Differential Effects of Peptide Diversity and Stromal Cell Type in Positive and Negative Selection in the

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Differential Effects of Peptide Diversity and Stromal Cell Type in Positive and Negative Selection in the Thymus1

Graham Anderson,2 Katharine M. Partington, and Eric J. Jenkinson

Thymocyte positive selection results in maturation to the single-positive stage, while negative selection results in death by apo- ptosis. Although kinetic analyses indicate only 3–5% of CD4؉8؉ cells reach the single-positive stage, the balance of positive and negative selection and the nature and quantity of cells mediating maximal negative selection are uncertain. Here, using a system where the number and type of stromal cells and thymocytes can be controlled, we investigated the maturation of CD4؉8؉ thymocytes in the presence or absence of thymic epithelium and dendritic cells (DC) from wild-type (wt) and H-2M؊/؊ mice expressing different peptide arrays. We find that titration of wt DC into reaggregates of wt epithelium has a dramatic effect on the number of CD4؉ cells generated, with 1% DC causing a maximal 80% reduction. Moreover, while addition of 1% wt DC into ؊/؊ ؉ cultures of H-2M epithelium causes a 90% reduction in CD4 cells, no effect was observed when similar numbers of wt thymic Downloaded from epithelium were added. Collectively, these data provide the first accurate indication of the quantity and quality of stromal cells required for maximal negative selection in the thymus, demonstrate the importance of peptide diversity in selection, and highlight a large degree of overlap between positive and negative selection events. The Journal of Immunology, 1998, 161: 6599–6603.

election of the ␣␤TCR repertoire is essential for the pro- versity is important in generating a fully diverse TCR repertoire http://www.jimmunol.org/ duction of a T cell population that is capable of efficient during positive selection (13–15). S recognition of self MHC molecules presenting nonself In addition to TCR ligation of the appropriate avidity, there is peptides but that is nonreactive to the same MHC molecules pre- also evidence that other factors are involved in determining the senting self peptides (1, 2). This process of repertoire selection outcome of thymic selection. Thus, efficient positive selection is takes place at the CD4ϩ8ϩ stage of T cell development in the dependent upon interaction with peptide/MHC complexes on thy- thymus and involves positive selection, triggering further matura- mic cortical epithelial cells (16), while negative selection is most tion to the single-positive CD4ϩ8Ϫ and CD4Ϫ8ϩ stages (3) and efficiently mediated by professional APCs of bone marrow origin, negative selection that induces programmed cell death in poten- namely dendritic cells (DC)3 (17, 18). At present, it is not clear tially autoreactive thymocytes (4). whether this cellular specialization for selection involves: differ- by guest on September 29, 2021 A key factor in determining whether a CD4ϩ8ϩ thymocyte un- ential expression of MHC-bound peptide arrays on positively or dergoes positive or negative selection is the avidity of its TCR- negatively selecting cells, the expression of a specialized peptide mediated interaction with peptide/MHC complexes on thymic stro- repertoire on positively selecting cells, or differential expression of mal cells (5, 6). CD4ϩ8ϩ thymocytes are known to have a finite costimulatory molecules that provide additional signals leading to lifespan of 3–4 days in the thymic cortex, during which time suc- either cell differentiation or cell death (19). cessive ␣␤TCR combinations, generated by ongoing TCR ␣-chain In this study, we explore the interaction of peptide diversity and gene rearrangements (7), are tested for interaction with stromal selecting cell type in T cell selection events, using reaggregate peptide/MHC ligands. Those cells that fail to interact with stromal thymus organ cultures (RTOCs) to recombine thymic epithelial MHC complexes during this time die from neglect (8), while low cells and DC from wild-type (wt) or H-2MϪ/Ϫ mice with a re- avidity interactions lead to positive selection and survival, and stricted presentation of MHC class II-bound peptides (9–11). Our high avidity interactions lead to negative selection through apo- results show that when any influence of negative selection by DC ptosis. Recent evidence suggests that there is a considerable degree is excluded, positive selection by epithelial cells bearing a limited of promiscuity in TCR-mediated peptide/MHC recognition, such MHC class II-bound peptide spectrum is reduced threefold com- that a single or a limited array of peptides can positively select pared with positive selection by wt epithelium. Importantly, this is cells that are able to recognize a range of other peptides presented not due to differences in MHC class II levels on H-2MϪ/Ϫ and wt by the same MHC molecule (9–12). However, although varied, the epithelial cells, and thus provides direct evidence for the role of diversity of such repertoires is limited, indicating that peptide di- 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 Ϫ/Ϫ Department of Anatomy, Medical School, University of Birmingham, Edgbaston, selection of T cell repertoires generated on both wt and H-2M ϩ Birmingham, United Kingdom. epithelial cells. Remarkably, we find that the number of CD4 Received for publication June 15, 1998. Accepted for publication August 28, 1998. cells generated by wt epithelium is reduced by 80% in the presence ϩ The costs of publication of this article were defrayed in part by the payment of page of small numbers of wt DC (1%), while the generation of CD4 charges. This article must therefore be hereby marked advertisement in accordance cells in the presence of H-2MϪ/Ϫ epithelium is even further re- with 18 U.S.C. Section 1734 solely to indicate this fact. duced (by 95%) by the same number of wt DC, emphasizing the 1 This work was supported by a Wellcome Trust project grant to G.A. 2 Address correspondence and reprint requests to Dr. Graham Anderson, Department of Anatomy, Medical School, University of Birmingham, Edgbaston, Birmingham, 3 Abbreviations used in this paper: DC, dendritic cells; RTOC, reaggregate thymus B15 2TT, U.K. E-mail address: [email protected] organ culture; wt, wild type; PE, phycoerythrin.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 6600 ROLE OF PEPTIDE DIVERSITY AND STROMAL CELLS IN T CELL SELECTION 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 posi- tively 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.

Materials and Methods Mice H-2MϪ/Ϫ mice, H-2b haplotype (10), obtained from The Jackson Labora- tories (Bar Harbor, ME), were bred and maintained at the Biomedical Ser- vices 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 Downloaded from

Anti-rat and anti-mouse IgG-coated Dynabeads (Dynal, Wirral, U.K.) were Ϫ Ϫ FIGURE 1. Positive selection by H-2M / thymic epithelium under- coated with the following Abs, as appropriate: anti-CD3 (clone KT3; a kind lines a role for peptide diversity in maturation of CD4ϩ T cells. Reaggre- gift of Dr. Julian Dyson, Medical Research Council Clinical Sciences Cen- ϩ ϩ Ϫ tre, London, U.K.), anti-CD8 (clone YTS 169.4; Sera Lab, Sussex, U.K.), gate cultures were made from equal numbers of CD4 8 TCR thymo- Ϫ Ϫ anti-CD45 (clone M1/9; American Type Culture Collection, Manas- cytes and either wt (a) or H-2M / thymic epithelial cells (b). Cultures sas,VA), anti-IAb (clone AF6–120.1; PharMingen, San Diego, CA), anti- were harvested after 5 days and analyzed for CD4CD8 expression by flow

DEC205 (clone NLDC-145; Serotec, Oxford, U.K.). The following Abs cytometry. c, Cell numbers of the CD4CD8 subsets shown in a and b.In http://www.jimmunol.org/ were used for flow cytometry of isolated stromal cells and thymocytes: the experiment shown, 8 ϫ 105 thymocytes were used as an input, with anti-CD8 FITC (clone 53–6.7), anti-CD4 phycoerythrin (PE) (clone ϫ 5 ϫ 5 Ϫ/Ϫ b b 1.6 10 and 1.1 10 collected from wt and H-2M RTOCs, respec- GK1.5), anti-IA PE (clone AF6–120.1; all from PharMingen). CLIP/IA tively. Similar results were obtained from three separate experiments. complexes were detected using 30–2 Ab (a kind gift of Dr. A. Y. Ruden- sky, 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). Results Effect of peptide diversity on the efficiency of positive selection

Cell separations by guest on September 29, 2021 Recent studies have suggested that when positive selection is con- ϩ ϩ Preparation of thymocytes. CD4 8 thymocytes at a preselection stage fined to a limited array of peptide/MHC class II complexes, the of development were prepared from newborn thymus cell suspensions by number of single-positive CD4ϩ cells and the diversity of reper- immunomagnetic separation, exactly as described (16, 17). Such a proce- dure results in a population of preselection CD4ϩ8ϩ69ϪTCRϪ thymocytes toire generated are reduced compared with that seen in selection in at Ͼ98% purity (20). a wt environment (14, 15). However, these studies are based on Isolation of thymic stromal cells. Thymuses from wt and H-2MϪ/Ϫ 15- models in which the selecting ligands are expressed on both pos- day mouse embryos were cultured in 2-deoxyguanosine for 5–7 days and itively (thymic epithelial cells) and negatively (thymic DC of bone trypsinized to form a single-cell suspension, as described (16, 17, 20). marrow origin) selecting cells, so that the overall outcome of se- After depletion of residual hemopoietic elements with anti-CD45-coated beads, such preparations consist of ϳ90% thymic epithelial cells (Refs. 16, lection reflects the balance of these two processes. Thus, while 17, and 20, and data not shown). positive selection on a limited peptide set may be highly efficient, Isolation of DC. DC were isolated from the pooled axial, inguinal, and this could be reduced by exposure to the same set of peptides on Ϫ/Ϫ mesenteric lymph nodes of 4- to 6-wk-old wt and H-2M mice by im- cells mediating negative selection. munomagnetic selection. Briefly, lymph node suspensions were subject to ϩ ϩ positive selection with anti-IAb-coated magnetic beads, followed by further Using RTOCs, when purified CD4 8 thymocytes can be ex- positive enrichment of DC using beads coated with anti-DEC205. Beads posed only to a single defined stromal cell type (cortical epithelial were then removed by pronase treatment, as described (21). Cells prepared cells), it is possible to look at the efficiency of positive selection in this manner have a characteristic dendritic-like appearance and act as mediated by these cells in the absence of any influence from DC. potent APCs in T cell stimulation assays (not shown). Therefore, we compared the efficiency of CD4ϩ cell generation from purified CD4ϩ8ϩ precursors with wt, as compared with Formation of RTOCs H-2MϪ/Ϫ, thymic epithelium in the absence of DC. RTOCs with ϩ ϩ b Freshly prepared thymocytes and appropriate stromal cells were mixed equivalent numbers of CD4 8 thymocytes and either wt (H-2 ) together in 1.5 ml Eppendorf tubes (Laser, Southampton, U.K.) at the nec- or H-2MϪ/Ϫ (H-2b) epithelial cells were prepared and harvested essary ratios and pelleted by centrifugation. Following removal of the su- after 5 days. Thymocyte yields were determined and positive se- pernatant, the cell pellet was carefully transferred to the surface of a 0.8 ␮ lection assessed by flow cytometric analysis of the number of m Nucleopore filter (Corning Costar U.K., High Wycombe, U.K.) in ϩ Ϫ Ϫ ϩ organ culture. Under such conditions, intact thymus lobes reform from CD4 8 and CD4 8 cells. these mixtures within 12–18 h (16, 17, 20). Fig. 1, a and c, shows that in RTOCs, as expected (20), CD4ϩ8Ϫ and CD4Ϫ8ϩ cells are generated in the presence of wt Flow cytometric analysis epithelium at a ratio of 3:1. However, in RTOCs initiated with H-2MϪ/Ϫ thymic epithelial cells, although normal numbers of Thymocytes harvested from RTOCs and freshly isolated stromal cells were Ϫ ϩ CD4 8 cells are generated (Fig. 1c), there is a marked reduction analyzed using a dual-laser Coulter Epics Elite machine (Coulter, Hialeah, ϩ Ϫ FL) with forward and side scatter gates set so as to exclude nonviable cells, in the development of CD4 8 cells, with a ratio between ϩ Ϫ Ϫ ϩ as described (17). CD4 8 and CD4 8 cells being ϳ1:1 (Fig. 1b), and an overall The Journal of Immunology 6601

FIGURE 3. Quantitative analysis of the effect of DC on positive selec- tion and the importance of peptide diversity. RTOCs of a 1:1 mixture of wt thymic epithelial cells and CD4ϩ8ϩTCRϪ thymocytes were set up either in the absence or presence of predetermined numbers of freshly prepared wt or H-2MϪ/Ϫ DC. The frequency of DC added is expressed as a proportion of the total number of cells per RTOC (epithelial cells and thymocytes

combined). After 5 days of culture, RTOCs were harvested, and viable Downloaded from FIGURE 2. Comparison of expression of TCR ligands on thymic epi- cells were counted and stained for CD4 and CD8. In the experiment shown, thelial cells and DC. Freshly trypsinized 2-dGuo-treated thymus lobes from 5 ϫ 105 thymocytes were used as an input, with the following yields Ϫ/Ϫ wt and H-2M embryos were analyzed for expression of I-A molecules obtained: no DC 16 ϫ 104, wt DC: 0.25% ϭ 8.5 ϫ 104; 0.5% ϭ 6 ϫ 104; (a and b) and CLIP/I-A complexes with the mAb 30–2 (c and d). Analysis 1% ϭ 3 ϫ 104; 2.5% ϭ 5 ϫ 104;5%ϭ 5 ϫ 104. H-2MϪ/Ϫ DC: 1% ϭ of I-A levels on lymph node DC was performed by two-color labeling of 12 ϫ 104; 10% ϭ 13 ϫ 104. This experiment was performed twice in its b lymph node suspensions for I-A PE and CD11c FITC. Histograms shown entirety with similar results. Ϫ/Ϫ are for wt (e) and H-2M (f) cells with forward/side scatter character- http://www.jimmunol.org/ istics of DCs, gating on CD11cbright cells. Negative controls were set up using an irrelevant isotype matched Ab. H-2MϪ/Ϫ DC. Cultures were harvested after 5 days and examined for the effects of DC number on generation of CD4ϩ cells. As shown in Fig. 3, the addition of wt DC to RTOCs with wt epithelial ϩ Ϫ fourfold reduction in the number of CD4 8 cells (Fig. 1c). Im- cells has a considerable impact on the number of single-positive ϩ Ϫ portantly, this reduction in CD4 8 numbers generated by CD4ϩ cells generated. This effect reached a plateau in the presence Ϫ Ϫ H-2M / epithelial cells is not due to lower levels of MHC class of 1% DC, when the yield of CD4ϩ cells was reduced by ϳ80%. II expression, since flow cytometric analysis showed MHC class II These observations confirm the potency of DC as mediators of

Ϫ Ϫ by guest on September 29, 2021 levels to be comparable on wt and H-2M / epithelium (Fig. 2, a negative selection and imply that even when DC represent as few and b). Interestingly, analysis of expression of CLIP/IAb com- as 1% of the total cell number per reaggregate culture, they can plexes using mAb 30-2 (22) showed that only a fraction of class II make contact with the majority of thymocytes. Such findings agree molecules are occupied by CLIP peptides in wt epithelium (Fig. well with the relative scarcity of DC in the thymus as compared Ϫ Ϫ 2c), while CLIP peptide expression is predominant on H-2M / with thymic epithelial cells (8, 17). They also show that more than thymic epithelial cells (Fig. 2d). These data exclude the possibility two-thirds of the repertoire positively selectable by wt epithelial ϩ Ϫ Ϫ that the reduced generation of CD4 cells in H-2M / mice is due cells is susceptible to removal by negative selection. Previous es- to the impact of negative selection by DC with a peptide/MHC timates of the effects of negative selection on the positively se- class II profile comparable to that on the epithelial cells mediating lected repertoire have ranged considerably (8, 12, 24–26), from as positive selection. Thus, we have obtained direct evidence that little as 5% (24) to as much as 50% (25). The results presented here ϩ peptide diversity plays a key role in the efficiency of CD4 T cell clearly provide further evidence for a high degree of overlap be- selection by thymic epithelium. tween T cell selection processes. In marked contrast to the effects seen with wt DC, H-2MϪ/Ϫ DC Differential effects of peptide diversity in negative selection expressing a limited array of MHC class II bound peptides had a Having established a system whereby positive selection on thymic much less dramatic effect on the number of CD4ϩ cells that were epithelium takes place unopposed by negative selection by DC, we produced in the presence of wt epithelium, causing a reduction of were able to examine the effects of DC-mediated negative selec- only 20% even when added at a frequency of 10% of total cell tion on the positively selected repertoire. Thus, we next analyzed number. This excludes the possibility that the extensive deletion the impact of negative selection by DC on the generation of pos- seen with wt cells is due to the nonspecific effects of incorporating itively selected CD4ϩ cells, both in terms of the efficiency of the DC into RTOC. Importantly, overall levels of MHC class II ex- deleting cells on a per cell basis and the effects of differences in pression on wt and H-2MϪ/Ϫ DC were found to be the same (Fig. peptide repertoire between cells mediating positive and negative 2, e and f), indicating that the magnitude of deletion seen with wt selection events. DC is a reflection of the wider spectrum of peptides presented by In an initial series of experiments, we first established the op- the MHC class II molecules on those cells. Conversely, the re- timal number of DC required to mediate negative selection. Pre- duced effect of H-2MϪ/Ϫ DC suggests that, although peptide rec- vious studies using chimeric organ cultures (23) have suggested ognition in thymocyte selection may be promiscuous to a degree that nonepithelial hemopoietic elements are efficient mediators of (14, 15), this is not sufficient to allow a limited array of peptides negative selection, although the precise identity and number of to have a predominant effect via negative selection on a repertoire cells mediating this effect were not defined. Therefore, we pre- positively selected on a wider range of peptides. pared RTOCs of wt epithelium, defined numbers of preselection When RTOCs were prepared in which positive selection oc- CD4ϩ8ϩ thymocytes, and titrated numbers of either purified wt or curred on H-2MϪ/Ϫ epithelium, addition of wt DC caused an even 6602 ROLE OF PEPTIDE DIVERSITY AND STROMAL CELLS IN T CELL SELECTION

FIGURE 5. Qualitative differences in the ability of thymic epithelial cells and DC in mediating deletion of CD4ϩ8ϩ thymocytes. Equal numbers of CD4ϩ8ϩTCRϪ thymocytes and H-2MϪ/Ϫ thymic epithelial cells were reaggregated either in the absence (a) or presence of 1% wt DC (b)or1% wt epithelial cells (c). After 4 days, cultures were harvested and viable

FIGURE 4. Introduction of wt DC has a dramatic impact on positive thymocytes analyzed for CD4CD8 expression by flow cytometry. In the Downloaded from selection by H-2MϪ/Ϫ thymic epithelium. RTOCs were made from 1:1 experiment shown, 1 ϫ 106 thymocytes were used as an input, with the mixtures of CD4ϩ8ϩTCRϪ thymocytes and H-2MϪ/Ϫ thymic epithelial following recoveries: control: 4.7 ϫ 105;1%DC0.5ϫ 105; 1% epithelial cells, and cultured for 5 days in the absence or presence of 1% H-2MϪ/Ϫ cells 5.6 ϫ 105. Similar results were obtained from three separate or wt DC. Viable thymocytes were harvested, counted, and analyzed for experiments. expression of CD4 and CD8. Data shown are the numbers of CD4ϩ8Ϫ thymocytes harvested from such cultures. Yields, from an input population Ϫ Ϫ of 8 ϫ 105 thymocytes are as follows: no DC ϭ 1.6 ϫ 105; 1% H-2M / http://www.jimmunol.org/ 5 5 DC ϭ 1.3 ϫ 10 ;1%wtDC0.3ϫ 10 . This experiment was performed ϩ ϩ three times with similar results. wt DC caused a reduction in the numbers of CD4 8 thymocytes, and both mature CD4ϩ8Ϫ and CD4Ϫ8ϩ subsets, a similar number of wt epithelial cells had no impact on the CD4ϩ8ϩ population or the generation of CD4ϩ8Ϫ cells (Fig. 5c). Again, this is unlikely to more dramatic reduction (95%) in the number of single-positive ϩ be due to differences in the overall level of MHC class II molecules CD4 cells generated than that seen when positive selection oc- on wt epithelium and wt DC, since these two cell types were found curred on wt epithelium (Fig. 4). This dramatic reduction, which is to display comparable MHC class II levels in flow cytometric anal- comparable to in vivo chimeric experiments (14), is unlikely to be

ysis (Fig. 2, a and e). These data therefore argue that thymic ep- by guest on September 29, 2021 solely due to the recognition of CLIP peptides on the wt DC, since Ϫ Ϫ ithelial cells and DC possess other qualitative differences that un- H-2M / DC (expressing predominantly CLIP/MHC class II derlie their different abilities in mediating positive and negative complexes) have a less profound effect on the generation of Ϫ Ϫ ϩ selection. H-2M / selected CD4 cells (Fig. 4). Thus, the most likely ex- planation for these findings is that many of the cells selected on peptide/self MHC complexes on H-2MϪ/Ϫ epithelial cells have Discussion sufficiently high TCR-mediated avidity for other peptide/self MHC In this study, we have provided direct evidence that diversity of complexes present on wt DC to trigger negative selection. This is peptide presentation by MHC class II molecules on thymic epi- consistent with previous observations that T cells selected on thelial cells increases the efficiency with which CD4ϩ cells are Ϫ Ϫ H-2M / epithelium and allowed to reach maturity display a con- positively selected. We also show that much of this selectable rep- siderable degree of “self” reactivity to homozygous wt DC in pro- ertoire is potentially autoreactive and is susceptible to negative liferation assays (9–11). On this note, it may well be the case that selection. This effect is particularly dramatic when positive selec- ϩ CD4 thymocytes positively selected by thymic epithelial cells in tion is mediated by epithelial cells expressing MHC class II mol- the absence of DC, show a degree of reactivity to self peptide/ ecules bearing a limited array of peptides, and negative selection is MHC complexes, since these cells would be negatively selected if mediated by wt DC presenting a more diverse peptide array. Thus, allowed to mature in the presence of DC. We are currently testing consistent with the reported promiscuity of TCR-mediated peptide/ this possibility in additional experiments. MHC recognition (9–12), there appears to be considerable overlap between positive and negative selection, even when they are me- Efficiency of different stromal cell types in mediating negative diated by different peptide arrays. Moreover, these effects imply selection that confining positive selection to a limited array of specialized Although a role for DC in negative selection is well established, peptides could place severe constraints on repertoire diversity. the ability of epithelial cells to mediate negative selection is still However, it should be noted that even when MHC class II mole- controversial (18, 27). The major impact of DC expressing a spec- cules are predominantly loaded with a single peptide, as in the case trum of wt peptides on the limited T cell repertoire positively se- of H-2MϪ/Ϫ mice and the CLIP peptide, Grubin et al. (28) have lected on MHC class II/limited peptide complexes in H-2MϪ/Ϫ shown that other MHC class II-bound peptides are present in these mice demonstrated in the previous section provided us with an mice that play a direct role in positive selection of CD4ϩ thymo- ideal model with which to compare the efficiency of DC and ep- cytes. Indeed, in agreement with the findings of Grubin et al. (28), ithelial cells in mediating negative selection. For this purpose, our analysis of CLIP expression on MHC class IIϩ thymic epithe- RTOCs were prepared from H-2MϪ/Ϫ epithelial cells and prese- lial cells from H-2MϪ/Ϫ mice shows that some of these cells do lection CD4ϩ8ϩ thymocytes with the further addition of either 1% not express CLIP (Fig. 2). Thus, while our data support the notion wt DC or 1% wt thymic epithelial cells. As shown in Fig. 5b, while that peptide diversity bound to MHC class II molecules on thymic The Journal of Immunology 6603 epithelial cells plays a key role in the efficiency of positive selec- 8. Surh, C. D., and J. Sprent. 1994. T-cell apoptosis detected in situ during positive tion, the degree of positive selection mediated by CLIP and other and negative selection in the thymus. Nature 372:100. 9. Miyazaki, T., P. Wolf, S. Tourne, C. Waltzinger, A. Dierich, N. Barois, non-CLIP peptides is uncertain. H. Ploegh, C. Benoist, D. Mathis. 1996. Mice lacking H2-M complexes, enig- Our findings also address the issue of stromal cell specialization matic elements of the MHC class II peptide-loading pathway. Cell 84:531. for positive and negative selection. The ability of small numbers of 10. Martin, W. D., G. G. Hicks, S. K. Mendiratta, H. I. Leva, H. E. Ruley, and L. van Kaer. 1996. H-2M mutant mice are defective in the peptide loading of DC (i.e., no more than 1% of total cell number per reaggregate class II molecules, , and T cell repertoire selection. Cell 84: ϩ lobe) to delete a substantial proportion of the CD4 cells select- 543. able on thymic epithelium emphasizes the efficiency of these cells 11. Fung-Leung, W. P., C. D. Surh, M. Liljedahl, J. Pang, D. Leturcq, P. A. Peterson, S. R. Webb, and L. Karlsson. 1996. Antigen presentation and T cell development as mediators of negative selection. in H2-M-deficient mice. Science 271:1278. In marked contrast to the ability of small numbers of wt DC to 12. Ignatowicz, L., J. Kappler, and P. Marrack. 1996. The repertoire of T cells shaped cause a dramatic reduction in CD4ϩ cells capable of positive se- by a single MHC/peptide ligand. Cell 84:521. Ϫ/Ϫ 13. Bevan, M. J. 1997. In thymic selection, peptide diversity gives and takes away. lection on H-2M epithelium, addition of the same number of Immunity 7:175. wt epithelial cells had no discernible effect. Since the overall levels 14. Surh, C. D., D. S. Lee, W. P. Fung-Leung, L. Karlsson, and J. Sprent. 1997. Thymic selection by a single MHC/peptide ligand produces a semidiverse rep- of MHC class II molecules expressed on wt DC and wt epithelium ϩ were found to be similar (Fig. 2), this argues that there are other ertoire of CD4 T-cells. Immunity 7:209. 15. Tourne, S., T. Miyazaki, A. Oxenius, L. Klein, T. Fehr, B. Kyewski, C. Benoist, qualitative differences underlying the differing ability of these cells and D. Mathis. 1997. Selection of a broad repertoire of CD4ϩ T-cells in to mediate negative selection. These differences may be overridden H-2MaϪ/Ϫ mice. Immunity 7:187. 16. Anderson, G., J. J. T. Owen, N. C. Moore, and E. J. Jenkinson. 1994. Thymic where avidity dependent on TCR alone is high, as in the case of ϩ ϩ epithelial cells provide unique signals for the positive selection of CD4 CD8 Downloaded from some TCR transgenic mice with higher than normal levels of TCR thymocytes in vitro. J. Exp. Med. 179:847. ϩ ϩ expression on CD4 8 thymocytes, especially where high levels 17. Jenkinson, E. J., G. Anderson, and J. J. T. Owen. 1992. Studies on T-cell mat- of cognate peptide were also present. Such factors may explain the uration on defined thymic stromal cells in vitro. J. Exp. Med. 176:745. 18. Volkmann, A., T. Zal, and B. Stockinger. 1997. Ag-presenting cells in the thymus reported ability of thymic epithelium, albeit from adult as com- that can negatively select MHC class II-restricted T cells recognizing a circulat- pared with our fetal sources, to mediate negative selection in some ing self antigen. J. Immunol. 158:693. models (18). However, within the range of TCR expression on 19. Kisielow, P., and H. von Boehmer. 1995. Development and selection of T cells: facts and puzzles. Adv. Immunol. 58:87. normal thymocytes, as used in this study, differences in the ability http://www.jimmunol.org/ 20. Anderson, G., N. C. Moore, J. J. T. Owen, and E. J. Jenkinson. 1994. Charac- to provide costimulatory signals may be one factor in determining teristics of an in vitro system of thymocyte positive selection. J. Immunol. 153: the differential efficiency of epithelium and DC in mediating neg- 1915. 21. Anderson, G., E. J. Jenkinson, N. C. Moore, and J. J. T. Owen. 1993. Both MHC ative selection. In this regard, it is interesting that B7 expression is ϩ class II thymic epithelial and mesenchyme cells are required for T-cell devel- absent from thymic epithelial cells but present on thymic DC (29), opment in the thymus. Nature 362:70. especially as its ligand, CD28, is expressed on cortical thymocytes, 22. Eastman, S., M. Deftos, P. C. DeRoos, D. -H. Hsu, L. Teyton, N. Braunstein, and B7/CD28 costimulation has been shown to influence negative C. J. Hackett, and A. Y. Rudensky. 1996. A study of invariant chain peptide CLIP:MHC class II complexes using new complex-specific monoclonal antibody. selection (30, 31). Similarly, differential expression of cell adhe- Eur. J. Immunol. 26:385. sion molecules affecting the overall avidity of T cell/stromal cell 23. Merkenschlager, M., C. Benoist, and D. Mathis. 1994. Evidence for a single- interaction may be important in determining outcomes of selection niche model of positive selection. Proc. Natl. Acad. Sci. USA 91:11694. by guest on September 29, 2021 24. Laufer, T. M., J. DeKoning, J. S. Markowitz, D. Lo, and L. H. Glimcher. 1996. and will be an area for further study. Unopposed positive selection and autoreactivity in mice expressing class II MHC only on thymic cortex. Nature 383:81. Acknowledgments 25. van Meerwijk, J. P. M., S. Marguerat, R. K. Lees, R. N. Germain, B. J. Fowlkes, and H.R. MacDonald. 1997. Quantitative impact of thymic clonal deletion on the We thank Dr. Alexander Rudensky for his kind gift of 30-2 Ab. T-cell repertoire. J. Exp. Med. 185:377. 26. Merkenschlager M., D. Graf, M. Lovatt, U. Bommhardt, R. Zamoyska, and A. G. Fisher. 1997. How many thymocytes audition for selection? J. Exp. Med. References 186:1149. 27. Bandeira, A., A. Coutinho, O. Burlen-Defranoux, I. Khazaal, M. Coltey, 1. Fink, P. J., and M. J. Bevan. 1995. Positive selection of thymocytes. Adv. Im- F. Jacquemart, N. Le Douarin, and J. Salaun. 1992. Thymic epithelium induces munol. 59:99. neither clonal deletion not anergy to Mls1a antigens. Eur. J. Immunol. 22:1397. 2. Benoist, C., and D. Mathis. 1997. Positive selection of T-cells: fastidious or promiscuous? Curr. Opin. Immunol. 9:245. 28. Grubin, C. E., S. Kovats, P. deRoos, and A. Y. Rudensky. 1997. Deficient pos- 3. Jameson, S. C., K. A. Hogquist, and M. J. Bevan. 1995. 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