In vivo confocal microscopy of conjunctiva-associated lymphoid tissue in healthy humans

Purpose. To investigate modifications with aging of the presence, distribution and morphologic features of conjunctiva-associated lymphoid tissue (CALT) in healthy human subjects using laser scanning in vivo confocal microscopy (IVCM). Methods. A total of 108 (age range, 17-75 years) subjects were enrolled. In vivo confocal microscopy of the tarsal and bulbar conjunctiva, and impression cytology (IC) with CD3 (intra-epithelial T-lymphocytes) and CD20 (intra-epithelial B-lymphocytes) antibody immunofluorescence staining were performed. The main outcomes were subepithelial lymphocyte density (LyD), follicular density (FD), and follicular area (FA). The secondary outcomes were follicular reflectivity (FR), and lymphocyte density (FLyD), and CD3 and CD20 positivity. Results. Conjunctiva-associated lymphoid tissue was observed in all subjects (97% only superior and 3% in both superior and inferior tarsum). Lymphocyte density ranged from 7.8 to 165.8 cells/mm2 (46.42 [18.37]; mean [SD]), FD from 0.5 to 19.4 follicles/mm2 (5.3 [3.6]), and FA from 1110 to 96, 280 mm2 (26, 440 [26, 280]). All three parameters showed a highly significant inverse cubic relationship with age (P < 0.001); that is, in the first and last parameters a steep decline up to 35 years and above 65 years of age, with a plateau phase between these ages, whereas FA had a gradually decreasing rate of loss over the studied age range. CD3 and CD20 IC were consistent with these results. Conclusions. In vivo confocal microscopy was effective in revealing CALT and modifications these structures undergo with aging. Aging correlated with an involution of all parameters defining lymphoid structures. These modifications may account for the decrease of mucosal immune response and increase of ocular surface diseases in the elderly.