Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1997 Feb 3;185(3):541-50.
doi: 10.1084/jem.185.3.541.

Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo

T Brocker  1 , M Riedinger, K Karjalainen
Affiliations

Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo

T Brocker et al. J Exp Med. .

Abstract

It is well established that lymphoid dendritic cells (DC) play an important role in the immune system. Beside their role as potent inducers of primary T cell responses, DC seem to play a crucial part as major histocompatibility complex (MHC) class II+ "interdigitating cells" in the thymus during thymocyte development. Thymic DC have been implicated in tolerance induction and also by some authors in inducing major histocompatibility complex restriction of thymocytes. Most of our knowledge about thymic DC was obtained using highly invasive and manipulatory experimental protocols such as thymus reaggregation cultures, suspension cultures, thymus grafting, and bone marrow reconstitution experiments. The DC used in those studies had to go through extensive isolation procedures or were cultured with recombinant growth factors. Since the functions of DC after these in vitro manipulations have been reported to be not identical to those of DC in vivo, we intended to establish a system that would allow us to investigate DC function avoiding artificial interferences due to handling. Here we present a transgenic mouse model in which we targeted gene expression specifically to DC. Using the CD 11c promoter we expressed MHC class II I-E molecules specifically on DC of all tissues, but not on other cell types. We report that I-E expression on thymic DC is sufficient to negatively select I-E reactive CD4+ T cells, and to a less complete extent, CD8+ T cells. In contrast, it only DC expressed I-E in a class II-deficient background, positive selection of CD4+ T cells could not be observed. Thus negative, but not positive, selection events can be induced by DC in vivo.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Restriction map of the transgenic construct CD11c-Eα d. MHC class II I-Eα d cDNA (striped box) was placed under the control of a mouse CD11c promoter–containing DNA segment (gray box). The rabbit β-globin gene fragment providing the cDNA with an intron and a polyadenylation signal (A(n)) is displayed as a white box. B, BamHI; Bs, BspHI; E, EcoRI; N, NotI; S, SstI; X, XhoI; restriction sites in parentheses have been destroyed by blunt-end cloning.
Figure 2
Figure 2
Serial cryostat sections of normal adult thymus stained with 14.4.4S specific for I-E (a, c, and e) and UEA-1 (b, d, and f  ), a lectin binding specific for medullary epithelial cells (28). (a and b) Serial sections of a wild-type B6 (I-E−) thymus. (a) I-E, no I-E staining can be detected in the B6 background, (b) UEA-1, staining is restricted to dense aggregates of epithelial cells in the medulla (M), while no staining can be detected in the cortex (C). (c and d) Serial sections of B6-Eα d (I-E+) thymus, class II I-E transgene staining is confluent on BM-derived and epithelial cells in the medulla (M), and reticular staining in the cortex (C) is typical of epithelial cells. UEA-1 staining (as in b) marking the medullary region (M) only. (e and f   ) Serial sections of B6CD11c-Eα d thymus. I-E (e) transgene under control of the CD11c promoter can be detected only in medulla (M) and medullary-cortical junctions. No I-E staining in cortex (C) was detectable. UEA-1 ( f  ) staining is the same as for b and d. All sections were photographed at ×100.
Figure 3
Figure 3
Immunofluorescence staining of B6-Eα d (a) and B6CD11c-Eα d (b) thymus with the mAb 14.4.4S specific for I-E. While B6-Eα d (a) shows a typical reticular staining of cortical (C) epithelial cells, the B6CD11c-Eα d thymus (b) does not display this pattern. Here, the I-E seems to be expressed only in the medulla (M) and on cells of the cortico-medullary junction. Photographs were taken at ×200.
Figure 4
Figure 4
Immunofluorescence staining of B6 (a and b), B6-Eα d (c and d), and B6CD11c-Eα d (e and f ) thymus with the medullary epithelial marker UEA-1 labeled with FITC (a, c, and e) or double immunofluorescence analysis with UEA-1–FITC plus 14.4.4S specific for I-E (biotinylated and detected with Avidin-Texas red) (b, d, and f ). The B6 thymus shows only green labeling (a and b) indicating the natural absence of I-E expression in this mouse strain. The B6-Eα d thymus (c and d) expresses both antigens in the wild-type pattern and the green UEA-1 positive cells (c) become yellow-orange in the double fluorescence filter (d). In addition, red single positive cells become evident and are most likely nonepithelial cells expressing the I-E transgene as well as UEA-1 weak positive cell processes of epithelial cells (36). In the B6CD11c-Eα d thymus (e and f ), both mAbs clearly stain two different cell types and no I-E staining is visible on the UEA-1–labeled (green single positives) medullary epithelial cells ( f ), while other nonepithelial I-E positive cells (red single positives) of dendritic shape become evident in the medulla (    f   ).
Figure 5
Figure 5
Expression of the CD11c-Eα d transgene leads to I-E expression on thymic DC, but not on thymic B cells. DC: Thymi of B6, B6-Eα d, and B6CD11c-Eα d mice were collagenase digested and DC were isolated after a low density gradient as described in Materials and Methods. Cells were stained with mAbs specific for CD11c (PE) and I-E or I-A (FITC), respectively. B cells: thymi of the three mouse types were complement depleted for CD4 and CD8 to enrich non–T cells (see Materials and Methods) and then stained with mAbs specific for the pan B cell antigen CD19 (PE) and I-E or I-A (FITC), respectively.
Figure 6
Figure 6
The majority of peritoneal lavage cells do not express the CD11c-Eα d transgene. Peritoneal washes of the mice indicated were performed 5 d after an initial intraperitoneal injection of 2 ml 3% thioglycollate. The isolated cells were incubated for 48 h in IFN-γ–containing medium (1,000 U/ml) to induce MHC class II expression, and then stained with mAbs specific for I-E (PE) and I-A (FITC). Shown are all cells from the cultures with the gates set on live cells only. The percentages of I-E positive cells in B6CD11c-Eα d mice varied from 2–7% in the four experiments done.
Figure 7
Figure 7
Clonal deletion of T cells as a consequence of Eα d transgene expression. Flow cytometric analysis of Vβ8, Vβ5, and Vβ11 expression among CD4+ and CD8+ LN T cells. B cell–depleted LN cells of the three indicated mouse strains (n = 8/strain) were triple stained with anti– CD4-PE, anti–CD8-R613, and either anti–Vβ8.1,8.2-FITC, Vβ5.1,5.2FITC, or Vβ11-FITC, respectively. Results are expressed as a percentage of Vβ+CD4+ and Vβ+CD8+ cells.
Figure 8
Figure 8
Influence of the Eα d transgenes on the number of CD4+ T cells. LN and thymus cell suspensions were stained with PE-labeled anti-CD4 and FITC-labeled anti-CD8 mAbs. The CD4/CD8 ratios in the LN T lymphocyte populations were determined from 5 mice per group and the following values were obtained: B6I-A+/+, 3.03 ± 0.07; B6I-A−/−, 0.031 ± 0.004; B6-Eα dI-A−/−, 2.94 ± 0.05; B6CD11c-Eα dI-A−/−, 0.04 ± 0.006. The percentages of CD4+CD8− thymocytes in the thymi of the same animals were: B6I-A+/+, 8.61 ± 0.71%; B6I-A−/−, 1.06 ± 0.03%; B6-Eα dI-A−/−, 6.96 ± 0.51%; B6CD11c-Eα dI-A−/−, 0.77 ± 0.132%.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jameson SC, Hogquist KA, Bevan MJ. Positive selection of thymocytes. Annu Rev Immunol. 1995;13:93–126. - PubMed
    1. Nossal GJ. Negative selection of lymphocytes. Cell. 1994;76:229–239. - PubMed
    1. Anderson G, Moore NC, Owen JJT, Jenkinson EJ. Cellular interactions in thymocyte development. Annu Rev Immunol. 1996;14:73–99. - PubMed
    1. Iwabuchi K, Nakayama K, McCoy RL, Wang F, Nishimura T, Habu S, Murphy KM, Loh DY. Cellular and peptide requirements for in vitro clonal deletion of immature thymocytes. Proc Natl Acad Sci USA. 1992;89:9000–9004. - PMC - PubMed
    1. Pircher H, Brduscha K, Steinhoff U, Kasai M, Mizuochi T, Zinkernagel RM, Hengartner H, Kyewski B, Muller KP. Tolerance induction by clonal deletion of CD4+8+ thymocytes in vitro does not require dedicated antigen-presenting cells. Eur J Immunol. 1993;23:669–674. - PubMed

Publication types