PMC

Rejection kinetics of corneal allografts in the BN-PVG strain combination. A: An example of a well healed corneal allograft approximately day 21. B: Typical appearance of a rejected PVG allograft, arrow indicates epithelial rejection line (occasionally observed). C: Rejection kinetics of the BN-PVG strain combination BN-PVG n=15, BN-BN n=9. Kaplan–Meier-Survival plot.

Flow cytometric analysis of helper T-cell activation markers in draining lymph nodes. A: Representative FACS plot of activation marker expression on CD4+ single positive T-helper cell in draining LN. Events acquired: 2×10⁵. B: Example of CD25 and CD134 expression pattern on CD4+CD8+ double positive T-cells. Bar diagrams: Cumulative results for the quantification of T-cell activation status of Helper T-lymphocytes. An asterisk (*) indicates statistical significance at p≤0.05 determined by Mann–Whitney U-Test.

Serum allo-antibody measurement in grafted animals. Bar diagram: Summary of allo-antibody screening in transplanted animals using a FACS based direct detection method. The presence of IgG1 infers a Th2 response, while the presence of IgG2a antibodies indicates a Th1-IFN-γ induced class switch in B-cells. For IgM detection IgM+ B-cell were excluded from analysis by CD45RA staining (see ); IgA isotypes could not be detected. FACS plots: Display of the measurement method for direct binding of allo-antibodies to allogeneic target cells (PVG donor splenocytes); negative controls: serum from PBS injected animals harvested after day 14, positive controls: serum from animals injected with 1×10⁶ γ-irradiated thymic dendritic cells collected after day 14.

Flow cytometric analysis of graft-infiltrating lymphocyte populations. A, B: Allo-grafted cornea analyzed at day 7 post-op. A: Forward-sideward scatter morphology of graft infiltrating cells. B: Representative image of CD161^dull expression on LGL. No other cell type could be detected. Recorded events: 2–5×10³. C-H: FACS results of allo-rejecting corneas: Events acquired: 5×10³ to 3×10⁴ per sample. C: FSC-SSC morphology of GIL. D: Detection of T-lymphocytes. E: Measurement of CD8 and CD161 NK markers on GIL. F: Sub-characterization of CD4+ T-cells and MHC-2 detection. G: Expression pattern of CD3 from populations gated in E. H: Measurement of CD25 T-cell activation markers on populations gated in D. Cut-off or positive CD25 expression was determined by measuring isotype FMO samples on lymphocytes in draining LN from the same animal. For scaling reasons data are not shown in histogram. Bar diagram: Summary of all lymphocyte specimens identified in rejected corneas (left diagram) and appropriate sub-characterizations (right diagram) n=5.

Qualitative and quantitative flow cytometric analysis of lymphocyte populations in draining lymph nodes. A: Representative FACS plot of CD4+ CD8+ staining used to count T-helper cells, cytotoxic T-cells and CD4+ CD8+ double positive T-lymphocytes. Events acquired: 2×10⁵. B: FACS plot example for B-cell detection. C: Representative FACS plot for NK cell assessment. NK-T cell were confirmed by CD3 expression (not shown). Bar diagrams: Cumulative results for the quantification of major and minor lymphocyte populations in draining LN of cornea transplanted animals. An asterisk (*) indicates statistical significance at p≤0.05 determined by Mann–Whitney U-Test. Allo-Tx-d7 - animals allo-grafted and analyzed at day 07 post op, n=6; allo-Tx-rej – animals displaying allo rejection of grafted corneas analyzed after the onset of rejection, n=5; syn-Tx-d7 – syngeneically grafted animals analyzed at day 7 post-op, n=3; syn-Tx-LT – syn-grafted long-term survivors analyzed at the end of the observation period at day 42; n=3.