Dendritic cell subsets and morphological characteristics in the thymus
Thymus tissue was digested into single-cell suspension, and dendritic cells in the thymus were analyzed by flow cytometry. Using lineage cocktail antibody to exclude thymocytes, T cells, B lymphocytes, NK cells, and macrophages. Cells with linage cocktail negative and HLADR positive were selected for further analysis. Plasmacytoid DCs (pDCs) were labeled by CD123 (Fig. 1, A). Another dendritic cell subset, myeloid DCs (mDCs) (Fig. 1, B), were labeled by CD11c and further divided into CD1c positive DC and CD141 positive DC (Fig. 1, B) by CD1c and CD141. Using CD123 and CD11c as markers in immunohistochemistry, the two subsets of DCs in thymus tissue were identified respectively (Fig. 1, C). PDCs were elliptic and granular, while mDCs were typical dendrite-like with irregular shapes and many projections. Though mDCs could be further labeled by CD1c and CD141 in poly colors flow cytometry, these two markers have poor specificity when used in immunohistochemistry (an observed phenomenon). Both pDCs and mDCs were mainly distributed in the thymus medulla (Fig. 1, D), and the density of pDCs in the thymus is higher than that of mDCs (Fig. 1, D).
The ratio between the medullary and cortex area in the thymus increases with an increase in age.
Normal thymus with different ages was collected, and HE staining was performed on the tissue slides. Four specific age groups (1 year - infant, 14 years - adolescent, 26 years - early adult, 40 years - middle adult) were selected to represent the characteristics of thymus changes with age (Fig. 2, A). The thymus gradually atrophies with increasing age and rapidly atrophies after puberty. By adulthood, the thymus has been replaced mainly by adipose tissue, but a tiny amount of thymus medulla and cortex remains. Although the thymus gradually atrophies with age increase and is replaced by adipose tissue, the atrophy degree of the thymic cortex and medulla is not uniform (Fig. 2, A). We calculated the area of the medullary and cortex in the stained thymus tissue and calculated the ratio (AreaM/AreaC, M/C) between the medullary area (AreaM) and the cortical area (AreaC) to establish the relationship between this ratio and age. With the increase of age, the ratio of M/C gradually increased (Fig. 2, B), and there was a significant correlation between them. This suggested that with aging, the thymic cortex atrophied more rapidly than the medulla.
The density of mDCs did not change significantly with aging, while pDCs' increased gradually with aging.
Using CD123 and CD11c to mark thymus tissues of different ages, we found the changes of pDCs and mDCs density. With the increase in age, most parts of the thymic medulla and cortex parts had been replaced by fat components in the typical 40 years old thymus, which was not suitable for the staining and density calculation of DCs. Therefore, normal thymus tissue over 40 years old was not included in the analysis. By analyzing the density of the two DC subsets in the thymic medulla and cortex, respectively, we found that the density of DC subsets in the thymic medulla and cortex changed with special tendency. As the age increases, the density of pDCs in the thymic medulla and cortex gradually increased, and the density of pDCs in the medulla was always much higher than that in the cortex. The density of mDCs in the medulla was significantly higher than that in the cortex regardless of age. Still, the density of mDCs in the medulla and cortex showed an opposite trend with age increase, with a gradual decrease in the medulla and a gradual increase in the cortex (Fig. 3, A). Combining the density of the two DC subsets in the medulla and cortex, we found that the density of pDCs still gradually increased with the age growth, thymus atrophy, and medulla proportion increase. Simultaneously, the density of mDCs also increased but did not change significantly (Fig. 3, B).