Antigen-Experienced CD4lo T Cells Are Linked to Deficient Contraction of the Immune Response in Autoimmune Diabetes
Figure 6
“CD4lo” T cells are present in Con A blasts produced from the spleen cells of NOD.BDC2.5 TCR transgenic mice. (a) Spleen cells from BDC2.5 TCR transgenic mice were stimulated with Con A for 72 hrs. The cells were then layered onto Ficol-Histopaque and centrifuged to recover an enriched population of the smaller “CD4lo” population. The Con A blasts and the enriched “CD4lo” population were cultured overnight in IL-2-supplemented medium and then stained with anti-CD4-PE and anti-CD69 FITC for FACS analysis. The numbers in the dot plots indicate the percentage of total cells that are within a region. A total of 20,000 events were collected for each plot. (b) “CD4lo”-enriched or “CD4hi”-enriched T cells were each adoptively transferred into 6 NOD.scid mice. As a control, untreated BDC2.5 TCR Tg spleen cells were also transferred into a control group of NOD.scid mice. Ten days later the pancreas was recovered from at least two mice per group, fixed, embedded, and cut into 5 micron sections that were subsequently stained with hematoxylin and eosin. The sections were viewed and photographed by a blinded observer. At least 50 islets were counted for each group to evaluate the pattern of insulitis. The arrows point to areas of inflammation, peri-insulits, which was predominant in “CD4lo” T cell recipients, and invasive insulitis, which was representative of most islets in recipients of “CD4hi” T cell recipients. The remaining mice were monitored for the development of diabetes as described in the Methods. (c) Spleen cells were recovered from the recipients in (b), stained with anti-CD4-PE and anti-TCR V beta 4-FITC, and then 100,000 events were collected during FACS analysis. The data in the dot plot were gated on viable cells (FSC SSC). The numbers indicate the percentage of cells in the region. The number in the parentheses for the “CD4lo” recipients indicates the percentage of “CD4lo” cells in the total CD4+ population.
