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The TCR C FG Loop Regulates T Cell Development¹

Maki Touma^*, Hsiu-Ching Chang^*,, Tetsuro Sasada^*,, Maris Handley^*, Linda K. Clayton^*, and Ellis L. Reinherz^2,*,

^* Laboratory of Immunobiology, Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115; and Department of Medicine, Harvard Medical School, Boston, MA 02115

	Abstract

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The TCR chain constant domain contains an unusually elongated, solvent-exposed FG loop. This structural element forms one component of an TCR cavity against which CD3 may abut to facilitate Ag-specific signaling. Consistent with this notion, in the present study we show that N15 TCR transfectants expressing a FG loop-deleted chain (FG) stimulate less tyrosine protein phosphorylation than those bearing a wild-type -chain (wt) upon TCR cross-linking. Furthermore, coimmunoprecipitation studies suggest a weakened association between the CD3 heterodimer and the -chain in TCR complexes containing the FG variant. To further investigate the biologic role of the C FG loop in development, we competitively reconstituted the thymus of Ly5 congenic or RAG-2^–/– mice using bone marrow cells from wt or FG transgenic C57BL/6 (B6) mice. Both wt and FG precursor cells generate thymocytes representative of all maturational stages. However, FG-expressing thymocytes dominate during subsequent development, resulting in an excess of FG-expressing peripheral T cells with reduced proliferative and cytokine production abilities upon TCR stimulation. Collectively, our results show that the unique C FG loop appendage primarily controls T cell development through selection processes.

	Introduction

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The TCR is a multisubunit complex on the surface of T cells and functions to convey information about the surrounding environment to the signaling machinery inside the cell (1, 2). The TCR also provides critical signals necessary for the completion of thymocyte development (3). As thymocytes mature, the composition of their surface receptor changes; the pT subunit is expressed in double-negative (DN)³ thymocytes along with the TCR chain to form the pre-TCR (4, 5). While it is currently a matter of debate as to whether the pre-TCR has a ligand, signaling through the pre-TCR triggers cessation of additional -chain rearrangements, resulting in allelic exclusion (6, 7). The pre-TCR also signals proliferation and transition from the DN3 to DN4 developmental stages (8). Upon rearrangement, the mature TCR chain replaces pT, permitting assembly of the TCR in double-positive (DP) thymocytes (5, 9). Development of a second T cell lineage, the T cells, does not depend on expression of the pre-TCR (reviewed in Ref. 8); this TCR is composed of rearranged - and -chains, which are equivalent to the - and -chains of the TCR, respectively (reviewed in Ref. 10).

Signal transduction by the pre-TCR, the mature TCR, and the TCR is conducted by the noncovalently associated invariant CD3 subunits (10). Based on thymocyte development in CD3 subunit knockout animals, the development of both and T cell lineages is dependent on CD3. In particular, in the absence of CD3, thymic development is blocked at the early DN stage (11, 12), suggesting a critical role for CD3 in pre-TCR signaling. Likewise, in the absence of CD3 (13) or CD3 (14, 15, 16), thymocyte development in both the and lineages is compromised severely. However, in contrast to the TCR, neither the pre-TCR nor TCR requires CD3 (10, 17). The stoichiometry of the TCR is thought to be one CD3 dimer paired with the TCR chain, one dimer paired with the TCR chain and a CD3 homodimer (10). While less well-defined, the TCR appears to exist as a complex with two CD3 heterodimers and a single CD3 homodimer. The pre-TCR may exist as a pT/TCR, CD3, CD3, and CD3 complex or perhaps with two CD3 dimers instead of one CD3 and one CD3 (10). The abundance of CD3 transcripts in DN thymocytes compared with DP thymocytes suggests that CD3 dimers are preferentially expressed in DN thymocytes over CD3 dimers. The levels of CD3 transcripts are approximately equal in DN and DP thymocytes (18); preferential formation of CD3 dimers over CD3 in DN thymocytes explains why the pre-TCR can function in the absence of CD3.

The central role of CD3 in all forms of TCR signaling suggests that the topology of CD3 in relation to the clonotypic subunits is important in this process. Three-dimensional structures of the TCR (19, 20, 21, 22) have shown that the FG loop of the TCR chain exists as an elongated, rigid element forming a sidewall of a cavity created by the asymmetric disposition of C and C domains, and whose size would encompass an unglycosylated Ig domain such as CD3 (22). Indeed, Ab-blocking experiments confirmed that a CD3 subunit lies in close proximity to the TCR chain FG loop (23). The role of this structure in TCR signaling was investigated by constructing transgenic (tg) mice whose TCR chain FG loop was deleted (FG) (24, 25, 26). Initial analysis noted less efficient expression of FG-containing TCRs in transfectants in vitro, but normal T cell development and TCR expression in tg T cells in vivo (24). It was later noted that the FG loop-deleted TCR V8.2-J2.1 chain paired less efficiently with the specific V14-J281 TCR chain, resulting in a reduction of NK1.1 cells in these mice compared with the wild-type V8.2-J2.1 TCR (25). Our previous paper (26) using the N15TCR on a RAG-2^–/– background to follow a single TCR identified further alterations in T cell development. In particular, we observed that pre-TCR function can be supported by the mutant FG chain. On the other hand, negative selection is reduced in the absence of the FG loop and T cells carrying this deletion respond less well to cognate Ags.

In this study, we have further delineated the role of the FG loop in mitogen-induced T cell proliferation and cytokine production in N15 chain tg mice. Biochemical analysis of transfectants expressing N15 TCRs with wild-type -chain (wt) or FG chains shows that the FG chains are more weakly associated with CD3, offering one potential basis for the reduction in the above activation measurements. In addition, in a RAG-2^–/– background, the pre-TCR containing a FG chain signals the DN3 to DN4 transition less efficiently than does the pre-TCR containing a wt chain. Nonetheless, when assayed competitively by coinjection in bone marrow (BM) chimeras, the FG loop-deleted tg progenitors are more proficient in repopulating the thymus and lymph node than their wt chain tg counterparts. We conclude that the presence of the C FG loop primarily regulates maturation of developing T cells through effects on selection.

	Materials and Methods

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Mice

N15wt and N15FG loop-deleted mutant mice were generated as described previously (26). The mice were backcrossed for more than 10 generations onto a C57BL/6 (B6) background. B6, B6 Ly5.1, B6 RAG-2^–/–, and BALB/c mice were purchased from Taconic Farms. To create RAG-2^–/– background N15 tg mice, N15wt RAG-2^+/+ and N15FG RAG-2^+/+ mice were bred with B6 RAG-2^–/– mice for two generations. For N15,FG TCR tg RAG-2^–/– mice, the N15FG mice were crossed with B6 N15 tg mice and then to B6 RAG-2^–/– mice. The N15 RAG-2^–/– mice were generated as described previously (27). All lines were maintained under specific pathogen-free conditions at the animal facility of Dana-Farber Cancer Institute under a protocol reviewed and approved by the Animal Care and Use Committee.

Abs and flow cytometric analysis

The following mAbs were used: FITC-conjugated anti-Ly5.2 (104), PE-, PE-Cy5- or PE-Cy7-conjugated anti-CD4 (H129.19), FITC-, PE-Cy5-, or allophycocyanin-conjugated anti-CD8 (53-6.7), PE-Cy5-conjugated anti-TCR C (H57-597), PE- or biotin-conjugated anti-V5.1, 5.2 (MR9.4), PE-conjugated anti-CD25 (PC61), FITC-conjugated anti-CD44 (IM7), FITC- or PE-conjugated anti-V 5.1, 5.2 (MR9.4), FITC-conjugated anti-CD69 (H1.2F3), FITC-conjugated anti-CD24 (HSA, M1/69), FITC-conjugated anti-CD5 (53-7.3), FITC-conjugated PNA, and PE-Cy5 or allophycocyanin-Cy7-conjugated streptavidin (BD Pharmingen). For flow cytometry, single-cell suspensions of thymocytes or lymph node (LN) cells were prepared at 5 x 10⁶ cells/ml in PBS containing 2% FCS and 0.05% NaN₃. Those cells were triple or five-color stained with the Abs at saturating concentrations according to standard procedures. A FACScan (BD Biosciences) and CellQuest software were used for analysis of triple stained samples, and FACSAria and FlowJo software (Tree Star) were used for five-color samples. Dead cells were excluded from the analysis by forward and side scatter gating.

Adoptive transfer of BM cells

BM cells were prepared from the tibia and femur of 2- to 3-mo-old N15wt or N15FG mice. T cells were eliminated from total BM cells by CD4^– and CD8^– negative separation using specific mAbs and magnetic beads. Equal numbers (5 x 10⁶) of BM cells from N15wt and N15FG mice were mixed and i.v. injected into irradiated (500 rad) B6 Ly5.1 mice or unirradiated B6 RAG-2^–/– mice. As controls, 5 x 10⁶ BM cells from N15wt or N15FG alone were injected. Four weeks after the injection, recipient mice were sacrificed, and thymus and LN cells were analyzed by FACS for donor-derived Ly5.2⁺ cells. Competition of N15wt cells and N15FG cells was judged by the ratio of MR9.4⁺H57⁺ (wild-type N15 T cells) to MR9.4⁺H57^– (FG loop-deficient N15 T cells).

Proliferation assay of T cells

CD8⁺ cells were sorted from N15wt or N15FG LN cells. Sorted cells (2 x 10⁵/well) were cultured for 48 h in the presence of complete RPMI 1640 medium with or without plate bound anti-CD3 (145-2C11, 5 µg/ml), anti-CD3 plus anti-CD28 (37.51, 10 µg/ml), Con A (2 µg/ml) plus irradiated spleen cells (2 x 10⁵/well), or PMA (50 ng/ml) plus ionomycin (200 ng/ml). One microcurie per well of [³H]thymidine (MP Biomedicals) was added for the last 18 h of culture, and the incorporated radioactivity was measured by scintillation counter.

Mixed leukocyte reaction

FACS-sorted LN CD8⁺ cells (2 x 10⁵/well) were cultured with irradiated spleen cells (2 x 10⁵/well) from syngeneic C57BL/6 mice or allogeneic BALB/c mice for 72 h in complete RPMI 1640 medium, and 1 µCi/well of [³H]thymidine was added for the last 18 h of the culture. Cells were harvested and incorporated radioactivity was measured.

Measurement of cytokine production

Cytokine production was induced under the same culture conditions used for proliferation assays (see Proliferation assay of T cells), except that the cell concentration was 5 x 10⁵/well. Supernatants were collected from 46 h cultures and kept at –80°C until used for cytokine multiplex analysis (Luminex).

Inhibition of intracellular signaling pathways

CD8⁺ cells from LN of N15wt and FG tg mice were prepared by depletion of CD4⁺ and B220⁺ (RA3-6B2) cells using magnetic beads. Purified CD8⁺ cells (2 x 10⁵/well) were incubated in an anti-CD3- and anti-CD28-coated plate for 15 h with various reagents that inhibit TCR signaling pathways. The following reagents were used: PP2 (10 µM), staurosporine (1 µM), Ly249002 (50 µM), wortmannin (100 nM), BAPTA-AM (25 µM), FTI-277 (40 µM), Raf1 (5 µM), and U73122 (0.5 µM). Golgi Plug reagent (BD Pharmingen) was added to the culture in the last 2 h of incubation, and cells were stained for cell surface CD8 and V5 and for intracellular IFN- to test inhibition of IFN- production by CD8⁺V5⁺ cells.

Transfectants

The N15N236A (C glycan addition site N236 mutated to A) and N15FG (deletion of 14 C FG loop region residues, aa 219–232) mutants were generated by PCR using N15wt cDNA as a template, and the mutant constant regions subcloned into the expression vector pSH encoding the entire N15 V and nonmutant component of the N15 C region to generate pSH-N15N236A and N15FG. To generate stable T cell transfectants, the N15 TCR wt or variant hybridomas were established by cotransfection of 25 µg of BglII/SalI-linearized pBJNeo-N15 and 10 µg of SalI-digested pSH-N15 into the TCR^– murine T cell hybridoma 58^––CD8⁺ cell line (28) by electroporation. Forty-eight hours after transfection, the cells were plated at 2 x 10⁴ cells/well in 24-well plates in selection medium (complete RPMI 1640 medium plus 0.1 mg/ml G418 and 2 mg/ml hygromycin). Two weeks later, the surviving clones were analyzed by FACS using the MR9.4 mAb. The cells expressing the highest levels were pooled to avoid clonal bias, and the polyclonal populations were sorted on several occasions to maintain comparable TCR surface expression levels.

Cell surface biotinylation, immunoprecipitation, and Western blotting

Cell surface biotinylation was performed by a modification of a described procedure (Current Protocols in Immunology). Briefly, 2 x 10⁷ cells were suspended in PBS containing Ca²⁺, Mg²⁺, and sulfo-NHS-biotin (50 µg/ml; Pierce) at 1 x 10⁷ cells/ml and incubated with gentle shaking at 4°C for 30 min. The cells were then washed and lysed (1% digitonin in TBS supplemented with 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM PMSF). The lysates were immunoprecipitated with 7D6 (anti-mouse CD3 mAb) coupled to GammaBind Plus Sepharose (Pharmacia Biotech). Immunoprecipitates were subjected to two-dimensional nonreducing/reducing diagonal SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated with HRP-conjugated streptavidin (ICN) in PBS containing 0.1% BSA and 0.1% Tween 20 at 37°C for 2 h. After washing, the blots were developed using ECL (Amersham Biosciences).

CD3 expression was analyzed by immunoprecipitation of the postnuclear supernatants with 13A1 (anti-mouse CD3 mAb)-coupled GammaBind Plus Sepharose, followed by Western blotting with Ab 387 (rabbit anti-mouse CD3).

Analysis of tyrosine phosphorylation

Immunoprecipitation with rabbit antisera against Zap70 (gift from J. Bolen, DNAX, Palo Alto, CA) was performed as described previously (29, 30). In brief, 1 x 10⁷ cells were stimulated by cross-linking the TCR with 20 µg/ml biotinylated anti-CD3 and 200 µg/ml streptavidin (Sigma-Aldrich) at 37°C for the indicated times. Cells were then lysed (1% Nonidet P-40, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, 1 mM Na₃VO₄, and 10 mM EDTA). Postnuclear lysates were mixed with 20 µl of GammaBind Plus Sepharose preincubated with rabbit antisera against Zap70. Immunoprecipitated samples were subjected to SDS-PAGE and transferred to a nitrocellulose membrane, then incubated with 4G10 mAb (anti-phosphotyrosine; gift from T. Roberts, Dana-Farber Cancer Institute, Boston, MA) or rabbit anti-sera against Zap70.

IL-2 production by transfectants activated by VSV8 peptide on H-2K^b APCs

To assess the capacity of the transfectants to be activated by the VSV8 peptide, R8 B cells were irradiated at 3200 rad then incubated with various peptide concentrations ranging from 10^–9 to 10^–4 M for 2 h at 37°C. A total of 1 x 10⁵ transfectants was then incubated with peptide-loaded R8 cells in medium supplemented with 10 ng/ml PMA. After 24 h, culture supernatants were harvested, and IL-2 concentrations in the supernatants were determined by MTT assay using the IL-2-dependent CTLL-20 cell line and recombinant human IL-2 as standards.

Analysis of phosphorylation of Erk1/2 MAPK

LN cells from N15,FG RAG-2^–/– or N15 RAG-2^–/– mice were incubated with 10^–5 M VSV8 cognate peptide at 4°C for 5 min, spun down, and then incubated at 37°C for the indicated time. Cells were chilled on ice and lysed in 1% Nonidet P-40, 50 mM Tris-HCl (pH 7.5), and 150 mM NaCl plus phosphatase and protease inhibitors. Lysates were run on SDS-PAGE and analyzed by Western blot with anti-Pan Erk1/2 (p42/44) and anti-phospho Erk1/2 (Cell Signaling Technology).

	Results

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Preferential generation of CD8⁺ thymocytes and T cells in C FG loop-deleted N15 tg mice

We previously described T lineage development in B6 background mice tg for the N15 TCR chain (N15wt) compared with mice tg for the N15 -chain in which the C FG loop (aa 219–232) was deleted (N15FG) (26). The N15 -chain is derived from a CD8⁺ cytotoxic T cell clone expressing a TCR specific for the vesicular stomatitis virus nucleoprotein aa 52–59 (VSV8) bound to the MHC class I H-2K^b molecule (28, 31). Cell surface TCR expression levels on T cells harboring the mutant N15FG chain are similar to those of N15wt-expressing cells. Flow cytometric analysis showed that early T cell development in N15FG mice as evidenced by the transition from the CD44^–CD25⁺ DN3 to the CD44^–CD25^– DN4 stage is similar to that of N15wt mice. However, the CD4/CD8 profiles of thymocytes and LN cells show a subtle but significant increase of CD8 single-positive (SP) thymocytes and CD8⁺ peripheral T cells in N15FG mice, suggesting enhanced positive selection (Fig. 1) (26). Consistent with this finding is the result of analysis of maturation markers on CD8 SP thymocytes. N15FG CD8 SP thymocytes express slightly higher levels of CD69 and CD5 but slightly decreased CD24 and PNA, all consistent with increased maturation/positive selection (Fig. 1). To further characterize these alterations in mice lacking the TCR FG loop, we have conducted competitive thymic reconstitution experiments in which BM cells from the N15wt and N15FG tg mice were mixed and used to create chimeras. We reasoned that compensatory mechanisms in the FG mutant mice could mask the effect of the FG loop deletion during development. However, such compensatory mechanisms are not possible when the mutant BM cells are forced to undergo competitive reconstitution in an irradiated Ly5 congenic or RAG-2^–/– host.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Increased generation of CD8+ SP thymocytes in N15FG mice. Thymocytes were prepared from N15wt and N15FG mice and stained for CD4 and CD8 and maturation markers, CD69, CD24, CD5, and PNA. The percentages of DN, DP, and SP subsets are indicated in the CD4/CD8 dot plot. Histograms represent the indicated markers on CD8SP cells gated as shown in the dot plot. The numbers in each histogram are the mean fluorescence intensity.

View larger version (53K): [in this window] [in a new window]   FIGURE 2. Competitive thymic reconstitution by N15wt and/or N15FG BM cells. Ly5.2 BM cells (5 x 106) from N15wt and N15FG mice were injected together or individually into irradiated B6 Ly5.1 mice. Four weeks after injection, total thymocytes were five-color stained with anti-Ly5.2-FITC, anti-CD4-PE-Cy7, anti-CD8-allophycocyanin, anti-TCR C (H57)-PE, anti-V5 (MR9.4)-biotin, and streptavidin-allophycocyanin-Cy7. For the analysis, dead cells were excluded by forward light scatter/side scatter (FSC/SSC), and Ly5.2-positive cells were gated to analyze donor-derived cells. CD4/CD8 profiles of Ly5.2+ thymocytes are shown. The percentages of each subset are indicated. Ly5.2+ DN, DP, and CD8 SP thymocytes were further analyzed for expression of MR9.4 (anti-V5) and H57 (anti-C). MR9.4low/H57low and MR9.4+/H57+ cells are N15wt-expressing cells while MR9.4low/H57– and MR9.4+/H57– are N15FG-bearing cells. Numbers indicate the percentages of cells in each subset.

View this table: [in this window] [in a new window]   Table I. Preferential development of N15FG vs N15wt T cells after adoptive transfer of BM cellsa

DN3 to DN4 transition affected by FG mutation

To further assess the function of the C FG loop in early T cell development, we analyzed DN cells in TCR tg mice in a RAG-2^–/– background; these mice express pT but not the mature TCR chain, allowing analysis of the function of the C FG loop in pre-TCR expression and signaling. TCR chain expression begins at the CD44^–CD25⁺ DN3 stage (18). In these cells, the -chain associates with pT instead of the mature TCR chain. Signals through the pre-TCR facilitate proliferation and development from the DN3 to DN4 stages. As shown previously (26) and in Fig. 3B, the ratio of DN4 to DN3 is similar for N15wt and N15FG tg thymocytes on a B6 RAG^+/+ background. In the RAG-2^–/– background, however, the N15FG DN4:DN3 ratio is lower than that in N15wt mice (Fig. 3B). DN thymocyte development in B6 RAG-2^–/– mice is arrested in DN3, so the ratio of DN4 to DN3 in B6 RAG-2^–/– mice is very low. Consequently, use of this genetic background is more sensitive for following early developmental changes. In addition, endogenous nontransgenic rearrangements cannot confuse the analysis. Note that both the N15wt RAG-2^–/– and N15FG RAG-2^–/– mice have comparable numbers of DP cells (Ref. 26 and data not shown), suggesting that the signaling capacity of pre-TCRs containing FG chains is adequate to support the DN to DP transition. Nevertheless, in the RAG-2^–/– background, the FG mutation results in an incomplete block in the DN3 to DN4 transition (Fig. 3A).

View larger version (31K): [in this window] [in a new window]   FIGURE 3. The transition from CD44–CD25+ (DN3) to CD44–CD25– (DN4) thymocytes is reduced in N15FG RAG-2–/– mice. Total thymocytes from N15wt RAG-2–/– and N15FG RAG-2–/– mice were stained with anti-CD4, anti-CD8, anti-CD25, and anti-CD44. The CD44/CD25 profiles of CD4–CD8– DN cells are shown in A. The percentages of cells in each quadrant are indicated. The ratio of DN4:DN3 cells in N15wt, N15FG, and C57BL/6 mice are shown in the left panel of B. The right panel shows the ratio of DN4/DN3 from comparable mice on a RAG-2–/– background mice (n = 4–7).

Impaired proliferation and cytokine production in N15FG T cells

To assess the functional role of the C FG loop in mature T cells, we tested the responsiveness of N15FG T cells to various stimuli in vitro. First, CD8⁺ and CD4⁺ T cells purified from LNs were stimulated with anti-CD3 (2C11), anti-CD3 plus anti-CD28, Con A, or PMA plus ionomycin and their proliferation assayed by [³H]thymidine incorporation. Proliferative responses of both CD8⁺ (Fig. 4) and CD4⁺ (data not shown) N15FG cells are approximately half the magnitude of those of N15wt cells when stimulated with anti-CD3, anti-CD3 plus anti-CD28, or Con A (Fig. 4A). PMA plus ionomycin stimulation produces a strong proliferative response in both N15wt and N15FG T cells, and the proliferation of the N15FG T cells is similar or even a little higher than that of N15wt T cells (Fig. 4A).

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Impaired proliferation and alloreactivity of N15FG T cells. A, CD8+ cells (2 x 105) were purified from N15wt or N15FG LN cells by FACS sorting and stimulated as indicated for 48 h. [3H]Thymidine was added for the last 18 h, and T cell proliferation was judged by [3H]thymidine incorporation. Mean and SD of triplicate cultures are shown. , N15wt; , N15FG CD8+ cells. Results are representative of four independent experiments. B, CD8+ cells (2 x 105) purified from N15wt or N15FG LN cells were cocultured with irradiated C57BL/6 (H-2b) spleen cells (2 x 105) or BALB/c (H-2d) spleen cells (2 x 105) for 3 days, and [3H]thymidine incorporation was measured as in A. Mean and SD of triplicate cultures are indicated. Results are representative of four independent experiments.

These findings suggest a clear deficit in TCR-triggered activation in N15FG-expressing mature T cells. To further analyze T cell function, we compared the IFN- production by CD8⁺ T cells from N15wt and N15FG mice. LN T cells were incubated on anti-CD3-coated plates and IFN--producing cells assessed by intracellular IFN- staining and FACS analysis. The number of IFN-⁺ cells in N15wt cultures is 3- to 10-fold more than the number in the N15FG cultures (data not shown). To confirm this analysis and to examine production of other cytokines in N15wt and N15FG T cells, we collected supernatants from N15wt or N15FG T cells stimulated with either anti-CD3 plus anti-CD28, Con A, or PMA plus ionomycin for 2 days and performed cytokine Luminex assays. In these assays, IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10 IL-12p40, IL-12p70, IL-13, IL-17, GM-CSF, IFN-, TNF-, MCP-1, RANTES, and KC were examined (Fig. 5). Those cytokines whose concentrations were <50 pg/ml were excluded from the figure. IL-1, IL-2, IFN-, TNF-, and RANTES production by stimulated N15FG T cells is lower than production by N15wt T cells. Although PMA plus ionomycin stimulation does not yield clear differences in the proliferation assay (Fig. 4A), production of RANTES and IFN- is impaired in the N15FG-expressing cells.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. Impaired cytokine production of N15FG T cells. CD8+ cells (5 x 105 cells/200 µl/well) were purified from N15wt or N15FG LN cells by FACS sorting and stimulated as indicated for 46 h. Culture supernatants were harvested, and cytokines were analyzed by multiplex assay (Luminex). and represent N15wt and N15FG cells, respectively.

Signaling pathways in N15FG T cells

Given that the N15FG T cells display obvious defects in proliferation and cytokine production, we compared signaling pathways in N15FG and in N15wt T cells by using various inhibitors in vitro and measuring the effects of each reagent on intracellular IFN- production revealed by flow cytometry. As shown in Fig. 6, PP2 (target = c-src), staurosporine (target = protein kinase C), Ly249002 (target = PI3K), wortmannin (target = PI3K), BAPTA-AM (target = Ca²⁺), and U73122 (target = phospholipase C) almost completely inhibit IFN- production in both N15wt and N15FG T cells stimulated with anti-CD3 plus anti-CD28. IFN- production in N15FG and N15wt T cells is partially inhibited by FTI-277 (target = K-ras) or Raf1 (target = Raf). Although the scales (reflected in y-axis) of the N15wt and N15FG responses are different, the former being 5- to 10-fold greater than the latter, the patterns of inhibition are very similar. We further investigated the effect of these inhibitors on the anti-CD3- plus anti-CD28-induced expression of CD69 or CD25 in N15wt and N15FG T cells. The effect of some reagents on the induction of CD69 or CD25 was different from that on IFN- production: PP2, staurosporine, BAPTA-AM, and U73122 almost completely inhibited expression of CD69 and CD25; Ly249002 and wortmannin showed weak inhibitory effects; and FTI-277 or Raf1 have no observable effect. Nonetheless, the patterns of inhibition of CD69 and CD25 expression by these reagents were comparable for both N15wt and N15FG T cells (data not shown). These data suggest that the intracellular signal pathways are largely identical in FG and wt T cells with the former attenuated relative to the latter.

View larger version (14K): [in this window] [in a new window]   FIGURE 6. Comparison of pharmacologic inhibition of IFN- production in N15wt and N15FG T cells. LN cells (2 x 105) were prepared from N15wt and N15FG mice and incubated on anti-CD3- plus anti-CD28-coated plates with indicated reagents for 15 h. Golgi Plug reagent was added for the final 2 h of incubation. Cells were surface stained with anti-CD8 and MR9.4, then stained for intracellular IFN-. The MR9.4+CD8+ IFN--producing cells were analyzed by flow cytometry. Samples were gated as live cells by forward light scatter vs side scatter. The results are representative of three independent experiments. The arrow and dotted line represent the background values for unstimulated cell cultures.

Deletion of the TCR C FG loop weakens the physical association between the CD3 dimer and the other TCR components

The signals induced by TCR stimuli are communicated to the cell interior via the CD3 subunits (10, 35). Given that the C FG loop and the glycan attached at CN236 form one wall of a TCR cavity within or adjacent to which the CD3 of a CD3 heterodimer might bind, deletion of either the glycan addition site at N236 (by an Asn to Ala mutation, N236A) or the FG loop would likely diminish the strength of the association between the TCR and the CD3 heterodimer. To test this possibility, N15wt-, N15N236A-, and N15FG TCR-expressing cell lines were generated. The parental 58^––CD8⁺CD3 T cell line (28) was cotransfected with a wild-type N15-chain cDNA and either wild-type or mutant N15-chain cDNAs. As shown in Fig. 7A, compared with the parental cell line, all transfectants express reactivity with the V5.2-specific mAb MR9.4 and the N15 chain anti-clonotype mAb R53 (28), although the fluorescence intensity in N15N236A and N15FG cells is 2- to 3-fold lower than in wt cells. As expected, the H57 C FG loop-specific mAb selectively binds the N15wt, as well the N15N236A variant, but is unreactive with N15FG. The level of surface CD3 as detected by the 2C11 mAb that recognizes CD3 in the context of both CD3 and CD3 dimers, as well as by 7D6 that selectively detects CD3, is slightly lower in both the N15N236A and N15FG transfectants.

View larger version (59K): [in this window] [in a new window]   FIGURE 7. Analysis of N15wt, N15N236A, and N15FG transfectants. A, FACS analysis of N15 wt and mutant transfectants. The TCR surface expression on N15wt or mutant transfectants was examined by indirect immunofluorescence staining using the specified mAbs and species-specific secondary Abs. The dark curve represents specific staining with mAb, and the light curve corresponds to background staining (second step Ab alone). Histograms were compiled from a minimum of 10,000 cells. The geometric peaks of the fluorescent intensities are presented. B, Biochemical analysis of the surface TCR complex on N15wt, N15N236A, and N15FG cells. Surface-biotinylated transfectants (2 x 107) were lysed with 1% digitonin buffer, and lysates were subjected to immunoprecipitation with the anti-CD3 mAb 7D6. The immunoprecipitates were then separated by two-dimensional nonreducing/reducing SDS-PAGE. Biotinylated proteins were detected by streptavidin. Inset, N15wt (lane 1), N15N236A (lane 2), and N15FG cells (lane 3) (2 x 107) were lysed, and samples were immunoprecipitated with anti-mouse CD3 mAb, 13A1. After nonreducing SDS-PAGE, CD3 proteins were visualized by Western blotting with the anti-CD3 heterosera. Results are representative of four independent experiments.

Disruption of TCR-stimulated phosphorylation by C FG loop deletion

To next determine the functional consequences of these alterations on biochemical association, the effect of TCR cross-linking by anti-CD3 mAbs on induced tyrosine phosphorylation was examined. Fig. 8A shows that, upon stimulation, there is rapid tyrosine phosphorylation of multiple proteins associated with active Zap70 in antiphosphorylation immunoblots of Zap70 immunoprecipitates from N15wt transfectants. The phosphorylated proteins include Zap70 at 70 kDa (whose identity was confirmed by stripping the 4G10 blot and reprobing with anti-Zap70 heteroantisera), as well as prominent bands at 145, 56, 46, 36, 25, 23, and 21 kDa. Although the identity of these substrates remains to be proven, their sizes are consistent with phospholipase C, lck, shc, linker for activation of T cells, CD3, and CD3, respectively. However, in N15N236A, the phosphorylation of Zap70 and these proteins is reduced in magnitude and more transient in duration and is essentially undetectable in N15FG.

View larger version (45K): [in this window] [in a new window]   FIGURE 8. The C FG loop deletion markedly diminishes TCR signaling function of transfectants. A, Analysis of TCR-stimulated protein-tyrosine phosphorylation of intracellular substrates in N15wt, N15N236A, and N15FG transfectants. N15wt and mutant cells (1 x 107) were stimulated with a combination of biotinylated anti-CD3 mAb 2C11 and streptavidin at 37°C for the indicated periods. The cell lysates were prepared and immunoprecipitated with antisera specific for Zap70. The immunoprecipitates were subjected to reducing SDS-PAGE followed by Western blotting with anti-phosphotyrosine (pTyr) mAb 4G10 (upper). Results are representative of three independent experiments. The blot was stripped and subsequently immunoblotted with Zap70 rabbit antisera with only the area of the Zap70 band shown (lower). The migration position of Zap70 in the pTyr blot is indicated by an arrow. B, IL-2 production induced by specific pMHCI in N15wt and variant TCR bearing cells. For activation, H-2Kb+ R8 cells (used as APCs) were loaded with 10–9–10–4 M VSV8 peptide. Mean ± SD of triplicate samples are indicated. Results are representative of four separate experiments.

To confirm the signaling defect in primary T cells, we used N15 RAG-2^–/– and N15,FG RAG-2^–/– animals as described in Materials and Methods. As shown in Fig. 9, there is a reduction in phosphorylation of Erk1/2 MAPK upon in vitro cognate peptide stimulation of LN T cells from N15,FG RAG-2^–/– as compared with N15 RAG-2^–/– mice. Note that T cells from these two strains of mice express equivalent levels of TCR as shown in Fig. 9A. Thus, the FG mutation disrupts TCR signaling leading to Erk1/2 phosphorylation.

View larger version (46K): [in this window] [in a new window]   FIGURE 9. Reduced Erk phosphorylation upon stimulation by cognate peptide in N15,FG RAG-2–/– vs N15 RAG-2–/– T cells. A, LN cells from N15 RAG-2–/– and N15,FG RAG-2–/– mice were analyzed for TCR expression by FACS with the anti-V5 Ab, MR9.4. The percentages of MR9.4+ T cells in total LN cells are given. Numbers in the upper left corner of the histograms are the mean fluorescence intensities. B, LN T cells from N15 RAG-2–/– and N15,FG RAG-2–/– mice were stimulated with 10–5 M VSV8 peptide for the indicated minutes, lysed, and analyzed for phospho Erk1/2 (upper panel) and total Erk1/2 (lower panel).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

In the present study, we have independently characterized the importance of the C FG loop in thymocyte development of T cells using N15wt and N15FG tg B6 mice. For this purpose, we used multicolor flow cytometry analysis and competitive adoptive transfer studies to reveal that removal of the loop favors the survival of thymocytes at all stages of intrathymic development. Collectively, these data suggest that removal of the C FG loop dramatically attenuates negative selection without preventing positive selection.

Competitive thymic reconstitution by adoptive transfer of BM cells from N15wt and N15FG tg mice allows direct comparison of the developmental potential of thymocytes expressing FG vs wt subunits. As shown in Fig. 2 and Table I, N15FG-bearing cells predominate over N15wt-expressing cells in all thymic subpopulations and in the peripheral CD8⁺ population in mixed wt and FG tg chimeras using Ly5 congenic or RAG-2^–/– recipient hosts. These data are directly reflective of the advantage of the FG-expressing over the wt-expressing thymocytes in development. Based on our observation that there is reduced negative selection in N15.FG RAG-2^–/– compared with N15 RAG-2^–/– mice (26), the FG mutation must reduce cell death during thymic development even in -chain tg thymocytes in which the -chain pairs with multiple endogenous -chains. In this regard, earlier in vivo BrdU labeling showed no difference in DNA synthesis among wt- and FG tg-expressing thymocytes (26). Thus, the increase in DP thymocyte populations is not because of enhanced proliferation but rather reduced negative selection. Negative selection requires a relatively strong TCR ligation (36). A reduction in cell death in FG mice suggests that TCRs containing the FG subunit are less effective at signal transduction than those containing the wt subunit.

T cells of the lineage mature through DP and SP stages that are associated with stringent selection events (3, 37). In contrast, some T cells of the lineage do not appear to be negatively selected in the thymus, but rather positively selected on cognate self-Ags (38). T cells differ from T cells in usage of CD3 (10), p56^lck (39), linker for activation of T cells (40), Vav (41), and p38 (42) among other signaling pathways. The crystal structure of human and murine TCRs shows that the human C FG loop is 12 residues shorter than the C FG loop, packing close to the core of the C domain (43, 44). Hence, if the C FG loop represents a key TCR structural component for negative selection, then its essential absence in C explains, at least in part, why some TCRs are not negatively selected in the thymus.

Pre-TCR expression at the level of DN thymocytes (DN3) promotes TCR development but not TCR development (45). In fact, pT^–/– mice are characterized by an excess number of cells (45). We further examined the effect of the FG loop deletion in pre-TCR function using N15wt RAG-2^–/– and N15FG RAG-2^–/– models since the CD3 heterodimer is thought to bind to a similar cave in the pre-TCR formed by C and pT ectodomains (22). The RAG-2^–/– background allows clonal analysis of pre-TCR function more readily than RAG-2^+/+ or RAG-2^+/– for two reasons. First, endogenous -chains cannot be expressed; hence, all -chains associate with pT rather than -chains (46). Second, a function of the pre-TCR is to signal productive -chain rearrangement, terminating further recombination machinery activity and resulting in -chain allelic exclusion (8). If the FG chain has less signaling capacity than wt, then a pre-TCR incorporating a FG subunit may not shut off rearrangements as efficiently as one incorporating wt. In the presence of the FG transgene, there may be more endogenous -chain expression than in the wt tg thymocytes (in the RAG-2^+/+ or RAG^+/– backgrounds). Assuming these endogenous -chains are incorporated into pre-TCRs, then the role of the FG loop in