PMC

Representative contour plots of CD90.1 versus FliC:I-A^b for CD4+ gated events from mixtures of 2.5 × 10⁸ B6 spleen and lymph node cells and the indicated numbers of SM1 TCR transgenic T cells analyzed directly (top row) or after tetramer-based enrichment (bottom row). SM1 cells are shown in the upper gate and polyclonal FliC:I-A^b-specific CD4+ T cells in the lower gate. Data is representative of two independent experiments.

A, Representative contour plot of FliC:I-A^b tetramer (APC) versus 2W1S:I-A^b tetramer (PE) for non-T cell lineage-, CD3+, CD4+ events in a spleen and lymph node cell population from a naive B6 mouse after enrichment with FliC:I-A^b and 2W1S:I-A^b tetramers. B, Representative contour plots of CD44 versus 2W1S:I-A^b for CD4+ or CD8+ gated events following 2W1S:I-A^b tetramer enrichment of total spleen and lymph node cells from a naive B6 mouse, or C, an OT-I RAG1^−/−, or D, an OT-II RAG1^+/+ TCR transgenic mouse. E, Representative contour plots of CD44 versus 2W1S:I-A^b for CD4+ gated events following 2W1S:I-A^b tetramer enrichment of total spleen and lymph node cells from OT-II RAG1^−/−, SM1 RAG1^−/−, and TEa RAG1^−/− TCR transgenic mice. Data is representative of at least 3 independent experiments.

A, Representative contour plots of CD44 versus tetramer for CD4+ events from total spleen and lymph node cells from naive B6 mice (top row) or B6 mice injected i.v. with 50 µg of the indicated peptide plus 5 µg LPS (bottom row) following enrichment with the indicated tetramers. B, Total numbers of tetramer+, CD4+ or CD8+ cells following tetramer enrichment of total spleen and lymph node cells from a naive B6 mouse or tetramer+, CD4+ cells from OT-II RAG1^−/−, TEa RAG1^−/−, or SM1 RAG1^−/− TCR transgenic mice. Symbols represent individual mice; horizontal bars indicate mean values. C, Mean total number (±S.D., n=2–8) of 2W1S:I-A^b+ (circles), FliC:I-A^b+ (triangles), or OVA:I-A^b+ (diamonds) cells in B6 mice over time following i.v. injection with 50 µg of the relevant peptide plus 5 µg LPS. D, Mean total number (±S.D., n=3) of 2W1S:I-A^b+ (circles), FliC:I-A^b+ (triangles), or OVA:I-A^b+ (diamonds) cells in B6 mice 4 days following i.v. injection with 5 µg LPS plus the indicated amount of relevant peptide.

A, 2W1S:I-A^b enrichment was performed on total spleen and lymph node cells from B6 mice injected i.v. 48 hours earlier with 5 µg LPS (left panels), or 5 µg LPS plus 250 µg 2W1S peptide (right panels). CD4+, 2W1S:I-A^b+ events were then analyzed for CD44 expression and blastogenesis (forward scatter). Numbers indicate percentages of events in each quadrant. Data is representative of at least 3 independent experiments. B, Contour plots of CD44 versus tetramer for 2W1S:I-A^b or FliC:I-A^b enriched spleen and lymph node cells from B6 RAG1^−/− hosts that received either 2W1S:I-A^b or FliC:I-A^b tetramer-depleted cells from naïve B6 mice, and were then injected i.v. with a mixture of 50 µg 2W1S peptide, 50 µg FliC peptide, and 5 µg LPS 8 days before analysis. The total number of tetramer+ cells for each population is shown below the relevant gate. Data shown is representative of three independent experiments.

A, Total lymph node and spleen cells from naive B6 mice (top) or B6 mice injected i.v. with 50 µg 2W1S peptide plus 5 µg LPS 4 days earlier (bottom) were stained with antibodies to TCR Vβ4 and Vα2. 2W1S:I-A^b- and 2W1S:I-A^b+ gated events are represented in histograms by shaded profiles and solid lines, respectively. B, TCR Vβ and Vα gene segment usage in total CD4+ T cells from naive mice (unfilled bars), or 2W1S:I-A^b+ CD4+ T cells from naive mice (filled bars) or mice injected i.v. with 50 µg 2W1S peptide plus 5 µg LPS 4 days earlier (hatched bars). Data are mean values ± S.D. from 4∓8 mice per group. C, Usage profile for TCR Vβ gene segments in total CD4+ T cells from naive B6 mice or tetramer+ CD4+ T cells from B6 mice injected i.v. with 50 µg of the indicated peptide plus 5 µg LPS 8 days earlier. Data are values from pooled samples taken from 4–10 mice per group, or D, samples taken from 4 individual mice per group.

A, Flow cytometry gates used in all figures to identify T cells from total spleen and lymph node cells: side scatter-width^low (left plot); CD3+, non-T cell lineage- (middle plot); and either CD4+ or CD8+ (right plot). B, Where indicated, B6 mice were injected i.v. with 50 µg of the indicated peptide plus 5 µg LPS. Eight days later, spleen and lymph node cells were harvested and stained with the indicated tetramer. Representative contour plots of CD44 versus the indicated tetramers are shown for non-T cell lineage-, CD3+, CD4+ events gated from 10⁶ total collected events. The total number of cells for each individual mouse shown is indicated below the relevant gate. Mean values ± S.D. were 2W1S:115,000 ± 40,000; FliC: 23,000 ± 20,000; OVA: 7,000 ± 3,000 (n=4 per group).