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The Journal of Immunology, 2004, 173: 4799-4805.
Copyright © 2004 by The American Association of Immunologists

The Role of Dendritic Cells in Selection of Classical and Nonclassical CD8+ T Cells In Vivo1

Michael A. Cannarile*, Nadege Decanis*, Joost P. M. van Meerwijk{dagger} and Thomas Brocker2,*

* Institute for Immunology, Ludwig Maximilians University, Munich, Germany; and {dagger} Tolerance and Autoimmunity Section, Institut National de la Santé et de la Recherche Médicale, Unité 563, Toulouse, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cell development is determined by positive and negative selection events. An intriguing question is how signals through the TCR can induce thymocyte survival and maturation in some and programmed cell death in other thymocytes. This paradox can be explained by the hypothesis that different thymic cell types expressing self-MHC/peptide ligands mediate either positive or negative selection events. Using transgenic mice that express MHC class I (MHC-I) selectively on DC, we demonstrate a compartmentalization of thymic functions and reveal that DC induce CTL tolerance to MHC-I-positive hemopoietic targets in vivo. However, in normal and bone marrow chimeric mice, MHC-I+ DC are sufficient to positively select neither MHC-Ib (H2-M3)- nor MHC-Ia (H2-K)-restricted CD8+ T cells. Thus, thymic DC are specialized in tolerance induction, but cannot positively select the vast majority of MHC-I-restricted CD8+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Our current view of thymic T cell development predicts interactions of thymocytes via their Ag-specific TCR with self-peptide/MHC complexes presented by different thymic cell types (reviewed in Ref.1). Studies in transplantation chimeras and in vitro thymus culture suggested that the vast majority of T cells are positively selected through interactions with cortical epithelial cells in the thymus, whereas bone marrow (BM)3-derived cells are rather specialized in tolerance induction by negative selection (reviewed in Refs.1, 2, 3). In contrast to conventional T cells, several nonclassical MHC class Ib (MHC-Ib)-restricted T cells, such as H2-M3-restricted Listeria monocytogenes-specific CD8+ T cells (4), CD1d-restricted NKT cells (5, 6), and Qa-1-restricted TCR-transgenic CD8+ T cells (7) seem to be routinely selected by as-yet-unidentified BM-derived thymic cells.

However, in many experiments with BM radiation chimeras, a low but significant frequency of T cell activity restricted by MHC-Ia of donor hemopoietic cells was detected (8). These findings were either interpreted as MHC cross-reactivity of T cells selected on host MHC, or the capacity of hemopoietic cells to actually induce positive selection (2). Also, more recent reports using BM transplantation into thymus-grafted nude mice (9), fetal liver cell transfer into MHC-I-deficient mice (10), reconstitution of irradiated mice with purified hemopoietic stem cells (11), or tetraparental chimerism (12) have established roles for BM-derived thymic cells in positive selection of conventional CD8+ T cells. In this context, dendritic cells (DC) were discussed as possible candidates inducing positive selection of CTL (10, 12). In addition, purified DC were shown to induce positive selection of TCR transgenic CD4+ T cells in vitro (13). However, this is in contrast to previous findings, demonstrating that thymic DC are responsible only for negative, but not positive selection of CD4+ thymocytes (14, 15, 16), and these opposing findings could eventually reflect differential requirements of CD8+ vs CD4+ thymocytes for positive selection.

To investigate the role of DC in CD8+ T cell selection in vivo, we created transgenic mice expressing {beta}2-microglobulin ({beta}2m) selectively on DC in a {beta}2m–/– background. In the present study, MHC-I+ DC were sufficient to induce CD8+ T cell tolerance, but could not positively select any functional MHC-Ia- or -Ib-restricted CD8+ T cells in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6-{beta}2m–/– and OT-1 mice (expressing transgenic TCR specific for chicken OVA257–264/H2-Kb) were obtained from The Jackson Laboratory (Bar Harbor, ME). K14-{beta}2m mice were described previously (17). CD11c-MHC-I-transgenic (CD11c-MHC-I), expressing {beta}2m under the control of the CD11c promoter, have been described previously (18). All mice were bred for >10 generations to the C57BL/6 background and maintained at the animal facility of Institute for Immunology (Ludwig Maximilians University, and Technical University, Munich, Germany). To create radiation BM chimeras, CD8-depleted BM cells (107) were injected i.v. into lethally irradiated (1060-rad gamma) mice.

mAbs and flow cytometry

mAbs and second-step reagent used were as follows: anti-H2-Kb (AF6-88.5), anti-H2-Kb/H2-Db (28-8-6), anti-CD4 (H129.19 and RM4-5), anti-CD8a (53-6.7), anti-CD11c (HL3), anti-CD45 (30-F11), anti-V{alpha}5.1,5.2 TCR (MR9-4), anti-V{beta}6 TCR (RR4-7), anti-V{beta}8.1,8.2 TCR (MR5-2), anti-V{beta}11 TCR (RR3-15), and allophycocyanin- or Cy5-streptavidin (all from BD Pharmingen, San Diego, CA). Anti-CD8{alpha} (5H10) from Caltag (Burlingame, CA) and FITC-UEA1 was from Vector Laboratories (Burlingame, CA). PE-labeled H2-M3- and H2-Kb tetramers were kind gifts from D. Busch (Technische University, Munich, Germany) and have been described previously (19). Cells were stained and analyzed (FACSCalibur; BD Biosciences, Mountain View, CA) as previously described (14).

Histological analysis

Fresh thymi were embedded in OCT medium (Miles, Elkhart, IN) and snap frozen, and 5-µm sections were cut with a cryostat (Jung Frigocut 2800 E; Leica, Bensheim, Germany). Staining was performed as previously described (20). Sections were analyzed on a Leica DMXA-RF8 microscope (Leica acquisition program QFISH) equipped with a Sensys CCD camera (Photometrix, Tucson, AZ).

Adoptive cell transfer and CFSE labeling

Preparation, CFSE labeling, and adoptive transfer of cells were conducted as described previously (18). Briefly, 1–50 x 106 erythrocyte-free cells from lymph nodes and spleens were washed twice with PBS and labeled with 5 µM CFSE (Molecular Probes, Eugene, OR) for 10 min at 37°C in PBS. After stopping the reaction (PBS, 2% FBS) and washing in PBS, cells were injected into the lateral tail vein of recipient mice.

Target cells for the in vivo cytotoxic activity assay were prepared similarly and labeled with a high (2.5 µM) or low (0.25 µM) concentration of CFSE, respectively. Equal numbers of CFSEhighMHC-I+ cells and CFSElowMHC-I–/– cells were mixed together, such that each mouse received a total of 35 x 106 cells in 250 µl of PBS. Twenty-four hours later, mice were sacrificed, spleen cell suspensions were analyzed by flow cytometry, and each population was detected by their differential CFSE fluorescence intensities. To detect OT-1 CD8 T cell expansion, we isolated OT-1 T cells by negative selection (CD8 T cell columns; R&D Systems, Minneapolis, MN) and injected 4 x 106 OT-1 T cells (>96% purity) into the lateral tail veins of recipient mice.

Bacteria and infections

Mice were infected with 2000 L. monocytogenes expressing recombinant chicken OVA by tail vein injection as previously described (21). Splenocytes were collected on day 6 after infection, bacterial counts were determined, and tetramer analyses were performed as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DC-selective MHC-I expression in CD11c-MHC-I transgenic mice

To restrict MHC-I expression to DC in vivo, {beta}2m-cDNA was expressed under control of the murine CD11c promoter in transgenic mice with a {beta}2m-deficient (MHC-I–/–) background (18). Although thymi of C57BL/6 mice (MHC-I+) showed the expected ubiquitous MHC-I expression pattern (Fig. 1A), in cryosections of CD11c-MHC-I thymi only DC were identified as MHC-I+ cells (A), whereas MHC–/– thymi were MHC-I– (A). The localization of thymic MHC-I+ DC in CD11c-MHC-I mice was not influenced by the presence of a {beta}2m transgene, and DC were confined to the medulla and corticomedullary junctions similar to MHC-I+ and MHC-I–/– animals (Fig. 1A).



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  		FIGURE 1. MHC-I expression selectively on thymic DC in CD11c-MHC-I mice. Immunohistological (A) and flow cytometry (B) analysis of thymi from MHC-I+, CD11c-MHC-I, and MHC-I–/– mice was performed on frozen thymic sections of the indicated mice. A, Sections were stained with reagents specific for medullary epithelial cells (UEA-1, green), DC (CD11c, red), and MHC-I (H2-Kb, blue). Cells double-positive for MHC-I+ (blue) and CD11c (red) appear purple. Photographs were taken with a x20 objective, resulting in a total magnification of x12. B, Thymic cell suspensions from MHC-I+ (solid black line), MHC–/– (dotted black line), or CD11c-MHC-I mice (red line) were analyzed by flow cytometry for MHC-I expression using an H2-KbDb-specific mAb. Small cells according to forward-scatter/side-scatter analysis were segregated according to their CD45 expression (data not shown) in lymphocytes (CD45+) and stromal cells (CD45–). Thymic DC were identified as forward-scatter/side-scatter large (data not shown) CD11c+ cells (CD11c+) and compared with CD11c–/CD11b+ (data not shown) thymic macrophages (CD11c–). Thymic B cells were present within the small and large cell populations (data not shown) and expressed CD19 (CD19+).

 
Flow cytometry of thymic cell suspensions confirmed the DC-specific expression of MHC-I and showed that neither CD45+ thymocytes (Fig. 1B, CD45+) nor CD45– thymus stroma cells (B, CD45–) nor CD11c–CD11b+ macrophages (B, CD11c–) from CD11c-MHC-I mice expressed the transgene. However, 95% of CD11c+ DC from CD11c-MHC-I mice expressed H2-Kb at wild-type levels similar to MHC-I+ mice (Fig. 1B, CD11c+). Furthermore, the majority of thymic B cells from CD11c-MHC-I mice were negative for MHC-I (Fig. 1B, CD19). However, a small MHC-I+ fraction (Fig. 1B, CD19; {approx}5–15%) showed CD11c expression (data not shown). Because these cells were also B220+, we could not exclude that they belonged to the plasmacytoid DC lineage (22). In contrast, it has been reported that plasmacytoid DC from the spleen are negative for CD19 expression, and therefore, it might be a small fraction of thymic cells (0.09 ± 0.02% per thymus) other than DC in CD11c-MHC-I mice that express H2-Kb. Because thymic B cells have no impact on selection of CD8+ thymocytes (16), we considered them to be negligible for this study on thymic selection.

No rescue of classical or nonclassical CD8 T cell repertoires by MHC-I-positive DC

As published previously, in MHC-I–/– mice, small numbers of CD8+TCR{alpha}{beta}+ T cells (23) are selected by low amounts of free ({beta}2m-negative) MHC-I H chains expressed on thymic epithelium (24). To test whether CD8 T cell numbers would increase by positive selection on MHC-I+ DC, flow cytometric analysis of thymus and lymph node cells was performed (Fig. 2). In contrast to MHC-I+ mice, CD11c-MHC-I mice lacked CD8+ thymocytes (Fig. 2A) and CD8+ lymph node T cells (B) to the same extent as MHC-I–/– mice (A and B). In lymph nodes, the CD4/CD8 ratio in CD11c-MHC-I (CD4/CD8 = 31.4) mice was comparable to MHC-I–/– mice (CD4/CD8 = 29.1) and different from the normal ratio of wild-type mice (MHC-I+, CD4/CD8 = 1.6). In contrast, the percentages and cell numbers of CD4+ or CD4+CD8+ thymocytes as well as the numbers of peripheral CD4+T cells were similar in all mice (Fig. 2B). Therefore, judged from the enumeration of CD8+ thymocytes and peripheral T cells, MHC-I+ DC are not sufficient to restore a normal CD8 T cell compartment in vivo by positive selection.



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  		FIGURE 2. Influence of MHC-I+ DC on frequencies of CD8+ thymocytes and peripheral T cells. A, Cell suspensions from thymus (upper panel) or lymph nodes (lower panel) were stained for CD4 and CD8 expression and analyzed by flow cytometry. Percentages of CD4+, CD4+CD8+, and CD8+ thymocytes (upper panel) or CD4+ and CD8+ T cells from lymph nodes (lower panel) are indicated. CD4/CD8 ratios in the lymph node T lymphocyte population were as follows: MHC-I+, 1.6; CD11c-MHC-I, 31.4; MHC-I–/–, 29.1. Calculated percentages (A) and total cell numbers (B) were obtained from n = 5 (MHC-I+, CD11c-MHC-I) or n = 4 (MHC-I –/–) mice per group with a SD of <15%. The differences of CD8+ thymocytes between CD11c-MHC-I and MHC-I–/– mice were not statistically significant (Student’s t test, p = 0.13 (percentages), and p = 0.28 (cell numbers)). Results are representative of at least six experiments with similar results.

 
To compare the influence of DC only (in CD11c-MHC-I+ mice) with the entirety of all thymic BM-derived cells (in MHC-I+ mice) on the CD8 T cell repertoire and to exclude any involvement ofthymic epithelial cells, we created BM chimeras using MHC-I–/– mice as hosts. The analysis of CD8+ T cells revealed that CD11c-MHC-I->MHC-I–/– chimeras did not contain significantly different percentages of CD8+ T cells compared with MHC-I–/–->MHC-I–/– chimeras (Fig. 3A, left panel). However, confirming previous studies (8, 9, 10, 11), MHC-I+->MHC-I–/– chimeras contained statistically significantly elevated CD8+ T cell percentages (Fig. 3A, left panel) and cell numbers (A, right panel), indicating a certain capacity of BM-derived cells in positive selection of CD8+ T cells. Lower total numbers of CD8+ T cells were found in CD11c-MHC-I->MHC-I–/– chimeras compared with MHC-I–/–->MHC-I–/– chimeras (Fig. 3A, right panel).



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  		FIGURE 3. MHC-I+ DC are insufficient to positively select a detectable repertoire of H2-M3- or H2-Kb-restricted CTL. A, Radiation BM chimeras were analyzed by flow cytometry (data not shown) and subsequent enumeration of CD8+ T cells. The percentages (left panel) and total CD8+ T cell numbers (right panel) from spleens of the different chimeras are shown as individual symbols. Total cell numbers of spleen cells were MHC-I+>MHC-I–/–, 74 ± 5 x 106; CD11c-MHC-I>MHC-I–/–, 43 ± 16 x 106; MHC-I–/–>MHC-I–/–, 62 ± 29 x 106. Asterisks indicate statistically significant differences as determined with the Student’s t test (**, p < 0.005; *, p < 0.05). B, Wild-type MHC-I+ control mice and the indicated BM chimeras were infected with 2000 CFU of L. monocytogenes-OVA i.v. at day 0 and sacrificed 6 days later. Splenocyte cell suspensions were stained for CD8– (not shown), CD44–, and H2-M3-f-MIGWII (B) or Kb-OVA MHC tetramer-positive cells (C). Numbers indicate the percentage of tetramer+ CTL in the indicated gates among all splenocytes to compare the chimeras with rare CD8+ T cells to normal MHC-I+ mice with abundant CD8+ T cells (MHC-I+, tetramer–CD44low cells). D, Analysis of adoptively transferred OT-1 CD8+ T cells 5 days after s.c. immunization with CFA only (CFA) or 300 µg of SIINFEKL emulsified in CFA (CFA/OVA). OT-1 cells from peripheral blood were identified by gating first on CD8+ cells (data not shown) and then further analyzed by staining for V{beta}5.1 and V{alpha}2. Numbers indicate the percentage of OT-1 cells within total PBL and are the mean of n = 5 mice per group. One representative experiment of at least five with similar results is shown.

 
Next, we determined whether the few CD8 T cells selected in the thymi of CD11c-MHC-I mice were functionally similar to normal CD8 T cells from MHC-I+ mice. To detect MHC-Ia and -Ib restriction in the different chimeras, mice were infected with recombinant L. monocytogenes expressing secreted OVA (21), and expansion of H2-Kb-restricted OVA-specific (25) as well as H2-M3-restricted f-MIGWII-specific CD8+ T cells (26) was monitored using the corresponding MHC tetramers. Although the background frequency of M3-f-MIGWII-tetramer+CD8+ T cells in uninfected animals was ~0.05% (data not shown), it increased in infected wild-type MHC-I+ mice and MHC-I+->MHC-I–/– chimeras by 5- 6-fold (Fig. 3B). However, neither in CD11cMHC-I->MHC-I–/– nor in MHC-I–/–->MHC-I–/– chimeras were significantly elevated levels of f-MIGWII-tetramer+CD8+ T cells detected (Fig. 3B). Also, the analysis of H2-Kb-restricted OVA-specific CTL revealed that only in wild-type MHC-I+ mice could a significant 6- to 10-fold increase of Kb-OVA-tetramer+CD8+ T cells be detected upon infection (Fig. 3C). In contrast, in all chimeras, tetramer+ T cell populations were rare (Fig. 3C) or undetectable (data not shown). An expansion of class I-restricted T cells in MHC-I–/–->MHC-I–/– chimeras was not expected due to MHC-I–/– APC. In contrast, the lack of tetramer+CD8+ T cells in CD11cMHC-I->MHC-I–/– cannot be explained by missing MHC-I+ APC, because peripheral MHC-I+ DC are present (18) and functional (Fig. 3D) in these mice. When TCR-transgenic CD8+ OT-1 T cells were adoptively transferred into naive CD11c-MHC-I or MHC-I+ mice, we measured almost identical >20-fold OT-1 expansion in both recipients when we immunized with SIINFEKL peptide/CFA (Fig. 3D, CFA/OVA) or OVA-expressing recombinant vaccines (data not shown). Together with previous data (18), these results exclude the remote possibility of nonfunctional {beta}2m expression in transgenic DC. Taken together, the above experiments demonstrate that thymic DC are not sufficient to positively select a detectable repertoire of H2-M3- or of H2-Kb-restricted CD8+ T cells.

Thymic MHC-I+ DC induce negative selection by clonal deletion of CD8+ thymocytes

Next, we wanted to analyze whether MHC-I+ thymic DC are sufficient to induce CD8 T cell tolerance toward self. Therefore, we first determined the TCRV{beta} usage as a potential sign for influence of MHC-I+ DC on the TCR repertoire. In the CD8 compartment, the relative percentage of T cells expressing TCRV{beta}6, V{beta}8, or V{beta}11 did not differ between CD11c-MHC-I and MHC-I–/– mice (Fig. 4A). However, we detected with statistical significance a ~2-fold higher percentage of TCRV{beta}5+CD8+T cells in CD11c-MHC-I as compared with MHC-I–/– mice (Fig. 4A), whereas their total cell numbers were similar (B). These reproducible and statistically significant, but subtle differences in the TCRV{beta}5 repertoire might indicate a certain influence of MHC-I+ thymic DC on the shape of the CTL repertoire in CD11c-MHC-I mice. However, because MHC-I–/– and CD11c-MHC-I mice contained only very low numbers of CD8 thymocytes and CD8 T cells due to absent positive selection (Fig. 2), V{beta} segment analyses were difficult to perform. Therefore, we took advantage of K14-{beta}2m mice, which express transgenic {beta}2m under control of the human K14 promoter selectively on thymic cortical epithelium (17). This transgenic cortical MHC-I expression induces positive selection of CD8 T cells in the absence of negative selection (17). As a result, 2- to 3-fold elevated frequencies (Fig. 4C, left panel; Refs.17 and 27) and total cell numbers of CD8+ thymocytes (C, right panel) are found in K14-{beta}2m mice as compared with wild-type MHC-I+ mice. However, when we analyzed CD11c-MHC-I x K14-{beta}2m double transgenic mice expressing MHC-I on both thymic cortical epithelial cells (17) and thymic DC (Fig. 1), these elevated frequencies and numbers of CD8 thymocytes were reduced to the normal levels of MHC-I+ mice (Fig. 4C). This decrease of CD8+ thymocytes in presence of MHC-I+ thymic DC directly demonstrates the capacity of thymic DC to negatively select CD8+ thymocytes by clonal deletion as previously speculated (17, 27).



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  		FIGURE 4. Negative selection of CD8+ T cells by MHC-I+ DC. A, LN cells of the indicated mouse strains were stained with anti-CD4, anti-CD8, and either anti-TCRV{beta}5.1/5.2, anti-TCRV{beta}6, anti-TCRV{beta}8.1/8.2, or anti-TCRV{beta}11, respectively, and analyzed by flow cytometry (data not shown). Results are presented as the percentage of CD4+V{beta}+ or CD8+V{beta}+ lymphocytes (A) and as total cell numbers of CD8+V{beta}+T cells (B). Total cell numbers of CD4+V{beta}+lymphocytes were similar in all mice and are not shown. C, Flow cytometry analysis of thymocytes from wild-type MHC-I+, K14-{beta}2m (17 ), and K14-{beta}2m x CD11c-MHC-I mice with anti-CD4 and anti-CD8 Abs. Numbers indicate the percentages of CD4+CD8– and CD4–CD8+ thymocytes. Total numbers of CD8+ thymocytes from MHC-I+ ({blacksquare}), K14-{beta}2m ({cjs2112}), and K14-{beta}2m x CD11c-MHC-I ({square}) are shown (right panel). Values were obtained from (n = 5) mice per group and are displayed as mean (±SD). *, Statistically significant differences as calculated with the Student’s t test (p = 0.001). Shown is one representative of three independent experiments with similar results.

 
MHC-I+ DC induce tolerance to MHC-I

The analysis of {beta}2m-deficient animals has revealed, in addition to decreased CD8 numbers (Fig. 2; Ref.23), dramatic functional consequences for {beta}2m–/– CD8+ T cells (24); they kill target cells expressing MHC-I at normal ligand density (24). Additional effector cells affected by the absence of MHC-I are {beta}2m–/– NK cells, which, in contrast to the wild-type counterparts, neither reject MHC-I–/– cells in vivo nor lyse them efficiently in vitro (28, 29, 30). Therefore, we wondered whether effector cells from CD11c-MHC-I mice exhibit similar properties as those from MHC-I–/– mice, or whether DC were sufficient to alter those functions. To analyze functions of {beta}2m–/– effector cells in CD11c-MHC-I mice, we used an in vivo cytotoxicity assay (31, 32). A fluorescent mix of MHC-I–/– and MHC-I+ target cells at a ratio of 1:1 was analyzed 24 h after i.v. transfer into the different recipient mice by gating first on cells positive for the life dye CFSE (data not shown) and then differentially quantified further, according to their low (MHC-I–/–) or high (MHC-I+) H2-Kb expression (Fig. 5A). As previously described (28, 29, 30), in control MHC-I+ hosts, a specific loss of MHC-I–/– cells was evident (Fig. 5A). As expected (24), control MHC-I–/– hosts specifically rejected MHC-I+ targets (Fig. 5A). However, in CD11c-MHC-I hosts neither MHC-I–/–, nor MHC-I+ targets were specifically reduced, resulting in an unaltered MHC-I–/–/MHC-I+ target ratio of 1 (Fig. 5A, lower panel). To monitor survival of the transferred targets over a longer period of time and to exclude the possibility of MHC-I+ targets lysing MHC-I–/– targets and vice versa, we transferred MHC-I+ targets alone into the different recipients (Fig. 5B, upper panel). Similar to the previous experiment, MHC-I+ grafts were accepted by MHC-I+ hosts and rejected by MHC-I–/– mice (Fig. 5B, upper panel). In contrast, CD11c-MHC-I mice accepted the MHC-I+ graft similar to wild-type MHC-I+ mice for a period of 20 days (Fig. 5B, upper panel) and longer (data not shown).



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  		FIGURE 5. CD11c-MHC-I mice are tolerant to MHC-I+ and MHC-I–/– grafts. CFSE+ splenocytes from both MHC-I+ and MHC-I–/– donors were i.v. transferred into the indicated recipients at an MHC-I–/–/MHC-I+ ratio of 1. A, Twenty-four hours later, mice were sacrificed, and CFSE+ cells in the spleens (not shown) were analyzed for their H2-Kb expression by flow cytometry. The numbers indicate relative percentages of MHC-I–/– (left region) vs MHC-I+ (right region) cells among all CFSE+ target cells. From these percentages, the MHC-I–/–/MHC-I+ ratio was determined (lower panel). Results indicate the average of n = 5 mice per group. B, To examine the class I reactivity of CD8 T cells in MHC-I+ ({blacksquare}), CD11c-MHC-I ({circ}), and MHC-I–/– ({triangleup}) hosts (upper panel), or MHC-I+ ({blacksquare}), K14-{beta}2m ({diamond}), and K14-{beta}2m x CD11c-MHC-I ({triangledown}) recipients (lower panel), CFSE-labeled MHC-I+ spleen cells were transferred i.v. into the indicated hosts. Flow cytometric analysis of peripheral blood was performed on the indicated days posttransfer. Percentages of CFSE+ cells within the live cell gate of total peripheral blood lymphocytes are shown for each day as a mean (±SD) of three individual mice per group (n = 3).

 
To test whether the reduction of CD8+ thymocytes seen in K14-{beta}2m x CD11c-MHC-I mice (Fig. 4C) would result in functional tolerance toward MHC-I, we performed the same assay with these animals (Fig. 5, lower panel). In contrast to K14-{beta}2m mice, which showed similar behavior as MHC-I–/– mice (Ref.17 , and Fig. 5B, lower panel), also K14-{beta}2m x CD11c-MHC-I mice accepted MHC-I+ targets for 15 days (Fig. 5B, lower panel) and longer (monitored up to 40 days; data not shown). This indicates that the significant deletion of {beta}2m–/– CD8+ thymocytes by MHC-I+ thymic DC in K14-{beta}2m x CD11c-MHC-I mice (Fig. 4C) resulted in efficient tolerance induction toward MHC-I. Also, individual analyses of the CD4+, CD8+, or B220+ subpopulations within the CFSE+ graft gave similar results (data not shown).

Taken together, expression of MHC-I on DC was sufficient to render MHC-I–/– and K14-{beta}2m mice tolerant to MHC-I.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data obtained with transgenic CD11c-MHC-I mice indicate that MHC-I+ thymic DC are not sufficient to induce positive selection in vivo (Figs. 2 and 3). These findings are in line with the current view that thymic hemopoietic cells in general, in contrast to thymic epithelial cells (Ref.33 ; reviewed in Ref.1), cannot mediate efficient positive selection of MHC-Ia-restricted CD8+ T cells. However, this view has been challenged recently by the demonstration that the CTL repertoire was dependent on the presence of the thymus, but MHC-I+ BM-derived cells determined its expansion and restriction (9). Because the readout leading to this conclusion was functional and therefore indirect, other interpretations such as selection by MHC cross-reactivity are possible. In fact, it was shown recently that by cross-reacting with nonclassical class I molecules expressed on Db–/–Kb–/– thymic epithelium, certain (34), but not all (10, 35), transgenic TCRs could be positively selected.

Together with our previous report on the inability of MHC-II-expressing thymic DC to positively select CD4+ T cells (14), the results presented in this study exclude DC as possible inducers of positive T cell selection in vivo. However, the reason for this inability remains still unclear. Is it a lack of special inherent properties, such as expression or lack of expression of costimulatory molecules or soluble factors, or rather the more noncortical location of DC? Common precursors give rise to thymocyte and DC progeny (36), with distinct intrathymic migration and repopulation patterns (37). Although appearance of thymocytes sensitive to positive selection signals was observed rather late in the cortex, DC appearance was restricted to medullary regions and preceded thymocyte appearance (37). Such a coordinated thymus repopulation pattern strictly avoids DC/CD4+CD8+ thymocyte contacts in cortical regions. There, cortical epithelial cells are, besides MHC-I+ thymocytes, which apparently cannot select MHC-Ia-restricted cells (38), the only cell type expressing high levels of MHC-I. Also, experiments using intrathymic transfer of fibroblast or epithelial cell lines (39, 40, 41), adenovirus-mediated intrathymic epithelial MHC-I expression (42), and transgenic expression of MHC-II (43) or class I (17) on cortical epithelial cells induced effective positive selection without participation of thymic APC.

This is in contrast to the established role of BM-derived cells in selection of nonclassical CD8+ T cells (4, 5, 6, 7). Data from our BM chimera experiments confirm that nonclassical class I molecules on hemopoietic cells induce efficient positive selection (Fig. 3, A and B). In chimeras expressing {beta}2m exclusively by bone-marrow derived cells and infected with L. monocytogenes, the amount of H2-M3-restricted f-MIGWII-specific CTL was nearly as high as that found in normal nonchimeric MHC-I+ mice. These data confirm the efficiency of positive selection of H2-M3-restricted CTL by BM-derived cells (4). Because in CD11c-MHC-I->MHC-I–/– chimeras these cells were not found (Fig. 4B), we can definitely exclude DC from the candidate list for mediators of positive selection for H2-M3-restricted CD8 T cells. It remains to be elucidated which hemopoietic cell types other than DC are actually responsible for positive selection of nonclassical CD8 T cells. It remains doubtful whether macrophages could have such functions within the thymus in addition to being scavengers (44). Also, thymic B cells were not able to mediate positive selection of CD4+ T cells in vivo (16). Selection of CD8+ T cells restricted to nonclassical MHC-I molecules may be mediated by CD4+CD8+ thymocytes, as described for NK T cells (6). Another possibility is that not a specific cell type induces positive selection, but the thymic environment has to provide a threshold percentage of available MHC-I+ cells. Then, irrespective of the cell type, if not enough MHC-I+ cells are present, selection cannot take place. Additional experiments are necessary to evaluate this possibility.

In contrast to their incapacity to positively select CD8+ T cells, DC were sufficient to induce tolerance; CD11c-MHC-I transgenic mice (unlike MHC-I–/– mice) accepted MHC-I+ grafts (Fig. 5). Thymic DC most likely have induced this functional CTL tolerance by clonal deletion, similar to what we have previously observed for MHC-II+ DC (14, 16). Direct evidence for this possibility comes from K14-{beta}2m x CD11c-MHC-I mice, where the presence of MHC-I+ thymic DC decreases the frequencies of mature CD8+ thymocytes in K14-{beta}2m thymi 3- to 5-fold down to wild-type levels (Fig. 4) and renders the resulting {beta}2m–/– CD8 T cells tolerant to MHC-I. In a recent report, the frequency of peripheral autoreactive CD8 T cells in K14-{beta}2m mice was calculated by limiting-dilution analysis to be 2% (17), which is in accordance with the frequency for autoreactive CD4+ T cells detected in a similar system (43). However, positive selection in absence of negative selection resulted in much (2- to 3-fold) higher total frequencies of CD8+ thymocytes (Fig. 4; Refs.17 and 27). One reason for this difference could be that limiting-dilution analysis is eventually not 100% efficient in detecting self-reactive T cells.

Interestingly, we found that DC do not influence NK cell tolerance, because CD11c-MHC-I mice do not reject an MHC-I–/– graft. Tolerance to both MHC-I+ and MHC-I–/– targets are apparently general features of mice expressing MHC-I in a mosaic pattern (45).

As a conclusion from the present study, it becomes clear that thymic DC are not able to induce positive selection of classical or nonclassical CD8 T cells, but can tolerize self-reactive CD8+ T cells. Therefore, DC are specialized APC in the thymus that deliver signals that protect the organism from T cell autoreactivity. Also, peripheral DC seem to induce tolerance of mature T cells during the steady state (46, 47). Therefore, DC might play at least a triple role during the lifetime of a T cell: 1) cognate thymocyte-DC interactions of certain strength will result in cell death (central tolerance by negative selection); 2) once exported to the periphery, naive T cells interact with DC to survive in the absence of specific Ag (20); and 3) finally, in peripheral lymphoid organs, the outcome of an Ag-specific cognate T cell-DC interaction can result in either Ag-specific immunity or peripheral tolerance (48).


   Acknowledgments
 
We thank A. Bol and W. Mertl for expert animal caretaking, and D. Busch and K. Linkemann for help with L. monocytogenes experiments and the kind gift of tetramers.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Deutsche Forschungsgemeinschaft SFB 456 Back

2 Address correspondence and reprint requests to Dr. Thomas Brocker, Institute for Immunology, Ludwig Maximilians University, Goethestrasse 31, D-80336 Munich, Germany. E-mail address: tbrocker{at}ifi.med.uni-muenchen.de Back

3 Abbreviations used in this paper: BM, bone marrow; MHC-I, MHC class I; DC, dendritic cell; {beta}2m, {beta}2-microglobulin. Back

Received for publication June 24, 2004. Accepted for publication August 3, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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