ARTICLES Late stages of T cell maturation in the thymus involve NF-B and tonic type I interferon signaling Yan Xing, Xiaodan Wang, Stephen C Jameson & Kristin A Hogquist Positive selection occurs in the thymic cortex, but critical maturation events occur later in the medulla. Here we defined the precise stage at which T cells acquired competence to proliferate and emigrate. Transcriptome analysis of late gene changes suggested roles for the transcription factor NF-B and interferon signaling. Mice lacking the inhibitor of NF-B (IB) kinase (IKK) kinase TAK1 underwent normal positive selection but exhibited a specific block in functional maturation. NF-B signaling provided protection from death mediated by the cytokine TNF and was required for proliferation and emigration. The interferon signature was independent of NF-B; however, thymocytes deficient in the interferon- (IFN-) receptor IFN-R showed reduced expression of the transcription factor STAT1 and phenotypic abnormality but were able to proliferate. Thus, both NF-B and tonic interferon signals are involved in the final maturation of thymocytes into naive T cells. T cell development occurs in the thymus, which provides a unique reside predominantly in the medulla; however, not all SP thymocytes microenvironment and presents ligands consisting of self peptide and are equivalent. major histocompatibility complex (MHC) molecules to T cell anti- CD24hiQa2lo SP thymocytes have been defined as ‘semi-mature’ gen receptors (TCRs). In the cortex of the thymus, low-affinity TCR and have been shown to be susceptible to apoptosis when triggered interactions initiate positive selection signals in CD4+CD8+ double- through the TCR6. In contrast, mature SP thymocyte and thymic positive (DP) immature thymocytes, which supports their survival emigrants proliferate when triggered through the TCR6,7. Over the and differentiation into CD4+ or CD8+ single-positive (SP) thymo- years, studies have shown that the expression of a small number of cytes. Positively selected cells move to the medullary region and, after other cell-surface proteins, including CD69 and various cytokine and several days, emigrate to the periphery. The term ‘positive selection’ chemokine receptors, changes during maturation8,9. However, the Nature America, Inc. All rights reserved. Inc. Nature America, is sometimes used to describe the entire process. However, in think- molecular mechanisms that control the maturation of SP thymocytes 6 ing about molecular mechanisms, it is helpful to break this down have remained unclear. into kinetically distinct processes, such as survival, allelic exclusion, Here we defined steps in the maturation of SP thymocytes through © 201 lineage commitment and functional maturation. which SP thymocytes became equipped with mature functions, such Cortical DP thymocytes require interaction of the surface TCR with as proliferation competency, emigration competency and cytokine selecting peptide-MHC complexes to induce survival. Expression of ‘licensing’. For this, we used comprehensive microarray analysis, the activation marker CD69, the TCR and the anti-apoptotic protein quantitative real-time PCR (qPCR) and flow cytometry, along with Bcl-2 is rapidly upregulated in cells of this population, which also combinations of several gene-deficient and transgenic mouse models. undergo changes in the expression of many other genes1,2. Genetic We found that SP thymocytes received signals via both the cytokine deficiency in TCRs, MHC molecules, the co-receptors CD4 or CD8 TNF and type I interferons in the thymus and that only TNF-resistant or molecules in the TCR signaling pathway blocks this process2. mature thymocytes survived and became emigration competent and Recombination-activating genes are rapidly repressed at this stage, licensed to produce cytokines. which facilitates allelic exclusion. Expression of the chemokine receptor CCR7 is upregulated somewhat later and facilitates the RESULTS migration of progenitor cells from the cortex to the medulla3. Lineage Three SP stages defined by function commitment occurs concurrently and involves downregulation of In this study, we sought to determine the ideal markers for flow the gene encoding the inappropriate co-receptor and the initiation of cytometry to define SP thymocyte stages by function. The ordered genetic remodeling that will ultimately determine if the cell has helper development of SP thymocytes has been characterized by the expres- or killer potential. Genetic deficiency in key transcription factors can sion of cell-surface makers (CD24, CD69, CD62L and Qa2)6,10; the block commitment to the CD4+ or CD8+ lineage4,5. Although line- carbohydrate epitope 6C10 on the glycoprotein Thy-1, recognized by age commitment is mechanistically independent of migration to the the monoclonal antibody SM6C10 (ref. 9); or chemokine receptors medulla, these processes are roughly concurrent. Thus, SP thymocytes CCR7 and CCR9 (ref. 11). However, these markers have not been well The Center for Immunology, Department of Laboratory Medicine & Pathology, University of Minnesota, Minneapolis, Minnesota, USA. Correspondence should be addressed to K.A.H. ([email protected]). Received 22 October 2015; accepted 19 February 2016; published online 4 April 2016; doi:10.1038/ni.3419 NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 1 ARTICLES Rag2-GFP+ + + a thymocytes CCR7 TCRβ b Medium CD3+CD28 c SM M1 M2 5 5 100 10 9.3 10 SM SM SM M1 M2 80 104 104 M1 60 M1 M2 0.0 1.4 54.0 3 3 ents CD4SP CCR7 10 10 CD69 40 Ev CD4SP 0 0 M2 20 0 0 103 104 105 0 103 104 105 0 103 104 105 S1PR1 105 TCRβ MHCI Rag2-GFP Events 60 104 SM M1 M2 CD8SP 3 1.7 4.3 58.0 5 40 10 10 20.7 CD8SP 72.7 69.7 66.2 20 0 104 4 3 27.7 0 10 30.5 3 4 5 CD 3 3 4 5 0 0 10 10 10 10 10 10 10 0 – CTV CD62L 0 103 104 105 d SM M1 M2 CD8 CD4SP 0.0 2.1 13.5 Figure 1 Three major SP stages defined by function. (a) Flow cytometry of thymocytes from Rag2GFP mice (n = 4), showing the expression of CCR7 and TCRβ by Rag2-GFP+ thymocytes (dump-gated as in Supplementary Fig. 1a) (top left), gates of three subsets of medullary thymocytes SSC 250K identified by expression of CD69 and MHC class I (top middle), and GFP expression by CD69+MHCI− 200K 150K + + − + CD8SP 0.1 1.8 12.9 (SM) cells, CD69 MHCI (M1) cells and CD69 MHCI (M2) cells (top right), as well as CD4- 100K and CD8-expression profiles of the populations above (bottom). Numbers adjacent to outlined 50K areas indicates percent cells in each. (b) Proliferation of the SM, M1 and M2 subsets of CD4SP 0 3 4 5 thymocytes (sorted from C57BL/6 mice; top row) and CD8SP thymocytes (sorted from MHC 0 10 10 10 TNF class II–deficient mice; bottom row) labeled with CellTrace Violet (CTV), left unstimulated (Medium) or stimulated for 3 d with anti-CD3 and anti-CD28 (CD3+CD28), and analyzed by flow cytometry. (c) Expression of S1PR1 and CD62L on the subsets of CD4SP and CD8SP thymocytes identified in a. Numbers adjacent to outlined areas indicate percent S1PR1+CD62L+ cells. (d) TNF production by CD4SP or CD8SP thymocytes 4 h after stimulation with anti-CD3 and anti-CD28. Numbers adjacent to outlined areas indicate percent TNF+ cells. SSC, side scatter. Data are representative of four (a) or three (b–d) independent experiments. correlated with functional maturation. For our comprehensive flow class I most precisely defined the boundary between proliferation- cytometry, we used mice with expression of green fluorescent protein incompetent cells and proliferation-competent cells. Thus, we (GFP) directed by the promoter of recombination-activating gene designated CD69+MHCI− population as semi-mature (SM) and des- 2 (Rag2) via a bacterial artificial chromosome transgene (Rag2GFP), ignated CD69+MHCI+ and CD69−MHC1+ populations as mature in which GFP expression (as Rag2-GFP) acts as a ‘molecular timer’ 1 (M1) and mature 2 (M2), respectively. To assess emigration and for differentiation events after positive selection10,12 and allows the trafficking competence, we assessed expression of the sphingosine exclusion of re-circulating mature T cells. To focus our analysis on 1-phosphate (S1P) receptor S1PR1 (ref. 15), L-selectin (CD62L)16 and Nature America, Inc. All rights reserved. Inc. Nature America, conventional αβ T cells, we used a ‘dump strategy’ to exclude γδ T the transcription factor KLF2, which is required for the expression 6 cells, invariant natural killer T cells (iNKT cells) and regulatory T cells of S1PR1 and CD62L in thymocytes15–17. There was high expression (Treg cells) (Supplementary Fig. 1a). We found that the combination of S1PR1 and CD62L on M2 cells but not on SM cells or M1 cells © 201 of staining for CD69 and MHC class I (MHCI) precisely defined SP (Fig. 1c). Likewise, there was high expression of KLF2 only on M2 thymocyte stages by function and could be used on cells of both the cells (Supplementary Fig. 1c). Thus, among proliferation-competent CD4+ lineage and CD8+ lineage; positively selected medullary thy- M1 and M2 thymocytes, only the most mature M2 cells were mocytes (TCRβ+CCR7+) clearly had three populations, on the basis competent to emigrate. Finally, we assessed at which stage SP of the expression of CD69 and MHC class I, including CD69+MHCI− thymocytes became licensed to produce the cytokine TNF18. cells, CD69+MHCI+ cells and CD69−MHCI+ cells13 (Fig. 1a). GFP Only the M2 subset had a substantial population of TNF-producing expression gradually decreased in this order (Fig. 1a, top right), which cells following stimulation via anti-CD3 and anti-CD28 (Fig. 1d), and indicated that CD69+MHCI− thymocytes preceded CD69+MHCI+ this fraction continued to increase among recent thymic emigrants cells and that CD69−MHCI+ cells were the most mature population. (data not shown), consistent with published reports18.