Thymic emigration revisited

Tom M McCaughtry, Matthew S Wilken, Kristin A Hogquist, Tom M McCaughtry, Matthew S Wilken, Kristin A Hogquist

Abstract

Conventional alphabeta T cell precursors undergo positive selection in the thymic cortex. When this is successful, they migrate to the medulla and are exposed to tissue-specific antigens (TSA) for purposes of central tolerance, and they undergo maturation to become functionally responsive T cells. It is commonly understood that thymocytes spend up to 2 wk in the medulla undergoing these final maturation steps before emigrating to peripheral lymphoid tissues. In addition, emigration is thought to occur via a stochastic mechanism whereby some progenitors leave early and others leave late-a so-called "lucky dip" process. However, recent research has revealed that medullary thymocytes are a heterogeneous mix of naive alphabeta T cell precursors, memory T cells, natural killer T cells, and regulatory T cells. Given this, we revisited the question of how long it takes naive alphabeta T cell precursors to emigrate. We combined the following three approaches to study this question: BrdU labeling, intrathymic injection of a cellular tag, and RAG2p-GFP reporter mice. We established that, on average, naive alphabeta T cell precursors emigrate only 4-5 d after becoming single-positive (SP) thymocytes. Furthermore, emigration occurs via a strict "conveyor belt" mechanism, where the oldest thymocytes leave first.

Figures

Figure 1.
Figure 1.
CD4 SPs are a heterogeneous population. (A) GFP expression in DP and bulk CD4 SP thymocytes. (B) A dump channel consisting of NK1.1, GL3, and CD25 was used to identify nonconventional αβ thymocytes, and CD44 was used to identify memory T cells. (left and right) GFPneg and GFPpos gated CD4 SP thymocytes, respectively. (C) GFP expression in CD1d/αGalCer tetramerpos CD3pos CD4 SP NKT cells compared with total CD4 SPs in the thymus. (D) GFP expression in Foxp3pos CD25pos CD4 SP T reg cells compared with total CD4 SPs in the thymus. Numbers on plots indicate the percentage of the parent gate with the SD from multiple experiments, where indicated. The mice used were 6–11 wk old.
Figure 2.
Figure 2.
GFP intensity decreases during thymocyte residency time. 2 mg of BrdU was injected at the indicated time point before harvest. (A) The expression of CD4 and CD8 on dumpneg BrdUpos thymocytes from the indicated time point. (B) The absolute number of dumpneg BrdUpos CD4 SP thymocytes was calculated for each time point. Data represent the mean ± the SD from two to three experiments. (C) The GFP MFI of dumpneg BrdUpos CD4 SPs from the indicated time points confirms that GFP intensity decreases with time.
Figure 3.
Figure 3.
GFPlo CD4 SPs are the most mature and have an “exit” phenotype. (A) Naive CD4 SP thymocytes were divided into three equal bins based on GFP intensity. The three bins, along with DP thymocytes, were then analyzed for their expression of the indicated surface molecule. Representative data from 4–5 experiments is shown. (B) Dumpneg cells from the indicated subset were FACS-sorted, RNA was isolated and used for cDNA synthesis, and quantitative RT-PCR was performed for KLF2 and S1P1. Data represent the mean fold change ± the SD from two independent sorts relative to CD69neg DPs and normalized using primers for β-catenin. GFPmid CD4 SPs were only sorted once.
Figure 4.
Figure 4.
Thymic emigration occurs after only 4–5 d. 50 μg of biotin was injected intrathymically and lymph node RTEs were visualized 12, 24, 48, and 72 h later by staining with streptavidin-conjugated Pacific Orange. The 12-h time point is shown. (A) GFP expression in bulk SApos CD4 SP and CD8 SP thymocytes and bulk CD4 and CD8 RTEs. (B) The expression of GFP in naive CD4 and CD8 RTEs compared with naive CD4 and CD8 SP thymocytes, respectively. Gates were drawn on naive CD4 and CD8 SP thymocytes, as seen in Fig. 3. The MFI for the indicated subsets is shown below the histogram. The CV values were as follows: CD4 SP, 57.59; CD4 RTE, 59.29; CD8 SP, 73.44; and CD8 RTE, 74.68. For comparison, DP thymocytes were 34.52. The MFI values were used to estimate the average difference in age between the youngest (GFPhi) SP thymocyte and a RTE, as described in the Materials and methods (shown on right, in days).
Figure 5.
Figure 5.
Continuous labeling shows rapid turnover of naive CD4 and CD8 SPs compared with CD44/dumppos CD4 SPs. 1 mg BrdU was injected intraperitoneally at the indicated time point before harvest, and mice were continuously given 0.8 mg/ml BrdU drinking water, which was changed daily until harvest. (A) The BrdU profile of the indicated subset of cells after 11 d of BrdU labeling. The percentage of BrdUneg cells is indicated. (B) The turnover kinetics of the indicated population of thymocytes over time.

References

    1. Ueno, T., F. Saito, D.H. Gray, S. Kuse, K. Hieshima, H. Nakano, T. Kakiuchi, M. Lipp, R.L. Boyd, and Y. Takahama. 2004. CCR7 signals are essential for cortex–medulla migration of developing thymocytes. J. Exp. Med. 200:493–505.
    1. Carlson, C.M., B.T. Endrizzi, J. Wu, X. Ding, M.A. Weinreich, E.R. Walsh, M.A. Wani, J.B. Lingrel, K.A. Hogquist, and S.C. Jameson. 2006. Kruppel-like factor 2 regulates thymocyte and T-cell migration. Nature. 442:299–302.
    1. Matloubian, M., C.G. Lo, G. Cinamon, M.J. Lesneski, Y. Xu, V. Brinkmann, M.L. Allende, R.L. Proia, and J.G. Cyster. 2004. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature. 427:355–360.
    1. Petrie, H.T., and J.C. Zuniga-Pflucker. 2007. Zoned out: functional mapping of stromal signaling microenvironments in the thymus. Annu. Rev. Immunol. 25:649–679.
    1. Tough, D.F., and J. Sprent. 1994. Turnover of naive- and memory-phenotype T cells. J. Exp. Med. 179:1127–1135.
    1. Lucas, B., F. Vasseur, and C. Penit. 1993. Normal sequence of phenotypic transitions in one cohort of 5-bromo-2'-deoxyuridine-pulse-labeled thymocytes. Correlation with T cell receptor expression. J. Immunol. 151:4574–4582.
    1. Egerton, M., R. Scollay, and K. Shortman. 1990. Kinetics of mature T-cell development in the thymus. Proc. Natl. Acad. Sci. USA. 87:2579–2582.
    1. Huesmann, M., B. Scott, P. Kisielow, and H. von Boehmer. 1991. Kinetics and efficacy of positive selection in the thymus of normal and T cell receptor transgenic mice. Cell. 66:533–540.
    1. Kelly, K.A., and R. Scollay. 1990. Analysis of recent thymic emigrants with subset- and maturity-related markers. Int. Immunol. 2:419–425.
    1. Derbinski, J., A. Schulte, B. Kyewski, and L. Klein. 2001. Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self. Nat. Immunol. 2:1032–1039.
    1. Baldwin, T.A., K.A. Hogquist, and S.C. Jameson. 2004. The fourth way? Harnessing aggressive tendencies in the thymus. J. Immunol. 173:6515–6520.
    1. Hogquist, K.A., T.A. Baldwin, and S.C. Jameson. 2005. Central tolerance: learning self-control in the thymus. Nat. Rev. Immunol. (Paris). 5:772–782.
    1. Berzins, S.P., F.W. McNab, C.M. Jones, M.J. Smyth, and D.I. Godfrey. 2006. Long-term retention of mature NK1.1+ NKT cells in the thymus. J. Immunol. 176:4059–4065.
    1. Bosco, N., F. Agenes, A.G. Rolink, and R. Ceredig. 2006. Peripheral T cell lymphopenia and concomitant enrichment in naturally arising regulatory T cells: the case of the pre-Talpha gene-deleted mouse. J. Immunol. 177:5014–5023.
    1. Reinhardt, R.L., A. Khoruts, R. Merica, T. Zell, and M.K. Jenkins. 2001. Visualizing the generation of memory CD4 T cells in the whole body. Nature. 410:101–105.
    1. Agus, D.B., C.D. Surh, and J. Sprent. 1991. Reentry of T cells to the adult thymus is restricted to activated T cells. J. Exp. Med. 173:1039–1046.
    1. Hale, J.S., T.E. Boursalian, G.L. Turk, and P.J. Fink. 2006. Thymic output in aged mice. Proc. Natl. Acad. Sci. USA. 103:8447–8452.
    1. Surh, C.D., J. Sprent, and S.R. Webb. 1993. Exclusion of circulating T cells from the thymus does not apply in the neonatal period. J. Exp. Med. 177:379–385.
    1. Yu, W., H. Nagaoka, M. Jankovic, Z. Misulovin, H. Suh, A. Rolink, F. Melchers, E. Meffre, and M.C. Nussenzweig. 1999. Continued RAG expression in late stages of B cell development and no apparent re-induction after immunization. Nature. 400:682–687.
    1. Boursalian, T.E., J. Golob, D.M. Soper, C.J. Cooper, and P.J. Fink. 2004. Continued maturation of thymic emigrants in the periphery. Nat. Immunol. 5:418–425.
    1. Fontenot, J.D., J.L. Dooley, A.G. Farr, and A.Y. Rudensky. 2005. Developmental regulation of Foxp3 expression during ontogeny. J. Exp. Med. 202:901–906.
    1. Gapin, L., J.L. Matsuda, C.D. Surh, and M. Kronenberg. 2001. NKT cells derive from double-positive thymocytes that are positively selected by CD1d. Nat. Immunol. 2:971–978.
    1. Benlagha, K., T. Kyin, A. Beavis, L. Teyton, and A. Bendelac. 2002. A thymic precursor to the NK T cell lineage. Science. 296:553–555.
    1. Pellicci, D.G., K.J. Hammond, A.P. Uldrich, A.G. Baxter, M.J. Smyth, and D.I. Godfrey. 2002. A natural killer T (NKT) cell developmental pathway involving a thymus-dependent NK1.1−CD4+ CD1d-dependent precursor stage. J. Exp. Med. 195:835–844.
    1. Scollay, R., and D.I. Godfrey. 1995. Thymic emigration: conveyor belts or lucky dips? Immunol. Today. 16:268–273.
    1. Le Campion, A., B. Lucas, N. Dautigny, S. Leaument, F. Vasseur, and C. Penit. 2002. Quantitative and qualitative adjustment of thymic T cell production by clonal expansion of premigrant thymocytes. J. Immunol. 168:1664–1671.
    1. Ernst, B., C.D. Surh, and J. Sprent. 1995. Thymic selection and cell division. J. Exp. Med. 182:961–971.
    1. Penit, C., and F. Vasseur. 1997. Expansion of mature thymocyte subsets before emigration to the periphery. J. Immunol. 159:4848–4856.
    1. Le Campion, A., F. Vasseur, and C. Penit. 2000. Regulation and kinetics of premigrant thymocyte expansion. Eur. J. Immunol. 30:738–746.
    1. Eck, S.C., P. Zhu, M. Pepper, S.J. Bensinger, B.D. Freedman, and T.M. Laufer. 2006. Developmental alterations in thymocyte sensitivity are actively regulated by MHC class II expression in the thymic medulla. J. Immunol. 176:2229–2237.
    1. DeVoss, J., Y. Hou, K. Johannes, W. Lu, G.I. Liou, J. Rinn, H. Chang, R. Caspi, L. Fong, and M.S. Anderson. 2006. Spontaneous autoimmunity prevented by thymic expression of a single self-antigen. J. Exp. Med. 203:2727–2735.
    1. Kurobe, H., C. Liu, T. Ueno, F. Saito, I. Ohigashi, N. Seach, R. Arakaki, Y. Hayashi, T. Kitagawa, M. Lipp, et al. 2006. CCR7-dependent cortex-to-medulla migration of positively selected thymocytes is essential for establishing central tolerance. Immunity. 24:165–177.
    1. Kotani, M., K. Seiki, A. Yamashita, and I. Horii. 1966. Lymphatic drainage of thymocytes to the circulation in the guinea pig. Blood. 27:511–520.
    1. Ernstrom, U., L. Glyllensten, and B. Larsson. 1965. Venous output of lymphocytes from the thymus. Nature. 207:540–541.
    1. Kato, S. 1997. Thymic microvascular system. Microsc. Res. Tech. 38:287–299.
    1. Ushiki, T. 1986. A scanning electron-microscopic study of the rat thymus with special reference to cell types and migration of lymphocytes into the general circulation. Cell Tissue Res. 244:285–298.
    1. Kamath, A.T., S. Henri, F. Battye, D.F. Tough, and K. Shortman. 2002. Developmental kinetics and lifespan of dendritic cells in mouse lymphoid organs. Blood. 100:1734–1741.
    1. Rybak, J.N., A. Ettorre, B. Kaissling, R. Giavazzi, D. Neri, and G. Elia. 2005. In vivo protein biotinylation for identification of organ-specific antigens accessible from the vasculature. Nat. Methods. 2:291–298.

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