© Rockefeller University Press, 0022-1007/2002/6/1419/ $5.00
The Journal of Experimental Medicine, Volume 195, Number 11, June 3, 2002 1419-1431
Involvement of the TCR Cß FG Loop in Thymic Selection and T Cell Function
Tetsuro Sasada1,2,
Maki Touma1,2,
Hsiu-Ching Chang1,2,
Linda K. Clayton1,2,
Jia-huai Wang1,3,4 and
Ellis L. Reinherz1,2
1 Laboratory of Immunobiology, Dana-Farber Cancer Institute
2 Department of Medicine, Harvard Medical School, Boston, MA 02115
3 Department of Pediatrics, Harvard Medical School, Boston, MA 02115
4 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115
Address correspondence to Dr. Ellis L. Reinherz, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Phone: 617-632-3412; Fax: 617-632-3351; E-mail: ellis_reinherz{at}dfci.harvard.edu
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Abstract
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The asymmetric disposition of T cell receptor (TCR) Cß and C
ectodomains creates a cavity with a side-wall formed by the rigid Cß FG loop. To investigate the significance of this conserved structure, we generated loop deletion (ß
FG) and ßwt transgenic (tg) mice using the TCR ß subunit of the N15 CTL. N15ßwt and N15ß
FG H-2b animals have comparable numbers of thymocytes in S phase and manifest developmental progression through the CD4-CD8- double-negative (DN) compartment. N15ß
FG facilitates transition from DN to CD4+8+ double-positive (DP) thymocytes in recombinase activating gene (RAG)-2-/- mice, showing that pre-TCR function remains. N15ß
FG animals possess
twofold more CD8+ single-positive (SP) thymocytes and lymph node T cells, consistent with enhanced positive selection. As an altered V
repertoire observed in N15ß
FG mice may confound the deletion's effect, we crossed N15
ß TCR tg RAG-2-/- with N15ß
FG tg RAG-2-/- H-2b mice to generate N15
ß RAG-2-/- and N15
ß.ß
FG RAG-2-/- littermates. N15
ß.ß
FG RAG-2-/- mice show an 810-fold increase in DP thymocytes due to reduced negative selection, as evidenced by diminished constitutive and cognate peptide-induced apoptosis. Compared with N15
ß, N15
ß.ß
FG T cells respond poorly to cognate antigens and weak agonists. Thus, the Cß FG loop facilitates negative selection of thymocytes and activation of T cells.
Key Words: negative selection thymocyte development TCR structure Ig superfamily transgenic mice
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Introduction
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The TCR is a multicomponent complex composed of a Ti disulfide-linked heterodimer in noncovalent association with the CD3 subunits (15). The majority of T cells express a Ti-encoded by
and ß TCR genes (Ti
ß) whereas a numerically minor but functionally significant subset expressed Ti encoded by
and
TCR genes (Ti
; for a review, see reference 6). The Ti
ß heterodimer whose overall shape and architecture are similar to an antibody Fab fragment represents the peptideMHC complex (pMHC)* ligand binding unit. Thus, Ti
ß determines the ligand specificity of an individual T cell. In contrast, the CD3 polypeptides mediate TCR-based signal transduction. The signaling function of the CD3 components involves a conserved motif, termed immunoreceptor tyrosine-based activation motif (ITAM), present at 13 copies in the cytoplasmic domain of each CD3 subunit (79). The various CD3 subunits exhibit different interactions with intracellular signaling factors and, acting in concert, lead to T cell activation (1015). However, how pMHC ligand binding to a Ti
ß or Ti
heterodimer subsequently initiates signaling via the CD3 molecules is currently unknown.
The TCR complex contains one copy of CD3
and CD3
and two copies of CD3
and CD3
configured as two CD3
and CD3
heterodimers and a disulfide-linked CD3
homodimer (1621). Recent crystallographic analysis (22) in conjunction with epitope mapping of a class Irestricted TCR (23) have identified a cavity within the Ti
ß constant (C) module of sufficient size to accommodate one nonglycosylated CD3
ectodomain. In this regard, the solution structure of a heterodimeric CD3
ectodomain complex reveals a unique side-to-side hydrophobic interface between the two C2-set immunoglobulin-like domains and indicates that a modular pair-wise association is shared between CD3
as well (24). CD3
has an elongated shape with length, width and depth being
40, 25, and 25 Å, respectively.
Herein, we have investigated the biological significance of the unusual structural arrangement of the Cß FG loop that forms the side wall of this C-module cavity. Mapping studies using the Cß FG loop-specific mAb H57 previously showed that one of two CD3
subunits resides adjacent to this structural element. To this end, we have created TCR transgenic mice bearing either intact or Cß FG loop-deleted ß chains. Our results show that the loss of the Cß FG loop attenuates negative selection without blocking positive selection within the thymus at the level of double-positive (DP) thymocytes. The sensitivity of mature T cells to cognate peptides as well as altered peptide ligands is also reduced. In contrast, pre-TCR facilitated transition from CD44-CD25+ to CD44-CD25- double-negative (DN) thymocytes and subsequent DP thymocyte generation is not altered. The implications of these findings for TCR and pre-TCR function are discussed.
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Materials and Methods
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T Cell Receptor Construct.
The FG loop-deleted mutant of the N15 TCRß chain constant region (Cß) was generated by PCR using N15ß wt cDNA as a template, subcloned into the pCRII vector, and verified by DNA sequencing analysis. The StuI digested DNA fragment containing the FG loop-deleted constant region of the TCRß chain was subcloned into the StuI site in the constant region of an N15 TCRß expression vector, which was previously used for the generation of N15 TCR
ß tg recombinase activating gene (RAG)-2-/- H-2b mice (25).
Mice.
Mice transgenic for the N15 wt or FG loop-deleted TCRß chain (hereafter for simplicity referred to as N15ßwt or N15ß
FG) were produced in the transgenic/gene targeting core facility at Dana-Farber Cancer Institute. The N15 TCRß shuttle constructs with the wt or FG loop-deleted mutant Cß were digested with PvuI/SalI, leaving the bacterial vector sequences attached to the TCR gene. These DNAs were injected into (SJL x C57BL/6) F1 fertilized eggs and implanted into foster mothers. Resulting progeny were screened for integration of the N15 wt or FG loop-deleted TCRß chain DNA by Southern blot analysis of tail DNA and by FACS® analysis of N15 TCRß chain expression in peripheral blood nucleated cells. Five transgenic founders were identified, two for N15ßwt, and three for N15ß
FG. The mice were backcrossed for 45 generations onto the C57BL/6 background before the experiments described below. To create N15ß
FG RAG-2-/- mice, the N15ß
FG mice were bred with RAG-2-/- C57BL/6 mice and the heterozygous N15ß
FG+/- RAG-2+/- F1 mice were intercrossed and offspring which were N15ß
FG+RAG-2-/- subsequently selected. The expression of the FG loop-deleted N15ß chain and lack of RAG-2 were confirmed based on the Southern blot analysis of tail DNA and FACS® analysis of peripheral blood cells. To introduce the N15
chain into N15ß
FG mice and generate Ag-specific T cells with the FG loop-deleted TCRß chain, the N15ß
FG RAG-2-/- (N15ß
FG+/-RAG-2-/-) mice were bred with N15 TCR
ß tg RAG-2-/- H-2b (N15
wt+/+ßwt+/+ RAG-2-/- H-2b) mice, and the resulting N15
wt+/-ßwt+/-RAG-2-/- H-2b or N15
wt+/- ßwt+/-ß
FG+/-RAG-2-/- H-2b (hereafter for simplicity referred to as N15
ß or N15
ß.ßFG mice) were used for the experiments. RAG-2-/- C57BL/6 mice were purchased from Taconic. All lines were maintained and bred under sterile barrier conditions at the animal facility of Dana-Farber Cancer Institute.
Peptide Synthesis.
Peptides were synthesized by standard solid phase methods on an Applied Biosystems 430A synthesizer at the Biopolymers Laboratory of the Massachusetts Institute of Technology. All peptides were purified by reverse phase HPLC (Hewlett Packard HPLC 1100) with a C4, 2-mm column. Peptides were analyzed for purity and correct molecular weight by electrospray mass spectrometry, amino acid (aa) analysis, and HPLC. Peptides are named to indicate the substituted aa and the position in the sequence of VSV8 (e.g., L4 denotes replacement of valine by leucine at the fourth residue of the VSV8 peptide). VSV8 is a cognate antigenic peptide agonist of the N15 TCR, which triggers thymic negative selection in N15 TCR
ß tg RAG-2-/- H-2b mice, whereas L4 and K1 peptides are altered peptide ligands (APLs) of VSV8 and weak agonists of the N15 TCR, which induce positive selection in N15 TCR
ß tg RAG-2-/-ß2M-/- H-2b mice (26).
Antibodies and Flow Cytometric Analysis.
The following mAbs were used: R-phycoerythrin (PE)- or Cychrome-conjugated antimouse CD4 (H129.19); FITC- or Cychrome-conjugated antimouse CD8
(536.7); Cychrome-conjugated anti-TCR Cß (H57597); PE-conjugated anti-CD25 (PC61); FITC-conjugated anti-CD44 (IM7); FITC-conjugated anti-Vß5.1, 5.2 (MR9.4); biotin-conjugated anti-CD69 (H1.2F3); biotin-conjugated anti-V
2 (B20.1), anti-V
3.2 (RR316), anti-V
8.3 (B21.14), anti-V
11.1, 11.2 (RR81), PE-conjugated streptavidin (BD PharMingen). For flow cytometry, single cell thymocyte or lymph node suspensions were prepared in PBS containing 2% FCS and 0.05% NaN3. Cells were stained at 5 x 106 cells per ml in PBS-2% FCS and 0.05% NaN3 containing the antibodies at saturating concentrations. Phenotypes and proportions of thymocyte and lymph node cell subsets were analyzed by two-color flow cytometry using a FACScanTM (Becton Dickinson) and the CELLQuestTM program. Dead cells were excluded from the analysis by forward and side scatter gating.
Bromodeoxyuridine Staining.
Bromodeoxyuridine (BrdU) incorporation was examined according to the manufacturer's protocol (BrdU Flow Kit; BD PharMingen). Mice were administered two intraperitoneal injections of 1 mg of BrdU in PBS at 4 h intervals. After 18 h, thymocytes were fixed/permeabilized and stained with FITC-conjugated anti-BrdU mAb following treatment with 30 µg/ml DNase. Live cells were gated and analyzed on a FACScanTM.
Apoptosis Assay.
Thymocytes from untreated, VSV8 peptide-injected (0.5 µg intravenously, 18 h previously) or dexamethasone-treated (0.5 mg intraperitoneally, 6 h previously) were examined. Apoptosis of thymocytes was evaluated by cellular DNA content determined by propidium iodide (PI) staining and alterations in plasma membrane permeability examined by Annexin V staining. For PI staining, after fixation with 40% ethanol at 4°C for 30 min, total thymocytes (106 cells) were resuspended in PBS containing 50 µg/ml RNase A and 2.5 µg/ml PI and incubated at 37°C for 30 min. PI incorporation was analyzed by flow cytometry. For annexin V staining, after washing with PBS, total thymocytes (106 cells) were resuspended in 1x binding buffer containing 0.5 µg/ml FITC-conjugated annexin V (ApoAlert Annexin V Apoptosis kit; CLONTECH Laboratories, Inc.) and 2.5 µg/ml PI to gate out dead cells, and incubated in the dark for 10 min at room temperature, followed by the analysis on a FACScanTM.
Proliferation Assay.
Lymph node cells from N15
ß or N15
ß.-ß
FG mice (105/well) were incubated at 37°C with 2 x 104 irradiated EL-4 thymoma cells, which had been preloaded for 2 h with the indicated doses of VSV8 or a weak agonist, L4, in AIM-V medium (Life Technologies) containing 50 µM 2-ME. After 48 h incubation, 0.4 µCi per well of [3H]TdR (ICN Biomedicals) was added, and after an additional 18 h of culture at 37°C, the cells were harvested and the incorporated radioactivity was measured.
Measurement of IFN-
.
IFN-
production was induced under the same culture conditions used for proliferation assays (see above). After 48 h of incubation, supernatants were collected and assayed for IFN-
using a mouse IFN-
ELISA kit (Mouse IFN-
OptEIA Set; BD PharMingen). The sensitivity of the assay was 31.32,000 pg/ml and results were calculated as the mean of duplicate wells.
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Results
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The TCR Cß FG Loop Structure and Sequence Comparison.
One remarkable feature of the TCR Cß domain is the striking elongation of the FG loop. Compared with other Ig-like structures (aside from the TCR C
domain), there is a 13 aa insertion within the FG loop (27). Fig. 1
A shows a view of the crystallographically resolved murine N15 class I MHC-restricted
ß TCR (Vß5.2 D2Jß2.6 Cß2/V
8 J
5C
; reference 22). The unique protrusion of the Cß domain's FG loop is apparent (bracketed in a black box). It was previously noticed that internally the loop is well structured (22). The Trp225 at the tip of the loop plays a key role. A hydrogen bond between the NE1 of Trp225 and the carbonyl group of Ser229 brings the bulky indole ring of Trp225 in position to make hydrophobic contacts with Leu219 and Pro232 at the base of the FG loop (Fig. 1 B, a close up view of the local region shown in the black box of Fig. 1 A). The hydrophobic patch composed of these three FG loop residues packs onto Leu119 and Val122 at the beginning of the Cß domain's A strand to form a mini-hydrophobic core. The Leu119 and Val122 themselves lie on a stable local strut created by a tight ß turn between Asp118 and Asn121. Moreover, a pair of mainchain hydrogen bonds between Thr123 on the A strand and Phe153 at the end of the B strand further stabilizes the local structure. Collectively, the Cß domain FG loop, albeit a long protrusion, is an integrated component of a rigid structural entity that connects the Vß and Cß domains. Not only are the residues mentioned above all invariant among mouse, rat, human, chimpanzee, rabbit, cow, dog, and pig as shown in the Fig. 1 C, but the three-dimensional structural feature depicted in Fig. 1 B is also conserved in all known TCR crystal structures, including class I MHC-restricted N15 and 2C from mouse (22, 28) and B7 from human (29), as well as the human class II MHC-restricted HA1.7 (30).
T Lineage Development in N15ß
FG Mice.
To analyze the role of the Cß FG loop in thymocyte development and mature T cell function in vivo, we established transgenic mice expressing the wild-type N15 TCRß chain or an FG loop deletion variant lacking aa 219232. For simplicity, these TCR transgenes will be referred to as N15ßwt and N15ß
FG, respectively. The N15ßwt subunit derives from the N15 CD8+ cytotoxic T cell clone whose TCR is specific for the vesicular stomatitis virus nucleoprotein aa 5259 (VSV8) (RGYVYQGL) bound to the MHC class I molecule H-2Kb (31). T lineage cells from the thymus and lymph nodes of N15ß
FG mice were then directly compared with those of the N15ßwt mice. Fig. 2
A shows a flow cytometric analysis of CD8+ single-positive (SP) lymph node T cells from 1 of 2 representative N15ßwt lines and 1 of 3 representative N15ß
FG lines. As expected, the anti-Cß FG loop specific mAb H57 (22, 32) reacts with virtually all CD8+ SP lymph node T cells in N15ßwt tg mice but almost no CD8+ SP lymph node T cells in N15ß
FG mice. Those few reactive T cells (6.8%) represent CD8+ SP cells that have rearranged endogenous ß chains due to incomplete TCRß chain allelic exclusion by the N15ß transgene, as previously reported in other transgenic systems (33). Fig. 2 A also indicates that despite the Cß FG loop deletion, N15ß
FG tg animals, like N15ßwt tg mice, predominantly express the transgenic Vß5.2 gene as detected by the MR9.4 mAb.
Perhaps more importantly, as shown in Fig. 2 B and Table I, comparison of thymocyte subsets reveals substantial differences between the N15ßwt and N15ß
FG tg mice. At 612 wk of age, for example, N15ß
FG mice show a subtle but significant reduction in the number of total thymocytes (N15ßwt, 12.05 ± 2.99 x 107 [n = 10]; N15ß
FG, 9.51 ± 2.09 x 107 [n = 15], P < 0.05; Table I). Furthermore, as shown by the representative CD4/CD8
double-staining profiles in Fig. 2 B, there is a detectable increase in the percentage of the CD8+ SP subset and a decrease in that of the DP subset in N15ß
FG thymocytes compared with N15ßwt mice. Therefore, N15ß
FG mice show a statistically significant increase in the absolute number of CD8+ SP thymocytes and a concommitant decrease in DP thymocytes (Table I). This increase in CD8+ SP cells is further reflected by the increase in CD8+ SP lymph node T cells (N15ßwt, 1.99 ± 0.77 x 107 [n = 10]; N15ß
FG 3.45 ± 0.82 x 107 [n = 15]). From these findings, we conclude that the absence of the FG loop in the constant region of N15 TCR ß chain influences T cell development in the tg mouse model.
There are two major checkpoints at which
ß thymocyte development is dependent upon the TCR ß chain. The earliest occurs during the transition from DN to DP thymocytes and is regulated by the pre-TCR complex comprised of the invariant pT
subunit disulfide linked to a TCR ß chain in noncovalent association with the CD3 subunits (34). The later checkpoint involves the
ß TCR and occurs during the transition from DP to SP thymocyte maturation. At that time, self-MHCrestricted, nonautoreactive T cells are selected for maturation and peripheral exportation (35). To investigate the mechanism of the altered thymocyte phenotype in N15ß
FG mice, we first compared early thymic development in the N15ßwt and N15ß
FG mice by examining the fraction of cells synthesizing DNA using BrdU incorporation in vivo. In this way, it is possible to test whether the FG loop deletion in the N15 TCRß chain affects proliferation and/or turnover of thymocytes. The N15ßwt and N15ß
FG tg mice were injected twice intraperitoneally with 1 mg of BrdU in PBS at 4 h intervals, and 18 h later, thymocytes were assayed for the incorporation of BrdU by flow cytometry. As shown in Fig. 3
A, the percentage of BrdU+ thymocytes in the N15ß
FG mice was comparable to that observed in control N15ßwt mice, indicating that the number of cells that had progressed through S phase is similar. In addition, we compared the developmental stages of immature thymocytes by examining the surface expression of CD25 and CD44 on DN thymocytes of these mice. The percentages of the four CD44/CD25 subsets (CD44+CD25-, CD44+CD25+, CD44-CD25+, and CD44-CD25-) in the N15ß
FG were equivalent to those of N15ßwt mice (Fig. 3 B), suggesting that the deletion of Cß FG loop from the N15 TCRß chain does not disrupt the pre-TCRfacilitated transition from CD44-CD25+ to CD44-CD25- DN thymocytes. To further assess the function of the Cß FG loop-deletion in pre-TCR signaling at the CD44-CD25+ DN stage, we established RAG-2deficient mice expressing the Cß FG loop-deleted N15ß chain (N15ß
FG RAG-2-/-). Introduction of a functionally rearranged wild-type TCRß chain overcomes the developmental arrest at the CD44-CD25+ DN stage observed in the RAG-2deficient mice, leading to strong proliferation of DN thymocytes and efficient generation of DP cells (36). As shown in Fig. 3 C, N15ß
FG RAG-2-/- mice generate DP thymocytes with essentially normal cellularity (6.6 x 107 thymocytes/mouse). These results suggest that signaling through the pre-TCR complex on CD44- CD25+ thymocytes is not blocked by the deletion of the Cß FG loop in the N15 TCRß chain.
Altered V
Usages by Peripheral Mature T Cells in the N15ß
FG Mice.
Given the nearly twofold increase in the absolute number of CD8+ SP lymph node T cells (Table I), we next examined whether deletion of the Cß FG loop of the N15 ß chain influences thymic repertoire selection. To this end, the TCR V
gene usage of CD8+ and CD4+ SP lymph node T cells in N15ßwt and N15ß
FG mice was assessed by flow cytometry using a panel of anti-V
mAbs. As shown in Table II, there are statistically significant differences in the TCR V
gene usage in peripheral lymph node T cells between N15ßwt and N15ß
FG mice. CD8+ lymph node T cells from N15ß
FG mice display an increased frequency of TCRs incorporating V
8.3 (mean = 4.37%, SD = 0.58 versus mean = 3.74%, SD = 0.54; P < 0.02) and V
11 (mean = 6.81%, SD = 0.70 versus mean = 5.46%, SD = 0.50; P < 5 x 10-5) gene segments compared with N15ßwt mice (Table II). CD4+ lymph node T cells from N15ß
FG mice also bear more TCRs incorporating V
2 (mean = 13.61%, SD = 0.95 versus mean = 11.77%, SD = 1.38; P < 0.005) than those from N15ßwt mice. These data suggest that lack of the Cß FG loop exerts an influence on selection of the TCR repertoire by affecting usage of functional V
gene segments, leading to the alterations in thymic and peripheral lymph node phenotypes observed in the N15ß
FG mice. That V
differences are observed in CD8+ SP thymocytes as well as lymph node cells is consistent with this view (data not shown), but does not exclude the possibility that impaired TCR signaling in the peripheral T cells in N15ß
FG animals further alters the repertoire.
Quantitative and Qualitative Alterations of Thymocytes and Peripheral T Cells in N15
ß.ß
FG Mice.
The differences between N15ßwt and N15ß
FG mice may have been modified by compensatory adaptations of V
gene expression. To analyze more clearly the role of the TCR Cß FG loop in thymocyte development and mature T cell function, we next crossed the N15ß
FG RAG-2-/- mice (N15ß
FG+/-RAG-2-/- H-2b) with N15
ß tg RAG-2-/- H-2b (N15
wt+/+ßwt+/+RAG-2-/- H-2b) to generate two mouse strains: N15
wt+/-ßwt+/- RAG-2-/- and N15
wt+/-ßwt+/-ß
FG+/-RAG-2-/- mice. These mice are referred to hereafter for simplicity as N15
ß and N15
ß.ß
FG, respectively. In these strains, the TCR
chain is fixed and on the RAG-2-/- background, so no confounding endogenous ß or
chains can be expressed.
T lineage cells from the thymus and lymph nodes of these animals are directly compared in Fig. 4, A and B
. As defined by the anti-Vß5 mAb MR9.4, using quantitative immunofluorescence on FACS®, the CD8+ SP lymph node T cells from N15
ß.ß
FG mice expressed the same level of N15 ß chain as CD8+ SP T cells derived from N15
ß mice. However, the fluorescence intensity of anti-TCR Cß H57 mAb staining in the CD8+ SP T cells from N15
ß.ß
FG mice is
50% that in N15
ß T cells, indicating that CD8+ SP T cells from N15
ß.ß
FG mice express both wild-type and FG loop-deleted N15 TCRß chains complexed with N15 TCR
chains at almost equal levels on their cell surface (Fig. 4 A). Although not shown, all peripheral T cells are CD8+ SP in both N15
ß and N15
ß.ß
FG mice with equivalent surface CD8 density as revealed by the anti-CD8
mAb 536.7.

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Figure 4. Quantitative and qualitative T lineage abnormalities in the N15 ß.ß FG mice. (A) TCR expression on lymph node T cells in the N15 ß and N15 ß.ß FG mice. Lymph node cells from 12-wk-old N15 ß and N15 ß.ß FG mice were triple-stained with anti-CD4, anti-CD8, and anti-TCR Cß FG loop-specific H57 or anti-Vß5 MR9.4 mAbs. The histograms of TCR Cß FG loop (left) and Vß5 (right) expression on the gated CD8+ SP lymph node T cells are shown. The numbers represent the values of mean fluorescence intensity. (B) The CD4/CD8 double-staining profile in thymocytes is altered in N15 ß.ß FG mice. The thymocytes from N15 ß and N15 ß.ß FG mice at 12 wk of age were double-stained with PE-anti-CD4 and FITC-anti-CD8 . The expression of CD4 (y axis) and CD8 (x axis) on thymocytes was detected by flow cytometry after gating on 10,000 live cells. The percentages of each subset are indicated. (C) The N15 ß.ß FG mice show a drastic increase in the total and DP thymocytes, but a decrease in the CD8+ SP lymph node T cells. The numbers of total, DP, CD8+ SP, and DN thymocytes and CD8+ SP lymph node T cells were calculated by quantifying the total numbers of thymocytes and lymph node cells from 812-wk-old N15 ß and N15 ß.ß FG mice and the percentages of each subset as determined by FACS® analysis. Open circles (N15 ß mice [n = 13]) and closed circles (N15 ß.ß FG mice [n = 9]) represent values of individual mice and bars represent average values for a given group. The total and DP thymocyte numbers are significantly higher, and CD8+ SP lymph node cell number is significantly lower in the N15 ß.ß FG mice, compared with the N15 ß mice (total and DP thymocytes, P < 0.0001; CD8+ SP lymph node cells, P < 0.0005).
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Comparison of the thymocyte cell number and subset distribution among N15
ß or N15
ß.ß
FG mice reveal striking differences. At 812 wk of age, N15
ß.ß
FG mice have a markedly larger thymus relative to that of N15
ß animals with an
sevenfold increase in the number of total thymocytes (Fig. 4 C). Furthermore, as shown by the CD4/CD8
double-staining profiles in Fig. 4 B, there is a substantial increase in the DP subset percentage and a decrease in the DN and CD8+ SP subset percentages in N15
ß.ß
FG thymocytes. As the total number of N15
ß.ß
FG thymocytes is
7 times more than N15
ß thymocytes, N15
ß.ß
FG mice show no significant differences in the absolute numbers of DN and CD8+ SP thymocytes, but
10-fold more DP thymocytes than N15
ß mice (N15
ß, 1.48 x 107 DP cells versus N15
ß.ß
FG, 13.2 x 107 DP cells; Fig. 4 C). These results indicate that the absence of the TCR Cß FG loop influences intrathymic T cell development in the N15 TCR
ß tg mouse system. In contrast to the difference in thymocyte number, the number of peripheral CD8+ SP lymph node T cells in N15
ß mice is
2 times more than that in the N15
ß.ß
FG mice (N15
ß, 1.71 x 107 cells versus N15
ß.ß
FG, 0.87 x 107 cells). This difference in peripheral CD8+ SP T cell number may result either from faulty homeostatic expansion of T cells in the peripheral lymphoid system of N15
ß.ß
FG mice as a consequence of impaired T cell activation (37) or represent a qualitative difference in CD8+ SP thymocyte maturation and/or release into the peripheral T cell compartment. CFSE dye tracking of the CD8+ SP transgenic T cells injected into B6 RAG-2-/- mice or irradiated B6 mice excludes detectable alteration in cell divisions, however (data not shown).
Decreased Thymic Negative Selection in the N15
ß.ß
FG Mice.
The T cell repertoire is shaped by both negative and positive thymic selection processes acting primarily at the level of the immature DP thymocytes (for reviews, see references 35 and 38). Previous studies showed that DP thymocytes are deleted during negative selection via a caspase-dependent apoptotic mechanism (39). To investigate the basis for the dramatic increase in DP thymocytes in N15
ß.ß
FG, we evaluated the efficiency of the negative selection process in the N15
ß versus N15
ß.ß
FG animals. Apoptotic cells were enumerated through measurement of DNA content in total thymocytes using propidium iodide and flow cytometric analysis. As shown in Fig. 5
A, the percentage of thymocytes with subdiploid DNA content, a typical feature of apoptotic cell death, is significantly higher in the N15
ß mice than in the N15
ß.ß
FG mice (N15
ß, 12.5% versus N15
ß.ß
FG, 1.1%). Consistent with this result, alterations in plasma membrane permeability, one of the earliest changes in cells undergoing apoptosis, is also altered differentially in N15
ß versus N15
ß.ß
FG mice. As shown in Fig. 5 B, about half (48.5%) of the thymocytes in the N15
ß mice are positive for annexin V staining, suggesting that a large number of DP thymocytes are being continuously deleted by apoptosis. In contrast, only a small percentage (10.0%) of thymocytes are annexin V positive in N15
ß.ß
FG mice. This finding suggests that the nature of constitutive ongoing negative selection in DP thymocytes is different between N15
ß and N15
ß.ß
FG mice.
To further probe differences in the negative selection threshold of DP thymocytes from N15
ß and N15
ß.ß
FG mice, we injected the cognate peptide VSV8 intravenously and compared the phenotypic changes in thymocytes. 18 h after intravenous injection with 0.5 µg of VSV8 peptide, N15
ß mice showed an
90% reduction in total thymocyte number (without VSV8 injection, 2.07 x 107 cells; after VSV8 injection, 0.26 x 107 cells), consistent with earlier observations (25). By contrast, there is no peptide-induced change in the total thymic cellularity in N15
ß.ß
FG mice (without VSV8 injection, 10.4 x 107 cells; after VSV8 injection, 10.6 x 107 cells). These results suggest that deletion of the Cß FG loop alters the sensitivity of immature DP thymocytes to negative selection induced by the cognate peptide in N15 TCRtg mice. Consistent with the thymic cellularity data, DNA staining with propidium iodide shows a significant increase in the percentage of subdiploid DNA-containing cells after VSV8 injection in the N15
ß (35.8%) but not in the N15
ß.ß
FG mice (1.3%; Fig. 5 A). Furthermore, as shown in Fig. 5 B, the N15
ß mice display a prominent increase in the annexin V+ thymocytes after 18 h of VSV8 (80.4%), compared with N15
ß.ß
FG mice (14.7%). Collectively, these results suggest that the increase in DP thymocytes in the N15
ß.ß
FG mice can be explained, at least in part, by the reduced sensitivity of those DP thymocytes to undergo peptide-triggered negative selection. Although not shown, higher doses of VSV8 peptide induce apoptotic cell death in both N15
ß and N15
ß.ß
FG thymocytes to a similar extent, suggesting that the N15
ß.ß
FG mice require a larger number of pMHC-ligated TCRs to trigger negative selection. That dexamethasone-induced DNA fragmentation of thymocytes is equivalent in both mouse strains implies no general apoptotic defect in N15
ß.ß
FG animals (data not shown). Note that the observed apoptosis of thymocytes is not secondary to peripheral cytokine production observed in other systems (40) as shown by the rapid kinetics of the N15 TCR tg system and prior parallel FTOC analysis (25, 39).
Impaired Proliferation and Cytokine Production upon Antigen-triggered Stimulation of Naive T Cells in the N15
ß.ß
FG Mice.
To examine the role of the Cß FG loop in mature T cell function, we next tested the antigen responsiveness of N15
ß.ß
FG T cells by culturing lymph node T cells with VSV8 cognate peptide or variant APLs in vitro. To first address whether the Cß FG loop deletion affects early phases of naive T cell activation, we assessed surface expression of the cell activation "markers," IL-2R
(CD25) and CD69, which represents a reliable method for determining the number of T cells that are activated in culture (41, 42). The lymph node T cells from N15
ß and N15
ß.ß
FG mice were stimulated in vitro for 18 h with varying molar concentrations of VSV8 or the APL variants, L4 and K1 (26), using irradiated EL-4 cells as H-2Kb-bearing APCs, and then were assayed for the expression of CD25 and CD69 by flow cytometry. As shown in Fig. 6
A, VSV8 peptide was capable of activating virtually almost the same percentages of T cells at the peptide concentration of 10-410-8 M in both N15
ß and N15
ß.ß
FG mice. However, a very subtle difference is observed in the activation responses of the N15
ß and N15
ß.ß
FG lymph node T cells after stimulation with a lower concentration of VSV8 (10-10 M) and more convincingly with the APLs L4 and K1, as assessed by CD69 (Fig. 6 A) and CD25 (data not shown) expression. At peptide concentrations of 10-510-7 M for L4 and 10-410-5 M for K1, the percentages of activated T cells from N15
ß mice are slightly higher than those from N15
ß.ß
FG mice; the doseresponse curve from N15
ß.ß
FG lymph node cells after treatment with L4 or K1 peptide is shifted to the right by a factor of
10. These findings indicate that in the mature lymphoid compartment, the early T cell activation by weak agonist ligation is modestly affected by the Cß FG loop deletion as measured by this assay.
We next examined and compared Ag-induced IFN-
production and proliferative responses of unprimed naive lymph node T cells from N15
ß and N15
ß.ß
FG mice. As shown in Fig. 6 B, when incubated for 48 h with VSV8 prepulsed EL-4 cells, both the N15
ß and N15
ß.ß
FG lymph node T cells secrete IFN-
into the culture supernatant at each peptide concentration tested (10-510-9 M). However, the values of IFN-
secreted from N15
ß lymph node T cells are higher than those from N15
ß.-ß
FG T cells at 10-510-8 M VSV8. In fact, compared with N15
ß T cells, the doseresponse curve from N15
ß.ß
FG lymph node T cells is shifted to the right by a factor of 1001,000. Furthermore, when incubated with EL-4 cells prepulsed with the weak agonist L4, the N15
ß T cells can produce readily detectable amounts of IFN-
at a peptide concentration of 10-4 M. In contrast, N15
ß.ß
FG lymph node T cells cannot induce IFN-
production after L4 stimulation at any peptide concentration tested (Fig. 6 B). Similarly, as shown in Fig. 6 C, significant differences are also observed in the IL-2dependent proliferative responses of the N15
ß and N15
ß.ß
FG lymph node T cells after stimulation with antigenic peptides as assessed by [3H]thymidine incorporation. When N15
ß and N15
ß.ß
FG lymph node T cells are cocultured with irradiated EL-4 cells prepulsed with VSV8 for 48 h, the amounts of incorporated [3H]thymidine in N15
ß.ß
FG lymph node T cells are clearly less than those in N15
ß T cells at peptide concentrations from 10-6 to 10-10 M (Fig. 6 C). In addition, compared with N15
ß T cells, N15
ß.ß
FG lymph node T cells show significantly decreased proliferative responses to L4 stimulation at peptide concentrations from 10-4 to 10-6 M, with the doseresponse curves from N15
ß.ß
FG lymph node T cells shifted to the right by a factor of 10100 (Fig. 6 C). Collectively, these results clearly demonstrate that responsiveness to antigenic peptides is impaired in the N15
ß.ß
FG T cells as determined by peptide antigen-specific induction of proliferation and cytokine production.
 |
Discussion
|
---|
In this study, we generated a TCR ß chain mutant (N15ß
FG) in which the entire Cß FG loop was deleted. The N15ß
FG construct or the normal N15ßwt cDNA was then introduced as a transgene into mice and differentiation/functional analysis of thymocytes conducted on the H-2b background. While N15ß
FG facilitated pre-TCR activity (Fig. 3, and Table I), our analysis reveals a distinct phenotype with respect to effects of the ß
FG mutation on further thymocyte maturation. N15ß
FG tg mice show a statistically significant increase in CD8+ SP thymocytes and lymph node T cells (Table I) in association with alteration of endogenous V
repertoires (Table II). These results imply that, in balance, enhanced positive selection is favored over negative selection. Given the potential confounding effects of repertoire alteration, we subsequently restricted V
expression to that of the N15
subunit on a RAG-2-/- background in a second set of animals. Thus, N15
ß TCR tg RAG-2-/- and N15ß
FG tg RAG-2-/- mice were crossed to generate N15
ß RAG-2-/- and N15
ß.- ß
FG RAG-2-/- littermates. N15
ß.ß
FG tg RAG-2-/- mice were striking with regard to an 810-fold increase in thymocyte cellularity as a consequence of an increase in DP thymocytes without alteration in cell numbers of DN or CD8+ SP compartments (Fig. 4). This increase was due to reduced negative selection with decreased hypodiploid cellular DNA content constitutively as well as following in vivo VSV8 cognate peptide injection in N15
ß.ß
FG tg RAG-2-/- compared with N15
ß tg RAG-2-/- H-2b mice (Fig. 5). Annexin V staining further confirmed the reduced apoptosis in N15
ß.ß
FG RAG-2-/- versus N15
ß RAG-2-/- mice.
At first glance, these data may appear in conflict with earlier studies by Degermann et al. (43), whose findings indicated that a TCR ß chain lacking the identical solvent-exposed Cß FG loop supported normal
ß T cell development in tg mice as well as normal function in cell transfectants. Their ß chain was derived from the 14.3.d TCR specific for HA110119 PR8 influenza hemagglutinin peptide complexed with I-Ed and comprised of Vß8.2 and Jß2.1 segments, unlike the N15ß
FG chain derived from the VSV8/Kb-specific N15 CD8+ SP CTL encoded by Vß5.2 Jß2.6 segments. In 14.3.d ß
FG tg mice, superantigen-induced proliferation, anti-H-2d CTL effector function, or helper T cell responses for anti-hapten antibody production were essentially normal. However, given that in the 14.3.d ß
FG tg mice, endogenous TCR
subunits pair with the introduced ß transgene product, the biologic system can readily overcome any deficiency mediated by the ß
FG deletion. Hence, polyclonal T cell responses are not a sensitive indicator of the functional consequences of the Cß FG loop deletion. Consistent with this notion, additional studies by the same authors in 14.3.d ß
FG mice show that
ß T cells coexpressing NK1.1 are severely depleted (44). This is particularly noteworthy, as
80% of NK1.1 T cells express the invariant (V
14-J
281) segment required for their development (45). Thus, even in the RAG-2+/+ background, a near single TCR specificity is generated with regard to NK1.1 T cells, permitting the effects of the Cß FG loop deletion to be recognized. Our results, in conjunction with those of Degermann, imply that the Cß FG loop is functionally significant for immune recognition-based signaling by both conventional
ß T cells and NK1.1 T cells of pMHC and CD1 specificities, respectively.
Prior mAb epitope mapping analysis showed that one of the two CD3
subunits within a TCR complex is in close proximity to the TCR Cß chain FG loop whereas the second CD3
subunit lies elsewhere. Crystallographic analysis of the N15 TCR
ß heterodimer reveals a striking asymmetry within the constant domain module such that virtually half of the Cß domain's ABED surface is exposed, yet uninvolved in C
domain contacts. This asymmetry creates a cavity beneath the ß chain with the Cß ABED surface as a ceiling and the plasma membrane as the floor. The two protruding C
loops (CD loop and EF loop) plus one C
(N185) and two Cß (N121, N186) glycans form one side-wall while the Cß FG loop and one Cß (N236) glycan form the second wall of this cave. In structural terms, the role of this extensive FG loop is to further rigidify the interface between Vß and Cß domains, thereby limiting the lateral movement of Vß. In addition, careful analysis of the Cß FG loop in several TCR structures (22, 28, 30, 46) reveals conservation of the three-dimensional structure of the loop, the adjacent Cß domain and the cave of both pMHCI- and pMHCII-restricted TCRs in human (29, 30) and mouse (22, 28). Such conservation implies an important role for this structural element on both CD4+ and CD8+ SP T cells. By forming a side wall of the cavity into which a CD3
subunit of one CD3 heterodimer can insert, the Cß FG loop presumably stabilizes the interaction between CD3
and the Ti
ß heterodimer. Not surprisingly, therefore, immunoprecipitation studies using the CD3
dimer-specific mAb 7D6 with N15
ß and N15
ß
FG transfectants reveals a markedly weakened association of CD3
with the TCR
ß heterodimer and CD3
in N15
ß
FG T cells (unpublished data). It is further noteworthy that the loop is negatively charged, perhaps influencing the electrostatics of the TCR surface in a manner similar to an N-linked glycan. Antedating the evolution of the FG loop in higher vertebrates, such a glycan localizes to the same region in primitive species (shark, chicken and axo; data not shown). Although the mouse has an adjacent N-linked glycan as well as the elongated FG loop in its TCR structure, both are not essential since the human TCR lacks an N-linked attachment site.
With these details in mind, we note that the TCR is a largely rigid structure: one component consists of the FG loop-"reinforced" Vß-Cß extracellular segment associated via an extensive (
2400 Å2) buried surface interface with the C
domain of the TCR
subunit, a second component. The Cß-C
interface contains multiple rigidifying salt bridges. Hence, the Vß-Cß-C
domains form a single conformationally rigid module. This Vß-Cß-C
module in turn is noncovalently associated with the CD3
and/or CD3
heterodimers such that the glycosylated CD3
and CD3
subunits extend away from the TCR heterodimer while the two nonglycosylated CD3
subunits are proximal. NMR structural studies show that CD3
and CD3
(and by extension CD3
) adopt C2-set IgSF folds, forming a stable heterodimeric complex by parallel strand pairing (G strand) and hydrophobic interaction (24). The CD3
-CD3
G strand pairing presumably creates a rod-like connector, permitting displacement of transmembrane helices of CD3
and CD3
upon TCR triggering, leading to intracellular kinase-dependent events and resulting in T lineage activation. Removal of the Cß FG loop, by eliminating one wall of the cave in the Cß domain would reduce one key component of the rigidifying architecture such that signaling might be impaired. Hence the altered negative selection in DP thymocytes of the N15
ß.ß
FG animals and the reduced capacity of their CD8+ SP lymph node T cells to proliferate to cognate antigens and APL are anticipated sequelae. That polyclonal thymocytes and
ß T cells in ß
FG tg mice (i.e. RAG-2+/+) can overcome this block, implies either that compensatory adaptations in the TCR
subunit or elsewhere readily occur or that additional protein interactions among TCR subunits (for example, within their transmembrane segments) maintain TCR-based signaling even in the absence of FG loop constraint. These possibilities are not mutually exclusive.
The prominent effect of the ß
FG transgene in N15
ß.ß
FG RAG-2-/- mice is the marked inefficiency of negative selection processes compared with that of N15
ß RAG-2-/- mice. Interestingly, the Cß FG loop deletion impairs negative selection without diminishing the capacity of DP thymocytes to differentiate to CD8 SP thymocytes; hence, the number of CD8+ SP thymocytes in N15
ß.ß
FG RAG-2-/- H-2b and N15
ß RAG-2-/- H-2b littermates is identical. In general, pMHC ligands of stronger affinity are negatively selecting while those with weaker affinity for a given TCR are positively selecting (for reviews, see references 47 and 48). Given that the Cß FG loop deletion will reduce rigidification and likely attenuate signaling from negatively selecting ligands, this result is not surprising. In contrast, positive selection must not be attenuated below a threshold required for initiating effective survival signaling. As other studies have demonstrated that positive and negative selection are under the control of different signaling pathways (for reviews, see references 38 and 49), this differential concept is entirely tenable. Nevertheless, in N15
ß.ß
FG RAG-2-/- mice, the numbers of CD8+ SP lymph node T cells are somewhat reduced compared with N15
ß RAG-2-/- mice on the same H-2 background. This observation in conjunction with increased HSA and PNA staining of CD8+ SP thymocytes in N15
ß.ß
FG RAG-2-/- mice (data not shown) suggests that positive selection may not be entirely unaffected by the Cß
FG mutation.
The pre-TCR consists of a TCRß chain disulfide-bonded to the thymus-restricted pT
subunit expressed on DN thymocytes in association with CD3
heterodimers and CD3
homodimers (34, 50). Although the single Ig-like ectodomain of pT
bears only 12% homology to TCR C
, key residues participating in the formation of the rigid interface between C
and Cß are preserved (22). This structural conservation suggests that the heterodimeric interface of pT
-Cß is similar to the interface of C
-Cß. From this conserved feature, we have inferred that a CD3
heterodimer slots into a pre-TCR cave comparable to that created within the TCR. That a CD3
knockout mutation disrupts pT
function (51) is consistent with this view. Furthermore, given that a CD3
knockout also disrupts pre-TCR function (52), it is tempting to speculate that CD3
is the cave-associated CD3
heterodimer. Limited glycosylation of CD3
relative to CD3
allows for ready modeled docking of the CD3
but not CD3
heterodimer at this cavity while still accommodating H57 mAb binding (unpublished results). Three additional facts also support the presumptive CD3
localization. First, it is known from chain association studies that CD3
pairs with TCR
whereas the CD3
heterodimer binds to TCRß (53). Second, these experiments show that CD3
and TCRß interact via their respective ectodomains whereas the associations of the CD3
,
, and
components with TCR
and ß are mediated via their transmembrane regions. Third, chemical cross-linking experiments in man and mouse imply proximity of TCRß and CD3
(18, 54). The above results notwithstanding, verification of these predictions still requires definitive structural elucidation.
The inability of the Cß FG loop deletion to influence the function of the pre-TCR is consistent with recent transgenic experiments in the pT
-/- background: pT
constructs lacking both the ectodomain and much of the cytoplasmic tail still support pre-TCR function (55). These data imply that pT
stabilizes the pre-TCR predominantly via transmembrane interactions, with CD3
heterodimer association not singularly dependent on the cave for pre-TCR function. Further support for this notion comes from three other observations: (a) mice transgenic for a TCRß chain lacking the Vß domain support DN to DP transition (56); (b) pT
and TCRß chains lacking their respective Ig-like domains still facilitate thymic differentiation in RAG-deficient mice (57); and (c) mutational analysis of the two tyrosines in the TCRß transmembrane region show that even conservative tyrosine to phenylalanine changes disrupt thymocyte survival, proliferation, and differentiation (58), further underscoring the importance of the transmembrane segment for pre-TCR function. Alterations within the TCR ectodomains are less well tolerated with regard to
ßTCR function, significantly impinging on TCR activity. For example, Cß FG loop deletion reduces the efficiency of negative selection in N15
ß.ß
FG RAG-2-/- mice. Mutations of the TCR
chain connecting peptide profoundly affect positive selection. The latter occurs through attenuation of ERK activation and a reduction of activated lck, Zap-70, phospho-CD3
, and phospho-LAT within lipid rafts (59). A similar phenotype with equivalent biochemical signaling aberrations is observed in CD3
-/- mice (60). If CD3
maps to the TCRß side and CD3
to the TCR
side of the
ß TCR complex, these findings can be rationalized.
In N15
ß.ß
FG RAG-2-/- H-2b mice, the level of N15 TCR as judged by both MR9.4 (anti-Vß5) or R53 (anti-Vß clonotype) is virtually identical to that found on T cells of N15
ß RAG-2-/- H-2b mice. However, in the former case,
50% of TCRs are wild-type and 50% incorporate the mutant ß
FG subunit. Yet mature CD8+ SP T cell function is significantly reduced in N15
ß.ß
FG RAG-2-/- mice. Thus, despite half of the TCRs being wild-type in structure, functional activation of these T cells vis-a-vis cytokine production or proliferation is dramatically reduced. In contrast, tetracycline regulatable TCR transgene expression shows that cells expressing as few as 1/20 of the normal levels of TCRs preserve their functional activity (61). Thus, paradoxically, the presence of TCRs with the ß
FG subunit is more detrimental to TCR-triggered activation than the complete absence of wild-type TCRs. Although the basis for this disparity remains to be elucidated, N15
ß.ß
FG TCRs may dysregulate wt TCRs by activating inhibitory pathways (phosphatases or others) in the absence of productive stimulation.
These data indicate an important functional role for the Cß FG loop in both thymic negative selection and mature T cell activation, T lineage activities requiring threshold stimulation to achieve a stringent set point. On the other hand, positive selection is only modestly altered. In view of evidence that positive selection requirements vis-a-vis pMHC interaction are less arduous with regard to affinity and avidity than those for negative selection (47, 48), this result is perhaps not surprising. Thus, attendant reduction in signaling strength through TCRs incorporating mutant ß
FG subunits may still be sufficient to achieve the more "relaxed" set point threshold for successful positive selection. Apparently, structural features of the
ß TCR have evolved to facilitate the "multi-tasking" requirements of TCR signaling function during development and mature T cell function. In sum, TCRß-linked transduction events are more critically involved in negative selection while it appears that TCR
-linked transduction processes are key for positive selection.
 |
Acknowledgments
|
---|
This work was supported by National Institutes of Health grants AI19807 to E.L. Reinherz, GM56008 to J.-H. Wang, and AI39098 to H.-C. Chang.
Submitted: January 23, 2002
Revised: March 29, 2002
Accepted: April 15, 2002
 |
Footnotes
|
---|
* Abbreviations used in this paper: aa, amino acid; APL, altered peptide ligand; BrdU, bromodeoxyuridine; DN, double negative; DP, double positive; PI, propidium iodide; pMHC, peptideMHC complex; RAG, recombinase activating gene; SP, single positive.
 |
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