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CD8αα intraepithelial lymphocytes arise from two main thymic precursors

Nature Immunology volume 18, pages 771–779 (2017)Cite this article

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Abstract

TCRαβ+CD4−CD8α+CD8β− intestinal intraepithelial lymphocytes (CD8αα IELs) are an abundant population of thymus-derived T cells that protect the gut barrier surface. We sought to better define the thymic IEL precursor (IELp) through analysis of its maturation, localization and emigration. We defined two precursor populations among TCRβ+CD4−CD8− thymocytes by dependence on the kinase TAK1 and rigorous lineage-exclusion criteria. Those IELp populations included a nascent PD-1+ population and a T-bet+ population that accumulated with age. Both gave rise to intestinal CD8αα IELs after adoptive transfer. The PD-1+ IELp population included more strongly self-reactive clones and was largely restricted by classical major histocompatibility complex (MHC) molecules. Those cells localized to the cortex and efficiently emigrated in a manner dependent on the receptor S1PR1. The T-bet+ IELp population localized to the medulla, included cells restricted by non-classical MHC molecules and expressed the receptor NK1.1, the integrin CD103 and the chemokine receptor CXCR3. The two IELp populations further differed in their use of the T cell antigen receptor (TCR) α-chain variable region (Vα) and β-chain variable region (Vβ). These data provide a foundation for understanding the biology of CD8αα IELs.

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Figure 1: A subset of TCRβ+DN cells is phenotypically and functionally mature.
Figure 2: Two main subsets of mature TCRβ+DN cells.
Figure 3: Both PD-1+ (type A) IELps and PD-1− (type B) IELps give rise to CD8αα IELs.
Figure 4: Thymic localization of type A and type B IELps.
Figure 5: The emigration of type A IELps is dominant over that of type B IELps.
Figure 6: No immediate precursor–product relationship between type A IELps and type B IELps.
Figure 7: Type A IELps and type B IELps have different antigen-receptor specificities.

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References

  1. Sheridan, B.S. & Lefrançois, L. Intraepithelial lymphocytes: to serve and protect. Curr. Gastroenterol. Rep. 12, 513–521 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Cheroutre, H., Lambolez, F. & Mucida, D. The light and dark sides of intestinal intraepithelial lymphocytes. Nat. Rev. Immunol. 11, 445–456 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Gangadharan, D. et al. Identification of pre- and postselection TCRαβ+ intraepithelial lymphocyte precursors in the thymus. Immunity 25, 631–641 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Mayans, S. et al. αβT cell receptors expressed by CD4−CD8αβ− intraepithelial T cells drive their fate into a unique lineage with unusual MHC reactivities. Immunity 41, 207–218 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. McDonald, B.D., Bunker, J.J., Ishizuka, I.E., Jabri, B. & Bendelac, A. Elevated T cell receptor signaling identifies a thymic precursor to the TCRαβ+CD4− CD8β− intraepithelial lymphocyte lineage. Immunity 41, 219–229 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Klose, C.S. et al. The transcription factor T-bet is induced by IL-15 and thymic agonist selection and controls CD8αα+ intraepithelial lymphocyte development. Immunity 41, 230–243 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Guo, X., Tanaka, Y. & Kondo, M. Thymic precursors of TCRαβ+CD8αα+ intraepithelial lymphocytes are negative for CD103. Immunol. Lett. 163, 40–48 (2015).

    Article  CAS  PubMed  Google Scholar 

  8. Lee, Y.J., Holzapfel, K.L., Zhu, J., Jameson, S.C. & Hogquist, K.A. Steady-state production of IL-4 modulates immunity in mouse strains and is determined by lineage diversity of iNKT cells. Nat. Immunol. 14, 1146–1154 (2013).

    Article  CAS  PubMed  Google Scholar 

  9. Xing, Y., Wang, X., Jameson, S.C. & Hogquist, K.A. Late stages of T cell maturation in the thymus involve NF-κB and tonic type I interferon signaling. Nat. Immunol. 17, 565–573 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Sanjo, H., Tokumaru, S., Akira, S. & Taki, S. Conditional deletion of TAK1 in T cells reveals a pivotal role of TCRαβ+ intraepithelial lymphocytes in preventing lymphopenia-associated colitis. PLoS One 10, e0128761 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Pobezinsky, L.A. et al. Clonal deletion and the fate of autoreactive thymocytes that survive negative selection. Nat. Immunol. 13, 569–578 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Stritesky, G.L. & Hogquist, K.A. Death diverted, but to what? Nat. Immunol. 13, 528–530 (2012).

    Article  CAS  PubMed  Google Scholar 

  13. Stritesky, G.L. et al. Murine thymic selection quantified using a unique method to capture deleted T cells. Proc. Natl. Acad. Sci. USA 110, 4679–4684 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Berlin, C. et al. α4β7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell 74, 185–195 (1993).

    Article  CAS  PubMed  Google Scholar 

  15. Hamann, A., Andrew, D.P., Jablonski-Westrich, D., Holzmann, B. & Butcher, E.C. Role of α4-integrins in lymphocyte homing to mucosal tissues in vivo. J. Immunol. 152, 3282–3293 (1994).

    CAS  PubMed  Google Scholar 

  16. Schön, M.P. et al. Mucosal T lymphocyte numbers are selectively reduced in integrin αE (CD103)-deficient mice. J. Immunol. 162, 6641–6649 (1999).

    PubMed  Google Scholar 

  17. Lee, Y.J. et al. Tissue-specific distribution of iNKT cells impacts their cytokine response. Immunity 43, 566–578 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bendall, S.C. et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332, 687–696 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Qiu, P. et al. Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE. Nat. Biotechnol. 29, 886–891 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kwan, J. & Killeen, N. CCR7 directs the migration of thymocytes into the thymic medulla. J. Immunol. 172, 3999–4007 (2004).

    Article  CAS  PubMed  Google Scholar 

  21. Ueno, T. et al. CCR7 signals are essential for cortex-medulla migration of developing thymocytes. J. Exp. Med. 200, 493–505 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Fan, X. & Rudensky, A.Y. hallmarks of tissue-resident lymphocytes. Cell 164, 1198–1211 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Boursalian, T.E., Golob, J., Soper, D.M., Cooper, C.J. & Fink, P.J. Continued maturation of thymic emigrants in the periphery. Nat. Immunol. 5, 418–425 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. McCaughtry, T.M., Wilken, M.S. & Hogquist, K.A. Thymic emigration revisited. J. Exp. Med. 204, 2513–2520 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Uehara, S., Song, K., Farber, J.M. & Love, P.E. Characterization of CCR9 expression and CCL25/thymus-expressed chemokine responsiveness during T cell development: CD3highCD69+ thymocytes and γδTCR+ thymocytes preferentially respond to CCL25. J. Immunol. 168, 134–142 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. Misslitz, A. et al. Thymic T cell development and progenitor localization depend on CCR7. J. Exp. Med. 200, 481–491 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Plotkin, J., Prockop, S.E., Lepique, A. & Petrie, H.T. Critical role for CXCR4 signaling in progenitor localization and T cell differentiation in the postnatal thymus. J. Immunol. 171, 4521–4527 (2003).

    Article  CAS  PubMed  Google Scholar 

  28. Hu, Z., Lancaster, J.N., Sasiponganan, C. & Ehrlich, L.I. CCR4 promotes medullary entry and thymocyte-dendritic cell interactions required for central tolerance. J. Exp. Med. 212, 1947–1965 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Drennan, M.B. et al. Cutting edge: the chemokine receptor CXCR3 retains invariant NK T cells in the thymus. J. Immunol. 183, 2213–2216 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. Gerner, M.Y., Kastenmuller, W., Ifrim, I., Kabat, J. & Germain, R.N. Histo-cytometry: a method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes. Immunity 37, 364–376 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Steinert, E.M. et al. Quantifying memory CD8 T cells reveals regionalization of immunosurveillance. Cell 161, 737–749 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. White, A.J. et al. An essential role for medullary thymic epithelial cells during the intrathymic development of invariant NKT cells. J. Immunol. 192, 2659–2666 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Matloubian, M. et al. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 427, 355–360 (2004).

    CAS  PubMed  Google Scholar 

  34. Carlson, C.M. et al. Kruppel-like factor 2 regulates thymocyte and T-cell migration. Nature 442, 299–302 (2006).

    Article  CAS  PubMed  Google Scholar 

  35. Odumade, O.A., Weinreich, M.A., Jameson, S.C. & Hogquist, K.A. Krüppel-like factor 2 regulates trafficking and homeostasis of gammadelta T cells. J. Immunol. 184, 6060–6066 (2010).

    Article  CAS  PubMed  Google Scholar 

  36. Zachariah, M.A. & Cyster, J.G. Neural crest-derived pericytes promote egress of mature thymocytes at the corticomedullary junction. Science 328, 1129–1135 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Fujiura, Y. et al. Development of CD8αα+ intestinal intraepithelial T cells in β2-microglobulin- and/or TAP1-deficient mice. J. Immunol. 156, 2710–2715 (1996).

    CAS  PubMed  Google Scholar 

  38. Ma, L.J., Acero, L.F., Zal, T. & Schluns, K.S. Trans-presentation of IL-15 by intestinal epithelial cells drives development of CD8αα IELs. J. Immunol. 183, 1044–1054 (2009).

    Article  CAS  PubMed  Google Scholar 

  39. Kunisawa, J. et al. Sphingosine 1-phosphate dependence in the regulation of lymphocyte trafficking to the gut epithelium. J. Exp. Med. 204, 2335–2348 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Moran, A.E. et al. T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse. J. Exp. Med. 208, 1279–1289 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Weinreich, M.A. et al. KLF2 transcription-factor deficiency in T cells results in unrestrained cytokine production and upregulation of bystander chemokine receptors. Immunity 31, 122–130 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lefrançois, L. & Lycke, N. Isolation of mouse small intestinal intraepithelial lymphocytes, Peyer's patch, and lamina propria cells. Curr. Protoc. Immunol. 3.19.1–3.19.11 (2001).

  43. Blair-Handon, R., Mueller, K. & Hoogstraten-Miller, S. An alternative method for intrathymic injections in mice. Lab Anim. 39, 248–252 (2010).

    Article  Google Scholar 

Download references

Acknowledgements

We thank J. Ding and J. Lam for technical assistance; and T.A. Baldwin, D.P. Golec and H. Borges de Silva for reading the manuscript and providing feedback and suggestions. B. Rudd inspired the use of Cd4CreERT2 mice to 'time-stamp' progenitor cells. Some results mentioned in the manuscript but not presented here were derived from experiments by H. Wang. Supported by the US National Institutes of Health (R37 AI39560 and PO1 AI35296 to K.A.H.; K99 AI114889 to Y.J.L.; and T35 AI118620-01 to R.L.K.).

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Authors and Affiliations

  1. The Department of Laboratory Medicine and Pathology and Center for Immunology, University of Minnesota, Minneapolis, Minnesota, USA

    Roland Ruscher, Rebecca L Kummer, Stephen C Jameson & Kristin A Hogquist

  2. Academy of Immunology and Microbiology, Institute for Basic Science, and Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Republic of Korea

    You Jeong Lee

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Contributions

R.R. and K.A.H. designed experiments; R.R., R.L.K. and Y.J.L. performed experiments and analyzed data; R.R. wrote the manuscript; S.C.J. provided input for interpretation; and K.A.H. conceptualized and directed the study and edited the manuscript.

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Correspondence to Kristin A Hogquist.

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Integrated supplementary information

Supplementary Figure 1 SPADE analysis of flow-cytometry data.

Thymocytes gated on CD5+TCRβ+ (a) were analyzed for the indicated markers (b–l) as explained in Figure 2.

Supplementary Figure 2 Gating strategy for recovered cells from IELp-transfer experiments and fate of transferred cells in the periphery.

(a) IELps and total DN (control) cells were transferred into Rag2–/– recipients and recovered from the IEL compartment 5-10 weeks after transfer. Shown is the gating strategy used to identify the transferred cells. (b) Frequency of CD4+, CD8ab+ or CD8aa+ cells among TCRβ+ cells recovered from the spleen of Rag2–/– recipients. ** P < 0.01, ANOVA with Bonferroni post-test. Graphs show mean (± s.d.)

Supplementary Figure 3 Back-gating of Nr4a1GFPhiPD-1hi and Tbx21GFPhiCD1dtet– thymocytes, and dependence of type B IELps on IL-15.

(a) Nr4a1GFPhighPD-1high cells (red) of total thymocytes were overlaid onto total thymocytes (grey), and their DN fraction was further gated as indicated. Value in brackets is mean ±s.e.m. from 3 independent experiments. (b) Tbx21GFPhighCD1dtet– cells (green) of total thymocytes were overlaid onto total thymocytes (grey), and their DN fraction further gated as indicated. Value in brackets is mean ±s.e.m. from 4 independent experiments. (c) Lethally irradiated WT or IL-15–/– mice were reconstituted with bone marrow from Tbx21GFP mice and proportions of mature PD-1+Tbx21GFPneg (type A, left graph) or PD-1–Tbx21GFPhigh (type B, right graph) thymocytes were determined at least 8 weeks post reconstitution. The exemplary histograms show CD122 expression by mature IELps in WT (solid) or IL-15–/– (dotted) chimeras. Each symbol represents an individual mouse. ** P < 0.01, Student’s t test.

Supplementary Figure 4 S1PR1 staining and intravenous labeling.

(a) Thymocytes from Klf2GFP mice were stained for S1PR1 and additional markers to identify IELps. The control shows mature IELps in an anti-S1PR1 antibody negative sample. Values in brackets indicate the mean ± s.e.m. combined from 3 independent experiments. (b) Proportion of mature IELps within the Klf2GFP+S1PR1+-gated CD5+TCRβ+CD1dtet–CD25–DN thymocytes (black) overlayed onto total CD5+TCRβ+ CD1dtet–CD25–DN cells. Values in brackets indicate the mean ± s.e.m. combined from 3 independent experiments. (c) Examples for gating on PE-negative (unlabeled) and PE-positive (labeled) cells in thymus or blood of CD45.2-PE intravenously injected mice. Enrichment for labeled cells was performed by MACS. (d, e): Proportions of various cell types within the unlabeled and labeled thymic fractions, and in the labeled blood. Graphs are combined from 11 (d, TCRβ+), 3 (d, I-Ab+CD19+ and CD11b+GR1+) or 5 (e) experiments with each symbol representing an individual mouse. * P < 0.05, *** P < 0.001, ANOVA with Bonferroni post-test.

Supplementary Figure 5 Intrathymic biotin injection and FTY720 treatment.

(a) Ultrasound images of intrathymic EZ-Link Sulfo-NHS biotin injections. 1.) Image prior to placing the injection 2.) Tip of the needle visible in the thymus. 3.) Biotin has been injected and is visible as a darker area. (b) 24h after intrathymic injections, splenic cells were analyzed. Accuracy of injections was validated by the low percentage of B220+ cells within streptavidin-counterlabeled cells. Only samples with <5% B220+ cells were considered for further analysis. The histogram shows GFP expression by streptavidin-counterlabeled TCRβ+ splenocytes of injected of Rag2GFP mice. Data are representative for 3 individual Rag2GFP mice. (c) Rag2GFP mice were injected i.p. with the S1PR1 antagonist FTY720 for six consecutive days. Spleens were analyzed on day seven for the presence of recently emigrated type A IELp. Data are representative of 2 control and 3 treated mice in 2 independent experiments.

Supplementary Figure 6 Accumulation of Tbx21GFPintα4β7+ IELps with FTY720 treatment.

(a) Tbx21GFP or C57BL/6 mice were treated with FTY720 for six consecutive days. Bin gates were set on thymic mature IELps gated PD-1high (1), PD-1int (2), PD-1low (3) and PD-1– (4) populations. In Tbx21GFP mice an additional gate was set on Tbx21GFPhighPD-1– cells. The graph shows the absolute number within each gate. (b) α4β7 expression within each bin gate was determined and is shown as histograms (left) and as frequencies (graph). (c) shows the α4β7 versus Tbx21GFP distribution of events within the mature IELp gate of control and FTY720 treated animals. Plots and histograms are representative of, and graphs are combined from 2 experiments with 4 (control Tbx21GFPhigh), 6 (FTY720 Tbx21GFPhigh and control bin gates) or 8 (FTY720 bin gates) mice. * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t test (a, b).

Supplementary Figure 7 Vβ analysis of type A and type B IELps.

TCR Vβ analysis of type A (red) and B (green) IELps. Graphs are combined from 2 experiments with 3 mice. * P < 0.05, ** P < 0.01, Student’s t test (mean ± s.d.).

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Ruscher, R., Kummer, R., Lee, Y. et al. CD8αα intraepithelial lymphocytes arise from two main thymic precursors. Nat Immunol 18, 771–779 (2017). https://doi.org/10.1038/ni.3751

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