Dendritic cells in the thymus contribute to T-regulatory cell induction - PubMed (original) (raw)

. 2008 Dec 16;105(50):19869-74.

doi: 10.1073/pnas.0810268105. Epub 2008 Dec 10.

Serani van Dommelen, Penghui Zhou, Alexandra Rizzitelli, Angela D'Amico, Raymond J Steptoe, Shalin H Naik, Mireille H Lahoud, Yang Liu, Pan Zheng, Ken Shortman, Li Wu

Affiliations

Dendritic cells in the thymus contribute to T-regulatory cell induction

Anna I Proietto et al. Proc Natl Acad Sci U S A. 2008.

Erratum in

Abstract

Central tolerance is established through negative selection of self-reactive thymocytes and the induction of T-regulatory cells (T(R)s). The role of thymic dendritic cells (TDCs) in these processes has not been clearly determined. In this study, we demonstrate that in vivo, TDCs not only play a role in negative selection but in the induction of T(R)s. TDCs include two conventional dendritic cell (DC) subtypes, CD8(lo)Sirpalpha(hi/+) (CD8(lo)Sirpalpha(+)) and CD8(hi)Sirpalpha(lo/-) (CD8(hi)Sirpalpha(-)) [corrected] which have different origins. We found that the CD8(hi)Sirpalpha(+) DCs represent a conventional DC subset that originates from the blood and migrates into the thymus. Moreover, we show that the CD8(lo)Sirpalpha(+) DCs demonstrate a superior capacity to induce T(R)s in vitro. Finally, using a thymic transplantation system, we demonstrate that the DCs in the periphery can migrate into the thymus, where they efficiently induce T(R) generation and negative selection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

MHCII+ DCs contribute to negative selection and TR induction. (A) The light density fraction of cells from the thymus of WT and MHCII−/− BM chimeras was analyzed to determine the % of donor-derived DCs. More than 98% of CD11c+ cells were CD45.2+ donor-derived DCs in both BM chimera groups. (B) MHCII expression on CD11c+ TDCs in WT (black line) and MHCII−/− (gray line) chimeras. (C) The % of double-negative, double-positive, CD4+, and CD8+ thymocytes (Upper) and CD4+CD25+Foxp3+ TRs (Lower). (D) The % and total number of CD4+ thymocytes in WT and MHCII−/− BM chimeras. (E) The % and total number of TRs in WT and MHCII−/− BM chimeras (n = 20–24 per group for D and E). (F) Suppressive activity of CD4+CD25+ thymocytes from MHCII−/− or WT chimeras. Data are the mean (error bars, ±SD) of triplicate cultures from one of two experiments. (G) The number of TRs (CD4+Foxp3+) in the thymus of B6 to B6, B6 to B7−/−, B7−/− to B6, and B7−/− to B7−/− BM chimeras was analyzed by flow cytometry. Data are the mean of three independent experiments (error bars, ±SD) (n = 68 for G). *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Fig. 2.

Fig. 2.

OVA expressing DCs induce OTII TRs and deletion of OTII CD4+ T cells. Irradiated WT CD45.1 mice were reconstituted with double-tg Rag2−/−OTII/CD11cOVA (Rag2−/−O/OVA) BM or Rag2−/−OTII BM as a control (n = 4–5 per group). (A) Total thymic cellularity of Rag2−/−O/OVA and control BM chimeras. (B) CD45.2+Vα2+ thymocytes were gated, and the % of CD4+ and CD8+ thymocytes was determined. To assess TR induction, CD4+ OTII+ thymocytes were gated for and expression of CD25 and Foxp3 was determined. The % and number of OTII+ CD4+ thymocytes (C) and the % and number of TRs (D) in Rag2−/−O/OVA and control BM chimeras.

Fig. 3.

Fig. 3.

Sirpα+ TcDCs are more mature and efficiently induce TRs in vitro. (A) Enriched TDCs and SDCs were segregated into pDCs (CD11cintCD45RA+) and cDCs (CD11c+CD45RA−) and further segregated into CD8loSirpα+ and CD8hiSirp− cDCs. Thymic and splenic Sirpα+ and Sirpα− cDCs (B) and thymic and splenic pDCs (C) were analyzed for the expression of costimulatory molecules, CD69, and MHCII. (D) TR (CD4+CD25+Foxp3+) induction by TDC subsets in the cocultures of TDCs and CD4+CD25− thymocytes for 5 days. Data shown are representative of six experiments. (E) The number of TRs induced was determined by flow cytometric analysis of the cultured cells of D using calibration beads (n = 6) (error bars, ±SD). (F) Suppressive capacity of TRs induced in cultures by Sirpα+ TcDCs. In vitro_–derived TRs were sorted as CD4+CD25+CD62L+ and subject to a suppression assay as per Fig. 1_F. The number of proliferating CFSE-labeled CD4+CD25− T cells was determined 3 days later (n = 2).

Fig. 4.

Fig. 4.

Sirpα+ TcDCs originate from peripheral blood and can migrate into the thymus. (A) DC generation from purified Lineage−Thy-1loc-kit+ intrathymic precursors (CD45.2) was analyzed 2 weeks after precursor transfer. The intrathymic precursor-derived cDCs were mainly CD8+Sirpα− (Right). A representative contour plot of the normal TcDC subsets is shown (Left) for comparison. (B) White blood cells (20 × 106) from CD45.1 mice were transferred i.v. into nonirradiated CD45.2 recipients. The phenotype of donor-derived cells in the thymus of recipients was determined 3 days later by gating for CD45.1+CD11c+CD45RAlo cDCs. Expression of Sirpα, CD11b, CD8, and MHCII was determined on this population. (C–E) Thymic lobes from OTII tg CD45.2+ mice crossed to CD45.1+ WT mice were grafted under the kidney capsule of CD45.2+ CD11cOVA tg or WT recipients. (C) The phenotype of recipient-derived CD45.2+CD45.1− DCs in the grafted thymic lobes from WT and CD11cOVA tg mice was determined. The recipient CD45.2+CD45.1−CD11c+CD45RA− cDCs were gated for, and the expression of CD8 and Sirpα was determined. The level of expression of MHCII was determined on Sirpα− and Sirpα+ cDCs. (D) The total number of CD45.1+CD4+Vα2+Vβ5+ cells (OTII) was calculated in OTII lobes grafted into WT or CD11cOVA tg recipients. Data are the mean of three independent experiments (error bars, ±SD) (n = 11–21). *, P < 0.05. (E) CD45.1+CD4+Vα2+Vβ5+ cells in the OTII lobes from WT and CD11cOVA tg recipients (as in D) were further analyzed for CD25 and Foxp3 expression. The total number of CD45.1+CD4+Vα2+Vβ5+CD25+Foxp3+ TRs was calculated. Data are the mean of three independent experiments (error bars, ±SD) (n = 11–21). *, P < 0.05.

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