Recognition mechanism of non-peptide antigens by human 
T cells
Seiji Yamashita1,
Yoshimasa Tanaka1,4,
Masashi Harazaki2,
Bunzo Mikami3 and
Nagahiro Minato1
1 Department of Immunology and Cell Biology, Graduate School of Biostudies and 2 Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan 3 Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Kyoto 611-0011, Japan 4 PRESTO, JST, Kyoto, Japan
Correspondence to: N. Minato; E-mail: minato{at}imm.med.kyoto-u.ac.jp
Transmitting editor: S. Koyasu
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Abstract
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The majority of 
T cells in adult human blood exhibit V
2/V
2-TCR and specifically respond to various kinds of non-peptide antigens. In this study, we comparatively analyzed the CDR3 repertoires of V
2-
and V
2-
chain genes in the adult and cord blood. It was confirmed that the vast majority of adult 
T cells exhibited V
2-
chains bearing a J
1.2 segment with no or short N-region and V
2-
chains with a conserved hydrophobic residue (leucine, valine or isoleucine) at position 97 encoded by N-region of V
/J
junction (
L97). The cord blood cells stimulated with pyrophosphomonoester antigen in vitro showed preferential expansion of the 
T cells expressing V
2- and V
2-TCR chains with these structural features as compared with those stimulated with a polyclonal mitogen phytohemagglutinin. TCR gene transfer studies indicated that alanine substitution of lysine at position 108 in J
1.2 (
K108) or
L97 abrogated the responsiveness of V
2/V
2-TCR to all kinds of the non-peptide antigens without affecting the response to anti-CD3 antibody. Furthermore, alanine substitution of arginine at position 51 in V
2 segment (
R51) adjacent to
K108 in the V
2/V
2-
TCR also abolished the antigen responsiveness. These results strongly suggested that a hydrophobic and two cationic residues (
L97,
K108 and
R51) clustered in a particular topology at the surface edge of the pocket structure of V
2/V
2-
TCR played essential roles in the recognition of non-peptide antigens.
Keywords: 
TCR, antigen recognition, antigenic selection, infection immunity, pyrophosphomonoester antigen
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Introduction
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T cells in adult human blood are activated and proliferate in response to non-peptide compounds such as isopentenyl pyrophosphate derived from pathogenic microbes including Mycobacterium tuberculosis (15) and isobutyl amine from various bacteria and certain plants (6). Recently, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, a precursor of isopentenyl pyrophosphate in microbial non-mevalonate metabolic pathway, was reported to be one of the most potent activators of human 
T cells (7,8). Substantial evidence has indicated that the activation of human 
T cells by these non-peptide compounds is mediated directly by the 
TCR composed of V
2 (also termed V
9) J
1.2 (also termed J
P)-
and V
2-
chains (913). Since the V
2/V
2-
T cells predominate in the 
T cell population of adult, but not in cord blood, epigenetic selection of the 
T cell subset after birth has been speculated (14).
While chemical features of the non-peptide compounds required for the effective activation of human 
T cells have been studied extensively (1517), how human 
T cells specifically recognize them remains largely unknown. Recently, crystallographic structure of a V
2/V
2 
TCR was resolved, and it was demonstrated that the TCR had a positively charged pocket structure formed by CDRs of TCR-
and -
chains (18). Although it was implied that the pocket structure might fit for the binding of pyrophosphomonoester antigens (18), there is no direct evidence for that at present. In the present study, we provide substantial evidence that the conserved three amino acid residues located at the surface edge of the pocket are essential for the recognition of all kinds of non-peptide antigens by TCR gene transfer analysis using mutated 
TCR genes. Based on these results, we propose a model in which different kinds of small molecular weight non-peptide antigens directly interact with a hydrophobic and two cationic residues located at the surface edge of the pocket structure in a fixed topology via hydrophobic and charge interactions, respectively.
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Methods
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Non-peptide antigens
Monoethyl pyrophosphate (EtPP) and 2-methy-3-butenyl-1-pyrophosphate (2M3B1-PP) were synthesized as described previously (1). Isobutyl amine (IBA) and pamidronate were purchased from Sigma-Aldrich Japan (Tokyo, Japan) and Novartis (Tokyo, Japan), respectively.
Cell cultures
Cord blood (three donors) and adult peripheral blood (four donors) were obtained from volunteers with informed consent. Cord blood mononuclear cells (CBMCs) and adult peripheral blood mononuclear cells (PBMCs) purified on Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) density centrifugation (600 g) were cultured in the modified Yssels medium (1) supplemented with 0.25% HSA at 12 x 106 cells/ml in the presence of either 1 µg/ml phytohemagglutinin (PHA)-L or 200 µM 2M3B1-PP. On day two, recombinant human IL-2 (100 U/ml) was added to the cell culture, and on day three, the medium was replaced by the modified Yssels medium supplemented with 10% pooled human AB serum and IL-2. The cells were cultured for 2 weeks. Jurkat transfectants (2 x 105 cells/well) were cultured in the modified Yssels medium supplemented with 0.25% HSA in the presence of varying concentrations of EtPP, IBA or EJ-1 tumor cells (2 x 105 cells/well) pulsed with varying concentrations of pamidronate for 24 h, and the supernatants were examined for IL-2 using the CTLL-2 assay (19).
Flow cytometric analysis
Cells were two-color stained with phycoerythrin-conjugated anti-CD3 mAb (Becton Dickinson Japan, Tokyo, Japan) and FITC-conjugated anti-TCR V
2 mAb (Immunotech, Marseille, France) and analyzed using a FACScan (Becton Dickinson Japan).
Sequence analysis
Fresh and cultured blood mononuclear cells were suspended in Isogen (Wako Pure Chemical Industries Ltd, Osaka, Japan) and total RNAs were extracted. cDNAs were synthesized using SuperScriptTM First-Strand Synthesis System (Invitrogen, San Diego, CA, USA), and CDR3 regions of V
2 TCR-
and V
2 TCR-
chain genes were amplified by RTPCR using the following primers. V
2 TCR-
chain: ggg-ctc-gag-gac-acc-gct-tta-caa-cga (XhoI 5') and ggg-tct-aga-gtg-agg-ttc-tct-gtg-t (XbaI 3'). V
2 TCR-
chain: ggg-ctc-gag-aac-act-tgt-gtg-ttg-gtt-ca (XhoI 5') and ggg-gga-tcc-agt-gta-tca-ctt-gta-gga-g (BamHI 3') or ccc-ctc-gag-aat-gac-ttt-cat-ata-ccc-ag (XhoI 5') and agt-gga-tcc-ctt-gga-tga-cac-gag-att-ta (BamHI 3'). The resulting products were digested with the indicated restriction enzymes, ligated into the relevant sites of pBluescriptKS and sequenced on an ABI Prism DNA sequencer (PE Applied Biosystems, Tokyo, Japan) using the following primers.
CDR3: act-ctc-acc-att-cac-aat-gta-gag-aaa-cag.
CDR3: ata-tca-act-ggt-aca-gga-aga-ccc-aag-gta-aca-caa-tga-c or ccc-ctc-gag-aat-gac-ttt-cat-ata-ccg-ag.
Gene transfer analysis
cDNAs of full-length TCR-
and TCR-
chains were inserted into a pEF-BOS expression vector, kindly provided by Dr S. Nagata, Osaka University, Suita, Osaka, Japan, using a BstXI adaptor (Invitrogen, San Diego, CA) to yield pEF-BOS-
and pEF-BOS-
. TCR-ß chain-deficient Jurkat cells, J.RT3-T3.5, were transfected with the pEF-BOS-
and pEF-BOS-
digested with ApaLI and ScaI, respectively, together with XhoI-digested pST-NeoB by electroporation and selected in the presence of 1 mg/ml of geneticin (Nacalaitesque, Osaka, Japan). The transfectants were cloned by limiting dilution, and CD3+ clones that produced IL-2 in response to anti-CD3 mAb and PMA (10 ng/ml) were selected.
Mutagenesis
TCR-
and -
chain cDNAs mutated by a single amino acid residue were generated using QuikChangeR Multi Site Directed Mutagenesis Kit (Stratagene, Cedar Creek, TX).
Statistic analysis
Statistic analysis was done by Students t-test. For the analysis of restricted diversity in CDR3 regions, the following quantity was introduced. Distance = (Smax S)/Smax. Smax is defined as lnN, and S as
PlnP, where N represents the total sample count (at least 50 in the present study) and P the probability of the occurrence of certain samples. The distance value ranges from 0 to 1, in which value 0 indicates the complete diversification and the degree of restriction becomes higher as the value approaches 1.
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Results
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Stimulation of cord blood with non-peptide antigens induces preferential expansion of 
T cells bearing V
2/V
2-
TCR with features common to those of adult 
T cells
We first compared the repertoire of V
2/V
2-
TCR of adult 
T cells before and after stimulation with non-peptide antigens, 2M3B1-PP and IBA, by random cDNA cloning and sequencing of the CDR3 regions. Since the proportions of 
T cells varied significantly among healthy adults, PBMC from two volunteers with low 
T cell proportions (1.6 and 1.7% in total CD3+ cells) and two with very high proportions (25.0 and 33.0%) were examined. Consistent with previous reports (10,20), the vast majority of 
TCRs in fresh PBMC from both groups exhibited preferential usage of J
1.2 with N-region of less than two amino acid residues in the TCR-
and a highly conserved hydrophobic residue with a short aliphatic side chain (L, V or I) at
97 position in the V
2/J
junctional region (Fig. 1B and C). Frequency of J
1 usage was also high (Fig. 1D). Stimulation with the non-peptide antigens for two weeks resulted in the marked expansion of V
2/V
2-
T cells (Fig. 1A). After the expansion, above features as well as the degree of diversity restriction in both TCR chains remained unchanged (Fig. 1B and DF), making the preferential outgrowth of any particular 
T cell clones unlikely.
Similar analysis was performed using CBMC from three donors. Because the proportions of V
2/V
2-
T cells in fresh CBMC were too small (<0.1% of total T cells) to obtain sufficient numbers of the cDNA clones, we compared the CBMC cultured in the presence of 2M3B1-PP with those of polyclonal mitogen PHA-L. 2M3B1-PP induced a marked increase in the proportions of V
2/V
2-
T cells as compared with PHA (Fig. 1A), which reflected their preferential expansion (data not shown). As shown in Fig. 1(B), TCR-
in the CBMC stimulated with 2M3B1-PP exhibited a significantly higher frequency of J
1.2 usage (mean
70%) than those stimulated with PHA-L (
20%), in which J
1.3 usage predominated (
70%). The mean frequency of TCR-
cDNA clones with L, V or I at
97 also tended to be higher in the former albeit less significant statistically (Fig. 3C), while J
1 frequencies were comparable between the two populations (Fig. 3D). Overall diversity restriction of both TCR-
and -
in the 2M3B1-PP-stimulated CBMC was significantly higher than in those stimulated with PHA, being comparable to that in adult 
T cells (Fig. 3E and F). These results implied that, unlike in adult PBMC, there was a significant antigen-driven selection of 
T cell clones in the CBMC.
Requirement of a hydrophobic residue with short aliphatic chain at
97 position in the V
2/J
junctional region for non-peptide antigen recognition
In order to investigate the role of conserved residue (L, V or I) at the
97 position encoded by the junctional N-region in antigen recognition, we selected three naturally occurring full-length TCR-
chain gene cDNAs. They were Cl.
263 with leucine at
97 in the context of short N-region (four residues) and Cl.
016 with leucine at
97 as a representative of long N-region (12 residues) isolated from the 2M3B1-PP-stimulated CBMC, and Cl.
255 with proline at
97 and long N-region (15 residues) from PHA-stimulated CBMC. We co-transfected each of them together with an identical TCR-
chain cDNA (Cl.
001) most frequently detected in the adult PBMC into J.RT3-T3.5 Jurkat cells, and obtained stable CD3+ clones that produced an IL-2 in response to anti-CD3 antibody stimulation. As shown in Fig. 2, both
001/
263 and
001/
016 clones produced IL-2 in response to both EtPP and IBA, while
001/
255 clone did not respond to either antigen at all at any concentration.
To confirm the role of
L97 more directly, we then generated
L97A and
L97S mutants using a TCR-
cDNA (Cl.
093) most frequently occurring in adult PBMC. As shown in Fig. 3, both
001/
093(L97A) and
001/
093(L97S) clones responded to anti-CD3 antibody at comparable levels to the
001/
093 clone. However, neither of them produced a detectable IL-2 in response to EtPP or IBA at any concentrations, while
001/
093 clone responded to both antigens. We also examined the response to the third group of non-peptide antigens, pamidronate (17). Since activation of 
T cells by pamidronate required cellular presentation (19,21), EJ-1 tumor cells pulsed with pamidronate were used. Again,
001/
093(
L97A) and
001/
093(
L97S) clones did not respond to pamidronate at all, whereas the
001/
093 clone did (Fig. 3). The results confirmed that hydrophobic residues at
97 position of TCR-
chains were essential for the recognition of all the three known types of non-peptide antigen irrespective of the length of N-region.
Critical involvement of two adjacent cationic residues,
K108 and
R51, at the surface edge of TCR pocket region in the recognition of non-peptide antigens
V
2J
1.2/V
2-
TCR has a positively charged pocket structure (18). We previously reported that the importance of
K108 and
K109 encoded by J
1.2 gene in antigen recognition by glutamate substitution (9). Based on the crystallographic structure, there is another cationic residue
R51 encoded by V
2 gene adjacent to
K108 (18). In order to verify the contribution of these cationic residues at a pocket structure, we introduced alanine mutation to them using Cl.
001 and Cl.
093 cDNA clones. Combinations of
001 (K108A)/
093,
001 (K109A)/
093, and
001/
093 (R51A) cDNAs were transfected into J.RT3-T3.5 Jurkat cells, and CD3+ clones that responded to anti-CD3 antibody were established. As shown in Fig. 4,
001 (K108A)/
093 responded to none of the non-peptide antigens at any concentrations. Unexpectedly, however,
001 (K109A)/
093 clone significantly responded to EtPP albeit to a lesser extent than the wild type control, and responded to IBA and pamidronate comparably to the control clone (Figs 3 and 4 for the control), indicating that
K109 was dispensable for the antigen recognition. As also shown in Fig. 4, mutation of arginine at
51 to either alanine (R51A) or glutamic acid (R51E) completely abrogated the response to all the antigens without affecting the response to anti-CD3 antibody. These results indicated that the two cationic residues at positions
108 and
51 at the surface edge of a pocket structure were essential for non-peptide antigen recognition.
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Discussion
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It was shown previously that 
T cells in CBMCs expressed 
TCRs composed of fairly diversified 
TCR, while the majority of 
T cells in adult PBMCs expressed highly restricted repertoire mostly utilizing V
2/V
2-
TCR (4,2225). This led to the postulation that the repertoire of adult 
T cells might be selected by environmental antigens after birth (20). V
2/V
2-
TCR of the vast majority of 
T cells in adult blood exhibit shared features in their CDR3 regions; preferential usage of J
1.2 segment with short N-region (mostly less than two residues) and J
1 segment, and a conserved hydrophobic residue (L, V or I) at the VJ junctional N-region (
97) of TCR-
. Present results indicated that the stimulation of CBMCs with a potent pyrophosphomonoester antigen, 2M3B1-PP, induced a preferential expansion of V
2/V
2-
T cells bearing these features as compared with PHA. The usage of J
1 was similarly high in both CBMC populations. Our unpublished results indicated that V
2/J
2- and V
2/J
3-chimeric TCR-
cDNA clones showed much poorer efficiency than V
2/J
1-TCR-
cDNA to be expressed functionally with a V
2/J
1.2-TCR-
cDNA in Jurkat cells (S. Yamashita et al., unpublished data). We thus tend to speculate that the high J
1 frequency in V
2-TCR-
may reflect the better paring efficiency with V
2-TCR-
in the cells, although unexpected bias of PHA stimulation could not be excluded. Actually, the stimulation with 2M3B1-PP resulted in the significant increase in overall restricted diversity in both
- and
-chains to the extent comparable with that in adult V
2/V
2- 
T cells as compared with that with PHA, indicating the antigen-driven selection of the repertoire in vitro. This is consistent with the previous in vivo observation, in which significant expansion of this particular T cell subset takes place within the first few years of life, presumably in response to repetitive microbial infections (26). Also, the majority of V
2/V
2-
T cells in adult PBMC was reported to show memory phenotype (27). These results may suggest a good possibility that the characteristic repertoire of the V
2/V
2-
T cell subset in adult blood may be formed epigenetically in part via selection by various non-peptide antigens derived from environmental microbes. Although the possibility remains to be verified, such a developmental repertoire selection may enable the 
T cells to respond to microbial infection rapidly in a manner resembling innate immunity.
Reported crystallographic structure indicates that two lysine residues (
K108 and
K109) unique for J
1.2 segment and a junctional leucine residue (
L97) of
-chain are located in close proximity at a positively charged pocket structure (18). Present results revealed that alanine substitution of
K108 of
-chain,
L97 or
R51 of
-chain in combination with native partner TCR chains completely abrogated the responsiveness to all the three different types of non-peptide antigens, with anti-CD3 antibody-response being retained. The same results were obtained by serine and glutamic acid substitution of
L97 and
R51, respectively. On the other hand, while we previously reported that mutation of
K109 to oppositely charged glutamic acid abolished the antigen responsiveness (9),
K109A only partially affected the response to EtPP with essentially no effect on that to IBA and pamidronate. We presume that
K109E, but not
K109A, mutation caused significant secondary topological changes in the adjacent
K108A via opposite charge effect. Since the relative topology of
K108 and
R51 may be critical for the interaction with pyrophosphate moiety of EtPP, possibly more so than for IBA or pamidronate (see below), we speculate that the partial effect of
K109E mutation on EtPP responsiveness may reflect the secondary effect on the topology of
K108. In any case, these results strongly suggested the critical involvement of these three residues in the recognition of non-peptide antigens irrespective of their structural differences.
All the three critical residues,
K108,
R51,
L97, are clustered at the surface edge of a positively charged pocket, while
K109 is located rather inside the pocket (Fig. 5, left). On the other hand, structural requirements for the antigenicity of pyrophosphomonoesters have been well documented in which the existence of a short aliphatic alkyl, alkenyl or hydroxyalkenyl chain is essential for activation of 
T cells (15). Based on this, we propose a model in which pyrophosphomonoester antigens bind to the TCR through the interactions of negatively charged pyrophosphate groups of the antigens with two adjacent cationic residues (
K108 and
R51) and hydrocarbon chains of the antigens with short aliphatic side chain of
L (V, I) 97 via hydrophobic interaction (Fig. 5, center). Alkyl amine antigens including IBA may bind to the TCR essentially in the same way, in which a positively charged amino group interacts with
R51 via bridging effect by an inorganic phosphate in the milieu and an aliphatic group of the antigens with
L (V, I) 97 (Fig. 5, right). The binding mode of nitrogen-containing bisphosphonate antigens such as pamidronate may be more complicated because of the requirement of cellular presentation for effective 
T cell activation (19,21,28), but charge interaction with the cationic residues and hydrophobic interaction with
L (V, I) 97 should be involved similarly. This model predicts that non-peptide antigens interact with the particular V
2J
1.2/V
2-
TCR at the very edge of the pocket structure rather than by being inserted inside the pocket.
In mice, it was suggested that the mode of antigen recognition of 
T cells was more similar to that of Ig than
ß T cells (29). For instance, an I-Ek-reactive 
T cell clone recognized an epitope of I-Ek molecule per se independently of the peptides in complex with I-Ek, and another 
T cell clone was reported to recognize herpes simplex virus type I glycoprotein directly without processing or presentation by MHC molecules (30). Specific recognition of microbial small non-peptide antigens by a subset of 
T cells is observed so far only in human and primates. Thus, unique recognition repertoires of 
T cells in different species may reflect distinct evolutional events in part related to the characteristic microbial infections.
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Acknowledgement
|
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This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, Sports, and Technology, Japanese Government.
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Abbreviations
|
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2M3B1-PP2-methy-3-butenyl-1-pyrophosphate
CBMCcord blood mononuclear cell
EtPPmonoethyl pyrophosphate
IBAisobutyl amine
PBMCperipheral blood mononuclear cell
PHAphytohemagglutinin
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