More than two to tango: mesenchymal cells are required for early T cell development

Before the mid-20^th century, the thymus was thought to be a vestigial organ, referred to by Nobel Laureate Sir Peter Medawar as “an evolutionary accident of no very great significance” (1). Fast-forward to 2019, when the prestigious Albert Lasker Basic Medical Research Award was conferred upon Dr. Jacques Miller for his identification of the thymus as the site of T cell development, and upon Dr. Max Cooper for his discovery that the bursa is the site of B cell development (2). Although it was long recognized that thymic stromal cells provided inductive cues for T cell development, the roles of distinctive stromal cell types were unknown. In this Pillars article, Graham Anderson and colleagues showed that thymic mesenchymal cells and thymic epithelial cells (TECs) are both essential for early T cell development, whereas TECs alone can mediate later events such as thymocyte selection and maturation (3) https://www.nature.com/articles/362070a0. This finding provided an initial appreciation for the contribution of mesenchymal cells to T cell development that inspired future studies with important implications for thymic atrophy and regeneration.

Appreciation for the immunological role of thymic stromal cells came from experiments as early as the 1950s and 1960s (4). While studying leukemia, Miller et al. made the critical observation that thymectomies at different times of life had profoundly different effects on T cell development and function in mice. Adult mice that were subjected to thymectomy displayed relatively normal T cell function, whereas neonatal thymectomy led to a failure of skin graft rejection and a high rate of mortality. Subsequently, examination of the embryonic day (E) 12 fetal thymus revealed a structured organ consisting of TECs surrounded by a thin layer of mesenchyme (5). E12 thymic lobes survived and grew in organ culture, but remained alymphoid. By contrast, implantation of E12 thymic lobes into the eyes of adult mice allowed host progenitors to colonize those lobes and give rise to T cells in the implant. However, when the mesenchymal capsule was removed prior to implantation, host colonization did not occur. Moreover, fetal mesenchyme from multiple tissue sources restored the ability of the TECs to support thymic development, but newborn mesenchyme did not. Thus, it became clear that fetal mesenchymal cells were required for T cell development, but the nature of their roles in supporting TEC and/or T cell development were unclear.

In 1993, Anderson et al. took a new approach towards addressing this question using an innovative modification of fetal thymic organ cultures (FTOCs) known as reaggregate organ culture (RTOC). Traditional FTOCs involve ex vivo placement of fetal thymic lobes at the air-media interface (6). To test the T cell potential of different hematopoietic precursors, thymic stroma can be depleted of endogenous thymocytes by treatment with deoxyguanosine (7). Treated lobes can then be placed in hanging drop cultures with progenitors for 1–2 days, followed by traditional FTOC. Anderson et al. took this approach a step further by separating thymic stromal components from deoxyguanosine treated lobe into TEC and mesenchymal fractions using magnetic beads, followed by reconstitution with thymocytes in various combinations. Remarkably, droplets consisting of thick cell slurries containing all thymic components were able to self-organize back into thymic structures with normal cortico-medullary architecture within twelve hours. Furthermore, T cell development was fully supported in these cultures, including the transition from CD4−CD8− (DN) to CD4+CD8+ (DP), and positive selection of DP cells into CD4+ single positive (SP) T cell receptor (TCR)β+ and CD8 SP TCRβ+ cells. These advances provided a fresh opportunity to assess the functions of different types of stromal cells in thymic formation and function.

In an elegant series of technically demanding experiments, Anderson et al. used the RTOC technique to examine the consequences of depletion or addition of stromal components on T cell development. To bypass the requirement for mesenchyme in TEC development at E12, fetal thymic lobes from E14 were used as the source of TECs. To isolate TECs, E14 thymic lobes were treated with deoxyguanosine, trypsinized, and depleted of the remaining mesenchymal and hematopoietic cells using magnetic beads. Thymocytes were isolated from fetal lobes using gentle disaggregation and anti-CD45 beads, while fetal lung was used as a source of mesenchyme. Deoxyguanosine-treated stroma depleted of TECs failed to reaggregate with fetal thymocytes, as expected. Isolated TECs reaggregated with fetal thymocytes formed small thymus-like structures, but failed to support lymphoid cell survival or development. By contrast, RTOCs consisting of purified TECs, mesenchymal cells, and thymocytes self-organized and fully supported T cell development. To assess the nature of the mesenchyme-derived signal, NIH3T3 fibroblasts or supernatant from these cells were used in place of fetal lung mesenchyme. Only intact NIH3T3 cells supported reaggregation and T cell development, indicating a requirement for cell-cell contact.

Anderson et al. went on to ask whether both TECs and mesenchyme were required for later stages of T cell development. A significant advantage of the RTOC technique is the ability to incorporate cells that lack the proper chemokine receptors for migration into the thymus. This feature enabled the use of DP cells from newborn thymus to examine requirements for selection and maturation by TECs and mesenchymal cells in RTOCs. Unlike RTOCs containing E14 thymocytes, which required mesenchyme to self-organize, RTOCs containing DPs and purified TECs alone were able to form intact thymic structures. Moreover, these RTOCs containing DP cells gave rise to mature CD4 TCRβ+ and CD8 TCRβ+ cells, indicating that positive selection and maturation of both SP subsets could occur independently of mesenchymal-derived signals. Taken together, these results showed for the first time that mesenchymal cells are essential for creating an environment conducive to early T cell development but are dispensable for the later stages of thymic selection and the DP to SP transition.

Subsequent studies by Anderson and others built upon these initial findings to decipher the molecular signals supplied by different stromal components during T cell development. The majority of these studies revealed more precise roles for mesenchymal cells in TEC differentiation and maintenance. Neural crest cell-derived mesenchymal cells supply bone morphogenic protein 4 to endodermal precursors during thymic organogenesis, and fibroblast/epidermal growth factors to promote expansion of TECs in the fetal thymus (8, 9). Thymic mesenchymal cells also differentiate into fibroblasts that secrete extracellular matrix (ECM) to maintain the 3D structure of the thymus. The ECM also improves adherence and increases the accessibility of TEC-derived growth factors to early T cell precursors. Thus, mesenchymal cells provide both direct and indirect support for hematopoietic progenitors at the early stages of T cell development.

Understanding the function of thymic mesenchymal cells has major relevance to the goal of inducing thymic regeneration. During aging, the thymus undergoes a decrease in size, accumulation of adipose tissue, degradation of cortico-medullary structure, and a decline in T cell output, leading to a less diverse T cell repertoire, and impaired responses to new pathogens (10). Thymic involution can also result from cancer treatments such as irradiation or chemotherapy, resulting in defective T-cell immunity that persists long after the treatment is completed. Thymic mesenchymal cells also can give rise to fibroblasts or adipocytes (11). It is therefore possible that differentiation of mesenchymal cells into adipocytes could be a major factor in the loss of thymic structure and function during involution. This possibility is supported by the ability of mesenchymal cells from various tissues to induce a degree of thymic regeneration after involution (12). Clearly, mesenchymal integrity is required for proper support of early T cell development, but whether adipocytes exert a negative influence on TECs, or T cell precursors remains a key question. Overall, the observations reported by Anderson et al. provided new insights and appreciation for the requirement for mesenchymal cells in endowing the thymus with its unique ability to support early T cell development.