Figure 5. Immunophenotype of resident GC cells by confocal microscopy.

A, Localization of CD3⁺ and CD4⁺ resident cells in germinal centers. Multiparameter confocal microscopy was used to identify CD3⁺ and CD4⁺ cells within germinal centers (PCNA⁺) of lymph nodes from SM and PM at baseline and 2 wpi by simultaneous localization of CD3 with Alexafluor 633 (blue), CD4 with Alexafluor 568 (red) and PCNA with Alexafluor 488 (green). CD4⁺ T lymphocytes (CD3⁺/CD4⁺) were identified by purple/pink co-localization of Alexafluors 633 (blue) and 568 (red). Macrophages (CD3^−/CD4⁺) were identified by localization of red Alexafluor 568. By default, CD8⁺ T lymphocytes (CD3⁺/CD4) were identified by localization of Alexafluor 633 (blue). Five randomly selected GC were imaged for most animals; however, all available GC were imaged when fewer than 5 GC were present per section. Magnitude of fluorescence (panels B through E) is reported as ratio of area fraction (AF) of fluorescent pixels within GC to area fraction of PCNA⁺ (total germinal center) fluorescent pixels, as calculated using an algorithm in the imaging software (Fovea Pro). Each point represents a germinal center, with horizontal bars indicating the mean. B, PM had greater numbers of CD4⁺ cells in GC at 2 wpi than SM (p = 0.02). C, No difference was noted in the mean number of CD3⁺ cells in GC between SM and PM at baseline and 2 wpi. However, numbers of CD3⁺ cells were higher at baseline than at 2 wpi in SM (p = 0.02). Values for CD8-depleted SM were excluded from the 2 wpi data set. D, CD3^−/CD4⁺ fluorescence (macrophages), calculated by subtracting CD3⁺ AF from the total CD4⁺ AF. Negative ratios reported as zero. PM had higher numbers of GC macrophages at 2 wpi compared to SM (p = 0.05). Mean number of GC macrophages increased in PM from baseline to 2 wpi (p = 0.003). E, CD3⁺/CD4⁻ fluorescence calculated by subtracting total CD4⁺ AF from CD3⁺ AF. Negative ratios reported as zero. Low numbers of CD3⁺/CD4⁻ cells (likely cytotoxic CD8⁺ T lymphocytes) were observed in GC for both species at all time points. SM have higher baseline numbers of (CD8⁺ cells) compared to 2 wpi (p = 0.02). Differences in mean area fractions between time points were analyzed using unpaired Student’s t-tests. Welsh’s correction was used if variances were unequal. Values for CD8-depleted SM were excluded from the2 wpi data set.