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Department of Anatomy, Medical School, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ßTCR repertoire is essential for the production of a T cell
population that is capable of efficient recognition of self MHC
molecules presenting nonself peptides but that is nonreactive to the
same MHC molecules presenting self peptides (1, 2). This process of
repertoire selection takes place at the CD4+8+
stage of T cell development in the thymus and involves positive
selection, triggering further maturation to the single-positive
CD4+8- and CD4-8+
stages (3) and negative selection that induces programmed cell death in
potentially autoreactive thymocytes (4).
A key factor in determining whether a CD4+8+
thymocyte undergoes positive or negative selection is the avidity of
its TCR-mediated interaction with peptide/MHC complexes on thymic
stromal cells (5, 6). CD4+8+ thymocytes are
known to have a finite lifespan of 3–4 days in the thymic cortex,
during which time successive ßTCR combinations, generated by
ongoing TCR -chain gene rearrangements (7), are tested for
interaction with stromal peptide/MHC ligands. Those cells that fail to
interact with stromal MHC complexes during this time die from
neglect (8), while low avidity interactions lead to positive selection
and survival, and high avidity interactions lead to negative selection
through apoptosis. Recent evidence suggests that there is a
considerable degree of promiscuity in TCR-mediated peptide/MHC
recognition, such that a single or a limited array of peptides can
positively select cells that are able to recognize a range of other
peptides presented by the same MHC molecule (9, 10, 11, 12). However, although
varied, the diversity of such repertoires is limited, indicating that
peptide diversity is important in generating a fully diverse TCR
repertoire during positive selection (13, 14, 15).
In addition to TCR ligation of the appropriate avidity, there is also evidence that other factors are involved in determining the outcome of thymic selection. Thus, efficient positive selection is dependent upon interaction with peptide/MHC complexes on thymic cortical epithelial cells (16), while negative selection is most efficiently mediated by professional APCs of bone marrow origin, namely dendritic cells (DC)3 (17, 18). At present, it is not clear whether this cellular specialization for selection involves: differential expression of MHC-bound peptide arrays on positively or negatively selecting cells, the expression of a specialized peptide repertoire on positively selecting cells, or differential expression of costimulatory molecules that provide additional signals leading to either cell differentiation or cell death (19).
In this study, we explore the interaction of peptide diversity and selecting cell type in T cell selection events, using reaggregate thymus organ cultures (RTOCs) to recombine thymic epithelial cells and DC from wild-type (wt) or H-2M-/- mice with a restricted presentation of MHC class II-bound peptides (9, 10, 11). Our results show that when any influence of negative selection by DC is excluded, positive selection by epithelial cells bearing a limited MHC class II-bound peptide spectrum is reduced threefold compared with positive selection by wt epithelium. Importantly, this is not due to differences in MHC class II levels on H-2M-/- and wt epithelial cells, and thus provides direct evidence for the role of peptide diversity in the efficiency of positive selection by thymic epithelial cells. Moreover, we have quantitated the frequency of bone marrow-derived DC required to induce maximal negative selection of T cell repertoires generated on both wt and H-2M-/- epithelial cells. Remarkably, we find that the number of CD4+ cells generated by wt epithelium is reduced by 80% in the presence of small numbers of wt DC (1%), while the generation of CD4+ cells in the presence of H-2M-/- epithelium is even further reduced (by 95%) by the same number of wt DC, emphasizing the impact of peptide diversity in negative selection in relation to that in positive selection. Finally, we show that in direct comparison on a per cell basis, wt thymic epithelial cells and wt DC, with similar overall levels of MHC class II expression, differ markedly in their ability to induce negative selection in a population of cells positively selected on a limited array of peptides, arguing that these two cell types are qualitatively different in the signals they provide for negative as well as positive selection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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H-2M-/- mice, H-2b haplotype (10), obtained from The Jackson Laboratories (Bar Harbor, ME), were bred and maintained at the Biomedical Services Unit, University of Birmingham. Embryos from these and wt C57/BL6 (H-2b haplotype) were obtained by timed matings, with the day of detection of the vaginal plug being designated as day zero.
Abs and immunoconjugates
Anti-rat and anti-mouse IgG-coated Dynabeads (Dynal, Wirral, U.K.) were coated with the following Abs, as appropriate: anti-CD3 (clone KT3; a kind gift of Dr. Julian Dyson, Medical Research Council Clinical Sciences Centre, London, U.K.), anti-CD8 (clone YTS 169.4; Sera Lab, Sussex, U.K.), anti-CD45 (clone M1/9; American Type Culture Collection, Manassas,VA), anti-IAb (clone AF6–120.1; PharMingen, San Diego, CA), anti-DEC205 (clone NLDC-145; Serotec, Oxford, U.K.). The following Abs were used for flow cytometry of isolated stromal cells and thymocytes: anti-CD8 FITC (clone 53–6.7), anti-CD4 phycoerythrin (PE) (clone GK1.5), anti-IAb PE (clone AF6–120.1; all from PharMingen). CLIP/IAb complexes were detected using 30–2 Ab (a kind gift of Dr. A. Y. Rudensky, Howard Hughes Medical Institute and Department of Immunology, University of Washington School of Medicine, Seattle, WA), followed by anti-mouse biotin (Caltag, South San Francisco, CA) and streptavidin APC (PharMingen).
Cell separations
Preparation of thymocytes. CD4+8+ thymocytes at a preselection stage of development were prepared from newborn thymus cell suspensions by immunomagnetic separation, exactly as described (16, 17). Such a procedure results in a population of preselection CD4+8+69-TCR- thymocytes at >98% purity (20).
Isolation of thymic stromal cells.
Thymuses from wt and H-2M-/- 15-day mouse embryos were
cultured in 2-deoxyguanosine for 5–7 days and trypsinized to form a
single-cell suspension, as described (16, 17, 20). After depletion of
residual hemopoietic elements with anti-CD45-coated beads, such
preparations consist of 
90% thymic epithelial cells (Refs. 16, 17,
and 20, and data not shown).
Isolation of DC. DC were isolated from the pooled axial, inguinal, and mesenteric lymph nodes of 4- to 6-wk-old wt and H-2M-/- mice by immunomagnetic selection. Briefly, lymph node suspensions were subject to positive selection with anti-IAb-coated magnetic beads, followed by further positive enrichment of DC using beads coated with anti-DEC205. Beads were then removed by pronase treatment, as described (21). Cells prepared in this manner have a characteristic dendritic-like appearance and act as potent APCs in T cell stimulation assays (not shown).
Formation of RTOCs
Freshly prepared thymocytes and appropriate stromal cells were mixed together in 1.5 ml Eppendorf tubes (Laser, Southampton, U.K.) at the necessary ratios and pelleted by centrifugation. Following removal of the supernatant, the cell pellet was carefully transferred to the surface of a 0.8 µm Nucleopore filter (Corning Costar U.K., High Wycombe, U.K.) in organ culture. Under such conditions, intact thymus lobes reform from these mixtures within 12–18 h (16, 17, 20).
Flow cytometric analysis
Thymocytes harvested from RTOCs and freshly isolated stromal cells were analyzed using a dual-laser Coulter Epics Elite machine (Coulter, Hialeah, FL) with forward and side scatter gates set so as to exclude nonviable cells, as described (17).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies have suggested that when positive selection is confined to a limited array of peptide/MHC class II complexes, the number of single-positive CD4+ cells and the diversity of repertoire generated are reduced compared with that seen in selection in a wt environment (14, 15). However, these studies are based on models in which the selecting ligands are expressed on both positively (thymic epithelial cells) and negatively (thymic DC of bone marrow origin) selecting cells, so that the overall outcome of selection reflects the balance of these two processes. Thus, while positive selection on a limited peptide set may be highly efficient, this could be reduced by exposure to the same set of peptides on cells mediating negative selection.
Using RTOCs, when purified CD4+8+ thymocytes can be exposed only to a single defined stromal cell type (cortical epithelial cells), it is possible to look at the efficiency of positive selection mediated by these cells in the absence of any influence from DC. Therefore, we compared the efficiency of CD4+ cell generation from purified CD4+8+ precursors with wt, as compared with H-2M-/-, thymic epithelium in the absence of DC. RTOCs with equivalent numbers of CD4+8+ thymocytes and either wt (H-2b) or H-2M-/- (H-2b) epithelial cells were prepared and harvested after 5 days. Thymocyte yields were determined and positive selection assessed by flow cytometric analysis of the number of CD4+8- and CD4-8+ cells.
Fig. 1
, a and c,
shows that in RTOCs, as expected (20), CD4+8-
and CD4-8+ cells are generated in the presence
of wt epithelium at a ratio of 3:1. However, in RTOCs initiated with
H-2M-/- thymic epithelial cells, although normal numbers
of CD4-8+ cells are generated (Fig. 1c), there is a marked reduction in the development of
CD4+8- cells, with a ratio between
CD4+8- and CD4-8+
cells being 1:1 (Fig. 1b), and an overall fourfold
reduction in the number of CD4+8- cells (Fig. 1c). Importantly, this reduction in
CD4+8- numbers generated by
H-2M-/- epithelial cells is not due to lower levels of
MHC class II expression, since flow cytometric analysis showed MHC
class II levels to be comparable on wt and H-2M-/-
epithelium (Fig. 2, a and
b). Interestingly, analysis of expression of
CLIP/IAb complexes using mAb 30-2 (22) showed that only a
fraction of class II molecules are occupied by CLIP peptides in wt
epithelium (Fig. 2c), while CLIP peptide expression is
predominant on H-2M-/- thymic epithelial cells (Fig. 2d). These data exclude the possibility that the reduced
generation of CD4+ cells in H-2M-/- mice is
due to the impact of negative selection by DC with a peptide/MHC class
II profile comparable to that on the epithelial cells mediating
positive selection. Thus, we have obtained direct evidence that peptide
diversity plays a key role in the efficiency of CD4+ T cell
selection by thymic epithelium.
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Having established a system whereby positive selection on thymic epithelium takes place unopposed by negative selection by DC, we were able to examine the effects of DC-mediated negative selection on the positively selected repertoire. Thus, we next analyzed the impact of negative selection by DC on the generation of positively selected CD4+ cells, both in terms of the efficiency of the deleting cells on a per cell basis and the effects of differences in peptide repertoire between cells mediating positive and negative selection events.
In an initial series of experiments, we first established the optimal
number of DC required to mediate negative selection. Previous studies
using chimeric organ cultures (23) have suggested that nonepithelial
hemopoietic elements are efficient mediators of negative selection,
although the precise identity and number of cells mediating this effect
were not defined. Therefore, we prepared RTOCs of wt epithelium,
defined numbers of preselection CD4+8+
thymocytes, and titrated numbers of either purified wt or
H-2M-/- DC. Cultures were harvested after 5 days and
examined for the effects of DC number on generation of CD4+
cells. As shown in Fig. 3, the addition
of wt DC to RTOCs with wt epithelial cells has a considerable impact on
the number of single-positive CD4+ cells generated. This
effect reached a plateau in the presence of 1% DC, when the yield of
CD4+ cells was reduced by 80%. These observations
confirm the potency of DC as mediators of negative selection and imply
that even when DC represent as few as 1% of the total cell number per
reaggregate culture, they can make contact with the majority of
thymocytes. Such findings agree well with the relative scarcity of DC
in the thymus as compared with thymic epithelial cells (8, 17). They
also show that more than two-thirds of the repertoire positively
selectable by wt epithelial cells is susceptible to removal by negative
selection. Previous estimates of the effects of negative selection on
the positively selected repertoire have ranged considerably (8, 12, 24, 25, 26), from as little as 5% (24) to as much as 50% (25). The
results presented here clearly provide further evidence for a high
degree of overlap between T cell selection processes.
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, e and f),
indicating that the magnitude of deletion seen with wt DC is a
reflection of the wider spectrum of peptides presented by the MHC class
II molecules on those cells. Conversely, the reduced effect of
H-2M-/- DC suggests that, although peptide recognition in
thymocyte selection may be promiscuous to a degree (14, 15), this is
not sufficient to allow a limited array of peptides to have a
predominant effect via negative selection on a repertoire positively
selected on a wider range of peptides.
When RTOCs were prepared in which positive selection occurred on
H-2M-/- epithelium, addition of wt DC caused an even more
dramatic reduction (95%) in the number of single-positive
CD4+ cells generated than that seen when positive selection
occurred on wt epithelium (Fig. 4). This
dramatic reduction, which is comparable to in vivo chimeric experiments
(14), is unlikely to be solely due to the recognition of CLIP peptides
on the wt DC, since H-2M-/- DC (expressing predominantly
CLIP/MHC class II complexes) have a less profound effect on the
generation of H-2M-/- selected CD4+ cells
(Fig. 4). Thus, the most likely explanation for these findings is that
many of the cells selected on peptide/self MHC complexes on
H-2M-/- epithelial cells have sufficiently high
TCR-mediated avidity for other peptide/self MHC complexes present on wt
DC to trigger negative selection. This is consistent with previous
observations that T cells selected on H-2M-/- epithelium
and allowed to reach maturity display a considerable degree of
"self" reactivity to homozygous wt DC in proliferation assays
(9, 10, 11). On this note, it may well be the case that CD4+
thymocytes positively selected by thymic epithelial cells in the
absence of DC, show a degree of reactivity to self peptide/MHC
complexes, since these cells would be negatively selected if allowed to
mature in the presence of DC. We are currently testing this possibility
in additional experiments.
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Although a role for DC in negative selection is well established,
the ability of epithelial cells to mediate negative selection is still
controversial (18, 27). The major impact of DC expressing a spectrum of
wt peptides on the limited T cell repertoire positively selected on MHC
class II/limited peptide complexes in H-2M-/- mice
demonstrated in the previous section provided us with an ideal model
with which to compare the efficiency of DC and epithelial cells in
mediating negative selection. For this purpose, RTOCs were prepared
from H-2M-/- epithelial cells and preselection
CD4+8+ thymocytes with the further addition of
either 1% wt DC or 1% wt thymic epithelial cells. As shown in Fig. 5b, while wt DC caused a
reduction in the numbers of CD4+8+ thymocytes,
and both mature CD4+8- and
CD4-8+ subsets, a similar number of wt
epithelial cells had no impact on the CD4+8+
population or the generation of CD4+8- cells
(Fig. 5c). Again, this is unlikely to be due to differences
in the overall level of MHC class II molecules on wt epithelium and wt
DC, since these two cell types were found to display comparable MHC
class II levels in flow cytometric analysis (Fig. 2, a and
e). These data therefore argue that thymic epithelial cells
and DC possess other qualitative differences that underlie their
different abilities in mediating positive and negative selection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Thus, while our data support the notion that
peptide diversity bound to MHC class II molecules on thymic epithelial
cells plays a key role in the efficiency of positive selection, the
degree of positive selection mediated by CLIP and other non-CLIP
peptides is uncertain. Our findings also address the issue of stromal cell specialization for positive and negative selection. The ability of small numbers of DC (i.e., no more than 1% of total cell number per reaggregate lobe) to delete a substantial proportion of the CD4+ cells selectable on thymic epithelium emphasizes the efficiency of these cells as mediators of negative selection.
In marked contrast to the ability of small numbers of wt DC to cause a
dramatic reduction in CD4+ cells capable of positive
selection on H-2M-/- epithelium, addition of the same
number of wt epithelial cells had no discernible effect. Since the
overall levels of MHC class II molecules expressed on wt DC and wt
epithelium were found to be similar (Fig. 2), this argues that there
are other qualitative differences underlying the differing ability of
these cells to mediate negative selection. These differences may be
overridden where avidity dependent on TCR alone is high, as in the case
of some TCR transgenic mice with higher than normal levels of TCR
expression on CD4+8+ thymocytes, especially
where high levels of cognate peptide were also present. Such factors
may explain the reported ability of thymic epithelium, albeit from
adult as compared with our fetal sources, to mediate negative selection
in some models (18). However, within the range of TCR expression on
normal thymocytes, as used in this study, differences in the ability to
provide costimulatory signals may be one factor in determining the
differential efficiency of epithelium and DC in mediating negative
selection. In this regard, it is interesting that B7 expression is
absent from thymic epithelial cells but present on thymic DC (29),
especially as its ligand, CD28, is expressed on cortical
thymocytes, and B7/CD28 costimulation has been shown to influence
negative selection (30, 31). Similarly, differential expression of cell
adhesion molecules affecting the overall avidity of T cell/stromal cell
interaction may be important in determining outcomes of selection and
will be an area for further study.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Graham Anderson, Department of Anatomy, Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cells; RTOC, reaggregate thymus organ culture; wt, wild type; PE, phycoerythrin.
Received for publication June 15, 1998. Accepted for publication August 28, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain genes maximize the production of useful thymocytes. J. Exp. Med. 178:615.This article has been cited by other articles:
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