Figure 1.
Identification of COL7-specific plasma cells (PCs).
Cells from lymph nodes, spleen and bone marrow were stained as described in the Methods section (gating strategy: Figure S1). A, Left plot: population within the lower quadrant not binding to GST resembles cells specific for mCOL7c only. Right plot: Staining was blocked by pre-incubation with unlabeled antigens (mCOL7c-HIS and GST). B, Frequencies of plasma cells specific for mCOL7c or GST after mCOL7c-GST immunization, from the draining lymph nodes, 14 weeks after immunization. C, Frequencies of plasma cells specific for mCOL7c only, from the indicated organs of mice (n = 3–5) immunized either with mCOL7c-GST or OVA (as a control group), 14 weeks after immunization. Similar distribution of mCOL7c-specific plasma cells were found also weeks 3, 7 and 21 after immunization. Data are representative for three independent experiments.
Figure 2.
Turn-over of plasma cell populations.
A, Frequencies of total plasma cells and mCOL7c-specific plasma cells among total plasma cells were determined at various time points after immunization as described in Fig. 2. B, from weeks 4 to 7 after immunization, mice were continuously fed with BrdU. Mice were analyzed directly after the pulse (week 7), as well as seven weeks (week 14) and 14 weeks (week 21) later. C, Left: GST-binding cells were excluded by electronic gating, allowing the identification of mCOL7c-specific plasma cells and plasma cells not binding to the immunizing antigens (bystander plasma cells neither specific for mCOL7c, nor GST). Right: Plasma cells specific for the indicated antigens were analyzed for BrdU incorporation directly after the feeding period at week 7, and 7 weeks later. Mean results (± SEM) are shown (n = 6). Data for week 7 and 14 are representative for two independent experiments.
Figure 3.
mCOL7c-GST specific CD4 T cells detected in spleen and draining lymph nodes after mCOL7c-GST immunization.
Draining lymph node and spleen cells were harvested 7 weeks after immunization and restimulated with OVA (negative control), mCOL7c-GST or PMA and Ionomycin (positive control). A, representative flow cytometric analysis of restimulated CD 4 T cells. Cells were pre-gated according to CD4 expression. B, Frequencies of mCOL7c-specific cells obtained from draining lymph nodes and spleens 7 weeks after mCOL7c-GST immunization are shown (n = 8). Data are representative for more than three independent experiments.
Figure 4.
CD4 T cells specific for various antigens detected after mCOL7c-GST immunization.
Draining lymph node cells were harvested 5 weeks after immunization and restimulated with no antigen, OVA, mCOL7c-HIS, GST or mCOL7c-GST, as indicated. T cells specific for the respective antigens were identified by electronic gating on CD4+ cells and the rapid up-regulation of CD154 [32]. A, representative flow cytometric analysis of cells stimulated with various antigens. Restimulation with PMA/Ionomycin was used as a positive control. B, Frequencies of antigen-specific cells obtained from draining lymph nodes 5 weeks after mCOL7c-GST immunization are shown (n = 6). Similar frequencies were determined at week 3 after immunization (data not shown). Data are representative for more than three independent experiments.
Figure 5.
T cells, plasma cells and antibodies specific for immunizing antigens.
Mice were immunized with either mCOL7c-GST or mCOL7c-HIS. A, CD4 T cells from draining lymph nodes specific for mCOL7c-HIS, mCOL7c-GST and GST were determined as CD154+ described in Fig. 1. Average background values of negative controls (unstimulated and OVA stimulated cells) were subtracted in all samples. Examples show T cell numbers at week 3 after immunization (n = 6). B, In the same experiment, the numbers of plasma cells specific for mCOL7c were determined as described in Fig. 2 (n = 6). C, mCOL7c-specific serum antibodies 8 weeks after immunization with mCOL7c-GST or mCOL7c-HIS determined by ELISA (n = 3–4). Data are representative for two independent experiments.