lymphocytes isolated from the lungs of chimeric mice at day 138 (day 6 postchallenge). (E) The mean percentage and standard deviation of CD8 cells that are antigen-specific (tetramer binding) in each group of chimeras is shown. The data are representative of two independent experiments. Immunity 2006 25, 643-654DOI: (10.1016/j.immuni.2006.08.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 Persistence of iBALT in WT and SLP Chimeric Mice after Influenza Infection (A–G) Groups of chimeric mice were infected with 100 EIU of PR8 on day 0 and lungs were harvested, embedded in OCT, and frozen on days 30 (A, B, and G), 50 (C and D), and 90 (E and F) after infection. Frozen sections were stained with Diff Kwik to visualize histopathology and with antibodies to CD3, B220, CD21, and CD11c to visualize T cells, B cells follicular dendritic cells, and dendritic cells/macrophages, respectively. Immunofluorescent sections were counterstained with DAPI (blue). Fluorescent images were obtained with a 10× objective and histology images were obtained with a 2.5× objective. Images are representative of 2–4 mice per group in 3 independent experiments. (H) The mean and standard deviation of the cross-sectional areas of individual iBALT areas were measured from seven lung midsections from each group. The numbers in parentheses above each bar indicates the total number of iBALT areas that were measured in seven sections. (I) The portion of the lung that contained iBALT was determined by totaling the iBALT areas and dividing by the total lung area in all seven sections. Immunity 2006 25, 643-654DOI: (10.1016/j.immuni.2006.08.022) Copyright © 2006 Elsevier Inc. Terms and Conditions