Examination of Thymic Positive and Negative Selection by Flow Cytometry

Abstract

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) α and β loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders(1-4). T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the αβTCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the αβTCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC(5). Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events(6). For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes(7-9). Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCRα chain at the DN stage, resulting in premature negative selection. Our lab has developed the HY(cd4) model, in which the transgenic HY TCRα is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice(10). Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HY(cd4) mouse model. While negative selection in HY(cd4) mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

Cutting Edge: Dual TCRα Expression Poses an Autoimmune Hazard by Limiting Regulatory T Cell Generation

“…We scrutinized positive selection using TCRβ and CD5 expression as surrogate markers for TCR signaling and progression through positive selection (where TCRβ lo CD5 lo -pre-selection, TCRβ lo CD5 int -initiating positive selection, TCRβ int CD5 hi -undergoing positive selection, and TCRβ hi CD5 hi -post-selection) (Fig. 4D) (39). We found that single TCR T cell and TCRα +/− double positive (DP) thymocytes were more likely to be classified as pre-selection and less likely to initiate and undergo positive selection relative to WT DP thymocytes, indicating less efficient positive selection (Fig.…”

Targeted loss of SHP1 in murine thymocytes dampens TCR signaling late in selection

“…A). Thymocytes were further analyzed based on defined maturation phases using TCRβ and CD69, with thymocytes progressing through selection by upregulating TCR and CD69 before downregulating CD69 upon completion of selection . Within the TCR and CD69 subsets, distinct thymocyte developmental stages were identified using CD4 and CD8α that included double negative (DN), double positive (DP), post‐selection DP, CD8α intermediate (CD8α int), CD4 single positive (CD4SP), and CD8α single positive (CD8SP) (Fig.…”

“…Within the TCR and CD69 subsets, distinct thymocyte developmental stages were identified using CD4 and CD8α that included double negative (DN), double positive (DP), post‐selection DP, CD8α intermediate (CD8α int), CD4 single positive (CD4SP), and CD8α single positive (CD8SP) (Fig. B) . When the individual subsets of maturing thymocytes were analyzed, the most mature (TCRβ Hi CD69 − ) stage revealed significantly reduced numbers of SHP1‐deficient cells in the post‐selection CD8αInt, CD4SP, and CD8αSP populations (Fig.…”

A defect in thymic tolerance causes T cell-mediated autoimmunity in a murine model of COPA syndrome

“…To identify at which stage of thymocyte selection the increase in SP cells occurred, we evaluated thymocytes by TCRb and CD69 and found a significant increase in the percentages of TCRβ + CD69 + and TCRβ + CD69 -SP cells that arise post-positive selection( Fig. 3C) (7,8). Taken together, the increase in SP cells suggests mutant Copa alters T cell development or selection as a precursor to spontaneous inflammation.…”

“…To carefully study the impact of thymic Copa on T cell selection we employed TCR transgenic mice to track the development and selection of MHC-restricted T cells (8,13). To exclude potential cell-intrinsic effects of mutant Copa on T cells, we generated chimeric mice by transplanting BM from wild-type OT-I (14) (H-2K b ), OT-II (15) (IA b ) and P14 (16) (H-2D b ) TCR 8 transgenic mice into irradiated Copa E241K/+ and WT hosts. Analysis of thymocytes demonstrated no significant differences in the percentages of major populations between Copa E241K/+ and WT hosts ( Fig.…”