Multicolor Flow Cytometry Approach for Analysis of Stromal and Thymocyte Cell Populations in the Mouse Thymus v1

The thymus is a primary lymphoid organ which contains stromal cell components that provide the necessary support for thymocyte development and maturation. Thymus stromal cells include, among others, thymic epithelial cells (TEC), endothelial cells, mesenchymal/fibroblast cells, and hematopoietic populations such as dendritic cells, B cells, and macrophages. TECs are particularly critical through all the stages of T cell development, contributing to the recruitment of hematopoietic precursors from the bone marrow, directing lineage specification, supporting proliferative expansion, and orchestrating both positive and negative selection processes (Cepeda & Griffith, 2018) (Han & Zúñiga-Pflücker, 2021). Anatomically, the thymus is organized into two distinct regions: the cortex in the outer zone, which contains the cortical TECs (cTECs) and the medulla in the inner zone, containing the medullary TECs (mTECs). cTECs are primarily involved in the positive selection of thymocytes. In contrast, mTECs participate in the negative selection, promoting central tolerance and supporting the development of Foxp3⁺ regulatory T cells and natural killer T cells (Han & Zúñiga-Pflücker, 2021) (Nitta & Suzuki, 2016). A young and fully functional thymus is essential for generating a pool of diverse naïve T cells, which supports robust immune responses. However, during aging, the thymus undergoes progressive involution characterized by structural and molecular modifications. These age-associated changes lead to a marked decline in the production of newly generated naïve T cells. In compensation, a homeostatic expansion of existing T cell populations occurs, resulting in reduced diversity within the peripheral T cell repertoire. Functionally, this adaptation contributes to diminished responsiveness to pathogens, increased susceptibility to autoimmune conditions, and impaired tumor surveillance (Liang et al., 2022) (Han & Zúñiga-Pflücker, 2021). The study of the thymic microenvironment and its influence on thymocyte outcome during aging provides valuable insights into the structural and functional changes that occur with senescence. One of the hallmark features of thymic aging is the decline in TEC cellularity, affecting both cTECs and mTECs subsets. TECs can be phenotypically identified with the pan-epithelial marker CD326 (EpCAM) within the CD45- thymic cell population. Further discrimination between TEC subsets could be achieved using the CD205 marker. cTECs subset could be characterized as CD45-CD326+CD205+, whereas CD45-CD326+CD205- cells correspond to mTECs (Jain & Gray, 2014). Aging is also associated with a reduction in MHC class II expression on TECs, reflecting a decline in TEC antigen-presenting ability (Gray et al., 2006). Within the TEC population, two subgroups, MHCIIlow and MHCIIhigh, can be distinguished based on the expression levels of MHC class II molecules (Jain & Gray, 2014) (Xing & Hogquist, 2014). A key molecular regulator of TEC function is the transcription factor FoxN1, which is indispensable for thymus organogenesis and TEC maintenance in adults. FoxN1 expression declines progressively with age, contributing to thymic involution and reduced naïve T cell output (Rode et al., 2015). Overexpression of FoxN1 has been shown to mitigate age-related thymic changes, restoring thymic mass, architecture, and the naïve T cell population in the spleen (Zook et al., 2011). Thymic medulla, besides containing mTECs, is populated by hematopoietic antigen presenting cells (APCs) including dendritic cells, B cells, and macrophages. Through expression and presentation of self-antigens, these APCs contribute to the process of shaping the normal T cell repertoire by negatively selecting thymocytes recognizing self-antigens. Senescence studies have demonstrated age-associated declines in APCs function and cell counts (Ki et al., 2014) (Cepeda & Griffith, 2018) (Wedemeyer & Griffith, 2025) (Van Dommelen et al., 2010). Thymocytes development starts from the double negative (DN) to simple positive (SP) T cells. At first, T cells are CD4-CD8- (DN), in the second stage they are double positive CD4+CD8+ (DP) and finally CD4+CD8- or CD4-CD8+ (SP) in the third stage. After positive selection, SP CD4+ or CD8+ T cells migrate to the thymus medulla to go through negative selection mediated by medullar TECs and APCs, where autoreactive SP T cells are deleted by apoptosis. This process of thymopoiesis results in population of peripheral blood and lymphoid sites with naive CD4+ and CD8+T cells and CD4+ regulatory T cells (Tregs) (Liang et al., 2022). Early T-lineage progenitors (ETPs) frequency declines with aging, and their potential ability to reconstitute the thymus is also reduced (Min et al., 2005). Both DN and DP population cell numbers are significantly modified with aging. DN subset frequency increases in aged mice compared with young, whereas the percentage of DP (CD4+CD8+) subpopulations significantly diminishes with age. Additionally, thymic aging is concomitant with the abnormal accumulation of CD3+ DN cells within the thymus (Aw et al., 2010). Aging also interferes with later stages of thymocyte development. DP and SP thymocytes in aged mice display deregulated CD3 expression, which may lead to attenuated TCR-dependent stimulation (Aw et al., 2010). Consistent with impaired thymocyte differentiation in the aged thymus, T-cell receptor excision circles (TRECs) within the thymus also significantly decline with aging in mice and humans (Ortman et al., 2002). Regarding thymocytes, the member of the high mobility group (HMG)-Box transcription factors TOX-1 has been identified as a mediator of thymic T cell expansion and the development of lymphoid inducer (Scott et al., 2019). The pattern of TOX expression in the thymus is highly regulated during the positive selection process. Downregulation of TOX-1 is observed in a later stage of positive selection, as cells take on a more mature SP phenotype (Wilkinson et al., 2002). Data about TOX expression in murine thymus during aging is scarce. Recently, it was published that Recent Thymic Emigrants (RTE) are distinguished by the expression of SOX4, IKZF2, and TOX and CD38 protein in humans. These cells significantly decrease with age (Bohacova et al., 2024). Interestingly, some TOX members have been related to T cell exhaustion in the periphery. In mice, TOX is required for the epigenetic remodeling and survival of exhausted CD8+ T cells in the chronic infection context (Yao et al., 2019). In contrast, a recent publication in human settings showed that TOX expression is related to anti-viral effective T cells (Sekine et al., 2020). Different markers related to thymocytes development, survival, and migration have been studied in murine settings. In this case, L-selectin (CD62L) is a cell adhesion molecule that is expressed on most circulating leukocytes. The expression of CD62L is increased in the late stage of thymocytes differentiation and allows mature thymocytes to enter from circulation into lymph nodes (Weinreich & Hogquist, 2008). Another important marker related to thymus development is the CD127 molecule (IL-7Ra chain). IL-7 augments survival of TN and SP thymocytes and is also essential for γδ T cells and thymic DC development (Lynch et al., 2009). Lymphocyte development is severely impaired in mice lacking IL-7 or IL7 receptor (IL-7R) or treated withanti-IL-7 antibodies (Peschon et al., 1994). In both syngeneic and allogeneic mouse models, administration of IL-7 leads to enhanced T-cell reconstitution through increased thymic T-cell development and homeostatic proliferation, as well as decreased T-cell apoptosis but increased T-cell function (Alpdogan et al., 2003). Regulatory T-cells (Tregs) were identified as sentinels of the immune response, keeping aberrant/exaggerated immune reactions in balance. After CD4 lineage commitment, some CD4 single-positive (SP) thymocytes, upon TCR stimulation, can develop into CD25+Foxp3+ Treg cells. The absolute number of Tregs in the thymus decreases with age, reflecting the reduction in cellularity that accompanies age-associated thymic involution (Chiu et al., 2007). Costimulatory and coinhibitory molecules affect cell fate decisions in the thymus by enhancing or dampening signaling through the TCR. Studies have shown that PD-1 is expressed by developing thymocytes (Nishimura et al., 1996). A recent publication has reported that PD-1 limits the production of IL-2 within the thymus, thereby restraining the development of Tregs (Caruso et al., 2024). The present protocol describes the development of flow cytometry panels tailored for the identification and characterization of thymocytes and thymus stromal cells. The panels could be useful to compare young and aged mice in senescence studies, offering valuable insights into age-associated changes in thymic architecture and immune function.