In vitro positive selection of αβ TCR transgenic thymocytes by a conditionally immortalized cortical epithelial clone

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1 iim$$$0304 International Immunology, Vol. 9, No. 3, pp Oxford University Press In vitro positive selection of αβ TCR transgenic thymocytes by a conditionally immortalized cortical epithelial clone Yujiro Tanaka, Owen Williams, Raquel Tarazona, Andreas Wack, Trisha Norton and Dimitris Kioussis Division of Molecular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK Keywords: reaggregate culture, TCR transgenic mouse, thymic cortical epithelial clone, thymic selection Abstract Development of mature CD4 and CD8 single-positive T cells requires a process known as positive selection, which depends on the specific recognition of self-peptide MHC complexes on thymic stromal cells by immature CD4 CD8 thymocytes. We have used an in vitro reaggregate system to study the positive selection of thymocytes by conditionally immortalized thymic epithelial clones. Thymocytes from mice transgenic for the F5 αβ TCR, specific for a peptide from the influenza nucleoprotein in the context of H-2D b, are positively selected in the H-2 b MHC background, but fail to mature in mice expressing the H-2 q haplotype. Development of embryonic day 15 F5 H-2 q transgenic thymocytes was followed in reaggregate cultures supplemented with H-2 b -expressing epithelial clones. A conditionally immortalized cortical epithelial clone, derived from H-2K b -tsa58 transgenic mice, was found to be as efficient as freshly isolated thymic stromal cells in positively selecting CD8 transgenic thymocytes. In contrast, an H-2 b -expressing kidney epithelial clone did not augment positive selection above background levels, implying that the effect of the thymic epithelial clone was not merely the presentation of selecting MHC molecules. Mature transgenic thymocytes generated in reaggregate cultures were able to differentiate into functionally competent cytotoxic T cells. This model provides an important in vitro system for the detailed study of the specific molecular interactions leading to positive selection of developing thymocytes. Introduction Immature thymocytes require interaction of their TCR with MHC complexes. Establishing the contribution of individual thymic MHC molecules in order to mature and eventually cell types to this process was greatly facilitated by the migrate to peripheral lymphoid organs. This process, known development of in vitro thymic reaggregation cultures (10). as positive selection, was originally described by analysing Studies using such cultures point towards a unique role for the capacity of selected T cells to recognize antigens in the thymic cortical epithelial cells in positive selection (11,12). context of selecting MHC molecules using bone marrow Identification of thymic epithelial molecules involved in such chimaeras and later by following the fate of bulk thymocyte a role is complicated by the inherent heterogeneity of thymic populations in TCR transgenic mice (1 4). stroma and the consequent difficulty in obtaining large num- Many studies attempting to identify the cell types respons- bers of pure cells. ible for thymocyte selection have indicated that thymic epithelial, We have previously reported the generation and character- fibroblast and haematopoietic cells could cause positive ization of conditionally immortalized thymic epithelial clones selection of thymocytes in vivo (5 9). However, since endogenous from H-2K b -tsa58/h-2 b transgenic mice (13). In the present epithelial cells were present in all these studies it was study we have used one such clone to induce positive impossible to rule out the importance of epithelial cell-specific selection of immature thymocytes from embryonic day 15 αβ signals distinct from expression of the selecting peptide TCR (F5) transgenic mice (14,15) in in vitro reaggregate The first two authors contributed equally to this work Correspondence to: D. Kioussis Transmitting editor: E. Simpson Received 20 June 1996, accepted 21 November 1996

2 382 Positive selection by an epithelial cell line in vitro Table 1. Phenotype of II-4 cells a Marker Staining of II4 cells H-2D b H-2I-A b Cytokeratin 4F1 NLDC-145 Mac-1 N418 CTLA-4 ligands a II-4 cells were stained for the various markers and analysed by flow cytometry or in the case of 4F1 and cytokeratin by immunohistochemistry. Plus and minus signs represent expression of particular markers and their relative staining intensity. cultures. We demonstrate that the thymic epithelial clone is more efficient at inducing thymocyte positive selection than a kidney epithelial clone bearing the restricting MHC molecule and that mature CD8 thymocytes generated in such cultures can give rise to functionally competent effector cells in vitro. Methods Mice Mice transgenic for the αβ TCR from the F5 cytotoxic T cell clone were generated in our laboratory as previously reported (15). Mice deficient in RAG-1 gene expression, generated by homologous recombination, were obtained from Dr Eugenia Spanopoulou (16). Inbred C57BL/10 and SWR mice were maintained in colonies at the institute. C57BL/10 mouse embryos at embryonic day 14 and F5 TCR/RAG-1 / /H-2 q mouse embryos at embryonic day 15, produced by timed matings, were used as a source of fetal stroma and fetal thymocytes respectively. Cell culture The thymic cortical epithelial clone II-4 and the kidney epithelial clone Kep1.2 were derived from H-2K b -tsa58 transgenic mice (17) generated on the C57BL/10 background (H-2 b MHC haplotype) (13). H-2K b -tsa58 transgenic mice carry a transgene which places the expression of a Fig. 1. The epithelial clones II4 and Kep1.2 express equivalent levels of MHC class I molecules. Single-cell suspensions of H-2 b stroma temperature-sensitive SV40 T antigen gene under the control from deoxyguanosine-treated fetal day 14 lobes (shaded plot), the of the MHC H-2K b promoter. Briefly, tissues were dissociated thymic epithelial clone II4 (solid line) and the kidney epithelial clone and cultured at 33 C with 5% CO 2 in RPMI 1640 (Gibco, Kep1.2 (broken line) were stained for MHC class I and II molecules, Grand Island, NY) supplemented with 10% heat inactivated and their forward scatter (cell size) analysed. FCS, 2 mm L-glutamine and antibiotics (RP10 medium), in the presence of 100 IU/ml IFN-γ (Genzyme, Boston, MA). One week later, adherent cells were removed by treatment C57BL/10 fetal stroma or epithelial clones. C57BL/10 fetal with 0.05% trypsin (Sigma, St Louis, MO) and 5 mm EDTA, stroma was obtained by treatment of embryonic day 14 and washed. Dynabeads (Dynal, Oslo, Norway) were used fetal thymic lobes, cultured for 7 days in RP10 medium to select cortical epithelial cells as described by Anderson containing 1.35 mm dguo (19), with 0.05% trypsin and 5 et al. (18). Three days prior to use in any experiment, the mm EDTA. The thymocyte input was derived by treatment epithelial clones were moved to 37 C. of embryonic day 15 F5 TCR/RAG-1 / /H-2 q thymic lobes Reaggregate cultures with 1.6 mg/ml collagenase (Worthington Biochemical, Freehold, NJ). Cell suspensions from these lobes, without Reaggregate cultures were performed as described (10) depletion of endogenous stroma, were washed twice in with modifications. The stromal component was either fresh RP10 medium. In a few experiments MHC class II

3 Positive selection by an epithelial cell line in vitro 383 Fig. 2. Mature F5 transgenic thymocytes fail to be positively selected on the H-2 q MHC haplotype. Thymocytes were isolated from 6- to 8- week-old F5/RAG-1 / /H-2 b (A) and F5/RAG-1 / /H-2 q (B) mice, and stained for CD4, CD8 and V β 11 molecules. Histograms represent V β 11 expression on total thymocytes. endogenous stroma was depleted by staining the cell Flow cytometry and immunohistochemistry suspensions with the NIMR-4 mab [anti-h-2a b (20)] and For flow cytometric analysis between and negatively selecting stained cells with sheep anti-rat Ig cells were stained with various combinations of mab. For coated magnetic beads (Dynal, Wirral, UK). Then, such analysis of the epithelial clones the cells were thymocytes were placed in 1.5 ml eppendorf tubes and harvested using 5 mm EDTA, without trypsin. The following different numbers of stromal cells added to some tubes. mab and second layer reagents were used: FITC-conjugated Where stromal cells were added, the ratio of stroma to YTS169.4 [anti-cd8α (21)], biotin-conjugated KT11 [antithymocytes was maintained between 1:8 and 1:16 for the Vβ11 (22)], IM7 [anti-cd44 (23)], anti-cd69 (PharMingen, cell lines and 1:4 for fresh stroma. The tubes were San Diego, CA), YBM5.10 [anti-hsa (24)], NLDC-145 centrifuged, supernatant removed and 1 2 µl of cell slurry [recognizing determinants on both dendritic cells and thymic transferred to a nucleopore filter for organ culture as cortical epithelial cells (25)], NIMR-4 mab (anti-h-2a b ), 28- described (19). Reaggregates were cultured for 11 days 14-8S [anti-h-2d b,d q,l d (26)], phycoerythrin-conjugated antiat 37 C with 5% CO 2 in RP10 medium. Thymocytes were CD4 (Boehringer Mannheim, Mannheim, Germany) and then harvested for analysis by gently disrupting the Streptavidin RED 670 (Gibco). FITC-conjugated CTLA-4 was reaggregates manually in 1.5 ml eppendorf tubes. In some used to determine expression of B7-1 and B7-2 molecules experiments reaggregate cultures were incubated for 4 h (27). BrdU-labelled thymocytes were stained for surface with 125 µg/ml BrdU (Sigma) and thymocytes then harvested. markers and then with a FITC-conjugated anti-brdu mab

4 384 Positive selection by an epithelial cell line in vitro Fig. 3. The thymic epithelial clone II4 positively selects F5 transgenic thymocytes in vitro. Reaggregate lobes were made from fetal thymic lobes of day 15 F5/RAG-1 / /H-2 q (FRQ) transgenic mice, reaggregated alone (A), with H-2 b stroma from deoxyguanosine-treated fetal day 14 thymic lobes (B), the kidney epithelial clone Kep1.2 (C) or the thymic epithelial clone II4 (D). Cultures were mechanically dissociated after 11 days, and single-cell suspensions stained for CD4, CD8 and V β 11 molecules. Histograms represent V β 11 expression on gated CD4 CD8 (shaded plot), CD4 CD8 (dark line) and CD4 CD8 (light line) thymocytes. (Becton Dickinson, San Jose, CA) according to published methods (28). Stained cells were analysed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) using Cellquest software (Becton Dickinson). Viable lymphocytes are shown after gating on forward and side scatter. Immunohistochemical analysis was carried out on epithelial cells grown on glass coverslips for 3 days at 37 C (13). Cells were stained with an anti-cytokeratin 1, 5-11 and 18 mab (K8.13; Sigma), and the 4F1 mab (29), specific for thymic cortical epithelial cells. For immunohistology, 6 µm thick cryosections of thymus or reaggregate cultures were fixed in acetone for 10 min at 20 C, air-dried and subsequently stained with a purified anti-h-2k b /H-2D b antibody (clone ; PharMingen, San Diego, CA) as first layer. Second layers were FITC-conjugated goat anti-mouse IgG2A and FITCconjugated goat anti-rat Ig (Southern Biotechnology Associates, Birmingham, AL). Generation and assay of CTL Dendritic cells were derived from C57BL/10 mouse bone Fig. 4. The thymic epithelial clone II-4 is more efficient at thymocyte marrow (30,31). Briefly, total bone marrow cells were cultured positive selection than the kidney epithelial clone Kep1.2. Reaggregate lobes were made from fetal thymic lobes of day 15 for 7 days in RP10 medium and 250 IU/ml granulocyte FRQ transgenic mice, reaggregated alone (open squares), with H- macrophage colony stimulating factor [kindly provided by Dr 2 b stroma from deoxyguanosine-treated fetal day 14 lobes (filled Gitta Stockinger (32)], after which non-adherent cells were squares), the kidney epithelial clone Kep1.2 (open triangles) or the collected. These dendritic cells were loaded with 10 µm of thymic epithelial clone II-4 (filled circles). Cultures were mechanically the antigenic NP68 peptide at 37 C for 2 h, washed twice dissociated after 11 days, and single-cell suspensions stained for CD4, CD8 and V and 100 µl aliquots of cells/ml plated out onto a round- β 11 molecules. Each symbol represents the absolute number of CD4 CD8 V β 11 hi thymocytes recovered from an individual bottomed 96-well plate. The NP68 [from the nucleoprotein reaggregate culture. The data is pooled from seven independent of influenza virus A/NT/60/68 (NP ): ASNENMDAM] experiments. Statistical analysis of the data gave the following P peptide was synthesized on an Applied Biosystems 430A values: FRQ alone compared with Kep1.2, P 0.11; FRQ alone with II-4, P ; FRQ alone with dguo B10, P ; Kep1.2 with peptide synthesizer. Thymocytes from duplicate reaggregate II-4, P ; Kep1.2 with dguo B10, P 0.01; II-4 with dguo cultures were pooled and plated out in triplicate 100 µl B10, P aliquots in RP10 medium, containing M 2-mercapto-

5 Positive selection by an epithelial cell line in vitro 385 Table 2. Absolute numbers and percentages of CD8 single-positive thymocytes generated in FRQ reaggregates Experiment No. of CD4 CD8 thymocytes ( 10 4 ) FRQ alone FRQ Kep1.2 FRQ II-4 FRQ dguob (15.7) a) 2.67 (27.2) 5.46 (37.4) 4.80 (27.3) 2.63 (28.6) 6.83 (50.2) (7.6) 2.36 (12.7) 3.15 (9.2) 3.32 (11.8) 2.12 (9.6) (15.3) 4.66 (15.4) 2.97 (11.8) 3.07 (14.2) 4.0 (15.7) 3.77 (15.4) (6.4) 2.13 (5.16) 5.5 (8.1) 2.05 (5.1) 3.38 (7.2) 3.7 (7.6) 3.11 (6.3) (13.2) 3.15 (15.1) 4.89 (24.7) 7.19 (16.3) (17.0) 1.97 (21.0) 4.03 (24.3) 4.80 (27.3) 2.71 (16.3) 1.63 (27.2) 3.36 (31.7) (16.5) 2.39 (14.9) 5.30 (54.1) 2.94 (22.6) 0.12 (6.0) 1.62 (19.7) 6.24 (25.2) (27.5) 1.12 (28.1) 1.96 (28.9) 2.63 (41.0) 1.11 (29.1) 2.08 (21.2) 1.67 (27.9) 1.30 (21.7) 2.95 (47.5) 1.32 (26.4) 2.15 (38.5) a Percentages are shown in parentheses. Table 3. Absolute numbers and percentages of CD4 CD8 double-positive thymocytes generated in FRQ reaggregates Experiment No. of CD4 CD8 thymocytes ( 10 4 ) FRQ alone FRQ Kep1.2 FRQ II-4 FRQ dguob (62.5) a 5.42 (55.3) 7.55 (51.7) 8.32 (47.3) 3.67 (39.9) 5.40 (39.7) (63.4) (66.1) (77.7) (56.7) (59.1) (61.0) (75.5) (55.7) (54.1) (72.5) (51.5) (71.5) (80.8) (79.2) (75.7) (80.7) (80.3) (83.0) (61.3) (57.5) (61.7) (54.9) (50.9) 5.05 (53.7) (62.3) 9.19 (52.2) (62.3) 2.55 (42.5) 5.75 (54.3) (27.4) 5.90 (36.9) 2.85 (29.1) 3.49 (26.9) 0.41 (20.4) 3.21 (39.1) (45.2) (28.1) 1.60 (40.0) 3.58 (52.6) 1.65 (25.7) 1.34 (35.2) 5.09 (51.9) 3.28 (54.7) 2.42 (40.3) 2.04 (32.9) 1.48 (29.6) 2.17 (38.7) a Percentages are shown in parentheses. ethanol, onto the plate containing the dendritic cells. Cells were cultured for 4 days and then harvested. Different numbers of thymocyte effector cells, depending on the yield of each culture, were added in 100 µl volumes to a 96 V-well plate and 3-fold serial dilutions made. EL4 cells were labelled with 50 µci [ 51 Cr]sodium chromate in IMDM-10 for 1 h at 37 C at a concentration of cells/ml, either alone or in the presence of 100 µm NP68. Cells were then washed three times in IMDM-10 and resuspended to cells/ml, and 100 µl volumes were plated out. After brief centrifugation the plates were maintained at 37 C with 5% CO 2 for 5 h, after which they were centrifuged again, 25 µl supernatant removed and spotted onto glass fibre Spot-on filter mats (Wallac). Filter mats were analysed on a 1205 Betaplate counter (Wallac). Percent specific lysis was determined as follows: % specific lysis [(experimental release spontaneous release)/(maximum release spontaneous release)] 100. In all experiments spontaneous release was 15%. Results Generation of conditionally immortalized thymic cortical epithelial and kidney epithelial cells An epithelial clone, II-4, was isolated from the adult thymus of H-2K b -tsa58 transgenic mice of the H-2 b MHC haplotype

6 386 Positive selection by an epithelial cell line in vitro

7 Positive selection by an epithelial cell line in vitro 387 carrying a temperature-sensitive T antigen gene (13,17) as (dendritic cells). These cells did not express the B7-1 and described previously (33). Briefly, the thymic tumour was B7-2 co-stimulatory molecules. Taken together, these data disrupted and cells were allowed to adhere on a culture dish. strongly suggest that the II-4 cells are indeed of thymic After 7 days, a population of adherent cells expressing class cortical epithelial origin. II MHC, but not haematopoietic (CD45) or medullar epithelial An epithelial cell line was also isolated from the kidneys of (A2B5) markers, was purified using immunomagnetic beads. the same T antigen transgenic mice. This cell line exhibited A population isolated in this manner from fresh thymus has the cobble stone appearance of epithelial cells and expressed been demonstrated to be mostly cortical-type epithelial cells high levels of MHC class I, similar to those found on the II-4 which can support T cell differentiation in vitro (10). Sub- cells, but low levels of MHC class II molecules (Fig. 1). sequently, the II-4 clone was derived from this population by limiting dilution. This clone was routinely cultured at 33 C H-2 b thymic stromal cells positively select F5 αβ transgenic (permissive temperature for the transgenic T antigen) with thymocytes in in vitro reaggregate cultures 100 IU/ml IFN-γ, and moved to 37 C (non-permissive temper- To examine positive selection of immature thymocytes in ature) for 72 h before its use in experiments or characterization in vitro cultures we made use of thymocytes from F5 αβ TCR with mab. As summarized in Table 1, the II-4 clone maintained transgenic mice (15) crossed onto RAG-1-deficient mice and its original phenotype of high level expression of class II MHC H-2 q MHC haplotype. The F5 TCR recognizes a nonamer and cytokeratin molecules after propagation in culture over a peptide from influenza nucleoprotein (NP68) in the context of period of several months. In addition, the II-4 cells stained MHC class I H-2D b (34). Thymocytes bearing this TCR are positive for a thymic cortical epithelial marker 4F1 and cortical positively selected in vivo in C57BL/10 mice (H-2 b haplotype), epithelial/dendritic cell marker NLDC-145, but not for other but fail to proceed further than the CD4 CD8 TCR int stage lineage markers such as Mac-1 (macrophages) and N418 in SWR mice (H-2 q haplotype), as judged by staining with the Fig. 6. Thymocytes proliferate to the same extent in reaggregates containing the II-4 and the Kep1.2 clones. Reaggregates were pulsed on day 7 of culture for 4 h with 125 µg/ml BrdU, harvested and thymocytes stained for CD4, CD8 and BrdU incorporation. Histograms show BrdU incorporation of gated CD8 single-positive thymocytes (numbers above markers refer to percent BrdU-positive cells). No significant difference was found in the amount of BrdU incorporated by CD8 single-positive thymocytes on days 6, 7, 8 and 9 of culture (data not shown). Fig. 5. The II-4 and Kep1.2 epithelial clones integrate and form similar patterns in reaggregate cultures. Reaggregates, made from day 15 FRQ fetal thymic lobes and the II-4 clone (C) or the Kep1.2 clone (D), were frozen on day 5 of culture and sections made. Reaggregate sections and control sections of adult FRQ (A) and H-2K b -tsa58 (B) thymic lobes were stained for H-2 b MHC class I expression.

8 388 Positive selection by an epithelial cell line in vitro anti-v β 11 mab (Fig. 2) (33,35). Using the in vitro reaggregation tional fetal thymic organ culture (data not shown). In addition, culture system (10) we were able to study the relative efficiency all reaggregate cultures, regardless of the type of stroma with of different H-2 b epithelial cell types in positively selecting which they were supplemented, also contained a small but thymocytes from day 15 F5/RAG-1 / /H-2 q (FRQ) embryos. At significant number of CD4 CD8 lo cells with immature charac- this point in gestation F5 transgenic thymocytes are mostly teristics (HSA and TCR lo ) (Fig. 3). Despite this unexpected CD4 CD8 TCR(V β 11), with a few (10 15%) having progressed background level of single-positive cells, inclusion of fresh to the CD4 CD8 TCR(V β 11) stage (data not shown). H-2 b thymic stroma in the reaggregate cultures resulted in Single-cell suspensions from day 15 F5/RAG-1 / /H-2 q the generation of significantly higher percentages of CD4 embryonic thymic lobes contain sufficient numbers of H-2 q CD8 V β 11 hi thymocytes (Fig. 3B) than were found in cultures stromal cells for lobes to reform within the first 14 h of culture containing only H-2 q stroma (Fig. 3A). From this data we without the addition of further stroma. Unexpectedly, after 11 conclude that fresh H-2 b thymic stroma can positively select days of culture, such reaggregate lobes contained a small F5 transgenic thymocytes in vitro. but significant number of CD4 CD8 single-positive cells Similarly, when included in reaggregates of fetal day 15 with a mature phenotype (HSA and TCR hi ) (Fig. 3A). The FRQ thymocytes, the II-4 clone described above was able to production of CD4 CD8 V β 11 hi thymocytes in the absence induce a significant increase in the percentages of CD4 of H-2 b -expressing stroma appears to be exclusive to the CD8 V β 11 hi thymocytes generated in such cultures (Fig. 3D). reaggregate culture system, as these cells were not found in Thus it appears that this conditionally immortalized thymic comparable levels in vivo in adult mice (33) or after conven- epithelial clone can substitute for fresh H-2 b stroma in the Fig. 7A. The F5 transgenic thymocytes generated in reaggregate cultures have a mature phenotype. Reaggregate lobes were made from fetal thymic lobes of day 15 FRQ transgenic mice, reaggregated with H-2 b stroma from deoxyguanosine-treated fetal day 14 lobes. Cultures were mechanically dissociated after 11 days, and single-cell suspensions stained for CD4, CD8, CD69, CD44 and HSA molecules. Histograms represent expression of markers on thymocyte populations gated in the dot plots.

9 Positive selection by an epithelial cell line in vitro 389 positive selection of F5 transgenic thymocytes. However, in the II-4 clone were considerably larger in size than the Kep1.2 contrast to fresh H-2 b stroma, the II-4 clone required the clone and expressed more MHC class II (Fig. 1). After 11 presence of endogenous H-2 q stroma for reaggregate forma- days of culture reaggregates incorporating kidney epithelial tion. In the absence of endogenous stroma, the clone formed cells developed similar subpopulations as the ones in the a small dense body and the culture failed to yield a significant reaggregates without additional epithelial cells (Fig. 3C). number of viable thymocytes (data not shown). However, quantitatively the generation of mature CD8 T cells It was possible that the II-4 clone promoted positive selec- in these cultures was less efficient than when fresh B10 tion of thymocytes simply by expression of the selecting H- stroma or the II-4 cells were included. 2 b MHC and that the putative specific epithelial signals (11,12) A better indication of the extent of positive selection was required for this process came from the endogenous FRQ provided by calculating the absolute numbers of cells pro- stroma. Thus, if the contribution of the II-4 clone to positive duced in different reaggregate cultures. A summary of the selection was limited to the expression of H-2 b MHC molecules, absolute numbers of CD4 CD8 V β 11 hi thymocytes generated any H-2 b -expressing cell would be expected to be as in reaggregate cultures from seven independent experiments efficient as the thymic epithelial clone in this process. To is presented in Fig. 4. Although there was a certain degree test this hypothesis, we compared the efficiency of positive of variability in the thymocyte yield from the reaggregate selection by the II-4 clone with that of a clone (Kep1.2) cultures, the II-4 clone was significantly more efficient than derived from kidney epithelium of H-2K b -tsa58 transgenic the Kep1.2 clone (P ) or the endogenous H-2 q mice bearing the selecting H-2 b MHC. Both clones expressed stroma (P ), and as efficient as fresh H-2 b stroma (P equivalent levels of MHC class I molecules, although cells of 0.99), at positively selecting the F5 transgenic thymocytes. On Fig. 7B. The F5 transgenic thymocytes generated in reaggregate cultures have a mature phenotype. Reaggregate lobes were made from fetal thymic lobes of day 15 FRQ transgenic mice, reaggregated with H-2 b stroma from the thymic epithelial clone II-4. Cultures were mechanically dissociated after 11 days, and single-cell suspensions stained for CD4, CD8, CD69, CD44 and HSA molecules. Histograms represent expression of markers on thymocyte populations gated in the dot plots.

10 390 Positive selection by an epithelial cell line in vitro the other hand, despite high level expression of the selecting MHC molecules, the Kep1.2 clone was unable to induce positive selection significantly above levels found in reaggregates of FRQ thymocytes alone (P ) (Fig. 4). This implies that the II-4 clone provides interactions, in addition to the appropriate MHC molecules, which are of critical importance in thymocyte positive selection. In addition to the II-4 clone, two independently derived thymic cortical epithelial clones were also found to be efficient at positively selecting FRQ thymocytes (data not shown). It is important to note that although there is variability in the numbers of CD4 CD8 double-positive thymocytes generated in the different reaggregate cultures, these are not reflected in the numbers of mature CD4 CD8 V β 11 hi thymocytes (Table 2 and 3). It is therefore unlikely that the more efficient generation of mature single-positive thymocytes in reaggregate cultures containing the II-4 clone was due to the clone promoting more efficient differentiation of thymocytes to the double-positive stage. The wide range of double-positive thymocyte formation may correlate with the number of viable precursor cells existing in each reaggregate culture, a parameter which is practically impossible to measure. In order to compare the efficiency of II-4 and Kep1.2 epithelial clone integration into the reaggregate cultures, sections were made of the reaggregates and stained for H- 2 b MHC class I expression (Fig. 5). Both clones showed a similar pattern of incorporation, forming the brightly stained patches which can be seen in the reaggregates. Since mature thymocytes have been shown to undergo some degree of proliferation in the thymus and in reaggregate cultures (36), it was possible that the II-4 clone stimulated such proliferation to a greater extent than the Kep1.2 clone and this resulted in greater numbers of mature thymocytes in reaggregates containing the former clone. To test this Fig. 8. The positively selected F5 transgenic thymocytes can give possibility, reaggregates were pulsed with BrdU for 4 h at rise to effector cytolytic cells in vitro. Transgenic thymocytes from different time points during the culture period and thymocytes reaggregates containing H-2 b stroma from deoxyguanosine-treated stained for BrdU incorporation. Although the proportion of fetal day 14 lobes (A), the thymic epithelial clone II4 (B) or the kidney epithelial clone Kep1.2 (C) were cultured with H-2 b dendritic cells, CD8 single-positive thymocytes which incorporated BrdU did loaded with 10 µm NP68 peptide. After 4 days cultures were harvested vary according to the day on which the BrdU pulse was and added at the indicated E:T ratios to 51 Cr-labelled EL4 cells, loaded made, no significant differences were seen between cultures with 100 µm NP68 peptide (filled circles) or unloaded (open circles). containing the II-4 clone and the Kep1.2 clone (Fig. 6). Thus, the mature thymocyte population contained equivalent numbers of cells which had undergone proliferation within the 4 h BrdU pulse in the reaggregate cultures containing the (CD69 CD44 HSA hi ) (Fig. 7). It is important to note that different epithelial clones. The more efficient generation of significant numbers of CD4 CD8 lo thymocytes were also mature thymocytes in cultures containing the II-4 clone is therefore more consistent with these cells promoting positive selection of immature thymocytes. observed in these cultures. These cells were immature (CD69 CD44 HSA hi ) and expressed lower levels of V β 11 than the CD4 CD8 V β 11 int thymocytes (Figs 3 and 7). Similar results were obtained for thymocytes from reaggregates The CD4 CD8 V β 11 hi transgenic thymocytes generated in containing Kep1.2 cells or those containing only H-2 q reaggregate cultures are mature and are functional stroma (data not shown). In order to establish the phenotype of thymocytes produced To determine the functional status of the thymocytes in the reaggregate cultures, these cells were stained for generated in reaggregate cultures, the capacity of these various markers. Such analysis revealed that the CD4 cells to differentiate into effector cytolytic lymphocytes was CD8 V β 11 hi thymocytes selected by fresh stroma or the II- examined. Thymocytes produced from reaggregate cultures 4 clone were of a mature phenotype, being CD44 lo HSA containing fresh H-2 b stroma, II-4 or Kep1.2 cells, gave (Fig. 7B). In addition, these cells expressed the CD69 rise to effector cytolytic T cells that specifically lysed target molecule, which is thought to be associated with thymocyte cells loaded with the nominal NP68 peptide (Fig. 8). This positive selection (37 39). As mentioned above, the was also the case for thymocytes generated in cultures CD4 CD8 V β 11 int thymocytes were phenotypically immature containing only H-2 q stroma (data not shown). Thus, mature

11 Positive selection by an epithelial cell line in vitro 391 and functionally competent thymocytes were generated in absence of endogenous, or fresh, epithelium the II-4 cells all reaggregate cultures, and the difference in efficiency formed dense bodies, rather than defined lobes, from which with which each stromal cell type was able to positively select only a small number of viable thymocytes could be retrieved. such thymocytes was apparent only in quantitative terms. It therefore appears that the conditionally immortalized clone lost the ability to reform the correct thymic epithelial architecture, whilst retaining the expression of molecules Discussion necessary for positive selection. Interestingly, analysis of reaggregates depleted of endogenous stroma revealed that We have demonstrated in this study that a conditionally cultures with II-4 cells contained more mature thymocytes immortalized thymic cortical epithelial clone can positively than those with Kep1.2 cells or no additional stroma (data select F5 αβ TCR transgenic thymocytes in vitro. Thymocytes not shown). However, such data must be viewed with generated by this process are phenotypically mature and caution due to the relatively low number of total viable have the capacity to differentiate into effector cytolytic cells thymocytes recovered in these experiments. in vitro. The II-4 thymic epithelial clone was significantly Although many different cell types have been demonstrated more efficient in thymocyte positive selection than an to induce positive selection in vivo (5 9), these epithelial clone from kidney, which expressed equivalent studies cannot rule out that the specific signals for this levels of the selecting MHC molecules. We conclude that process came from endogenous thymic epithelia. In these the II-4 clone has retained expression of a putative molecule cases the only contribution of each particular cell type (or molecules) that plays an important role in positive would be the introduction of the selecting MHC molecules. selection of thymocytes. The co-stimulatory molecules B7- Such an explanation is supported by experiments in vitro 1 and B7-2 are unlikely to plays a role in this process which demonstrate a unique role for thymic epithelium in because they are not expressed by the II-4 clone (Table positive selection (11). It is interesting to note that in our 1). This is in agreement with a recent study showing that experiments, the kidney epithelial clone Kep1.2, which B7 is not essential for positive selection (40). expressed high levels of the selecting MHC molecules, did Reaggregate cultures which did not contain the selecting not significantly improve the yield of mature transgenic H-2 b MHC molecules, but only the non-selecting H-2 q MHC thymocytes over background levels. One explanation is that of the endogenous stroma, nevertheless did generate positive selection of F5 thymocytes is dependent on a transgenic thymocytes that were phenotypically mature, unique peptide(s) specifically expressed in combination functionally competent and indistinguishable from mature with MHC molecules on thymic cortical epithelial cells but transgenic cells produced by bona fide positive selection not on kidney epithelial cells. In other studies using different on epithelium expressing H-2 b MHC molecules. TCR transgenic models, the selecting ligand may have The relatively high background numbers of mature been ubiquitously expressed so that even fibroblasts could transgenic thymocytes in reaggregate cultures only containing mediate positive selection. Another possibility is that the II- H-2 q stroma is unique to the reaggregate culture 4 cells, being of thymic origin, are able to co-operate more system, since such numbers were not found in vivo in efficiently with endogenous epithelial cells in reaggregate adult thymi or in conventional fetal thymic organ cultures formation than the Kep1.2 cells, which originate from the from F5/RAG-1 / /H-2 q mice. Recently, it was reported that kidney. However, the structures of the reaggregate lobes MHC class I-deficient stroma was able to support the formed by these two cell lines are grossly indistinguishable. inefficient generation of CD8 TCR hi cells in reaggregate Finally, production of cytokines, by the II-4 cells, which cultures (41). It is possible that disaggregation of fetal promote differentiation of CD8 cells cannot be excluded at thymic lobes results in a signalling event occurring in the the moment. thymocytes. Such a signal in combination with low-affinity Regardless of the mechanism, the II-4 clone appears to interactions between the F5 TCR and H-2 q MHC class I promote positive selection of F5 thymocytes as efficiently molecules, which are not normally sufficient to positively as fresh thymic stromal cells. Our data strongly suggest select CD8 /F5 T cells, could now allow the appearance that II-4 cells contribute to the positive selection by of such cells. A signalling event such as the one postulated more than simple MHC presentation. This clone therefore above is unlikely to be connected with the CD4 and CD8 constitutes a source of homogeneous material for the accessory molecules or the TCR, since the majority of identification of molecules necessary for positive selection thymocytes did not express these molecules at embryonic of developing thymocytes. Taken together with the fact that day 15 and similar results were obtained with fetal day large numbers of II-4 cells are readily obtainable, this clone 14 thymocytes which are negative for these molecules. is ideal for such studies. Alternatively, the high backgrounds may be the result of the elimination of negatively selecting elements in reaggregate cultures, that would be present in vivo and in fetal thymic Acknowledgements lobe cultures. However, addition of splenic dendritic cells We thank Dr Kyuhei Tomonari for kindly providing us with the KT11 from RAG-1 / /H-2 q mice did not significantly alter the antibody, Dr Mary Ritter for the 4F1 antibody, Dr Gitta Stockinger numbers of mature thymocytes produced in these cultures for granulocyte macrophage colony stimulating factor supernatant and Dr Eugenia Spanopoulou for the gift of RAG-1 / mice. In (data not shown). addition, we would like to thank Dr Graham Anderson, Professor The II-4 cells required the presence of endogenous Eric J. Jenkinson and Professor John J. T. Owen for showing us epithelium in order to form reaggregate lobes. In the how to set up the reaggregate culture system and for helpful

12 392 Positive selection by an epithelial cell line in vitro discussions, and Dr Rose Zamoyska and Dr Gitta Stockinger for immunoglobulin transgenes guide ordered B-cell differentiation critical reading of the manuscript. This work was supported by in Rag-1-deficient mice. Genes Dev. 8:1030. the Medical Research Council. Y. T. and O. W. were supported 17 Jat, P. S., Noble, M. D., Ataliotis, P., Tanaka, Y., Yannoutsos, by fellowships from the Leukaemia Research Fund, and A. W. by N., Larsen, L. and Kioussis, D Direct derivation of a grant from the Boehringer Ingelheim Fonds. conditionally immortal cell lines from an H-2K b tsa58 transgenic mouse. Proc. Natl Acad. Sci. USA 88: Anderson, G., Jenkinson, E. J., Moore, N. C. and Owen, J. J. Abbreviations T MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus. FRQ F5 TCR/RAG-1 / /H-2 q transgenic mice Nature 362: Jenkinson, E. J., Franchi, L. L., Kingston, R. and Owen, J. J. 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