RAGE expression in human T cells: a link between environmental factors and adaptive immune responses

Abstract

The Receptor for Advanced Glycation Endproducts (RAGE) is a scavenger ligand that binds glycated endproducts as well as molecules released during cell death such as S100b and HMGB1. RAGE is expressed on antigen presenting cells where it may participate in activation of innate immune responses but its role in adaptive human immune responses has not been described. We have found that RAGE is expressed intracellularly in human T cells following TCR activation but constitutively on T cells from patients with diabetes. The levels of RAGE on T cells from patients with diabetes are not related to the level of glucose control. It co-localizes to the endosomes. Its expression increases in activated T cells from healthy control subjects but bystander cells also express RAGE after stimulation of the antigen specific T cells. RAGE ligands enhance RAGE expression. In patients with T1D, the level of RAGE expression decreases with T cell activation. RAGE+ T cells express higher levels of IL-17A, CD107a, and IL-5 than RAGE- cells from the same individual with T1D. Our studies have identified the expression of RAGE on adaptive immune cells and a role for this receptor and its ligands in modulating human immune responses.

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. Phenotype of RAGE+ T cells.

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. Phenotype of RAGE+ T cells.

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.