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Science 27 March 2009:
Vol. 323. no. 5922, pp. 1726 - 1729
DOI: 10.1126/science.1168676
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Reports

Visualizing Antigen-Specific and Infected Cells in Situ Predicts Outcomes in Early Viral Infection

Qingsheng Li1, Pamela J. Skinner2, Sang-Jun Ha3, Lijie Duan1, Teresa L. Mattila2, Aaron Hage2, Cara White2, Daniel L. Barber4, Leigh O'Mara3, Peter J. Southern1, Cavan S. Reilly5, John V. Carlis6, Christopher J. Miller7, Rafi Ahmed3 and Ashley T. Haase1*

1 Department of Microbiology, Medical School, University of Minnesota, Minneapolis, MN 55455, USA.
2 Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, MN55108, USA.
3 Emory Vaccine Center, Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA.
4 Immunobiology Section, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
5 Division of Biostatistics, School of Public Health, University of Minnesota, Minneapolis, MN 55455, USA.
6 Department of Computer Science and Engineering, Institute of Technology, University of Minnesota, Minneapolis, MN 55455, USA.
7 Center for Comparative Medicine, California National Primate Research Center, University of California, Davis, CA 95616, USA.


Figure 1 Fig. 1. ISTH analysis. (A) Cervical tissues, 21 dpi. The image of a whole section of cervix was reconstructed as a montage in Photoshop from separate confocal images, using the red channel for the Gag-tetramer+ cells and green for the SIV RNA+ cells. The blue arrow from the region enclosed by the blue rectangle points to a red Gag-tetramer+ cell–green SIV RNA+ cell conjugate in the enlarged inset. The excess of Gag-tetramer+ cells is shown in the inset in a region enclosed by the red rectangle. (B) Lymphatic tissues, 21 dpi. Montage of a whole lymph node section reconstructed in Photoshop from separate images, using the red channel for the Gag-tetramer+ cells, blue for the SIV RNA+ cells, and green for CD8+ T cells. The red arrow in the enlarged inset (upper left) points to a red Gag-tetramer+ cell; the light blue arrow points to a blue SIV RNA+ cell. The two lightened separate images with an encircled distinctive V-shaped constellation of Gag-tetramer+ cells provide a frame of reference for Fig. 2. Scale bars for whole sections, 50 µm; insets, 10 µm. [View Larger Version of this Image (99K GIF file)]
 

Figure 2 Fig. 2. Spatial relationships between SIV-tetramer+ and SIV RNA+ cells in the lymph node shown in Fig. 1B, and relationship between E:T ratios and reduction in viral replication. (A) Gag-tetramer+ cells are shown against a gray-white mask of the section, with the V-shaped encircled constellation shown in Fig. 1B. Scale bar, 50 µm. (B) SIV RNA+ and tetramer+ cells were mapped by plotting the x and y coordinates of their centers (centroids) onto a two-dimensional grid measured from a fixed starting position (0,0). Upper panel, Gag-tetramer+ cells; middle panel, SIV RNA+ cells; lower panel, superimposition of upper and middle panels revealing the close spatial proximity of the virus-specific tetramer+ cells with SIV RNA+ throughout the lymph node. The white crosses and encircled areas in the panels are included as points of reference. (C) E:T ratio correlates with reduction in viral load in cervical vaginal and lymphatic tissues. Natural log–transformed viral load fold reductions are plotted against E:T ratios for Gag- and Tat-tetramer+ cells in cervical (CX) and vaginal (Vag) tissues and lymph nodes (LN) from five animals at 20 to 28 dpi. E:T ratios were determined by ISH as described (13). [View Larger Version of this Image (73K GIF file)]
 

Figure 3 Fig. 3. Time course comparison of cells infected by LCMV-Armstrong or LCMV–clone 13. Outer columns: LCMV RNA+ cells detected by ISH with LCMV-specific probes have a greenish appearance in developed radioautographs viewed in reflected light. Spleen white pulp is counterstained dark blue. Inner columns: LCMV-antigen+ cells are stained red with polyclonal antibody to LCMV; FRCs in white pulp are stained green with the FRC marker ER-TR7. Both viral strains at 1 dpi (d1) infect macrophages in the marginal zone and FRCs in the white pulp, but LCMV–clone 13 infects larger numbers of both cell types. LCMV–clone 13 continues to infect large numbers of both cell types over the 8-day time course of the experiment, whereas the numbers of infected cells rapidly decline in LCMV-Armstrong infections. Scale bars (bottom row), 10 µm. [View Larger Version of this Image (139K GIF file)]
 

Figure 4 Fig. 4. (A) FACS analysis of the immunodominant epitope response to acute LCMV-Armstrong and LCMV–clone 13 infections in spleen. Flow cytometric quantification is presented as proportions and numbers of stained tetramer+ cells from disaggregated spleens from groups of three mice per time point after LCMV-Armstrong or LCMV–clone 13 infection. Representative plots gated on CD8+ T cells are shown by MHC class I tetramer (y axis) for each immunodominant epitope (GP33-41, GP276-286, and NP396-404) and CD44 (x axis). (B) E:T ratios in acute LCMV-Armstrong and LCMV–clone 13 infections in spleen. E:T ratios at the times shown were determined by ISTH as described (13). [View Larger Version of this Image (52K GIF file)]
 

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