Figure 1.
Expression of RAGE on human APC's and T cells.
A: Surface RAGE expression was studied on CD11c+ PBMC before (top) and after (bottom) culture with LPS for 7 days. (solid line = staining with anti-RAGE antibody, dashed line = staining with isotype control) B: Cell surface (L and intracellular RAGE expression was studied on CD4+ T cells before (top) and after 7 days in culture with anti-CD3 mAb (bottom). A single experiment representative of cultures with more than 4 donors is shown. C: PBMC were activated with anti-CD3 mAb for 48 hrs and lysed or separated into CD4+ and CD8+ T cells with magnetic beads and lysed. A blot of the lysates was probed with anti-RAGE antibody. The arrow identifies RAGE in the cells.
Figure 2.
RAGE is seen in a granular pattern in T cells and colocalizes with endosomes.
A: Jurkat cells were transfected with GFP-RAGE (A) or control GFP vector (B) and photographed. A granular pattern of staining is seen within the RAGE transfected cells. C–E: HEK293 cells were transfected with GFP-RAGE (D and E) and fixed and stained with RhoB (C and E). Panel E shows the merged staining. The arrows indicate cells+ for RhoB and RAGE.
Figure 3.
RAGE is expressed on antigen specific CD8+ T cells during culture.
Peripheral blood cells from HLA-A2.1+ healthy control subjects that were cultured with or without EBV peptide were stained with Class I MHC tetramer loaded with EBV peptide and for intracellular RAGE. Culture with the peptide increased the proportion of tetramer+ T cells increased 2.5-fold. On the tetramer+ T cells, the proportion that were RAGE+ increased 2.5 fold (p = 0.02). Data from 3 individuals are shown.
Figure 4.
RAGE ligand enhances RAGE expression on T cells.
Peripheral blood cells were cultured with or without EBV peptide and IL-2 with or without S100b. After 7 days, CD4+ and CD8+ T cells were analyzed for the expression of RAGE. The percentages shown in each panel indicate the percentage of RAGE+ T cells (minus background staining with control Ig) of CD4+ or CD8+ cells. A single experiment representative of 3 is shown.
Figure 5.
Expression of RAGE on T cells from patients with T1D and T2D.
A:PBMC were isolated from patients with T1D (top two rows) and T2D (bottom row). They were stained with CD4+ or CD8+ Abs and for surface or intracellular RAGE. Two single experiments, representative of 7 are shown. B. The level of RAGE expression in unmanipulated CD4+ (solid symbols) and CD8+ (open symbols) T cells from patients with T1D, T2D, and healthy control subjects (HC) or patients with rheumatoid arthritis (RA) and Sjogren's syndrome (SS) are shown (*p<0.05, *** p<0.01). C. The relationship between hemoglobin A1c levels and the percentage of RAGE in CD4+ T cells in patients with T1 and T2D is shown (p = ns).
Figure 6.
Changes in RAGE expression on activated T cells from patients with T1D and healthy control subjects.
RAGE expression was studied on CD4+ or CD8+ T cells before and 48 hrs after culture with anti-CD3 mAb. RAGE expression was higher on CD4+ (p<0.001) and CD8+ (p<0.001) T cells from patients with T1D vs healthy controls. While the level of RAGE expression increased in CD4+ and CD8+ T cells from healthy control subjects (p<0.05), it decreased in the patients with T1D (p<0.05).
Figure 7.
CD8+ T cells, that were not activated from a patient with T1D (R column) or a CD8+ T cells from a HLA-A2+ healthy control subject, activated with anti-CD3+28 mAbs (L column) or from the same HC subject activated with EBV peptide (middle column) were compared. The RAGE+ T cells from the patient with T1D do not express CD25, are CCR7+ and have a more uniform distribution of CD45RA. Results from a single donor representative of 3 is shown.
Figure 8.
A: The phenotype of RAGE+ and − PBMC from patients with T1D were studied by flow cytometry (n = 4). PBMC were activated with PMA/ionomycin for 6 hours and the percentage of cytokine+ RAGE+ or RAGE− T cells was determined in the same individual and compared by paired t-test. The percentages that are shown (mean± SEM) represent the percent of the RAGE+ or RAGE− T cells that were cytokine+. (*p<0.05). B. A single representative experiment showing staining with RAGE and CD107a and IL-17 are shown. Gates were placed around CD4+ T cells. The inserts show the the percentage of total CD4+ cells in each quadrant.