1. Memory CD8+ T Cells Undergo Peripheral Tolerance
Huub T.C. Kreuwel, Sandra Aung, Cheryl Silao, Linda A. Sherman 
Immunity 
Volume 17, Issue 1, Pages (July 2002)
DOI: /S (02)

2. Figure 1
HA-Specific CD8+ T Cell Responses Gradually Wane in Neonatal InsHA Mice
Adult (8-week-old) BALB/c (A and B) and InsHA+/− (C and D) or neonatal (2-week-old) InsHA+/− mice (E and F) were immunized with 500 HAU of influenza virus. Three (A, C, and E) or eleven (B, D, and F) weeks later, 7 × 106 splenocytes were cultured with HA-presenting APCs, and after 6 days specific lysis was tested in a 5 hr chromium release assay, using B10.D2 targets pulsed with 25 μM KdHA peptide. Background lysis on target cells without antigen was below 15% at the highest E:T ratio. Data shows two adult and seven neonates per group and is representative of two independent experiments.
Immunity  , 73-81DOI: ( /S (02) )

3. Figure 2
Memory CD8+ T Cells Can Also Undergo Peripheral Tolerance by Exogenous Administration of Soluble Peptide
BALB/c mice (8 weeks of age) were immunized with 500 HA of influenza virus. Three weeks later, primed animals were then either immunized i.v. with 250 μg KdHA peptide in PBS (open circles), PBS only (closed circles), or 107 pfu recombinant vaccinia expressing the KdHA epitope (open squares). Two weeks after peptide treatment, splenocytes were cultured with APCs and pulsed with KdHA peptide (A) or KdNP peptide as an internal control (B). After 6 days, specific lysis was tested in a 5 hr chromium release assay, using B10.D2 targets pulsed with 25 μM KdHA peptide (A) or KdNP peptide (B). Background lysis on target cells without antigen was below 5% at the highest E:T ratio. Data show the mean specific lysis of eight individual mice per data point pooled from two independently performed experiments.
Immunity  , 73-81DOI: ( /S (02) )

4. Figure 3
Phenotypic and Functional Analysis of Naive and Memory CD8+ Clone-4 TCR Cells
(A) Naive, effector, and memory Clone-4 TCR T cells were stained with anti-CD8-FITC versus anti-Thy1.1-PE antibodies, and each of the following CyChrome-labeled antibodies: anti-CD25, anti-CD62L (L-selectin), anti-CD69, and anti-Ly-6C. Data show expression on gated Thy1.1+/+CD8+ T cells and are representative of two independent experiments with similar results.
(B) Naive and memory Clone-4 TCR T cells were incubated with KdHA peptide for 6 hr at 37°C. Cells were then stained for CD8, Thy1.1, and intracellular IFN-γ and analyzed by FACS. Data show percentage of IFN-γ positive cells gated on CD8+Thy1.1+ cells. Data show one out of two independent experiments with similar results.
Immunity  , 73-81DOI: ( /S (02) )

5. Figure 4
The Response of Memory CD8+ Clone-4 TCR Cells to HA Crosspresented in the Pancreatic Lymph Nodes
5 × 106 purified naive CFSE-labeled CD8+Thy1.1+ Clone-4 TCR cells (left panels) or 2 × 106 purified memory CFSE-labeled CD8+Thy1.1+ Clone-4 TCR cells (right panels) were injected into InsHA mice. As a control, cells were also injected in normal InsHA-negative BALB/c mice (data not shown). Mice were sacrificed 4 days after adoptive transfer, and cells from pooled lymph nodes were analyzed by FACS. Histograms represent the amount of CFSE label gating on CD8+Thy1.1+ cells. Data are representative of one out of two independent experiments.
Immunity  , 73-81DOI: ( /S (02) )

6. Figure 5
HA-Specific Memory Clone-4 TCR Cells Undergo Tolerance in InsHA Recipients but Not BALB/c Mice
3 × 103 memory Clone-4 TCR Thy1.1+/+ cells were adoptively transferred into nonirradiated Thy1.2+/+ BALB/c (A) and InsHA+/+ (B) recipient mice. Nine weeks later, recipients were immunized with 500 HAU of influenza virus to permit the clonal expansion and detection of the Thy1.1+/+ CD8+ Clone-4 TCR cells. Nine days after immunization, splenocytes were stained with antibodies specific for CD8 and Thy1.1 and analyzed by flow cytometry. Data represent one out of two experiments with similar results.
Immunity  , 73-81DOI: ( /S (02) )