Thymic positive and negative selection of developing T lymphocytes confronts us with a paradox: How can a T-cell antigen receptor (TCR)-major histocompatibility complex (MHC)/peptide interaction in the former process lead to transduction of signals allowing for cell survival and in the latter induce programmed cell death or a hyporesponsive state known as anergy? One of the hypotheses put forward states that the outcome of a TCR-MHC/peptide interaction depends on the cell type presenting the selecting ligand to the developing thymocyte. Here we describe the development and lack of self-tolerance of CD8+ T lymphocytes in transgenic mice expressing MHC class I molecules in the thymus exclusively on cortical epithelial cells. Despite the absence of MHC class I expression on professional antigen-presenting cells, normal numbers of CD8+ cells were observed in the periphery. Upon specific activation, transgenic CD8+ T cells efficiently lysed syngeneic MHC class I+ targets in vitro and in vivo, indicating that thymic cortical epithelium (in contrast to medullary epithelium and antigen-presenting cells of hematopoietic origin) is incapable of tolerance induction. Thus, compartmentalization of the antigen-presenting cells involved in thymic positive selection and tolerance induction can (at least in part) explain the positive/negative selection paradox.

Because of the random nature of T-cell receptor alpha (TCRα) and TCRβ gene rearrangements, the developing T-cell repertoire needs to undergo positive and negative selection processes. Thymic positive selection is thought to enrich for T cells expressing a clonotypic TCRαβ capable of interaction with self-major histocompatibility complex (MHC) heterodimers, although alternative views exist. Thymic negative selection eliminates or inactivates self-reactive T lymphocytes through induction of apoptosis and anergy. Although negative selection probably rids the developing T-cell repertoire of the majority of self-reactivity, peripheral tolerance mechanisms survey remaining self-specific T-cell clones. The resulting peripherally circulating T-cell repertoire is capable of recognition of foreign, but not self-peptides presented by self-MHC molecules.1-5 

Because thymic positive and negative selection both involve TCR-MHC/peptide interactions, an important issue is what determines the outcome of such an interaction. Initially, studies have focused on the thymic stromal cell types involved in thymic selection. It has been known that MHC/peptide complexes expressed on cortical epithelium are capable of positive selection of conventional TCRαβ+thymocytes, whereas medullary epithelium as well as antigen-presenting cells (APCs) of hematopoietic origin cannot,6-8 although quantitatively minor exceptions to this rule have been reported.9,10 On the other hand, negative selection is known to be mediated primarily by APCs of hematopoietic origin (reviewed in Anderson et al1 and Laufer et al11).

Given the fact that thymic epithelium can induce positive selection while APCs cannot, it was conceivable that differences in the repertoire of presented peptides play a role in the outcome of a TCR-MHC/peptide interaction. Although peptide elution studies from epithelial cells and APCs have thus far failed to confirm this hypothesis,12 several lines of evidence indicate that thymic epithelial cells have specialized antigen presentation properties and may present different peptides from those presented by professional APC.13-18 Therefore, an interaction with MHC/peptide ligands expressed by thymic epithelial cells may allow for positive selection, and these cells would subsequently not be negatively selected because the same ligand is not expressed on cells capable of negative selection. However, in mice expressing MHC class II molecules loaded with a single peptide CD4+ T lymphocytes develop,19-23 demonstrating that possible differences in the repertoire of peptides presented by positively and negatively selecting cell types alone cannot fully explain the positive/negative selection paradox.

An alternative hypothesis on the mechanism of thymic positive/negative selection states that the intensity of TCR triggering determines the selection outcome. A high avidity interaction would lead to negative selection, a very weak signaling to death by neglect, whereas an intermediate TCR-MHC/peptide avidity would allow for positive selection. In fetal thymus organ cultures, it has been shown that the low level expression of agonist MHC/peptide ligand allows the survival of developing thymocytes, supporting the avidity model.24,25 However, when tested, the surviving mature T cells could not be activated by using the same ligand, suggesting that even low avidity interactions do not allow for functional positive selection.26,27 Recent data on the capacity of modified peptide ligands to differentially induce certain, but not other effector functions, suggested that these ligands may also play a role in positive selection (reviewed by Jameson et al28 and Germain and Stefanova29). Studies on TCR-transgenic fetal thymus organ cultures supplemented with modified peptides have suggested a role for these peptides in positive selection.30 However, the fact that altered peptide ligands can also inhibit positive selection,31 and an unexpected MHC/modified peptide ligand-induced mismatch between MHC class specificity and CD4/CD8 lineage outcome32 has complicated the interpretation of altered peptide studies. Finally, a differentiation-state dependent “interpretation” of TCR-mediated signals has been proposed to play a role in positive/negative selection.33 34 

In an alternative approach to the paradox of positive/negative selection, we and others have initiated a dissection of the 2 selection processes.8 35-38 We have previously generated irradiation bone marrow chimeras in which radioresistant cells (which are required for positive selection) express MHC molecules, but APCs of hematopoietic origin do not. Although a 2- to 3-fold increase in the rate of development of mature thymocytes was observed, syngeneic reactivity of chimera-derived T cells was rather limited, implying that a significant level of negative selection was still operating in the absence of MHC expression on APCs. Here we report data on transgenic mice expressing MHC class I ligands exclusively on thymic cortical epithelial cells. Although positive selection seems to occur normally in these transgenic mice, no evidence for any negative selection could be observed using in vitro and in vivo assays. Therefore, positive and negative selection seem to be mediated by different cell types expressing self-MHC/peptide ligands.

Mice

C57BL/6 and DBA/2 mice were obtained from Harlan Netherlands (Zeist, The Netherlands), whereas H-2Kb mutant B6.C-H2bm1- and β2-microglobulin (β2m)-deficient C57BL/6-β2m° mice39 were purchased from Jackson Laboratories (Bar Harbor, ME). The construct used to generate K14-β2m transgenic mice was made as follows: A 0.6-kilobase (kb)SalI/NsiI β2m complementary DNA (cDNA) fragment (generously provided by Dr D. Margulies, National Institutes of Health, Bethesda, MD) was inserted into the BamHI site of pG3Z.K14 (generously provided by Dr E. Fuchs, Howard Hughes Medical Institute, University of Chicago, IL), a vector containing the human keratin 14 (K14) promoter, βglobin intron, and the K14 poly A addition signal.40 The 3.6-kb transgene was excised withKpnI and HindIII and microinjected into (B6 β2m° x C57BL/6)F1 zygotes. Transgenic founders were backcrossed to C57BL/6-β2m° mice to obtain K14-β2m transgenic, endogenous β2m-deficient mice.

Immunohistology

Thymi were snap frozen in liquid nitrogen and subsequently placed in OCT (Miles, Elkhard, IN). Cryostat sections (5 μm) were air-dried on frosted microscope slides for at least 2 hours, placed in acetone at room temperature (RT) for 5 minutes, and rehydrated in phosphate-buffered saline (PBS). Blocking was performed for 15 minutes with antibody diluent (Dako, Glostrup, Denmark). Sections were then incubated with monoclonal antibody 6C3 (Pharmingen, San Diego, CA) at a saturating concentration for 30 minutes. After washing with PBS/0.01% Tween20 for 30 minutes, slides were incubated with FITC-labeled antirat IgG F(ab′)2 antibody fragment (Immunotech, Marseille, France) for 30 minutes and subsequently washed for 30 minutes. After blocking with mouse Ig (Pierce, Rockford, IL), slides were incubated with biotinylated H-2Kb and H-2Db specific antibody 28-8-6 (Pharmingen), washed, and finally incubated with rhodamine-conjugated streptavidine (Immunotech). After extensive washing, slides were mounted in Faramount (Dako) and analyzed by immunofluorescence microscopy.

Flow cytometric analysis

Single cell suspensions were prepared from thymus, spleen, and pooled mesenteric, brachial, inguinal, and axillary lymph nodes isolated from K14-β2m mice and β2m+/° littermates, as well as from bone marrow from hematopoietic chimeras. Cells were preincubated with 2.4G2 culture supernatant to block Fc receptors,41 washed, and subsequently incubated with antibody for 30 minutes in PBS/2% fetal calf serum (FCS). The following antibodies were used: PE-conjugated anti-CD62L and anti-B220 (Caltag, Burlingame, CA); FITC-conjugated anti-TCRβ and anti-H-2Kb, PE-conjugated anti-CD4, anti-CD44, anti-CD69, anti-Mac1, and anti-CD11c, and Cy-chrome–labeled anti-CD8 (all from Pharmingen). After 2 washes in PBS/2% FCS, cells were analyzed with a FACScan flow cytometer (Becton Dickinson, San Jose, CA).

Cytotoxic T lymphocyte assays

Unseparated splenocytes derived from K14-β2m transgenic or control (C57BL/6, B6.C-H2bm1 or DBA/2) mice were cultured for 6 days in the presence of T-cell–depleted (anti-Thy1 antibody AT8342 plus complement) irradiated (1000 Rad γ) splenocytes in the presence of 30 U/mL IL-2 (EL4 supernatant43). For lysis of RMA and EL-4 targets (C57BL/6 origin43,44), C57BL/6 APC-stimulated effector cells were used, whereas P815 (DBA/2 origin45) lysis assays were performed by using T cells stimulated with DBA/2 APCs. Targets (2000 cells per well) were labeled with 51Cr, extensively washed, and mixed with effector cells in duplicate at effector (viable cell)-to-target (E/T) ratios indicated. 51Cr release in the supernatant was measured 4 hours later. Specific lysis is51Cr release above background as a percentage of maximum (as determined by acid lysis of targets). For antibody-blocking experiments, effectors were preincubated (30 minutes) with the indicated concentrations of anti-CD4 (GK1.546) or anti-CD8 (H3547) antibodies, then mixed with51Cr-labeled targets (2000 cells per well) at an E/T ratio required for half maximum lysis.

Limiting dilution assays

Splenocytes from K14-β2m and control (C57BL/6 and B6.C-H2bm1) mice were preincubated with 2.4G2 supernatant,41 and subsequently doubly stained with PE-conjugated anti-CD8 and FITC-conjugated anti-TCRβ antibodies (Pharmingen). CD8+TCR+ cells were then sorted (with a FACStar Plus sorter, Becton Dickinson, San Jose, CA) directly into 96-well plates containing 2.5 × 106 irradiated (1000 Rad γ) C57BL/6 splenocytes per well. Cultures were maintained for 2 weeks in 200 μL Dulbecco modified Eagle medium supplemented with 30 U/mL exogenous IL2 (EL4 supernatant). Half (100 μL) of the cultures were used for standard cytotoxic assays, performed with51Cr-labeled RMA targets. Lysis was considered positive if51Cr release exceeded mean + 3 SD of spontaneous release (measured in 48 control wells containing targets and APCs). The frequency of RMA lysing precursors was calculated as described.48 

Hematopoietic chimeras

Irradiation bone marrow chimeras were prepared essentially as described previously.49 Briefly, anti-NK1.1 antibody-treated hosts (100 μg PK136 intraperitoneally50) were lethally irradiated (1000 Rad γ) using a 137Cs source and intravenously reconstituted with 107 C57BL/6 plus C57BL/6-β2m° bone marrow cells (ratio 1:1) that had previously been depleted of T cells using anti-Thy1 antibody AT8342 plus complement. Unseparated lymph node cells (107) from transgenic or control mice were coinjected. In some experiments, transgenic lymph node cells were depleted of CD4+ and/or CD8+ T cells before transfer by using anti-CD4 antibody RL172.451 or anti-CD8 antibody 3.16852 plus complement. Chimeras were kept on antibiotic-containing drinking water (0.2% Bactrim, Roche, Basel, Switzerland) for the duration of the experiment (2 weeks).

Major histocompatibility complex class I expression in K14-β2m transgenic mice

We have generated transgenic mice that express murine β2m under the control of the human keratin K14 promoter known to be exclusively active in the basal layer of stratified squamous epithelia.40,53 Transgenic mice were crossed to C57BL/6 β2m-deficient animals39 and K14-β2m transgenic, endogenous β2m-deficient mice identified (K14-β2m). Flow cytometric analysis of K14-β2m T cells, B cells, macrophages, and dendritic cells from bone marrow, spleen, liver, and thymus demonstrated that these cells do not express detectable levels of MHC class I molecules (Figure 1A; data not shown). In contrast, cortical (but not medullary) epithelial cells in K14-β2m transgenic thymi express MHC class I molecules at levels approaching those observed in wild-type controls (Figure 1B).

Fig. 1.

MHC class I expression in K14-β2m mice.

(A) Lack of H-2Kb expression on K14-β2m splenic B cells, T cells, macrophages, and dendritic cells. Splenocytes from K14-β2m mice (black lines) and β2m+/° littermates (gray lines) were stained with anti–H-2Kb combined with anti-B220, anti-TCRβ, anti–Mac-1, or anti-CD11c antibodies. Histograms of H-2Kb expression on electronically gated B220+ (B cells), TCRβ+ (T cells), Mac-1+ (macrophages), or CD11c+ (dendritic cells) subsets are representative of 3 independent experiments. (B) Thymic cortical epithelial expression of MHC class I in K14-β2m mice. Sections of wild-type C57BL/6, C57BL/6-β2m°, and K14-β2m thymi were doubly stained with FITC-conjugated antibody 6C3, which detects cortical epithelial cells, and rhodamine-labeled H-2Kb– and H-2Db–specific antibody 28-8-6. Green and red fluorescence of the same fields is shown.

Fig. 1.

MHC class I expression in K14-β2m mice.

(A) Lack of H-2Kb expression on K14-β2m splenic B cells, T cells, macrophages, and dendritic cells. Splenocytes from K14-β2m mice (black lines) and β2m+/° littermates (gray lines) were stained with anti–H-2Kb combined with anti-B220, anti-TCRβ, anti–Mac-1, or anti-CD11c antibodies. Histograms of H-2Kb expression on electronically gated B220+ (B cells), TCRβ+ (T cells), Mac-1+ (macrophages), or CD11c+ (dendritic cells) subsets are representative of 3 independent experiments. (B) Thymic cortical epithelial expression of MHC class I in K14-β2m mice. Sections of wild-type C57BL/6, C57BL/6-β2m°, and K14-β2m thymi were doubly stained with FITC-conjugated antibody 6C3, which detects cortical epithelial cells, and rhodamine-labeled H-2Kb– and H-2Db–specific antibody 28-8-6. Green and red fluorescence of the same fields is shown.

Close modal

Thymic cortical epithelial expression of major histocompatibility complex class I allows development of CD8+ T lymphocytes

We next analyzed the development of mature CD8+ T lymphocytes in K14-β2m transgenic mice. Compared with wild-type controls, thymi from transgenic mice contained a 2- to 3-fold increased percentage and an absolute number of mature CD4CD8+TCRhigh thymocytes (Figure2A). In K14-β2m lymph nodes and spleens, normal numbers of CD8+ T lymphocytes were detected (Figure 2A). Moreover, on the basis of the expression of the activation/memory markers CD44, CD62L, and CD69, most CD8+ splenocytes from transgenic as well as wild-type littermates had a naive phenotype (Figure 2B). This result is surprising in view of recent reports indicating that peripheral naive CD8+ T lymphocytes require TCR interactions with MHC class I for their survival.54 55 

Fig. 2.

Development and peripheral maintenance of CD8+ T lymphocytes in K14-β2m mice.

(A) Thymocytes from K14-β2m mice and β2m+/° littermates were triply stained with anti-CD4, anti-CD8, and anti-TCRβ antibodies. Percentages (mean ± SD, n = 6) of CD4CD8, CD4+CD8+, CD4+CD8TCRhigh and CD4CD8+TCRhigh cells are indicated. Thymi from K14-β2m mice and β2m+/° littermates contained 127 ± 45 × 106 and 116 ± 25 × 106 cells, respectively. Spleen and lymph node cell suspensions were stained with anti-CD8 and anti-TCRβ antibodies (n = 3). K14-β2m mice and β2m+/° littermates had 98 ± 26 × 106 and 107 ± 25 × 106 splenocytes and 20 ± 6 × 106 and 28 ± 2 × 106 lymph node cells, respectively. (B) Expression of memory/activation markers by CD8+ splenocytes from K14-β2m mice and β2m+/° littermates. Cells were stained with anti-TCRβ, anti-CD8, and anti-CD44, anti-CD62L or anti-CD69 antibodies. Representative histograms of electronically gated TCRβ+CD8+ cells are shown.

Fig. 2.

Development and peripheral maintenance of CD8+ T lymphocytes in K14-β2m mice.

(A) Thymocytes from K14-β2m mice and β2m+/° littermates were triply stained with anti-CD4, anti-CD8, and anti-TCRβ antibodies. Percentages (mean ± SD, n = 6) of CD4CD8, CD4+CD8+, CD4+CD8TCRhigh and CD4CD8+TCRhigh cells are indicated. Thymi from K14-β2m mice and β2m+/° littermates contained 127 ± 45 × 106 and 116 ± 25 × 106 cells, respectively. Spleen and lymph node cell suspensions were stained with anti-CD8 and anti-TCRβ antibodies (n = 3). K14-β2m mice and β2m+/° littermates had 98 ± 26 × 106 and 107 ± 25 × 106 splenocytes and 20 ± 6 × 106 and 28 ± 2 × 106 lymph node cells, respectively. (B) Expression of memory/activation markers by CD8+ splenocytes from K14-β2m mice and β2m+/° littermates. Cells were stained with anti-TCRβ, anti-CD8, and anti-CD44, anti-CD62L or anti-CD69 antibodies. Representative histograms of electronically gated TCRβ+CD8+ cells are shown.

Close modal

Activated CD8+ T lymphocytes from K14-β2m mice efficiently lyse syngeneic major histocompatibility complex class I expressing targets in vitro

To analyze whether T lymphocytes derived from K14-β2m transgenic mice had undergone negative selection, splenocytes were cultured with irradiated syngeneic MHC class I and II expressing (C57BL/6) APCs for 6 days in the presence of exogenous IL-2, followed by an in vitro lysis assay that used C57BL/6-derived RMA or EL-4 lymphomas as targets. K14-β2m–derived effector T cells lysed syngeneic targets as efficiently as (allogeneic) B6.C-H2bm1- or DBA/2-derived T cells (Figure 3A). Similar results were obtained with T lymphocytes derived from a second independent K14-β2m transgenic mouse line (data not shown). Lysis by both K14-β2m– and B6.C-H2bm1–derived effector T cells was completely CD8-dependent, as shown by anti-CD8 antibody blocking (Figure3B).

Fig. 3.

Activated CD8+ T lymphocytes from K14-β2m mice lyse MHC class I+ syngeneic targets in vitro.

(A) Splenocytes from DBA/2, C57BL/6, B6.C-H2bm1 and K14-β2m mice were stimulated in vitro with irradiated C57BL/6 spleen cells in the presence of exogenous IL-2. After 6 days of culture, effector cells were assayed for cytolytic activity using C57BL/6-derived RMA lymphoma target cells at E/T ratios indicated. Data are representative of 3 independent experiments. Similar results were obtained by using C57BL/6-derived EL-4 thymoma cells as targets (data not shown). (B) In vitro–stimulated effector cells from K14-β2m and B6.C-H2bm1 mice were incubated for 30 minutes with titrated concentrations of anti-CD4 or anti-CD8 antibodies before addition of labeled RMA target cells. Data are depicted as percentage of lysis in the absence of antibody. (C) Precursor frequency analysis of syngeneic target lysing CD8+ T lymphocytes. Indicated numbers of CD8+TCRβ+ splenocytes were electronically sorted into 96-well plates seeded with C57BL/6 APCs. Lysis of RMA targets was assessed 2 weeks later.

Fig. 3.

Activated CD8+ T lymphocytes from K14-β2m mice lyse MHC class I+ syngeneic targets in vitro.

(A) Splenocytes from DBA/2, C57BL/6, B6.C-H2bm1 and K14-β2m mice were stimulated in vitro with irradiated C57BL/6 spleen cells in the presence of exogenous IL-2. After 6 days of culture, effector cells were assayed for cytolytic activity using C57BL/6-derived RMA lymphoma target cells at E/T ratios indicated. Data are representative of 3 independent experiments. Similar results were obtained by using C57BL/6-derived EL-4 thymoma cells as targets (data not shown). (B) In vitro–stimulated effector cells from K14-β2m and B6.C-H2bm1 mice were incubated for 30 minutes with titrated concentrations of anti-CD4 or anti-CD8 antibodies before addition of labeled RMA target cells. Data are depicted as percentage of lysis in the absence of antibody. (C) Precursor frequency analysis of syngeneic target lysing CD8+ T lymphocytes. Indicated numbers of CD8+TCRβ+ splenocytes were electronically sorted into 96-well plates seeded with C57BL/6 APCs. Lysis of RMA targets was assessed 2 weeks later.

Close modal

We next analyzed the frequency of autospecific CD8+K14-β2m transgenic T lymphocytes by limiting dilution analysis. The minimal estimate of the H-2b reactive cytotoxic T lymphocyte (CTL) precursor frequency among K14-β2m–derived CD8 T lymphocytes was 1 of 48, ie, 7-fold higher than that observed among allogeneic B6.C-H2bm1 CTL (1 of 320), and almost 100-fold higher than that of syngeneic wild-type C57BL/6 cells (1 of 3372) (Figure 3C). These data confirm the high level of autoreactivity of K14-β2m–derived CTL.

CD8+ T cells from K14-β2m mice kill syngeneic major histocompatibility complex class I–expressing hematopoietic targets in vivo

T lymphocytes that can be activated by syngeneic MHC ligands in vitro are not necessarily reactive to syngeneic cells in vivo.38 56-58 To assess whether K14-β2m–derived T cells were capable of lysing syngeneic targets in vivo, we cotransferred C57BL/6 and C57BL/6-β2m° bone marrow as well as K14-β2m or control C57BL/6 lymph node T cells into lethally irradiated C57BL/6 hosts. Survival of coinjected C57BL/6 and β2m° bone marrow cells was analyzed by flow cytometry 2 weeks after transfer. In K14-β2m T-cell–injected hosts, no MHC class I positive bone marrow precursor cells had survived, and only β2m° bone marrow had reconstituted the hosts (Figure 4A). Moreover, antibody-depletion experiments demonstrated that CD8+ T cells from the K14-β2m lymph node cell inoculum were capable of in vivo C57BL/6 bone marrow lysis (Figure 4B). Therefore, CD8+T lymphocytes developing in K14-β2m transgenic mice are capable of lysing targets that express syngeneic MHC class I ligands in vivo as well as in vitro.

Fig. 4.

CD8+ T lymphocytes from K14-β2m mice kill syngeneic MHC class I+ hematopoietic cells in vivo.

Lethally irradiated C57BL/6 hosts were reconstituted with a mixture of C57BL/6 and C57BL/6-β2m° bone marrow cells that were cotransferred with C57BL/6 or K14-β2m lymph node (LN) cells. Where indicated, the K14-β2m lymph node cells were depleted of either CD4+ (LN ΔCD4) or CD4+ plus CD8+ (LN ΔCD4ΔCD8) cells (by antibody plus complement treatment) before transfer. Reconstitution by C57BL/6 bone marrow cells was assessed 2 weeks later by 2-color flow cytometry of bone marrow with the use of anti-CD43 (detecting B cell precursors) and anti–H-2Kbantibodies.

Fig. 4.

CD8+ T lymphocytes from K14-β2m mice kill syngeneic MHC class I+ hematopoietic cells in vivo.

Lethally irradiated C57BL/6 hosts were reconstituted with a mixture of C57BL/6 and C57BL/6-β2m° bone marrow cells that were cotransferred with C57BL/6 or K14-β2m lymph node (LN) cells. Where indicated, the K14-β2m lymph node cells were depleted of either CD4+ (LN ΔCD4) or CD4+ plus CD8+ (LN ΔCD4ΔCD8) cells (by antibody plus complement treatment) before transfer. Reconstitution by C57BL/6 bone marrow cells was assessed 2 weeks later by 2-color flow cytometry of bone marrow with the use of anti-CD43 (detecting B cell precursors) and anti–H-2Kbantibodies.

Close modal

Analysis of the mechanisms responsible for thymic positive and negative selection would be greatly facilitated by the dissection of these processes. Here we have reported data on mice expressing MHC class I molecules under control of the human K14 promoter. In the thymus, MHC class I expression was limited to cortical epithelial cells, and no expression by medullary epithelial cells, T or B lymphocytes, dendritic cells, and macrophages was observed. CD8+ T lymphocytes developed apparently normally and populated peripheral lymphoid organs. These cells could be stimulated to lyse syngeneic targets in vitro as well as in vivo. Therefore, dissection of the thymic positive and negative selection mechanisms can be achieved by using transgenic mice that express MHC class I molecules exclusively on cortical epithelial cells.

Expression of transgenic β2m under control of the human K14 promoter in the thymus was limited to cortical epithelial cells, consistent with the results obtained by Laufer and colleagues8 who used transgenic mice that expressed the I-Aβ chain under control of the same promoter construct. In transgenic mice expressing the costimulatory molecule B7-1 under control of a human K14 promoter construct, the expression pattern seemed very different: Medullary (but not cortical) epithelial cells expressed the transgene.59However, the promoter construct was not identical to the one used by us and by Laufer and colleagues.8,60 Finally, analysis of murine K14 expression in the thymus revealed its exclusive localization in medullary regions.61 Taken together, these data suggest that cortical targeting achieved by us and by Laufer and colleagues critically depends on the promoter construct and is not an inherent characteristic of K14 expression.

Thymic cortical epithelial expression of MHC class I molecules in our transgenic mice allowed for efficient positive selection of mature CD8+ thymocytes. Similarly, MHC class II expression by thymic cortical epithelial cells leads to positive selection of mature CD4+ thymocytes.6,8 In contrast, in mice in which MHC class II expression was limited to thymic medullary epithelium, no positive selection of CD4+ T lymphocytes occurred.6 Moreover, MHC class I expression by APCs leads to positive selection of only a minute population of cytotoxic T cells, whereas MHC class II expression by APCs does not allow detectable positive selection of CD4+ T cells.7,9,10 37It therefore appears that only thymic cortical epithelium supports positive selection of significant numbers of both CD4+6,8and CD8+ T lymphocytes (our results).

In our K14-β2m mice, thymic cortical epithelial expression of MHC class I led to the development of 2- to 3-fold increased numbers of mature CD8+ thymocytes. In the absence of MHC class I expression by professional APCs (and therefore of an important fraction of thymic clonal deletion), this increase in mature CD8+thymocytes was to be expected. Indeed, we have previously shown that in bone marrow chimeras expressing MHC by radioresistant thymic epithelial cells, but lacking MHC expression by APCs, a 2- to 3-fold increase in the rate of generation of mature CD8+ thymocytes occurs.37 

Because peripheral naive CD8+ T lymphocytes are believed to require TCR interactions with MHC class I for their survival,54,55 the persistence of normal numbers of peripheral K14-β2m CD8+ T cells in the absence of MHC class I expression by professional APCs was rather unexpected. In contrast to naive T cells, memory CD8+ T cells are thought to survive in the absence of continuous TCR-MHC class I interactions,55 though alternative views exist.54 Nevertheless, flow cytometric analysis of K14-β2m peripheral CD8+ T cells revealed that the vast majority expressed a naive resting phenotype. Therefore, it appears that even in the absence of MHC class I expression by professional APCs,62 a quantitatively normal CD8+ T-cell repertoire is maintained. The peripheral CD8+ T lymphocyte pool in K14-β2m transgenic mice may be maintained by increased thymic egress of mature cells, or alternatively may persist because of TCR interactions with MHC class I expressed extrathymically on epithelial cells.40 53 Additional experiments will be required to distinguish between these 2 possibilities.

A significant proportion of peripheral CD8+ T lymphocytes from K14-β2m transgenic mice were autospecific and could be induced to lyse syngeneic MHC-expressing target cells in vitro. In bone marrow chimeras lacking MHC class I (J.V.M.V.M. and H.R.M.D., unpublished data, 1997) or MHC class I and II expression38 by hematopoietic cells, but expressing MHC on radioresistant elements (eg, thymic cortical and medullary epithelium), T lymphocytes lysing host-type MHC-expressing target cells in vitro could readily be generated as well. Syngeneic H-2b targets were approximately 10-fold less efficiently lysed by chimera-derived CD8+ T lymphocytes than by allogeneic DBA/2 T cells (our unpublished results, 1997, and van Meerwijk and MacDonald38). In contrast, K14-β2m and allogeneic DBA/2 or B6.C-H2bm1 CTL lysed H-2b targets equally efficiently. Moreover, although K14-β2m transgenic CTL lysed H-2b targets in vitro as well as in vivo, in the case of P→F1 and MHC°→wild-type chimeras, as well as in transgenic mice expressing tolerizing ligands exclusively on medullary epithelium, in vitro T-cell reactivity to host or transgene-type MHC was accompanied by in vivo tolerance, a phenomenon termed “split tolerance.”38,56-58 Taken together, these data confirm a role for medullary epithelium in negative selection.16,17,58,63,64 Moreover, because mature CD4+ T lymphocytes that had artificially developed in the absence of any MHC-TCR interaction (and therefore MHC-dependent positive or negative selection) were equally reactive to H-2b and H-2d APCs,65 the fact that H-2b targets are lysed equally efficiently by K14-β2m as by allogeneic B6.C-H2bm1– and DBA/2-derived T cells strongly suggest that cortical epithelium does not support negative selection.

Our data indicating that cortical epithelial cells are incapable of CD8+ T lymphocyte tolerance induction are apparently contradictory to earlier reports showing that in mice expressing transgenic MHC class I restricted TCRs autospecific (cortical) CD4+CD8+ thymocytes were absent (reviewed by Stockinger66). However, deletion observed in these mice is not necessarily due to MHC expressed by cortical epithelial cells and may be mediated by cortical macrophages, a hypothesis consistent with the relatively poor TCR-transgenic CD4+CD8+thymocyte deletion by ligand-expressing thymic epithelial cells.67 Data suggesting that other mechanisms may be involved in the depletion of autospecific TCR transgenic CD4+CD8+ thymocytes have also been reported.68 69 

The frequency of autoreactive CTL precursors in K14-β2m mice (as determined by limiting dilution analysis) was 1 of 48, a value similar to that obtained by Laufer and colleagues for autoreactive CD4+ T-cell precursors.8 These data suggest that only 2% of positively selectable thymocytes normally undergo tolerance induction, a value well below the 50% to 70% obtained earlier by us and by others analyzing T-hybrid specificity and kinetics of mature CD4+ or CD8+ thymocyte development.22,37 65 However, values obtained by limiting dilution analysis are necessarily minimal estimates, and (in the absence of a valid correction for plating efficiency) cannot be compared with kinetic and T-hybrid data.

In conclusion, our data indicate that MHC class I expressed by cortical epithelial cells cannot induce negative selection of CD8+ T lymphocytes, a conclusion consistent with earlier data on reactivity of CD4+ T cells that had developed in mice expressing MHC class II exclusively on cortical epithelium.8,35Therefore, cortical epithelial cells appear to be specialized in thymic positive selection, whereas medullary epithelial cells and intrathymic APCs of hematopoietic origin are specialized in negative selection. Although certain in vitro cultured thymic epithelial cell lines are capable of both positive and negative selection in vitro and when injected intrathymically,70,71 the physiologic relevance of these findings remains unclear. Whatever the explanation, the data presented here and elsewhere8 35 on K14-MHC transgenic mice clearly point to a general lack of tolerance induction by cortical epithelial cells. The ability to dissect thymic positive and negative selection by using these mice should facilitate analysis of the responsible mechanisms.

We thank Dr E. Fuchs for the K14 promoter construct, Dr D. Margulies for β2m cDNA, Dr C. L. Blanchard for constructing the K14-β2m vector, Dr M. Guttinger for helpful suggestions, and R. Lees and G. Enault for expert technical help.

Supported in part by grants from ARC (#7287), Région Midi Pyrénées (RECH/97001940), FRM (10000121-10), and by institutional funds from INSERM and University Toulouse III.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

1
Anderson
G
Moore
NC
Owen
JJT
Jenkinson
EJ
Cellular interactions in thymocyte development.
Annu Rev Immunol.
14
1996
73
99
2
Guidos
CJ
Positive selection of CD4+ and CD8+ T cells.
Curr Opin Immunol.
8
1996
225
232
3
Sebzda
E
Mariathasan
S
Ohteki
T
et al
Selection of the T cell repertoire.
Annu Rev Immunol.
17
1999
829
874
4
Anderson
G
Hare
KJ
Jenkinson
EJ
Positive selection of thymocytes: the long and winding road.
Immunol Today.
20
1999
463
468
5
Matzinger
P
Why positive selection?
Immunol Rev.
135
1993
81
117
6
Cosgrove
D
Chan
SH
Waltzinger
C
Benoist
C
Mathis
D
The thymic compartment responsible for positive selection of CD4+ T cells.
Int Immunol.
4
1992
707
710
7
Markowitz
JS
Auchincloss
HJ
Grusby
MJ
Glimcher
LH
Class II-positive hematopoietic cells cannot mediate positive selection of CD4+ T lymphocytes in class II-deficient mice.
Proc Natl Acad Sci U S A.
90
1993
2779
2783
8
Laufer
TM
DeKoning
J
Markowitz
JS
Lo
D
Glimcher
LH
Unopposed positive selection and autoreactivity in mice expressing class II MHC only on thymic cortex.
Nature.
383
1996
81
85
9
Bix
M
Raulet
D
Inefficient positive selection of T cells directed by haematopoietic cells.
Nature.
359
1992
330
333
10
Zerrahn
J
Volkmann
A
Coles
MC
et al
Class I MHC molecules on hematopoietic cells can support intrathymic positive selection of T cell receptor transgenic T cells.
Proc Natl Acad Sci U S A.
96
1999
11470
11475
11
Laufer
TM
Glimcher
LH
Lo
D
Using thymus anatomy to dissect T cell repertoire selection.
Semin Immunol.
11
1999
65
70
12
Marrack
P
Ignatowicz
L
Kappler
JW
Boymel
J
Freed
JH
Comparison of peptides bound to spleen and thymus class II.
J Exp Med.
178
1993
2173
2183
13
Oukka
M
Andre
P
Turmel
P
et al
Selectivity of the major histocompatibility complex class II presentation pathway of cortical thymic epithelial cell lines.
Eur J Immunol.
27
1997
855
859
14
Kasai
M
Hirokawa
K
Kajino
K
et al
Difference in antigen presentation pathways between cortical and medullary thymic epithelial cells.
Eur J Immunol.
26
1996
2101
2107
15
Marrack
P
McCormack
J
Kappler
J
Presentation of antigen, foreign major histocompatibility complex proteins and self by thymus cortical epithelium.
Nature.
338
1989
503
505
16
Oukka
M
Cohen-Tannoudji
M
Tanaka
Y
Babinet
C
Kosmatopoulos
K
Medullary thymic epithelial cells induce tolerance to intracellular proteins.
J Immunol.
156
1996
968
975
17
Klein
L
Klein
T
Ruther
U
Kyewski
B
CD4 T cell tolerance to human C-reactive protein, an inducible serum protein, is mediated by medullary thymic epithelium.
J Exp Med.
188
1998
5
16
18
Nakagawa
T
Roth
W
Wong
P
et al
Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus.
Science.
280
1998
450
453
19
Miyazaki
T
Wolf
P
Tourne
S
et al
Mice lacking H2-M complexes, enigmatic elements of the MHC class II peptide-loading pathway.
Cell.
84
1996
531
541
20
Martin
WD
Hicks
GG
Mendiratta
SK
et al
H2-M mutant mice are defective in the peptide loading of class II molecules, antigen presentation, and T cell repertoire selection.
Cell.
84
1996
543
550
21
Liu
CP
Parker
D
Kappler
J
Marrack
P
Selection of antigen-specific T cells by a single IEk peptide combination.
J Exp Med.
186
1997
1441
1450
22
Ignatowicz
L
Kappler
J
Marrack
P
The repertoire of T cells shaped by a single MHC/peptide ligand.
Cell.
84
1996
521
529
23
Fung-Leung
W-P
Surh
CD
Liljedahl
M
et al
Antigen presentation and T cell development in H2-M-deficient mice.
Science.
271
1996
1278
1281
24
Ashton-Rickardt
PG
Bandeira
A
Delaney
JR
et al
Evidence for a differential avidity model of T cell selection in the thymus.
Cell.
76
1994
651
663
25
Sebzda
E
Wallace
VA
Mayer
J
et al
Positive and negative thymocyte selection induced by different concentrations of a single peptide.
Science.
263
1994
1615
1618
26
Sebzda
E
Kuendig
TM
Thomson
CT
et al
Mature T cell reactivity altered by peptide agonist that induces positive selection.
J Exp Med.
183
1996
1093
1104
27
Hogquist
KA
Jameson
SC
Bevan
MJ
Strong agonist ligands for the T cell receptor do not mediate positive selection of functional CD8+ T cells.
Immunity.
3
1995
79
86
28
Jameson
SC
Hogquist
KA
Bevan
MJ
Positive selection of thymocytes.
Annu Rev Immunol.
13
1995
93
126
29
Germain
RN
Stefanova
I
The dynamics of T cell receptor signaling: complex orchestration and the key roles of tempo and cooperation.
Annu Rev Immunol.
17
1999
467
522
30
Sloan-Lancaster
J
Allen
PM
Altered peptide ligand-induced partial T cell activation: molecular mechanisms and role in T cell biology.
Annu Rev Immunol.
14
1996
1
27
31
Spain
LM
Jorgensen
JL
Davis
MM
Berg
LJ
A peptide antigen antagonist prevents the differentiation of T cell receptor transgenic thymocytes.
J Immunol.
152
1994
1709
1717
32
Volkmann
A
Barthlott
T
Weiss
S
Frank
R
Stockinger
B
Antagonist peptide selects thymocytes expressing a class II major histocompatibility complex-restricted T cell receptor into the CD8 lineage.
J Exp Med.
188
1998
1083
1089
33
Lucas
B
Stefanova
I
Yasutomo
K
Dautigny
N
Germain
RN
Divergent changes in the sensitivity of maturing T cells to structurally related ligands underlies formation of a useful T cell repertoire.
Immunity.
10
1999
367
376
34
Davey
GM
Schober
SL
Endrizzi
BT
et al
Preselection thymocytes are more sensitive to T cell receptor stimulation than mature T cells.
J Exp Med.
188
1998
1867
1874
35
Laufer
TM
Fan
L
Glimcher
LH
Self-reactive T cells selected on thymic cortical epithelium are polyclonal and are pathogenic in vivo.
J Immunol.
162
1999
5078
5084
36
DeKoning
J
DiMolfetto
L
Reilly
C
et al
Thymic cortical epithelium is sufficient for the development of mature T cells in relB-deficient mice.
J Immunol.
158
1997
2558
2566
37
van Meerwijk
JPM
Marguerat
S
Lees
RK
et al
Quantitative impact of thymic clonal deletion on the T cell repertoire.
J Exp Med.
185
1997
377
383
38
van Meerwijk
JPM
MacDonald
HR
In vivo T lymphocyte tolerance in the absence of thymic clonal deletion mediated by haematopoietic cells.
Blood.
93
1999
3856
3862
39
Zijlstra
M
Li
E
Sajjadi
F
Subramani
S
Jaenisch
R
Germ-line transmission of a disrupted beta 2-microglobulin gene produced by homologous recombination in embryonic stem cells.
Nature.
342
1989
435
438
40
Vassar
R
Fuchs
E
Transgenic mice provide new insights into the role of TGF-alpha during epidermal development and differentiation.
Genes Dev.
5
1991
714
727
41
Unkeless
JC
Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors.
J Exp Med.
150
1979
580
596
42
Sarmiento
M
Loken
MR
Fitch
FW
Structural differences in cell surface T25 polypeptides from thymocytes and cloned T cells.
Hybridoma.
1
1981
13
26
43
Farrar
JJ
Fuller-Farrar
J
Simon
PL
et al
Thymoma production of T cell growth factor (Interleukin 2).
J Immunol.
125
1980
2555
2558
44
Ljunggren
HG
Paabo
S
Cochet
M
et al
Molecular analysis of H-2-deficient lymphoma lines: distinct defects in biosynthesis and association of MHC class I heavy chains and beta 2- microglobulin observed in cells with increased sensitivity to NK cell lysis.
J Immunol.
142
1989
2911
2917
45
Ralph
P
Nakoinz
I
Direct toxic effects of immunopotentiators on monocytic, myelomonocytic, and histiocytic or macrophage tumor cells in culture.
Cancer Res.
37
1977
546
550
46
Dialynas
DP
Quan
ZS
Wall
KA
et al
Characterization of the murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to the human Leu-3/T4 molecule.
J Immunol.
131
1983
2445
2451
47
Golstein
P
Goridis
C
Schmitt-Verhulst
A-M
et al
Lymphoid cell surface interaction structures detected using cytolysis-inhibiting monoclonal antibodies.
Immunol Rev.
68
1982
5
42
48
Taswell
C
Limiting dilution assays for the determination of immunocompetent cell frequencies, I: data analysis.
J Immunol.
126
1981
1614
1619
49
van Meerwijk
JPM
O'Connell
EM
Germain
RN
Evidence for lineage commitment and initiation of positive selection by thymocytes with intermediate surface phenotypes.
J Immunol.
154
1995
6314
6324
50
Koo
GC
Peppard
JR
Establishment of monoclonal anti-Nk-1.1 antibody.
Hybridoma.
3
1984
301
303
51
Ceredig
R
Lowenthal
JW
Nabholz
M
MacDonald
HR
Expression of interleukin-2 receptors as a differentiation marker on intrathymic stem cells.
Nature.
314
1985
98
100
52
Sarmiento
M
Dialynas
DP
Lancki
DW
et al
Cloned T lymphocytes and monoclonal antibodies as probes for cell surface molecules active in T cell-mediated cytolysis.
Immunol Rev.
68
1982
135
169
53
Vassar
R
Rosenberg
M
Ross
S
Tyner
A
Fuchs
E
Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice.
Proc Natl Acad Sci U S A.
86
1989
1563
1567
54
Tanchot
C
Lemonnier
FA
Perarnau
B
Freitas
AA
Rocha
B
Differential requirements for survival and proliferation of CD8 naive or memory T cells.
Science.
276
1997
2057
2062
55
Murali-Krishna
K
Lau
LL
Sambhara
S
et al
Persistence of memory CD8 T cells in MHC class I-deficient mice.
Science.
286
1999
1377
1381
56
Sprent
J
Kosaka
H
Gao
E-K
Surh
CD
Webb
SR
Intrathymic and extrathymic tolerance in bone marrow chimeras.
Immunol Rev.
133
1993
151
176
57
Sprent
J
Hurd
M
Schaefer
M
Heath
W
Split tolerance in spleen chimeras.
J Immunol.
154
1995
1198
1206
58
Hoffmann
MW
Allison
J
Miller
JF
Tolerance induction by thymic medullary epithelium.
Proc Natl Acad Sci U S A.
89
1992
2526
2530
59
Burns
RP
Jr
Nasir
A
Haake
AR
Barth
RK
Gaspari
AA
B7–1 overexpression by thymic epithelial cells results in transient and long-lasting effects on thymocytes and peripheral T helper cells but does not result in immunodeficiency.
Cell Immunol.
194
1999
162
177
60
Nasir
A
Ferbel
B
Salminen
W
Barth
RK
Gaspari
AA
Exaggerated and persistent cutaneous delayed-type hypersensitivity in transgenic mice whose epidermal keratinocytes constitutively express B7–1 antigen.
J Clin Invest.
94
1994
892
898
61
Klug
DB
Carter
C
Crouch
E
et al
Interdependence of cortical thymic epithelial cell differentiation and T-lineage commitment.
Proc Natl Acad Sci U S A.
95
1998
11822
11827
62
Brocker
T
Survival of mature CD4 T lymphocytes is dependent on major histocompatibility complex class II-expressing dendritic cells.
J Exp Med.
186
1997
1223
1232
63
Schonrich
G
Momburg
F
Hammerling
GJ
Arnold
B
Anergy induced by thymic medullary epithelium.
Eur J Immunol.
22
1992
1687
1691
64
Burkly
LC
Degermann
S
Longley
J
et al
Clonal deletion of V beta 5+ T cells by transgenic I-E restricted to thymic medullary epithelium.
J Immunol.
151
1993
3954
3960
65
Zerrahn
J
Held
W
Raulet
DH
The MHC reactivity of the T cell repertoire prior to positive and negative selection.
Cell.
88
1997
627
636
66
Stockinger
B
T lymphocyte tolerance: from thymic deletion to peripheral control mechanisms.
Adv Immunol.
71
1999
229
265
67
Carlow
DA
Teh
SJ
Teh
HS
Altered thymocyte development resulting from expressing a deleting ligand on selecting thymic epithelium.
J Immunol.
148
1992
2988
2995
68
Takahama
Y
Shores
EW
Singer
A
Negative selection of precursor thymocytes before their differentiation into CD4+CD8+ cells.
Science.
258
1992
653
656
69
Martin
S
Bevan
MJ
Antigen-specific and nonspecific deletion of immature cortical thymocytes caused by antigen injection.
Eur J Immunol.
27
1997
2726
2736
70
Hugo
P
Kappler
JW
Godfrey
DI
Marrack
PC
Thymic epithelial cell lines that mediate positive selection can also induce thymocyte clonal deletion.
J Immunol.
152
1994
1022
1031
71
Vukmanovic
S
Jameson
SC
Bevan
MJ
A thymic epithelial cell line induces both positive and negative selection in the thymus.
Int Immunol.
6
1994
239
246

Author notes

Joost P. M. van Meerwijk, INSERM U395 CHU Purpan, BP 3028, 31024 Toulouse Cedex 3, France; e-mail:joost.van-meerwijk@purpan.inserm.fr.

Sign in via your Institution