αβtcrs Differ in the Degree of Their Specificity for the Positively Selecting MHC/Peptide Ligand

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1 This information is current as of December 16, αβtcrs Differ in the Degree of Their Specificity for the Positively Selecting MHC/Peptide Ligand Piotr Kraj, Rafal Pacholczyk and Leszek Ignatowicz J Immunol 2001; 166: ; ; doi: /jimmunol References Subscription Permissions Alerts This article cites 72 articles, 23 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 16, 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 TCRs Differ in the Degree of Their Specificity for the Positively Selecting MHC/Peptide Ligand 1 Piotr Kraj, Rafal Pacholczyk, and Leszek Ignatowicz 2 We have tested the peptide specificity of positive selection using three transgenic TCRs, originally selected on class II MHC (A b ) covalently bound with one peptide E (52 68) (Ep). The transgenic TCR specific for the cytochrome c-derived (43 58) peptide was selected on A b bound with different arrays of endogenous peptides or the analogue of Ep covalently bound to A b, but not on the original A b Ep complex. In contrast, transgenic TCRs specific for two different analogues of the Ep peptide and A b did not mature as CD4 T cells in various thymic environments, including the A b EpIi mice. These results show that TCRs can be promiscuous or specific for the selecting MHC/peptide complex, and suggest that in mice described in this study transgenic expression of the TCR changes the original requirements for the positively selecting MHC/peptide complex. Future studies will determine whether the latter phenomenon is general or specific for this system. The Journal of Immunology, 2001, 166: The majority of T lymphocytes develop in the thymus, where their receptors for Ags ( TCRs) confront multiple MHC/peptide complexes present on thymic stromal cells. TCRs are inherently biased to recognize MHC and can discriminate between peptides bound to MHC within a wide range of avidity (1 4). Low-avidity interactions between TCRs on immature T cells and self MHC/peptide ligand(s) result in positive selection, which rescues selected thymocytes from programmed cell death (5, 6). High-avidity interactions between TCRs and self MHC/peptide complex(es) have an opposite outcome, i.e., the thymocyte is classified as self-reactive and is induced to die during negative selection (7, 8). The lack of recognition of self MHC/ peptide complexes by immature thymocytes allows programmed cell death to occur and neglected immature T cells to die (9). While the specificity of negative selection for a defined MHC/peptide has been shown in multiple experiments, the contribution of self peptide(s) presented by MHC during positive selection of T cells is less known. Using mice deficient in intracellular chaperones involved in Ag processing, it was demonstrated that efficient positive selection of a wide repertoire of TCRs expressed on T cells requires many different peptides to be associated with MHC molecules (10 14). However, a noticeable number of CD8 T cells differentiated in fetal thymic organ cultures of TAP1 or 2 -microglobulin-deficient mice when the expression of class I MHC was restored by adding a single peptide (15, 16). Likewise, class II MHC molecules bound with a dominant class II-associated invariant chain peptide (CLIP) 3 or single E (52 68) peptide (Ep) could select a number of TCRs, suggesting that positive selection of Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA Received for publication June 27, Accepted for publication November 21, 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 National Institutes of Health basic research grants (AI A1 and HD ) to L.I. 2 Address correspondence and reprint requests to Dr. Leszek Ignatowicz, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA address: ignatowi@immagene.mcg.edu 3 Abbreviations used in this paper: CLIP, class II-associated invariant chain peptide; PCC, pigeon cytochrome c-derived peptide. some TCRs may be promiscuous (17 20). Moreover, TCRs selected by a single MHC/peptide complex could recognize antigenic peptides that were different from the selecting peptide, implying that some of these receptors may engage distinct MHC/ peptide ligands (21 23). Peptide specificity of positive selection of T cells has been commonly studied using TCR transgenic mice in which all T cells can express the same TCR. Transgenic expression of TCR preserves its original specificity for antigenic peptide and sensitivity to negative selection (7, 24 26). However, whether transgenic expression conserves the TCR s original requirements for positively selecting MHC/peptide complex is unknown. Indeed, thymic development of TCR transgenic T cells in a genuine thymic milieu can be manifested by highly enhanced or severely impaired positive selection (27 31). Since the TCR genes for transgenic mice are cloned from wild-type mice that express multiple MHC/peptide complexes, the natural, positively selecting MHC/peptide ligand(s) for a native TCR is not known and, therefore, its role in the selection of transgenic TCR could not be evaluated. In this study, we investigated the flexibility of positive selection of several transgenic TCRs originally selected in vivo in mice expressing one detectable class II MHC/peptide complex. We cloned three TCRs selected in vivo on thymic epithelium expressing A b molecules covalently bound with a single peptide (Ep), and specific for different peptides presented by A b (19). These TCRs were expressed as transgenes using two different expression systems under the CD2 or TCR promoters (32, 33). We then followed their in vivo positive selection on thymic epithelium that expressed A b bound with many or few endogenous peptides or covalently linked single peptides. Transgenic expression of each of these TCRs arrested the development of transgenic T cells toward the CD4 lineage on the original, positively selecting thymic epithelium expressing the A b Ep complex. However, one of the transgenic TCRs, specific for antigenic peptide with no sequence homology to Ep peptide, was efficiently selected on thymic epithelium expressing A b bound with different arrays of self peptides or a covalently bound analogue of Ep. In contrast, transgenic TCRs specific for two different Ep analogues were not selected as CD4 T cells in any of the tested thymic environments. Instead, transgenic T cells bearing these TCRs differentiated as CD8 or CD4 CD8 T cells, similarly to misselected MHC class II-restricted transgenic TCRs (such as AND, DO11.10) that commit to Copyright 2001 by The American Association of Immunologists /01/$02.00

3 2252 SPECIFICITY OF T CELL-POSITIVE SELECTION the wrong lineage in the absence of the optimal selecting class II MHC/peptide ligand or coreceptor (34 38). These results demonstrate that the repertoire of TCRs selected by the A b Ep complex contains TCRs with high or low specificity for the positively selecting MHC/peptide ligand, and indicate that transgenic expression of TCR changes the original requirement for the positively selecting MHC/peptide ligand. Materials and Methods Animals Mice expressing A b covalently bound with the single peptide derived from E (52 68) (Ep) were described previously (19). We have produced mice expressing covalently bound analogues of Ep (Ep58K and Ep63K) and pigeon cytochrome c-derived peptide PCC (43 58) (Pacholczyk, manuscript in preparation). All single A b /peptide mice were crossed to invariant chain knockout mice (A b Ii ) (kindly provided by E. Bikoff, Harvard Medical School, Boston, MA and R. Germain, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD) and mice deficient for wild-type A b (kindly provided by D. Mathis, Harvard Medical School). The DM knockout mice were kindly provided by L. van Kaer (Vanderbilt University, Nashville, TN). C57BL/6 (A b wt), A bm12, and TCR -chain knockout mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed under specific pathogen-free conditions in the animal care facility at the Medical College of Georgia (Augusta, GA). To generate chimeric mice, 8- to 12-wk-old recipient animals were lethally irradiated (1100 rad) and reconstituted on the same day with fetal liver cells from A b fetuses (day 15, gestational age). Alternatively, recipient mice were reconstituted with the bone marrow of adult mice treated with anti-thy-1.2 mab (H.O ) and complement. Chimeras were used for experiments not earlier than 8 wk after reconstitution. At least three mice or chimeras of each type were analyzed. Generation of Ag-specific T cell hybridomas Hybridomas specific for E (52 68) analogues and PCC were generated as described (22, 39). Briefly, single peptide mice expressing A b covalently bound with peptide derived from the E molecule (A b EpIi ) were irradiated and reconstituted with fetal liver cells from C57BL/6 mice. Chimeras were immunized with synthetic peptides Ep58K(ASFEAQKALANIAV DKA), Ep63K(ASFEAQGALANKAVDKA), and PCC(AEGFSYTDA NKNKGIT) in CFA (2.5 mg/ml). CD4 T cells from the draining lymph nodes were converted into T cell hybridomas, as described (39). The PCCspecific hybridoma responded better to the PCC(50V54A) analogue, and this peptide was then used as Ag in proliferation assays. Hybridomas were analyzed using flow cytometry for the expression of CD4 and TCR. Double-positive hybridomas were tested for peptide-specific response using a standard IL-2 release assay (40). In short, a T cell hybridoma ( ) cell was stimulated in the presence or absence of a specific peptide using irradiated A b wt splenocytes. After 24 h, supernatants were tested for the presence of IL-2 by measuring proliferation of IL-2-dependent cells (HT-2) using an MTT assay (Sigma, St.Louis, MO) (41). Cloning TCRs from T cell hybridomas and generation of TCR transgenic mice Total RNA was isolated using the Ultraspec RNA isolation system (Biotex, Edmonton, Canada) based on the guanidium thiocyanate method (42). The RNA was subjected to affinity chromatography on oligo(dt) resin (Qiagen, San Francisco, CA). Sequences of the TCR genes were obtained using two independent cloning strategies. We used V - and V -specific primers to amplify and clone parts of the - and -chains. Alternatively, we used a linker PCR method to clone V regions of the - and -chains starting with the 5 untranslated sequences. In the first approach, the first strand of the cdna was synthesized with AMV reverse transcriptase (Promega, Madison, WI) and an oligo(dt) primer. Aliquots of the cdna reaction were used in the PCR reaction with a collection of V -orv -specific oligonucleotides as 5 primers and primers specific for and (TCR-C 3) C regions as 3 primers. We used modified linker PCR as the second approach to clone TCR genes (42). Using this method, we were able to obtain the sequences of V - and V chain transcripts starting with the 5 untranslated region. The two approaches used yielded the same results: that is, the - and -chain clones isolated by amplification with V - and V -specific primers had the identical complementarity-determining region 3 sequences as isolated by the linker PCR method. All hybridomas had nonproductive rearrangement of one of the loci. The DNA sequences obtained by the linker PCR method contained the complete V and V sequences, starting 5 from the start codon. The sequences obtained were used to search the GenBank database. The PCC(50V54A)-specific TCR is encoded by V 4.5-J 23/V 8.1-D 2- J 2.6 genes, the Ep58K-specific receptor uses V 3.4-J 27/V 3-D 1- J 1.2 genes, and the Ep63K-specific receptor uses V 2-J 26/V 14-D 2- J 2.6 genes. Based on sequence information, we designed primers to amplify and clone the - and -chains in TCR expression vectors pt and VA-hCD2 (kindly provided by D. Mathis and D. Kioussis, National Institute of Medical Research, London, U.K.). The PCC(50V54A)-specific TCR was cloned in both pt and VA-hCD2 cassettes. The Ep58K- and Ep63K-specific TCRs were cloned only into pt cassettes. The integrity of constructs was checked by transfection using a variant of the 5KC hybridoma deficient in TCR expression. Transfectants were analyzed by a stimulation assay with the respective antigenic peptides and by staining with mab specific for the respective TCR V (if available) and V segments (data not shown). All TCR transgenic mice were made by comicroinjection of the respective TCR and TCR constructs into fertilized eggs of F 1 (C57BL/6 CBA/Ca) mice. Two founder mice were generated for PCC(VA), one for PCC(pT), three for Ep58K(pT), and two for Ep63K(pT) constructs. No significant differences in the phenotypes among the different mouse lines expressing the same transgenic TCRs were noticed. In particular, founder lines expressing a particular TCR transgene did not differ in the level of TCR expression on thymocyte populations. Selected founder mice were crossed at least four times with C57BL/6TCR / mice. Mice used as bone marrow donors were TCR transgenics that expressed wildtype A b and were deficient in the expression of endogenous TCR -chain. Flow cytometry analysis mabs specific for CD4, CD8, and different V and V segments of TCRs were self-prepared or purchased from PharMingen (San Diego, CA). Standard staining procedures were used as previously described (39). Cells were analyzed using a FACSCalibur instrument (Becton Dickinson, San Jose, CA) and CellQuest software. Spleens were strained through nylon mesh. Erythrocytes were lysed by treatment with ammonium chloride solution. Cells were stained on ice with anti-cd45r (B220) Ab and with Y3P or YAe. Gating on forward and side scatter identified viable cells. Expression of total A b or A b bound to Ep peptide was determined on gated CD45R-positive cells. Ag response of TCR transgenic cells Proliferation of lymph node cells isolated from various chimeras was measured in response to Ag. Lymph node cells from transgenic mice and radiation chimeras reconstituted with bone marrow from PCC(VA) or PCC(pT) TCR C57BL/6 transgenic mice were used in proliferation assays. Cells were stimulated with the antigenic peptide PCC(50V54A) (50 g/ml) presented by the wild-type A b APCs. A total of irradiated APCs was mixed with , , and responder cells. Lymph node cells from Ep58K(pT) and Ep63K(pT) TCR transgenic mice and Ep58K(pT)TCR 3A b EpIi and Ep63K(pT)TCR 3A b EpIi radiation chimeras were stimulated with soluble antigenic peptides Ep58K and Ep63K, respectively, (50 g/ml) that were presented by spleen cells expressing A b and lacking class I MHC expression due to knockout of the 2 -microglobulin gene. In 96-well plate, irradiated APCs were mixed with and responder cells. IgGVH (59 74) peptide was used as a negative control, and proliferation was measured after 3 days by the MTT assay (41). Results Selection and cloning of Ag-specific TCRs from CD4 T cells positively selected in vivo by a single A b Ep complex We have followed the in vivo development of CD4 T cells expressing transgenic TCRs that were originally positively selected on class II MHC (A b ) bound with the known peptide (Ep). In mice exclusively expressing the A b Ep complex, more than two-thirds of TCRs on CD4 T cells were found not tolerant to self peptides bound to A b, which led to their profound negative selection upon exposure to wild-type A b /peptide complexes (19, 39). In vivo studies of the development of CD4 T cells expressing such TCRs would have to be limited to the original A b EpIi mice, and their promiscuity or specificity for the selecting MHC/peptide ligand cannot be determined in mice expressing A b bound with peptides different from Ep. Therefore, to isolate genes encoding TCRs tolerant to self peptides and A b, we produced radiation chimeras by

4 The Journal of Immunology 2253 reconstituting lethally irradiated A b EpIi mice with fetal liver cells from 15-day-old fetuses of C57BL/6 mice. In these chimeras, the TCR repertoire of CD4 T cells was positively selected by radioresistant thymic epithelium expressing the A b Ep complex and was purged of TCRs reactive with the natural set of self peptides by the donor s APCs derived from fetal liver. Expression of donor wild-type A b molecules in the periphery allowed us to prime CD4 T cells positively selected on A b Ep on thymic epithelium with antigenic peptides, and to establish CD4 T cell hybridomas specific for antigenic peptides presented by A b (39). To have a better representation of the repertoire originally positively selected by A b Ep complex, we cloned TCR genes from three different CD4 T cell hybridomas and used them to make TCR transgenic mice. All three hybridomas had the second TCR locus nonfunctionally rearranged, so the TCR expressed on the surface had to mediate positive selection of the parental CD4 T cell (Ignatowicz, Kraj, unpublished). All three hybridomas were also tolerant to covalent A b Ep complex and A b bound with self peptides. The first hybridoma, 2.1.4, recognized the PCC (43 58) peptide or its analogue PCC(50V54A), which had enhanced binding and stimulatory properties over the original PCC peptide (43). The TCR cloned from this CD4 T cell hybridoma was expressed in vivo using either the CD2 promoter (VA cassette) or genuine TCR promoters (pt /pt cassettes) (32, 33). We refer to these two transgenic TCRs as PCC(VA) and PCC(pT). Two other CD4 T cell hybridomas that we selected as TCR gene donors expressed TCRs specific for two analogues of the Ep peptide that each had a single TCR contact residue changed (22, 44). Hybridoma was specific for the Ep analogue with isoleucine replaced by lysine in position 63 (Ep63K). Another hybridoma, 123.1, was specific for the Ep analogue with glycine replaced by lysine in position 58 (Ep58K) (39). Both of these TCRs were expressed as transgenes using pt /pt expression cassettes. We refer to these two transgenic TCRs as Ep63K(pT) and Ep58K(pT). The DNA sequences of TCRs isolated from the transgenic mice were confirmed by DNA sequencing to exclude a possibility of mutation. The progeny of founder mice for each transgenic TCR were backcrossed to TCR -chain-deficient ( ) H-2 b mice. Four selected mouse lines with exclusive expression of the respective transgenic TCRs were produced and used in all further studies. Transgenic PCC(VA) and PCC(pT) TCRs are efficiently selected on A b bound with different repertoires of self-derived peptides, some A b /covalent peptide complexes, but are not positively selected by the original A b Ep complex We first analyzed two transgenic TCRs that recognized the PCC(50V54A) analogue. As shown in Fig. 1, the expression levels of the transgenic PCC(VA) or PCC(pT) TCRs on CD4 CD8 and CD4 subpopulations of thymocytes were slightly different from the expression level of endogenous TCRs in wild-type mice. The expression of the PCC(VA) TCR was higher then the expression of native TCRs on CD4 CD8 thymocytes, while the same TCR cloned in the (pt) cassettes was expressed at lower levels. Moreover, thymocytes and peripheral CD4 T cells partially downregulated expression of the PCC(pT) TCR. This phenomenon was not associated with negative selection as tested by TUNEL assay, staining with annexin V, or thymus cellularity (data not shown). Noticeably, the PCC(VA) or PCC(pT) TCRs were efficiently positively selected by wild-type A b /peptide complexes in the absence of A b Ep complex, and isolated transgenic CD4 T cells vigorously responded to antigenic peptide (shown later in Fig. 4, A and B). Positive selection of the PCC(VA)- or PCC(pT)-expressing thymocytes specifically depended on the presence of A b molecules. FIGURE 1. Expression of PCC(VA) and PCC(pT) TCRs on thymocytes and peripheral lymph node cells. A, Thymocytes from C57BL/6 mice (left), PCC(VA) (middle), and PCC(pT) TCR transgenic mice (right) were stained for CD4 and CD8 expression. The total numbers of thymocytes in C57BL6, PCC(VA), and PCC(pT) TCR transgenic mice were respectively , , At least three mice of each kind were analyzed. B, TCR expression on gated double-negative CD4 CD8 (left), double-positive CD4 CD8 (middle), and single-positive CD4 CD8 thymocytes (right) of PCC(VA) ( ), PCC(pT) TCR mice ( ), and C57BL/6 mice (OOO). C, Peripheral lymph node cells of C57BL/6 mouse (left), PCC(VA) (middle), and PC- C(pT) (left) TCR transgenic mice were stained for CD4 and V 8 expression. Numbers indicate the percentage of different subpopulations of thymocytes or peripheral T cells. Lethally irradiated recipient mice expressing A d E d (BALB/c) or A k E k (CBA/Ca) molecules or lacking A b expression and which were reconstituted with the bone marrow from these TCR transgenic mice did not generate mature thymocytes (data not shown). Following these initial observations, we investigated the peptide specificity of positive selection of these two transgenic TCRs by reconstituting lethally irradiated mice expressing A b molecules bound with different arrays of endogenous or covalently bound peptides with T cell-depleted, transgenic bone marrow. As shown in the upper panel in Fig. 2A, nocd4 transgenic T cells were found in the thymus and in the periphery of the original A b EpIi mice lethally irradiated and reconstituted with transgenic bone marrow. Development of the PCC(VA) thymocytes was arrested at the CD4 CD8 stage, and no negative selection was detected by TUNEL assay (data not shown). Similarly, thymocytes bearing the PCC(pT) TCR controlled by the genuine TCR regulatory elements were not positively selected by the original thymic epithelium expressing the A b Ep complex (Fig. 2B). An interpretation that native CD4 T cells expressing this TCR were originally selected in A b EpIi chimeras by undetectable A b /self-derived complexes rather than the A b Ep complex could not explain why the same low-abundant peptides did not continue to positively select the PCC(VA) or PCC(pT) TCRs (45). In contrast, the PCC(VA)-bearing CD4 T cells were positively selected in radiation chimeras that express, on thymic epithelium,

5 2254 SPECIFICITY OF T CELL-POSITIVE SELECTION FIGURE 2. Flow cytometric analysis of thymocytes from recipient mice reconstituted with bone marrow from PCC(VA) and PCC(pT) TCR transgenic mice. Eight- to ten-week-old recipient mice were lethally irradiated and reconstituted i.v. with at least bone marrow cells from mice expressing PCC(VA) or PCC(pT) TCRs crossed to TCR C57BL/6. Donor bone marrow was treated with anti-thy-1.2 and complement. Panels on the left show thymocytes stained with anti-cd4 and anti-cd8 Abs, and panels on the right show peripheral lymph node cells stained with anti-cd4 and anti-v 8 Ab. Bone marrow from PCC(VA) and PCC(pT) TCR transgenic mice was used to reconstitute the following recipient mice: A, B,A b EpIi ; C,A bm12 ; D, E,A b Ii ; F,A b DM. Numbers represent the percentage of different subpopulations of thymocytes or peripheral T cells. The numbers of cells recovered from thymuses were: A, ; B, ; C, ; D, ; E, ; F, Three mice in each group were analyzed. a variant of A b with an altered peptide-binding grove (A bm12 ) (Fig. 2C). The CD4 T cells from A bm12 mice are not tolerant to APCs from A b wild-type mice, and vice versa, due to the different array of self peptides bound to A b vs A bm12 (46 48). This suggested that different self peptides might select the PCC(VA) TCR. To test whether A b molecules occupied with a more limited set of self peptides would support positive selection of the PCC(VA)- or PCC(pT)-expressing CD4 T cells, we made radiation chimeras using invariant chain-deficient mice (A b Ii ) as recipients. In these mice, peptides bound to low levels of A b are distinct from peptides bound to A b in wild-type mice (49 51). Nevertheless, the PCC(VA) TCR and PCC(pT) TCRs were positively selected by A b /self peptide complexes expressed on thymic epithelium of A b Ii mice (Fig. 2, D and E). In contrast, the development of transgenic T cells was arrested at the CD4 CD8 stage when bone marrow from PCC(VA) or PCC(pT) mice was used to reconstitute H2-DM knockout mice (A b DM ), in which the majority of A b molecules is occupied with CLIP peptide (17, 18, 52) (Fig. 2F and data not shown). In conclusion, we found that, regardless of the expression system used, the TCR expressed as a transgene is not positively selected by its original, positively selecting class II MHC/peptide ligand, but instead is selected on high or low levels of A b bound with different endogenous peptides. Since both TCRs tested above were not selected in the original A b EpIi mice, we made new transgenic mice that express A b covalently bound with two different variants of Ep. Each of these variants has a single amino acid mutation in the residues identified as TCR contact residues. Glycine at position 58 and isoleucine at position 63 were substituted with lysine to produce Ep58K and Ep63K, respectively. We also made transgenic mice that express PCC (43 58) peptide covalently bound to A b. This peptide has previously been found to have the lowest affinity to A b molecules of all peptides tested in this study (22). We used mice expressing the PCC/A b complex to determine whether low-abundant peptides that might have replaced the covalently linked peptide positively selected transgenic CD4 T cells. None of the covalently bound Ep variants or PCC peptides were recognized by the original T cell hybridoma. Transgenic mice expressing Ep variants or PCC covalently bound to A b were separately bred to obtain mice without endogenous A b and invariant chain (11, 53). These mice, which we refer to as A b Ep58KIi,A b Ep63KIi, and A b PCCIi (Pacholczyk, manuscript in preparation), were then lethally irradiated and separately reconstituted with bone marrow from the PCC(VA) or PCC(pT) TCR transgenics. Development of the PCC(VA)- or PCC(pT)-bearing thymocytes was arrested at the double-positive stage in A b PCCIi and A b Ep63KIi radiation chimeras reconstituted with bone marrow from the relevant TCR transgenic mouse (Fig. 3, A and B, and data not shown). In contrast, both PCC(VA) and PCC(pT) TCRs were positively selected on thymic epithelium expressing the A b Ep58K complex (Fig. 3, C and D). The CD4 T cells selected in A b Ep58KIi recipients repopulated the peripheral lymphoid organs and responded in vitro to antigenic peptide (Fig. 4, A and B). Transgenic TCRs specific for analogues of Ep peptide are misselected as CD4 CD8 or CD8 T cells in different thymic environments Two transgenic TCRs specific for the Ep63K and Ep58K analogues were expressed in pt /pt cassettes since this system provides genuine TCR regulatory sequences and because only quantitatively different outcomes of positive selection were recorded for previously analyzed PCC(VA) vs PCC(pT) transgenic T cells. As shown in Fig. 5, mice expressing wild-type A b /peptide complexes and lacking endogenous TCR -chain had higher expression levels

6 The Journal of Immunology 2255 FIGURE 3. Analysis of PCC(VA) or PCC(pT) TCR transgenic thymocytes and peripheral lymph node cells isolated from single peptide mice. Recipient mice used were: A, A b Ep63KIi ; B, A b PCCIi ; C, D, A b Ep58KIi. These mice were reconstituted with bone marrow from PC- C(VA) TCR (A, B, C) or PCC(pT) TCR (D) transgenic mice. Left panels, Show thymocytes stained for CD4 and CD8 expression. Right panels, Show expression of the V 8 transgenic -chain and CD4 on peripheral lymph node cells. Numbers represent the percentage of different subpopulations of thymocytes or T cells. Total number of thymocytes were: A, ; B, ; C, ; D, Data represent analysis of at least three mice in each group. of the Ep63K(pT) or Ep58K(pT) TCRs on some double-positive thymocytes, compared with the expression of native TCRs. A similar expression pattern of these two transgenic TCRs was a common feature found in five different founder lines (2 for Ep63K(pT) and 3 for Ep58K(pT)). Thymocytes bearing the Ep63K(pT) or Ep58K(pT) TCRs that developed in mice expressing wild-type A b bound with self peptides differentiated toward CD8 or CD4 CD8 subpopulations (Fig. 5). The Ep58K(pT) and Ep64K(pT) transgenic T cells proliferated in vitro in response to A b and Ag, but showed no cytotoxic activity (Fig. 4C, and data not shown). To investigate whether the original thymic epithelial cells expressing the A b Ep complex are capable of reverting the lineage commitment of Ep63K(pT) and Ep58K(pT) TCR-expressing thymocytes, we lethally irradiated the original A b EpIi mice and reconstituted them with transgenic bone marrow. Regardless of the genuine composition of the A b bound with covalent Ep, we found FIGURE 4. Functional analysis of peripheral transgenic T cells isolated from PCC(VA), PCC(pT), Ep58K(pT), and Ep63K(pT) TCR transgenic mice or different radiation chimeras. x-axis represents cell number and y-axis represents OD 570 nm reading. Filled symbols (f, F,, Œ) represent the responses to antigenic peptides. Open symbols (, E,, ) refer to the responses to control peptide IgGVH (59 74). A, Proliferative response of transgenic T cells isolated from PCC(VA) TCR transgenic mouse (f, ), PCC(VA)TCR3A b Ep58KIi chimeras (F, E), and PCC(VA)TCR3A b Ii (, ) chimeras. B, Proliferative response of transgenic T cells isolated from PCC(pT) TCR transgenic mouse (f, ), PCC(pT)TCR3A b Ep58KIi chimeras (F, E), and PCC(pT)TCR3A b Ii chimeras (, ). C, Proliferative response of transgenic T cells isolated from Ep58K(pT) TCR transgenic mouse (, ), Ep63K(pT) TCR transgenic mouse (F, E), Ep58K(pT) TCR 3A b EpIi chimeras (f, ), and 63K(pT) TCR 3A b EpIi (Œ, ) chimeras. no CD4 T cells bearing a transgenic TCR in the original selecting environment. Instead, as in the wild-type mice, the transgenic T cells emerged from the thymus as CD4 CD8 or CD8 T cells (Fig. 6, A and C). To compensate for the elevated expression of TCR that may skew transgenic T cells toward the CD4 cells, we have seeded the

7 2256 SPECIFICITY OF T CELL-POSITIVE SELECTION mice were irradiated and reconstituted with T cell-depleted bone marrow from the relevant TCR transgenics. In the A b Ep63KIi irradiated host, both transgenic thymocytes matured again as CD8 or CD4 CD8 T cells, while in chimeras expressing the A b Ep58K complex on thymic epithelium, 58K(pT) TCR-bearing thymocytes were negatively selected (Fig. 6B). Differentiation of the transgenic Ep63K(pT) and Ep58K(pT) TCRs also progressed toward CD4 cells in other tested chimeras, e.g., A b Ii,A b DM,A bm12, similarly to other misselected, class II MHC-restricted transgenic TCRs (data not shown) (34). Importantly, the down-regulation of CD4 on transgenic T cells bearing the Ep63K(pT) TCR was not a result of the lack of tolerance to wild-type A b /peptides expressed on bone marrow-derived thymic APCs. Bone marrow from mice expressing this TCR and devoid of A b (Ep63K(pT)A b ) was used to reconstitute lethally irradiated hosts expressing A b bound with self peptides or covalent Ep, and transgenic thymocytes still differentiated only as CD4 T cells (data not shown). FIGURE 5. Expression of the Ep58K(pT) and Ep63K(pT) TCRs on thymocytes and peripheral lymph node cells. A, Thymocytes from Ep58K(pT) TCR mice (left), Ep63K(pT) TCR transgenic mice (right) were stained for CD4 and CD8 expression. B, TCR expression on gated doublepositive CD4 CD8 (left), and single-positive CD4 CD8 thymocytes (right) of Ep58K(pT) ( ) and Ep63K(pT) TCR ( ) and C57BL6 mice (OOO). C, Peripheral lymph node cells of Ep58K(pT) (left) and Ep63K(pT) (right) TCR transgenic mice were stained for CD8 and transgenic V expression. The total number of thymocytes in Ep58K(pT) mice was , and in Ep63K(pT) mice Three mice were analyzed in each group. transgenic bone marrow into mice that express less A b Ep complex on thymic epithelium than homozygous A b EpIi mice (Fig. 7). The T cells expressing the original TCRs specific for Ep63K and Ep58K were derived from chimeras homozygous for A b Ep complex. In mice heterozygous for the A b Ep transgene, A b Ep expression on thymic epithelium was reduced by approximately 30% (assessed by comparing mean fluorescence intensities) in comparison with homozygous mice (data not shown). The expression of A b Ep complex is diluted by expression of other A b /self peptide complexes in mice that have endogenous A b -chain, lack an invariant chain, and are transgenic for one copy of the A b Ep chain (A b wta b Ep / Ii mice) (Fig. 7). We have also crossed A b Ep transgenic mice with H2-M-deficient mice to get A b CLIPAb EpDM mice. In these mice, A b wt and transgenic A b molecules are dominantly bound by CLIP peptide. The covalent Ep is cleaved in the presence of Ii, which results in further reduction of A b Ep expression (Fig. 7). Finally, in mice that express the A b Ep complex in the absence of endogenous A b -chain, but in the presence of Ii, the expression of A b Ep complex was the lowest of all mice tested (Fig. 7). All the mice expressing various levels of the A b Ep complex were lethally irradiated and reconstituted with T cell-depleted bone marrow from either Ep63K(pT) or Ep58K(pT) transgenic mice. Regardless of the different amounts of the A b Ep complex, the transgenic T cells differentiated as CD8 and CD4 CD8 in all the mice described above (data not shown). We also tested whether the Ep63K(pT) or Ep58K(pT) TCRs would be selected in vivo toward the CD4 lineage by analogues of Ep covalently bound to A b. The A b Ep63KIi and A b Ep58KIi Transgenic T cells are not positively selected on the thymic epithelium expressing A b Ep complex in mixed bone marrow chimeras Positive selection of T cells expressing transgenic TCRs depends on the availability of the selecting MHC/peptide ligand. To determine whether the block in positive selection of our transgenic TCRs results from competition for a very small amount of the selecting A b /peptide ligand, we have made mixed bone marrow chimeras. For that purpose, we reconstituted lethally irradiated A b EpIi mice with bone marrow cells derived as follows: 80% from TCR knockout, 10% from PCC(VA) (or Ep63K(pT)), and 10% from B6.PL donors. In this experiment, the majority of CD4 CD8 thymocytes did not express a functional TCR, and only 5 20% expressed either the transgenic TCR (and Thy-1.2) or endogenous TCRs (and Thy-1.1). As shown in Fig. 8, the reduced frequency of thymocytes bearing rearranged transgenic TCRs did not restore their positive selection. Based on this result, we concluded that the lack of selection of bulk transgenic TCRs does not result from competition for the limiting amount of the low-abundant MHC/peptide ligand. Discussion To our knowledge, this is the first study that investigated the development of T cells expressing TCRs originally selected in vivo on a defined class II MHC/peptide complex. In this system, all T cells expressed transgenic TCRs positively selected by thymic epithelium expressing A b Ep complex, and all detectable class II MHC molecules were preloaded with the selecting peptide. One may have expected that our model would constitute an optimal selecting niche (54 56). Instead, T cells expressing three different transgenic TCRs originally positively selected on thymic epithelium with class II MHC molecules covalently bound with one peptide were unable to follow the ontogeny of the same native TCRs in the genuine thymic environment. The lack of positive selection of three transgenic TCRs in the original thymic milieu does not contradict the results of the experiments of others in which the selection of many transgenic TCRs cloned from wild-type mice followed the normal development of T cells. Instead, our results suggest that selection of the transgenic TCR in wild-type mice depends on: 1) TCR flexibility for the selecting MHC/peptide ligand; and 2) complexity of self peptides bound to MHC. In wildtype mice bearing a transgenic TCR, these two conditions often compensate for different patterns and levels of expression of the transgenic TCR, which results in the anticipated differentiation of transgenic T cells. In mice coexpressing a transgenic TCR and

8 The Journal of Immunology 2257 FIGURE 6. Flow cytometric analysis of thymocytes (upper) and peripheral lymph nodes (lower) from recipient mice reconstituted with bone marrow from Ep58K(pT) and Ep63K(pT) TCR transgenic mice. A, Cells from A b EpIi recipient mouse reconstituted with Ep58K(pT) TCR mouse bone marrow were stained with anti-cd4 and anti-cd8 Abs (upper) and anti-cd8, and anti-v 3 (lower). B, Negative selection of Ep58K(pT) TCR on Ep58K peptide covalently bound to A b.a b Ep58KIi mice were reconstituted with Ep58K(pT) TCR bone marrow, and cells were analyzed as above. C, Cells from A b EpIi recipient mouse reconstituted with 63K(pT) TCR mouse bone marrow were stained with anti-cd4 and anti-cd8 mabs (upper) and anti-cd8, anti-v 14 Abs (lower). Total number of thymocytes was: A, ; B, ; C, Three mice were analyzed in each group. MHC occupied with covalent peptide, the thymocytes have no choice of an alternative selecting MHC/peptide ligand, and the development is altered. Therefore, we postulate that transgenic expression of the TCR may affect its original requirements for the positively selecting MHC/peptide ligand(s?). Several possible rationales can be offered to resolve the lack of selection of the transgenic TCRs on the A b Ep complex. One may argue that the TCRs investigated in this study were selected on contaminating peptides that replaced some of the covalently attached Ep, or wild-type self peptides derived from donor bone marrow (45, 57). However, selection of the transgenic TCRs was followed in A b wt3a b EpIi chimeras, that is, in the same environment that still contains the contaminating or bone marrow-derived peptides. The experiments conducted to date did not detect the presence of endogenous peptides in the original A b EpIi mice (58, 59). The development of the majority of CD4 thymocytes was blocked in A b EpIi fetal thymic organ culture with YAe Ab specific for the A b Ep complex (Pacholczyk, manuscript in preparation). Lowering the precursor frequency in experiments with mixed bone marrow chimeras could not restore positive selection of transgenic T cells. Together these results suggest that the lack of selection of the transgenic cells is not due to the limited availability of MHC/peptide ligand or growth factors (55, 56). Conceivably, TCRs positively selected on one highly abundant MHC/peptide ligand may represent abnormal CD4 T cells. It has been shown previously that T cells isolated from single peptide mice have a normal surface phenotype and respond to antigenic peptides in an MHC-restricted manner (39). Also, the T cells expressing transgenic TCRs specifically respond to their cognate antigenic peptides presented by A b. We have also defined the selecting MHC backgrounds for the PCC-specific TCR and show that transgenic CD4 cells remained functional. In conclusion, we did not find evidence that T cells selected on one MHC/peptide ligand differ from T cells present in normal mice. Therefore, we favor the explanation that transgenic expression of an TCR frequently requires the use of an alternative peptide bound to the selecting MHC haplotype. This is often accomplished in mice expressing MHC bound with a wild-type repertoire of peptides, but not in mice expressing a single class II MHC/peptide complex. In several in vitro studies, chemically synthesized or naturally derived peptides bound to MHC positively selected a defined transgenic TCR in fetal thymic organ cultures (5, 6, 60, 61). However, when some of these peptides were genetically manipulated to bind to the selecting MHC molecules in vivo, the same transgenic TCRs were not positively selected (62, 63). Failure of the in vivo induced, positive selection of the transgenic TCR by the in vitro defined, positively selecting peptide indicates that manipulation of any of the TCR/MHC/peptide components may alter the final outcome of the selection processes. These results support the hypothesis that positive selection depends on the subtle avidity of the involved TCR/MHC/peptide complexes (64). Possibly, natively expressed TCRs adjust to the quantity of the selecting MHC/peptide ligand differently than the transgenic TCRs, which may explain the difficulty of achieving positive selection of the transgenic TCR on the original selecting MHC/peptide ligand. Multiple lines of evidence indicate that recognition of MHC by developing thymocytes influences the T cell lineage commitment (28, 65 67). However, the ability of the TCR to engage class I or II MHC is not the only factor that determines coreceptor expression on mature T cells. Transgenic T cells expressing AND or DO11.10 TCRs differentiated as CD8 or CD4 CD8 T cells in the absence of the optimal selecting MHC/peptide ligand (34, 68). Ligation of CD3 on C5 TCR transgenic thymocytes in fetal thymic organ cultures by anti-cd3 F(ab ) 2 reagents or an antagonistic peptide led to the differentiation of mature CD8 thymocytes, irrespective of the MHC class II restriction specificity of this TCR. The tyrosine phosphorylation patterns in these thymocytes resembled patterns found in mature CD8 T cells stimulated with antagonistic peptides (37). Hence, it has been proposed that the relative balance of signals delivered by TCR engagement and by p56 lck activation is responsible for lineage commitment (69). The phenotype of Ep58K(pT) and Ep63K(pT) transgenic cells indicates that these cells did not receive an optimal selecting signal and were selected with the mismatched CD8 coreceptor. Recently, it was shown that such class II MHC-restricted TCR transgenic cells with the aberrant expression of a coreceptor methylate the CD8 gene, down-regulate its surface expression, and gradually convert to CD4 CD8 T cells that poorly survive in the periphery (38). If transgenic expression unintentionally tests TCR flexibility for positive selection, our results show that the repertoire of TCRs selected on the single class II MHC/peptide complex consists of TCRs promiscuous or specific for the A b and selecting peptide. The PCC-specific TCR was selected in different thymic environments, which correlated with its ability to bind unrelated MHC/

9 2258 SPECIFICITY OF T CELL-POSITIVE SELECTION FIGURE 7. Expression of A b and A b Ep in different recipients reconstituted with bone marrow from Ep63K(pT) or Ep58K(pT) TCR transgenics. Spleen B cells (B220 ) isolated from different recipient mice were stained with Y3P Ab specific for all A b molecules (A) or with YAe Ab specific only for A b bound to Ep peptide (B). A, Total level of A b class II molecules expressed on splenic B cells in C57BL/6 mice (filled histogram), A b Ep / Ii homozygote (OOO), A b Ep / Ii heterozygote ( ), A b CLIPA b EpDM ( ), A b Ep / Ii ( ), and in A b - wta b Ep / Ii ( ) recipient mice determined by staining with Y3P. B, The level of A b Ep class II complex expressed on splenic B cells in C57BL/6 mice (filled histogram), A b Ep / Ii homozygote (OOO), A b Ep / Ii heterozygote ( ), A b CLIPA b EpDM ( ), A b Ep / Ii ( ), and in A b wta b Ep / Ii ( ) recipient mice determined by staining with YAe. peptide ligands (such as PCC v Ep). Positive selection of this transgenic TCR in mice different in the complexity of peptides bound to A b, such as C57BL/6, A bm12,a b Ii, and A b Ep58KIi, suggests that this receptor is selected by different peptides bound to A b rather than a single peptide ubiquitously present in all these mice. On the other hand, the only peptides we identified that were recognized by Ep58K(pT) and Ep63K(pT) TCRs were Ep or its close analogues. We hypothesize that these TCRs may have narrow requirements for positive selection. Various levels of A b Ep ligand did not select both of these TCRs. In addition, these TCRs were not able to adopt an alternative, positively selecting ligand when tested in the same set of mice as the PCC-specific receptors. Therefore, we postulate that the TCR repertoire consists of TCRs specific and promiscuous in their choice of the positively selecting MHC/peptide ligand. The crystallographic data showing that the peptide contribution to the interaction between TCR and MHC/ peptide complex varies support this conclusion (70 72). Acknowledgments We thank Drs. P. Kisielow, P. Jensen, B. Chmielowski, and A. Mellor for helpful discussion, and R. Markowitz for editing the manuscript. We also thank H. Ignatowicz and G. Pacholczyk for their help in producing transgenic mice, and Drs. D. Mathis, C. Benoist, E. Bikoff, R. Germain, L. Glimcher, C. Janeway, D. Kioussis, and L. van Kaer for their generous gifts of knockout mice, DNA constructs, and Abs. All work involving animals was conducted under protocols approved by the Animal Care and Use Committee at Medical College of Georgia. FIGURE 8. Transgenic T cells expressing PCC(VA) or Ep63K(pT) are not selected in A b EpIi recipients reconstituted with mixed bone marrow. The lethally irradiated A b EpIi mice have been reconstituted with mixed bone marrow cells (80% TCR /, 10% of either PCC(VA) or Ep63K(pT) (all expressing Thy-1.2), and 10% of B6. PL expressing Thy- 1.1). Three chimeras of each type were analyzed 8 wk after reconstitution. Thymus reconstitution with TCR cells varied between 5 and 20% of total thymocytes. Expression of the transgenic receptor is shown vs Thy-1.1 on gated TCR T cells in the thymus (left) and in the periphery (right). A, TCR-positive thymocytes and lymph node cells from PCC(VA) mixed bone marrow chimera stained with anti-v 8 and anti-thy-1.1 Abs. B, TCR-positive thymocytes and lymph node cells from Ep63K(pT) mixed bone marrow chimera stained with anti-v 2 and anti-thy-1.1 Abs. References 1. Merkenschlager, M., D. Graf, M. Lovatt, U. Bommhardt, R. Zamoyska, and A. G. Fisher How many thymocytes audition for selection? J. Exp. Med. 186: Zerrahn, J., W. Held, and D. H. Raulet The MHC reactivity of the T cell repertoire prior to positive and negative selection. Cell 88: Jameson, S. C., K. A. Hogquist, and M. J. Bevan Positive selection of thymocytes. Annu. Rev. Immunol. 13: Allen, P Peptides in positive selection: a delicate balance. Cell 76: Hogquist, K. A., S. C. Jameson, W. R. Heath, J. L. Howard, M. J. Bevan, and F. R. Carbone T cell receptor antagonist peptides induce positive selection. Cell 76: Ashton-Rickardt, P. G., A. Bandeira, J. R. Delaney, L. Van Kaer, H.-P. Pircher, R. M. Zinkernagel, and S. Tonegawa Evidence for a differential avidity model of T cell selection in the thymus. Cell 76: Kisielow, P., H. Bluthmann, U. D. Staerz, M. Steinmetz, and H. von Boehmer Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4 8 thymocytes. Nature 333: Kappler, J. W., N. Roehm, and P. Marrack T cell tolerance by clonal elimination in the thymus. Cell 49: Surh, C. D., and J. Sprent T-cell apoptosis detected in situ during positive and negative selection in the thymus. Nature 372: Kaer, L., P. G. Ashton-Rickardt, H. L. Ploegh, and S. Tonegawa TAP1 mutant mice are deficient in antigen presentation, surface class molecules, and CD4 8 cells. Cell 71: Bikoff, E. K., L. Y. Huang, V. Episkopou, J. van Meerwijk, R. N. Germain, and E. J. Robertson Defective major histocompatibility complex class II assembly, transport, peptide acquisition, and CD4 T cell selection in mice lacking invariant chain expression. J. Exp. Med. 177: Kenty, G., W. D. Martin, L. Van Kaer, and E. K. Bikoff MHC class II expression in double mutant mice lacking invariant chain and DM functions. J. Immunol. 160: Elliott, E., J. Drake, S. Amigorena, J. Elsmore, P. Webster, I. Mellman, and R. Flavell The invariant chain is required for intracellular transport and function of major histocompatibility complex class II molecules. J. Exp. Med. 179: Shachar, I., E. A. Elliott, B. Chasnof, I. S. Grewal, and R. A. Flavell Reconstitution of invariant chain function in transgenic mice in vivo by individual p31 and p41 isoforms. Immunity 3: Ashton-Rickardt, P. G., L. V. Kaer, T. N. M. Schumacher, H. L. Ploegh, and S. Tonegawa Peptide contributes to the specificity of positive selection of CD8 T cells in the thymus. Cell 73: Pawlowski, T., M. D. Singleton, D. Y. Loh, R. Berg, and U. D. Staerz Permissive recognition during positive selection. Eur. J. Immunol. 26:851.