Maturation of T Cells Expressing Fc εriγ Containing TCR/CD3 Complexes

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1 This information is current as of December 17, Early TCR αβ Expression Promotes Maturation of T Cells Expressing Fc εriγ Containing TCR/CD3 Complexes Karin Petersson and Fredrik Ivars J Immunol 2001; 166: ; ; doi: /jimmunol References Subscription Permissions Alerts This article cites 55 articles, 27 of which you can access for free at: Why The JI? Submit online. Rapid Reviews! 30 days* from submission to initial decision No Triage! Every submission reviewed by practicing scientists Fast Publication! 4 weeks from acceptance to publication *average Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: Downloaded from by guest on December 17, 2018 The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD Copyright 2001 by The American Association of Immunologists All rights reserved. Print ISSN: Online ISSN:

2 Early TCR Expression Promotes Maturation of T Cells Expressing Fc RI Containing TCR/CD3 Complexes 1 Karin Petersson and Fredrik Ivars 2 In a previous study we presented data indicating that the expanded population of CD4 CD8 (DN) T cells in TCR -chaintransgenic mice was partially if not entirely derived from T cell lineage cells. The development of both T cells and DN T cells is poorly understood; therefore, we thought it would be important to identify the immediate precursors of the transgeneinduced DN T cells. We have in this report studied the early T cell development in these mice and we show that the transgenic TCR -chain is expressed by precursor thymocytes already at the CD3 CD4 CD8 (triple negative, TN) CD44 CD25 stage of development. Both by using purified precursor populations in reconstitution experiments and by analyzing fetal thymocyte development, we demonstrated that early TN precursors expressing endogenous TCR -chains matured into DN T cells at several stages of development. The genes encoding the -chain of the high affinity receptor for IgE (Fc RI ) and the CD3 protein were found to be reciprocally expressed in TN thymocytes such that during development the Fc RI expression decreased whereas CD3 expression increased. Furthermore, in a fraction of the transgene-induced DN T cells the Fc RI protein colocalized with the TCR/CD3 complex. These data suggest that similarly to T cells and NKT cells, precursors expressing the TCR early in the common developmental pathway may use the Fc RI protein as a signaling component of the TCR/CD3 complex. The Journal of Immunology, 2001, 166: Most of the T cells in peripheral lymphoid organs have differentiated in the thymus. Their precursors that enter the thymus from the bone marrow are CD44, Thy-1, and c-kit low (1). These cells generate the CD4 CD8 (DN) 3 thymocytes, which can be divided into four phenotypically distinct subpopulations based on the expression of the CD25 and CD44 markers (2). Some of the CD44 CD25 DN thymocytes are precursors of NK, B, and dendritic cells (3, 4). In this population there are also mature NK or T cells that carry TCR or heterodimers and CD3 complexes composed of the invariant CD3-chains,,, and (5, 6). These mature cells may also express disulfide-linked homodimers of the Fc RI protein or Fc RI /CD3 heterodimers in their TCR/CD3 complexes, because this has been observed in peripheral NK and T cells (7, 8). Fetal CD44 CD25 thymocytes also express the Fc RII/III (CD32/ CD16) molecules on the cell surface (9). At the stage when the CD25 protein starts to be expressed (CD44 CD25 ), the expression of the pt -chain gene is also initiated (10). Provided the TCR -chain locus is successfully rearranged, the pt protein is brought to the cell surface together with the TCR -chain protein and the CD3 complex (11). This pre-tcr complex transmits a TCR -selection signal via the CD3 complex Section for Immunology, Department of Cell and Molecular Biology, Lund University, Lund, Sweden Received for publication August 1, Accepted for publication March 16, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants to F.I. from the following foundations: Anna Greta Crafoords Stiftelse för reumatologisk forskning, Greta och Johan Kocks Stiftelser, Konung Gustaf den V:s 80 års fond, Kungliga Fysiografiska Sällskapet i Lund, the Alfred Österlunds stiftelse, and the Medical Faculty of Lund University. 2 Address correspondence and reprint requests to Dr. Fredrik Ivars, Section for Immunology, Lund University, Sölvegatan 19, SE Lund, Sweden. address: Fredrik.Ivars@immuno.lu.se 3 Abbreviations used in this paper: DN, CD4 CD8 ; FTOC, fetal thymus organ culture; TN (triple negative), CD3 CD4 CD8 ; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; DP, double positive; TG, transgenic; HSA, heat-stable Ag. proteins and as a consequence the thymocyte proceeds to proliferation and maturation (reviewed in Refs. 12 and 13). Subsequently, rearrangements of the TCR -chain gene and expression of the CD4 and CD8 genes (14, 15) are induced, leading to the formation of CD4 CD8 TCR low cells, which ultimately give rise to mature CD4 or CD8 T cells. The developmental pathway of T cells is still poorly understood. The TCR - and TCR -chain genes are probably rearranged simultaneously with or even slightly before the TCR -chain genes (16 18). Currently, there are conflicting views as to when and how the T cell lineage deviates from the differentiation pathway of conventional T cells; one reason for this is that there are currently no reliable markers defining T cell precursors. It is believed that and T cells share a common precursor cell, because mature cells of both subsets generally have rearranged the genes for both kinds of TCR (reviewed in Ref. 19). Genetically altered mice have been particularly useful as tools to study the molecular events involved in / T cell lineage commitment (reviewed in Refs ). Such studies also showed that expression of TCR transgenes or TCR gene inactivation did not always lead to complete ablation of a particular T cell lineage. Thus, in the absence of the particular TCR, cells belonging to the (22, 23) or (24 26) lineages still developed by the aid of the and TCRs, respectively. Although the simplest interpretation of these data would be that the precursors maturing under these conditions are precommitted to a lineage, the role of the TCR signaling in T cell lineage commitment is still controversial (reviewed in Refs ). We have previously studied the expanded population of DN TCR T cells in TCR -chain-transgenic (TG) mice (26). These cells are phenotypically and functionally more similar to T cells than to T cells (27). A small subpopulation of T cells possessing functional TCR rearrangements is found in normal mice (28). This population is drastically diminished in the transgenics; therefore, we suggested that the TG DN T cells might arise from precursors of these cells as the result of competition in surface TCR expression during development (26). This may not be Copyright 2001 by The American Association of Immunologists /01/$02.00

3 The Journal of Immunology 6617 the only source of these cells, as they are numerically many more than the TCR T cells in normal mice. Because it is possible that the surface marker phenotype and function of these DN T cells is determined by their precursor origin, we thought that further studies on the development of these cells might be informative for understanding the mechanisms of lineage commitment. To identify the precursors of these DN T cells we have in this report studied TCR expression by thymus precursor populations in TCR -TG mice. Our data show that early TCR expression promotes the generation of DN T cells at several early stages of thymocyte development. These TCR -chain induced cells acquire the DN cell phenotype, and some of them express unconventional CD3 complexes containing the Fc RI protein. Our present data further support the view that the phenotype and function of lineage cells are imposed on the precursor cell already at the commitment event (29). Materials and Methods Mice C57BL/6 mice and 2B4 TCR -chain-tg mice (30) inbred on the C57BL/6 background, were kept in a specific pathogen-free animal facility at Lund University. RAG1-deficient mice, provided by William Agace (Lund University, Lund, Sweden) were originally bought from The Jackson Laboratory (Bar Harbor, ME). Cell enrichment and isolation The thymus, lymph nodes, or spleen were dissected, and single-cell suspensions were prepared in HBSS (Life Technologies, Paisley, U.K.) and washed twice in the same buffer. After centrifugation, thymocytes were resuspended at cells/ml in HBSS containing anti-cd4 (RL172.4) and anti-cd8 (3.155) Abs. Low-tox Rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada) and DNase (10 g/ml; Sigma, St. Louis, MO) were added, and the suspension was incubated at 37 C for 45 min. Dead cells were removed by density centrifugation (Lympholyte; Cedarlane Laboratories). The remaining lymphocytes were washed in RPMI 1640 medium supplemented with 100 g/ml penicillin, 100 g/ml streptomycin, 0.05 mm 2-ME, 1 mm sodium pyruvate, and 10% FCS (cell culture medium; all obtained from Life Technologies). The cells were then resuspended in cell culture medium containing appropriate amounts of either anti-cd4 (GK1.5) and anti-cd8 ( ) Abs, or biotinylated anti- CD4 (RM4-5; PharMingen, San Diego, CA) and anti-cd8 ( ) Abs. After incubation at 8 C for 15 min the cells were washed in PBS supplemented with 2% FCS and magnetic beads conjugated with anti-rat- Abs or streptavidin (Miltenyi Biotec, Bergisch Gladbach, Germany) were added. The cells were incubated at 8 C for 20 min, washed, and loaded on a MACS column (Miltenyi Biotec). DN cells were isolated according to the guidelines of the manufacturer and resuspended in appropriate medium for later experiments. Immature single positive thymocytes were prepared by depleting thymocytes of CD4 cells by magnetic cell sorting. CD8 CD4 TCR TCR CD44 cells were thereafter isolated using flow cytometric cell sorting (see below). Flow cytometry and cell sorting For flow cytometric analyses, cells were resuspended in HEPES-buffered HBSS containing 0.1% NaN 3 and 2% FCS. The following Abs were used: PE-conjugated anti-tcr (H57), anti-tcr (GL3), anti-c-kit, FITC-conjugated anti-cd25 (7D4), anti-tcr (H57), anti-tcr (GL3), and biotinconjugated anti-cd44 (IM7) (all obtained from PharMingen). PE-conjugated goat F(ab ) 2 -anti-rat IgG and FITC-conjugated goat F(ab ) 2 -anti-rat IgG were bought from Caltag (Burlingame, CA). FITC-conjugated anti- FcRII/III Abs (2.4G2) were purified and conjugated in our laboratory. Cy5-conjugated anti-2b4 (A-24B-2) and CD3 (KT3) were prepared in our laboratory by using a Cy5 labeling kit (Amersham Pharmacia Biotech, Uppsala, Sweden). PerCP-conjugated streptavidin (BD Biosciences, Mountain View, CA) or Red613-conjugated streptavidin (Life Technologies) were used as second-step reagents. Anti-FcRII/III Abs (2.4G2) were used to block binding to Fc-receptors except those in Fig. 4. Normal rat serum was used for blocking remaining binding sites of anti-rat Ig Abs when appropriate. Intracellular staining was performed essentially as previously described (27). The analyses were performed using FACSort or FACSCalibur flow cytometers (BD Biosciences). For cell sorting, cells were resuspended in PBS supplemented with 2% FCS. Sorting was performed using a FACSVantage cell sorter (BD Biosciences). The purity of sorted cells was 98%. Cell cultures The BW and WEHI 3 cell lines as well as DN T cell hybridomas previously described (26) were cultured in cell culture medium at 37 C in humidified atmosphere conditioned with 5% CO 2. RNA extraction and RT-PCR analysis Total cellular RNA was prepared from cells using Trizol (Life Technologies) according to the manufacturer s recommendations. cdna was prepared using oligo(dt) primer (Promega, Madison, WI) and Superscript II reverse transcriptase (Life Technologies). RT-PCR analysis was performed using a MJ Technologies PTC-100 thermal cycler (MJ Research, Watertown, MA). To standardize cdna preparations, aliquots were amplified using hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-specific (5 - CACAGGACTAGAACACCTGC-3, 5 -GCTGGTGAAAAGGACCTCT3 ) primers and amplification cycles of 94 C for 30 s, 55 C for 30 s, and 72 C for 1 min for 35 cycles. CD3 (5 -AGAAGCCTACACTGAGATCG-3, 5 - GGATGACGTTCTGTGTTCAG-3 ) and Fc RI (5 -CACTTCTAAT TCTCTCCGAGCC-3, 5 -CATTGTTTAGTGAGAGTCGAGG-3 ) primers were used to amplify the corresponding cdnas using cycles of 94 C for 30 s, 60 C for 30 s, and 72 C for 1 min for 35 cycles. PCR products were separated on 1.5% agarose gels. The intensity of ethidium bromide-stained bands was quantitated using the Gelpro software (Media Cybernetics, Silver Spring, MD). The CD3 and Fc RI products were transferred to a nitrocellulose membrane by vacuum blotting in 0.4 M NaOH, 0.6 M NaCl solution. The membrane was cross-linked by UV light and blocked for1hat40 C in 6 SSC phosphate/edta buffer supplemented with Denhardt s solution and 100 g/ml carrier DNA. Oligonucleotide probes (CD3 5 -TGCTGATGT CACTTGTGAAG-3 ; Fc RI 5 -GCTAGCTAGGCT-CTACATCA-3 ) hybridizing to sequences amplified by the PCR primers were labeled with [ - 32 P] ATP (Amersham Pharmacia Biotech) using T4 kinase (Life Technologies). After ethanol precipitation, the probe was added to the prehybridization mixture, and the blots were hybridized overnight at 40 C. The membrane was then washed in 5 standard saline citrate phosphate/edta at room temperature for 10 min and exposed to x-ray film for 1 12 h. Fetal thymus organ cultures (FTOCs) C57BL/6 and 2B4 TG fetuses were taken from timed pregnant females at day 14 of gestation. Thymi were dissected and placed on Nucleopore Track-Etch membranes (Corning Separations Division, NY) in RPMI 1640 medium supplemented with 10% FCS (Sigma), 100 g/ml penicillin, 100 g/ml streptomycin, 0.05 mm 2-ME, 1 mm sodium pyruvate, and 2% MEM nonessential amino acids (Life Technologies). The 2B4 TG thymic lobes were cultured for various days at 37 C at 5% CO 2. The C57BL/6 thymic lobes were first irradiated (3000 rad) in a cesium source and transferred to Terasaki plates (Nunclon; Nunc, Roskilde, Denmark). The lobes were then seeded with CD3 CD4 CD8 (triple negative, TN) CD44 CD25 or CD44 CD25 thymocytes sorted from the 2B4 TG mice by adding sorted cells in 20 l medium per well and lobe. The plates were then inverted for 2 days (hanging drop cultures). On day 2 the lobes were transferred to Nucleopore Track-Etch membranes and incubated as above until analysis by flow cytometry. TCR/Fc RI cocapping Enriched DN thymocytes and peripheral cells from TCR TG mice were incubated with biotinylated A-2B4-2 TG TCR -chain-specific Ab in precapping solution (PBS, 1% BSA) at 4 C. The cells were washed and streptavidin-pe (PharMingen) was added. The cells were then either incubated on ice or at 37 C for min to induce capping. After this the capping was inhibited by adding 1.5 ml ice-cold capping solution (PBS, 1% BSA, 0.1% NaN 3 ). The cells were then fixed in 2% paraformaldehyde, and intracellular staining of the Fc RI protein was performed. The method of intracellular staining was as previously described (27), with the exception that the cells were blocked intracellularly with normal rabbit serum before adding anti-fc RI serum (Upstate Biotechnology, Lake Placid, NY) and FITC-conjugated goat anti-rabbit-ig (PharMingen). The cells were loaded on to Polysine microslides (Menzel-Gläser, Freiburg, Germany) and fixed in Fluorescent Mounting Media (Dako, Carpinteria, CA) overnight before they were analyzed using a confocal microscope (MRC 1024; Bio-Rad, Hercules, CA) equipped with filters set for FITC and Texas Red. The images were analyzed using Lasersharp software (Bio-Rad).

4 6618 EARLY MATURING T CELLS Immunoprecipitations Lysates from the TG DN T cell hybridomas expressing Fc RI mrna were subjected to immunocoprecipitations of CD3 - ortgtcr -associated proteins. Cells ( ) were lysed for 1hinNonidet P-40 buffer (1% Nonidet P-40, 150 mm NaCl, 50 mm Tris, ph 7.5, and 1 mm PMSF) at 4 C. The lysates were centrifuged at 13,000 rpm at 4 C for 15 min, and the supernatants were mixed overnight at 4 C with anti-cd3 (145.2C11) or A-2B42-conjugated Sepharose beads (a gift from M. Gullberg, Umeå University, Umeå, Sweden). The beads were then washed twice in 1 ml 0.1% Nonidet P-40 solution, twice in 1 ml 150 mm NaCl, 50 mm Tris (ph 7.5), and twice in 1 ml 50 mm Tris (ph 7.5). The samples were then boiled in 30 lof3 Tricine-SDS loading buffer (4% SDS, 12% glycerol, 50 mm Tris, 10% 2-ME, 50 mm DTT, 0.01% Serva Blue G, ph 6.8) for 10 min and loaded on a 10% Tricine-SDS-PAGE gel (31). The separated proteins were transferred to membranes (Hybond-C Extra; Amersham Pharmacia Biotech) using a Trans-Blot SD SemiDry Transfer Cell (Bio-Rad). The membranes were blocked for 45 min in PBS with 5% dry milk and 1% Antifoam (Sigma). Abs to the Fc RI (Upstate Biotechnology) and CD3 -chain (PharMingen) proteins were added in 50 mm Tris (ph 7.5), 150 mm NaCl, 2.5 mm EDTA, 15% FCS, and 0.02% NaN 3 for 1 2 h and the membranes were then washed three times for 15 min each in PBS with 0.1% Tween 20. Proteins were revealed using the ECL system (Amersham Pharmacia Biotech). The Kaleidoscope Prestained Standard protein marker (Bio-Rad) was used to estimate the molecular mass of revealed proteins. A TG DN T cell clone that was negative for Fc RI mrna was used as a specificity control. The conditions for detecting Fc RI protein were optimized using lysates of the WEHI-3 macrophage cell line precipitated with the 2.4G2 mab. Results The TG TCR -chain induces early expression of the TCR Previous results have suggested that the expanded population of DN T cells in TCR -chain-tg mice originate from lineage precursors (26). As a first step to identify the immediate precursors of these cells we analyzed expression of the TG TCR in the four DN thymocyte precursor populations defined by CD44 and CD25 expression (2). As shown in Fig. 1A, a fraction of the cells in all these populations expressed the TG TCR -chain-positive TCR. Most importantly, DN TCR cells were detected in the TG CD44 CD25 and CD44 CD25 DN populations, whereas very few TCR cells were detected in these populations in normal thymocytes (Fig. 1B). The latter result was expected because these populations do not express the TCR -locus (32). It has previously been reported that the CD44 CD25 precursors have not yet rearranged their TCR genes (33, 34). However, subdivision of the DN and DN T cells using the CD24 (heatstable Ag, HSA) marker provided a possible explanation to the presence of TCR cells in the CD44 CD25 population. As shown in Fig. 1C, both in normal and TG mice the majority of the CD24 cells of both and lineages were CD25 and expressed low or intermediate levels of CD44. The majority of the CD24, presumably more mature DN and DN T-cells in normal thymus, expressed higher levels of CD44 and a fraction of the cells expressed the NK.1.1 and CD122 markers (data not shown). The TG CD24 DN cells expressed slightly lower levels of CD44 than those in normal thymus, and, as previously shown (26), the TG cells were NK1.1 and CD122. These data indicated that the TCR cells in the CD44 CD25 population might be derived from CD25 that had up-regulated their CD44 expression after TCR expression. This possibility was also suggested by previous data (35). To better understand the role of the various TCR proteins in the development of the TG DN T cells, we next tested whether the TG TCR -chain protein would be expressed also in TCR cells. To this end DN TG thymocytes were stained intracellularly with Abs toward the TCR protein and surface TCR /TCR -negative cells analyzed. The TG TCR protein was expressed intracellularly by variable proportions, but not by all cells, of the four TN populations (Fig. 1D). We consistently observed low 2B4 -chain expression in the CD44 CD25 population, but the reason for this phenomenon is not known. In similar experiments we also observed that virtually all the thymocytes in these populations expressing intracellular TCR -chains also expressed the TCR on the membrane, as revealed by surface TG TCR staining (Fig. 1E). These data indicated that TCR expression is a limiting factor for the development of the TG DN T cells. TG DN T cells develop at several DN stages In an attempt to identify when the TG DN T cells appear during intrathymic development, we purified TN CD44 CD25 and CD44 CD25 precursors from TCR TG mice by cell sorting and studied their differentiation potential in FTOCs. At day 3 of culture, we found TG DN T cells of the CD44 CD25 phenotype in lobes seeded with CD44 CD25 precursors (Fig. 2A). In addition, TG DN T cells were found in the subsequent developmental stages during the following 2 days of culture. This indicates that the seeded CD44 CD25 precursors differentiated in the FTOCs such that they both expressed the TCR and by time acquired the CD44/CD25 phenotype of later developmental stages. We have not formally addressed in reconstitution experiments, however, the possibility that cells that have already expressed the TCR may change their CD44/CD25 phenotype and hence be allocated to a certain downstream population. Nevertheless, because CD44 CD25 precursors gave rise to TG DN T cells (data not shown), we conclude that these cells can develop at several stages. As expected (2), both of these populations also gave rise to the development of T cells (data not shown). As an independent approach to study the kinetics of appearance of TG DN T cells in the various DN populations, we cultured day 14 fetal TCR TG thymic lobes in vitro. The proportion of TG TCR cells in the CD44 CD25 and CD44 CD25 populations decreased by time in culture (Fig. 2B), as did the overall number of cells in these populations (data not shown). In contrast, the proportion of TG TCR cells in the CD44 CD25 population increased as did also the overall proportion of this population. These data provided further support for the conclusion that TG DN T cells develop at several stages of differentiation. The Fc RI gene is expressed by developing T cells in the adult thymus It has previously been shown that the Fc RI gene is expressed in the early fetal thymus, whereas the CD3 gene is expressed later during gestation and in the adult thymus (36). Studies of Fc RI gene expression during T cell development in the adult thymus have shown that this gene is expressed primarily in CD25 DN thymocytes (37), whereas the CD3 gene is expressed mostly in the CD25 DN population and in later stages of development. Furthermore, fetal thymocytes express the Fc-receptors for IgG of both the Fc RII and Fc RIII isoforms (9). In our experiments, we also detected Fc RII/III-positive cells in the CD44 CD25 population of adult TG thymocytes both by RT-PCR analyses and flow cytometry using the 2.4G2 Ab (data not shown). Taken together, these previous observations suggested that DN precursor T cells may express the Fc RI gene. To clarify this issue, we isolated the four TN populations defined by CD44 and CD25 expression from adult thymocytes of C57BL/6 and TCR -TG mice by cell sorting and compared their expression of the Fc RI and CD3 genes using RT-PCR. In accordance with previous data (37, 38), we found that the steadystate level of CD3 mrna was relatively low in the CD44 CD25 population but increased during development (Fig.

5 The Journal of Immunology 6619 FIGURE 1. The TCR transgene induces early TCR expression. Thymocytes from TCR -chain TG mice (A) or C57BL/6 mice (B) were depleted of CD4- and CD8-expressing cells and stained with 2B4 (CD3 in B), TCR, CD44, and CD25 Abs as indicated and analyzed by flow cytometry. C, DN TCR -TG and C57BL/6 thymocytes (WT) were prepared as in A and stained with 2B4 (TG), TCR (WT), CD44, CD25, and CD24 (HSA) Abs. CD24-positive (HSA ) and CD24-negative (HSA ) cells were gated and analyzed separately. D, DN TCR -TG (solid lines) and C57BL/6 thymocytes (dashed lines) prepared as in A were stained with TCR, TCR (both PE-labeled), anti-cd25, and anti-cd44 Abs on the membrane and intracellularly with 2B4 -chain-specific Abs. TN cells were then gated and analyzed. E, TG DN cells prepared as in A were stained with 2B4, CD44, and CD25 Abs on the membrane and intracellularly with TCR Abs. The numbers indicate the percentage of total, DN, and TN thymocytes, respectively. 3A). Conversely, the steady-state level of Fc RI mrna was relatively high in this population, whereas the level decreased in the following TN populations. The TCR transgene did not influence the expression pattern of these two genes as the data obtained from normal and TG thymocytes were essentially identical. To confirm that the Fc RI gene is expressed in bona fide precursors we also analyzed various thymocyte populations from RAG 1-deficient mice. As can be seen in Fig. 3B, the Fc RI and CD3 genes were also reciprocally expressed in CD44 and CD25 precursors in this case. Taken together, these data suggested that the Fc RI gene may be expressed in the subpopulations that give rise to both and TG DN T cells. In addition, they suggested that early maturing T cells might use the Fc RI protein instead of the CD3 protein in their TCR/CD3 complexes.

6 6620 EARLY MATURING T CELLS afc RI mrna-negative clone (Fig. 4D). The fact that the anti- Fc RII/III Ab precipitated a protein of approximately the same size from lysates of the macrophage cell line WEHI-3 (Fig. 4E) further confirmed the identity of the Fc RI protein. This experiment also excluded the possibility that the precipitation of the Fc RI protein by anti-cd3 Abs would be due to binding of these Abs to Fc-receptors. Taken together, these data demonstrated that some of the TG DN T cells express the Fc RI protein in TCR/CD3 complexes. FIGURE 2. TG DN T cells are generated at several developmental stages. Purified TN TCR -TG CD44 CD25 thymocytes ( cells) were seeded into irradiated d14 fetal thymic lobes from C57BL/6 mice. A, Expression of CD25 and CD44 molecules by DN TG TCR cells was analyzed by flow cytometry at indicated days of culture. B, Day 14 fetal TCR -TG thymus lobes were analyzed immediately or cultured in vitro for the indicated days. The fraction of TG TCR cells was determined in the indicated populations as in A. TG DN T cells can use an unconventional TCR/CD3 complex We observed that a fraction of peripheral TG DN and T cells expressed the Fc RII/III marker by flow cytometry (Fig. 4A). These data appear contradictory to previously published results (7, 39). The reason for this discrepancy is not known, but might be related to the low expression level of these molecules. However, we have also confirmed the expression of the Fc RII/III molecules in both these cell types by RT-PCR analyses (data not shown). In those experiments we also investigated whether the membrane Fc RII/III expression by peripheral DN T cells would correlate with Fc RI mrna expression. As shown in Fig. 4B, this gene was expressed in TG DN T cells, T cells, and NK T cells. We also analyzed Fc RI expression in 10 randomly picked clones of TG DN T cell hybridomas. In accordance with the above flow cytometry analyses, 2 of 10 clones (20%) were Fc RII/III by flow cytometry and expressed the Fc RI gene by RT-PCR analysis (data not shown). Because some TG DN T cells may develop from early precursors, expressing the Fc RI gene (Figs. 2 and 3), we wanted to know whether the Fc RI expression at the mrna level would correlate with the use of the Fc RI -chain in TCR/CD3 complexes of these cells. To investigate this possibility, we analyzed the same hybridomas by immunoprecipitation experiments. Anti-CD3 Abs coprecipitated both the Fc RI (10 kda) and the CD3 protein (14 kda) from lysates of the two clones that were positive for Fc RI mrna (Fig. 4C). Abs to the TG TCR -chain coprecipitated the CD3 protein but, for unknown reasons, did not efficiently precipitate the Fc RI protein. As would be expected, the Fc RI protein was not detected in an anti-cd3 Ab precipitate from a lysate of The Fc RI protein is associated with the TCR complex in DN TG TCR thymocytes We also wanted to confirm that the Fc RI protein would be expressed as a part of TCR/CD3 complexes of DN TCR thymocytes from the TCR -TG mice. To test this possibility, the TCR/CD3 complexes of TG DN thymocytes were stained with biotin-labeled anti-tg TCR Abs and PE-streptavidin. Thereafter the cells were fixed and stained intracellularly to detect the intracytoplasmic part of the Fc RI protein. The thymocytes showed a somewhat patchy distribution of both the TCR proteins (red) and the Fc RI proteins (green) (Fig. 5A). This pattern of TCR staining is very similar to that observed when precursor thymocytes were stained with pt Abs (40). The fluorescence of the TCR and Fc RI staining overlapped on some cells, suggesting that these proteins partially colocalized in the membrane (top cell in Fig. 5A, middle and lower panels). However, areas with Fc RI staining alone were also seen on such cells, suggesting that they may also express Fc-receptors. The reason why overlapping these two images did not always yield the expected yellow color (Fig. 5A, top panel) could be due to the relatively low intensity of the Fc RI staining and that not all TCR/CD3 complexes contained the Fc RI protein. We reasoned that if the Fc RI protein would indeed be associated with the TCR then one would also expect these proteins to colocalize after capping the TCR with specific Abs. Capping of the surface TCRs was induced by incubating the cells at 37 during the staining with PE-streptavidin. The incubation was interrupted at various times, and a sample where a large number of the cells had visible cap formation by fluorescence microscopy was further analyzed. The capping greatly increased the overlap of the TCR and Fc RI -specific staining (Fig. 5B, top left cell), further supporting that these molecules colocalized in the membrane. Note that this cell still had areas with Fc RI staining only indicating that it also carried Fc-receptors. As a specificity control, capped cells were stained with irrelevant rabbit Abs and the FITC-conjugated second step Abs (Fig. 5C). In this sample we detected only red fluorescence from the bound anti-tcr Abs and no green fluorescence. We also detected thymocytes that expressed TCRs that were not detectably associated with the Fc RI protein, as well as thymocytes that stained for the Fc RI protein only (see Fig. 5B; cell to the right). The latter were numerous and probably represented TCR cells and T cells. The above results indicate that a fraction but not all of the DN TCR thymocytes expressed TCR/CD3 complexes containing the Fc RI protein. These data taken together with the observation that the Fc RI and CD3 genes are expressed in a reciprocal fashion in early precursor thymocytes are consistent with the view that early developing TG DN T cells may express these unconventional TCR/CD3 complexes. Discussion The TCR -chain gene is expressed later than the other TCR genes; therefore, the TCR -chain protein is an important limiting factor in T cell development. In this report we present data indicating

7 The Journal of Immunology 6621 FIGURE 3. Fc RI and CD3 genes are reciprocally expressed in adult TN thymus subpopulations. A, RT-PCR analysis was performed using RNA from sorted TN thymocyte subpopulations from C57BL/6 and TCR -TG mice. cdnas were amplified with HGPRT-, Fc RI -, and CD3 -specific primers, as indicated. The Fc RI - and CD3 -specific products were blotted to filters and hybridized with oligonucleotide probes. The diagrams show the ratios between the signal obtained by phosphorimager analysis and the HGPRT signal quantitated from ethidium bromide-stained gels. The difference in ratios between cdnas from normal and TG mice is due to the differential ethidium bromide signal in different agarose gels. B, RT-PCR analysis of cdnas from various precursor populations sorted from RAG-deficient (RAG / ) thymocytes. cdnas were equalized using HGPRT-specific primers. that expression of the TCR -chain protein by transgenesis induced expression of the TCR at developmental stages at which very few TCR cells could be detected in the normal thymus. Thus, in the TCR TG mice we found TCR expressing cells already in the CD44 CD25 and CD44 CD25 DN thymocyte subpopulations. The development of such cells was also detected both by analysis of fetal TG thymi at various days of development and in normal fetal thymi reconstituted with adult TG TN CD44 CD25 and TN CD44 CD25 precursors. Other investigators have previously reported that these precursor populations from normal mice are also capable of reconstituting the development of T cells (2), and we have confirmed these data using TG precursors (K.P., unpublished observations). The TG TCR -chain protein could potentially interfere with the development of both - and lineage cells. As shown in our previous report, the proportion and absolute number of double positive (DP) thymocytes is decreased in TCR -TG mice (26). The pre-tcr is important both for the progression of T cell development and for the expansion of -lineage cells (reviewed in Ref. 41). The decrease in DP thymocytes observed in the transgenics might then be due to the fact that TG TCR -chain proteins compete with pt proteins in binding to the TCR -chains, thereby leading to suboptimal formation of the pre-tcr in the precursor cells. Furthermore, our previous report (26) also demonstrated that TCR -expressing T cells were depleted in the TCR -transgenics, suggesting that the TCR -chain expression interfered with T cell development as well. In this report we showed that the CD3 and Fc RI genes were expressed in a reciprocal manner in adult TN thymocytes both in normal and in TG mice. The expression of the Fc RI gene was high during the early CD44 CD25 stage after which it declined, whereas expression of the CD3 gene was low in that population and increased thereafter. Most importantly, the expression of the TCR transgene induced the maturation of early precursors to DN TCR T cells expressing the Fc RI protein as a component of the TCR/CD3 complex. Thus, our data suggest that the usage of this protein in the TCR/CD3 complex of mature peripheral T cells might be developmentally controlled and fixed upon expression of the TCR heterodimer. If this possibility would be correct one would expect that T cells maturing at later stages of development might use the CD3 rather than the Fc RI protein. Indeed, many peripheral T cells do not express the Fc RI gene nor do

8 6622 EARLY MATURING T CELLS FIGURE 4. TG DN T cells can use an unconventional TCR/CD3 complex. A, B cell-depleted DN TCR -TG spleen cells were analyzed by flow cytometry. The histograms show the fraction of DN 2B4 and TCR cells expressing Fc RII/RIII. B, cdnas from sorted TG DN T cells, T cells, and NK T cells from normal C57BL/6 thymus were equalized using HGPRT-specific primers. Equalized cdnas were amplified with Fc RI - and CD3 -specific primers, blotted to filters, and hybridized with oligonucleotide probes. Lysates from Fc RI mrna T cell hybridomas (clones K15 and K23) were precipitated either with anti-tg TCR (C, lanes 1 and 2) or anti-cd3 (C, lanes 3 and 4) Abs and separated on a 10% Tricine SDS-PAGE gel. Immunoblots were performed with either anti-cd3 or anti-fc RI Abs (lanes 1 2 and 3 4, respectively). D, Lysates from a control Fc RI mrna T cell hybridoma K1 was precipitated and blotted in the same way as in C. The blot was overexposed to confirm the absence of the Fc RI protein. E, Anti-CD3 (lane 1) and anti-fc RII/III (2.4G2) (lane 2) immunoprecipitates of a lysate from the WEHI 3 macrophage cell line were immunoblotted with Fc RI Abs to demonstrate the specificity of the detection system. The Fc RI (10 kda) and CD3 proteins (14 kda) and dimers thereof are indicated. the majority of TG DN T cells (data not shown). However, this hypothesis could not be directly tested in the FTOC experiments because the yield of mature T cells is so low that the association of the Fc RI protein with the TCR/CD3 complex in these cells cannot be firmly established. The settlement of this point will have to await more elaborate experiments in which T cell are recovered from the FTOCs, expanded, and analyzed with either of the two approaches used here. The CD3, CD3, and Fc RI proteins all have in common that they participate in the assembly of the TCR (42, 43). Fc RI deficient mice appear normal in T cell development, even though the peripheral and thymic DN TCR cells have a TCR low phenotype in these mice (44). The functional performance of TCRs carried by T cells of Fc RI -deficient mice was not reported (44). However, another study demonstrated that T cells from TCR -TG CD3 -deficient mice responded to TCR stimulation but not to Ag-specific stimulation (45), indicating that the Fc RI protein at least in some situations cannot functionally fully replace the CD3 protein. Indeed, the association of the tyrosine kinase fyn and the downstream signaling from Fc RI -containing TCR/CD3 complexes was shown to be distinct from those containing CD3 (46). Thus, T cells carrying the unconventional TCR/CD3 complexes might be functionally different from those carrying conventional CD3 -containing complexes. Interestingly, not only mature conventional T cells but also NK T cells were depleted in CD3 -deficient mice (47). The deficiency in NK T cells might either be due to the lack of CD3 signaling or to the few DP thymocytes in these mice, because the DP cells are crucial for the development of NK T cells (48 50). In contrast, NK T cell numbers were increased in these mice. We favor the view that and NK T cells are both able to develop in the absence of CD3 expression, but that NK T cells are independent of DP thymocytes for their maturation. Our previous report suggested that some of the TG DN T cells derive from T cell precursors that had successfully rearranged the TCR chain (26). However, due to the large number of the TG DN T cells, this may not be the only source of these cells. NK1.1 DN T cells are also depleted in the thymus but not in the liver of these mice (Ref. 26 and data not shown). Therefore, some of the TG DN T cells might originate from precursors of NK T cells. Such cells may not express an autoreactive TCR due to the forced expression of the TG TCR -chain, and would then not acquire the characteristic NK T cell phenotype (8). The observation that TG DN T cells developed at several TN stages and that most of these cells probably originate from precursors of T cells (24, 26), suggests the possibility that T cells in the normal thymus will also mature at these stages provided they express the TCR. As shown in here (Fig. 1B), very

9 The Journal of Immunology 6623 FIGURE 5. Early thymocytes can use an unconventional TCR/CD3 complex. A, DN transgenic thymocytes were stained with biotinylated anti-tg TCR -Abs and PEstreptavidin. The cells were incubated on ice (A) or for 15 min at 37 C to induce capping of the TCR (B and C) then fixed and labeled intracellularly with anti-fc RI (A and B) or control rabbit Abs (C). These were detected by FITClabeled anti-rabbit Ig antiserum (see Results for details). The cells were then analyzed by confocal microscopy. Top, Overlay of images obtained of the green (FITC, middle row) and red (PE, bottom row) fluorescences. few surface CD3-positive TCR -negative cells were found in the normal thymus at the CD44 CD25 and CD44 CD25 stages. Importantly, however, we detected a low but significant number of T cells in these populations in in vitro cultured fetal thymi (data not shown). Thus, the use of transgenesis revealed the capacity of lineage cells to develop at these stages of differentiation. Furthermore, our present data suggested that lineage cells developing in the thymus may express the Fc RI protein as a component of their TCR/CD3 complexes. Because we have previously shown that the TG DN T cells studied here also up-regulated CD8 -expression upon activation (27) exactly as T cells, these data taken together indicate that T cells phenotypically similar to those found in gut epithelium (37) can also be produced in the thymus. The pre-tcr plays an important role in the allelic exclusion of the TCR locus (51, 52). Furthermore, it was claimed that the pre-tcr delivers a signal directing the vs lineage split toward the development of lineage cells, as more T cells expressing intracellular TCR -chains were generated in pt -deficient mice than in normal mice (53). These data were interpreted to suggest that the pre-tcr would remove precursors from the lineage. However, virtually all CD25 TN precursors have been shown to express the pt protein (53, 54) and, if the above explanation were correct, one would not expect to find in normal mice lineage cells expressing the TCR protein intracellularly. Alternatively, lineage commitment might be induced before TCR expression and the role of the pre-tcr would be to confirm that decision. It is clear that in the absence of a functional pre- TCR, lineage cells still develop. Thus, both the -TCR and the -TCR can compensate for the role of the pre-tcr in development, even though the production of DP thymocytes is much less efficient (23, 25) in these cases. Furthermore, our present and previous results as well as those from another group indicated that lineage precursors could be induced to maturation by the TCR (24, 26). These data are clearly compatible with the alternative possibility and suggest that the role of the TCR in these cases would be rather to induce survival/maturation signals in already committed precursors. The apparent inefficiency of pre-tcr-deficient mice in generating DP thymocytes, i.e., lineage cells, is probably a result of the relatively poor capacity of the and TCRs in inducing the expansion of early precursors. The reason why more than lineage cells develop in the adult thymus as compared with the fetal thymus could rather be due to the efficient expansion of lineage cells induced by the developmental stagespecific expression of the pre-tcr than to a direct role in lineage commitment. Thus, the role of the TCR signaling in lineage commitment is still controversial and will require more experimentation to be settled. Note. While this manuscript was under revision, Terrence et al. (55) provided further evidence for the lineage origin of the transgene-induced DN T cells studied here. Furthermore, Trop et al. (56) demonstrated that TCR -chains efficiently displace the pt protein and thereby prevent pre-tcr expression, as proposed in this manuscript. Acknowledgments We thank Dr. Martin Gullberg for his expert technical advice and generous gift of reagents, Stefan Seth for operating the confocal microscope, Dr. Su Ling Li for helpful advice, Dr. Susanna Cardell and Dr. William Agace for gift of RAG1-deficient mice and for improving the manuscript, and Prof. Tomas Leanderson for support. References 1. Amagai, T., M. Itoi, and Y. Kondo Limited development capacity of the earliest embryonic murine thymus. Eur. J. Immunol. 25: Godfrey, D. I., J. Kennedy, T. Suda, and A. Zlotnik A developmental pathway involving four phenotypically and functionally distinct subsets of CD3 CD4 CD8 triple-negative adult mouse thymocytes defined by CD44 and CD25 expression. J. Immunol. 150: Wu, L., M. Antica, G. R. Johnson, R. Scollay, and K. Shortman Developmental potential of the earliest precursor cells from the adult mouse thymus. J. Exp. Med. 174: Wu, L., C. L. Li, and K. Shortman Thymic dendritic cell precursors: relationship to the T lymphocyte lineage and phenotype of the dendritic cell progeny. J. Exp. Med. 184:903.

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