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The Journal of Immunology, 2005, 175: 6344-6351.
Copyright © 2005 by The American Association of Immunologists

CD1a-, b-, and c-Restricted TCRs Recognize Both Self and Foreign Antigens1

Michael S. Vincent2, Xiaowei Xiong, Ethan P. Grant, Wei Peng and Michael B. Brenner2

Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham & Women’s Hospital and Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Individual CD1-restricted T cells can recognize either endogenous or foreign lipid Ags, but the extent to which the same CD1-restricted TCR can react to both self and microbial lipids is unknown. In this study, we have identified CD1a-, CD1b-, and CD1c-restricted T cells from normal human donors that induce cytolysis and secrete copious IFN-{gamma} in response to self-CD1 expressed on monocyte-derived dendritic cells. Remarkably, microbial Ags presented by CD1 are even more potent agonists for these same T cells. The {alpha}{beta} T cell receptors from such clones are diverse and confer specificity for both self-CD1 and foreign lipid Ags. The dual reactivity of these CD1-restricted cells suggests that the capacity for rapid responses to inflammatory stimuli without memory coexists with the capacity for strong Ag-specific responses and the generation of memory in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The T cell repertoire generated in the thymus, once thought to be purged of T cells capable of responding to self-peptide Ags, is now recognized to comprise T cells for which the self-reactivity is of physiologic importance. The spectrum of peptide-MHC self-reactivity ranges from responses detectable only at the biochemical level in the form of partial {zeta}-chain phosphorylation (1) to overt production of inhibitory cytokines by self-reactive regulatory T cells (2). Experiments using adoptive transfer of transgenic T cells have demonstrated the specificity of weak peripheral T cell reactivity for self-MHC-peptide complexes inapparent in standard cellular assays. Such specificity is manifested in the distinct MHC backgrounds required for the maintenance of both naive and fully functional memory T cells (3, 4, 5). Further evidence for this specificity arises from the analysis of homeostatic expansion by transgenic or polyclonal T cells in lymphopenic environments with limited MHC-peptide diversity (6, 7).

T cells self-reactive to the CD1 family of Ag-presenting molecules also display Ag and CD1 isoform specificity, but unlike most weak TCR interactions with peptide-MHC, many TCR interactions with self-CD1 are strong enough to be demonstrable in standard cellular assays or in combination with cytokines (8, 9, 10, 11, 12). CD1a-, CD1b-, and CD1c-restricted T cells recognizing mycobacterial lipid Ags have also been clearly delineated (13, 14, 15, 16, 17, 18, 19, 20, 21, 22). CD1d-reactive cells have been implicated in tumor surveillance (23, 24, 25), early host defense (12, 26, 27, 28), contact hypersensitivity (29), experimental forms of tolerance (30), and the regulation of autoimmune disease (31, 32, 33). In contrast, the most compelling evidence available for CD1a, CD1b, and CD1c function points to a role in Ag-specific anti-mycobacterial host defense (12, 34, 35).

CD1d-restricted T cells bearing activation markers and invariant TCR{alpha} chains are thought to represent partially activated, self-reactive T cells (12); however, recently several studies have found microbial foreign Ags that stimulate these cells, including {alpha}-sphingolipids, lipophosphoglycans, and phosphatidyl inositol mannosides (36, 37, 38, 39, 40).

Foreign lipid Ag-reactive CD1a-, CD1b-, and CD1c-restricted T cells seem much like foreign peptide-specific MHC class I- and class II-restricted T cells, except for the recognition of lipid-containing Ags rather than peptides. It has been proposed that this subset participates in the adaptive immune response (14). Both the CD1d-reactive and the CD1a-, CD1b-, CD1c-reactive T cells that are self-reactive may participate early in the immune response in the absence of foreign Ag recognition, producing cytokines (26), instructing dendritic cell (DC)3 maturation (11, 41), and activating other cells such as NK cells (42) and macrophages (27) that participate in host defense. Here we considered the possibility that CD1-reactive T cells with diverse TCRs might recognize both self and foreign Ags. We show that CD1a-, CD1b-, and CD1c-reactive T cells are overtly self-reactive, but unexpectedly, these same T cells react even more strongly to specific foreign Ags. Unlike other cells that participate in innate immunity, this latter class of CD1-restricted cells retains the ability to specifically respond to foreign Ags by virtue of their somatically rearranged Ag receptors. We suggest that this dual reactivity may impart the capacity for CD1a, b, and c reactive T cells to participate in both early and subsequent adaptive immunological reactions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Monocyte-derived DCs and derivation of T cell clones

The use of human mononuclear cells described in these studies was reviewed and approved by the Brigham and Women’s Hospital institutional review board. Monocyte-derived DCs were prepared essentially as described (10), beginning with leukapheresis-enriched mononuclear cells cultured in GM-CSF and IL-4 at 300 and 200 U/ml, respectively. Cells were harvested after 72–96 h unless otherwise noted and were irradiated with 5000 rad using a 137Cs source. Nonadherent mononuclear cells were washed repeatedly and used for subsequent studies. Complete medium consisted of RPMI 1640 supplemented with 10% FCS, HEPES, L-glutamine, essential and nonessential amino acids, sodium pyruvate, 2-ME, and penicillin/streptomycin. Depletion of CD4 cells was performed using a VarioMacs apparatus according to the manufacturer’s specifications. Cell lines were established in 24-well plates with 106 CD4-depleted cells and 106 irradiated monocyte-derived DCs and 1–5 µg/ml microbial detergent extracts from Mycobacterium tuberculosis, Escherichia coli, or Yersinia enterocolitica generated by Triton X-114 phase separation. The preparation from the H37Rv strain of M. tuberculosis (43) was generously supplied by Dr. J. T. Belisle (Colorado State University, Fort Collins, CO; National Institutes of Health, National Institute of Allergy and Infectious Diseases, Contract NO1 AI-75320). For E. coli (strain B/r; American Type Culture Collection) and Y. enterocolitica (serotype O:3; a clinical isolate provided by E. Marker-Hermann, Dr.-Horst-Schmidt-Kliniken, Wiesbaden, Germany), bacteria were grown to log phase in Luria-Bertani medium. Bacterial pellets adjusted to 10 mg/ml were sonicated with a Branson probe sonicator for 5 min at an output of 30%. Triton X-114 phase separation was performed essentially as described (44), beginning with a 1 mg/ml sonicate. The detergent phase was extracted three times, and the hydrophobic material was precipitated with 10 volumes of acetone before addition to the T cell cultures.

A second stimulation with autologous monocyte-derived DCs and Ag was performed at 10–14 days at the same cell ratios, and IL-2 (Chiron) was added to 0.3 nM. The IL-2 concentration was gradually raised to 1 nM before a third stimulation 10–14 days later and was maintained at that level. The third stimulation occurred under the same conditions. The fourth stimulation was again performed in 10–14 days, but allogeneic monocyte-derived DCs were used in place of autologous monocyte-derived DCs. Limiting dilution cloning was performed when cells reached a resting state after the second and fourth stimulations. Cells were plated at 3, 1, and 0.3 cells per well with 2 nM IL-2, PHA-P (Difco) at 1/4000, 5 x 104 irradiated (7500 rad) EBV feeders, and 1 x 105 irradiated (5000 rad) nonadherent PBMC feeders. Clones were expanded in 1–2 nM IL-2 initially and then according to the method of Riddell et al. (45). The KHO.8 clone included in our panel was derived similarly from a line that was CD1a-restricted and self-reactive after several stimulations of CD4-depleted T cells with immature DCs and an organic extract of M. tuberculosis.

mAbs and cell lines

The C1R (13) and HeLa (46) CD1 transfectants have been described previously. J.RT3-T.3 and HT-2 cells were from the American Type Culture Collection. For CD1a, CD1b, and CD1c detection and blocking, the mouse IgG1 Abs 10D12.2 (47), BCD1b3.1 (48), and F10/21A3.1 (S. A. Porcelli, unpublished results), respectively, were used. The nonbinding mouse IgG1 Ab P3 served as an isotype control in some experiments. The OKT4 and OKT8 hybridomas were from the American Type Culture Collection. The anti-TCR{alpha}{beta} Ab BMA031 was from Endogen, and the CD8{alpha}{beta}-specific 2ST8-5H7 Ab was from BD Biosciences.

T cell assays

Monocyte-derived DCs at 5 x 104 cells per well in 96 flat-well plates were preincubated with Abs at least 15 min before addition of 5 x 104 T cell clones in the absence of IL-2. The total volume was 200 µl. For proliferation assays, [3H]TdR 1µCi (6.7 Ci/mMol or 2.0 Ci/mMol; New England Nuclear) was added for the final 6 or 18 h, respectively, of a 72 h incubation. Cells were harvested on a Tomtec 96-well plate harvester and counted in a Betaplate scintillation counter (both from Wallac). For cytotoxicity assays, targets in 200 µl were incubated with 200 µl of 51Cr at 37°C for 2 h. After extensive washing, 2000 target cells were placed in 96-V wells before addition of T cells with or without Abs in a total volume of 150 µl. Maximal release was determined by addition of 1% IGEPAL CA-630 (Sigma-Aldrich). After a 4-h incubation at 37°C, 25 µl of the supernatants was spotted onto a filter mat, dried, and counted in a Betaplate scintillation counter. Specific release was calculated as follows: (experimental cpm – spontaneous cpm)/(total cpm – spontaneous cpm) x 100%. To measure cytokine production, T cell clones at a density of 106/ml in 48-well plates were incubated alone, with PMA (10 nM) and ionomycin (1 µg/ml) (both from Sigma-Aldrich), or with monocyte-derived DCs with or without the indicated Abs used at 20 µg/ml. Supernatants from triplicate wells were harvested after centrifugation and frozen in several aliquots at –20°C. Cytokines were measured by ELISA using matched Ab pairs (Endogen) according to the manufacturer’s instructions.

TCR cloning, transfection, and transfectant assays

RNA was extracted from 2 x 106 T cell clones as described (49). Inverse PCR was performed as described (50, 51). Briefly, double-stranded cDNA was generated by standard methods, and T4 DNA polymerase was used to create blunt ends before circularization with T4 DNA ligase. PCR-amplified products generated using the primer pair described (51) were gel purified and cloned directly into pCR2.1 using TOPO TA cloning (Invitrogen Life Technologies) and were sequenced on an automated system (Applied Biosystems 373 A). Full-length cDNA for the TCR{beta} chain was amplified from whole cDNA using Pfu polymerase (New England Biolabs) and the previously described primers and PCR protocol (51). The TCR{alpha}-chains for Mt1.50 and Mt2.21were similarly amplified using the forward primers for AV3S1 (5'-gggggtaccatttcaggtcttctgtga-3') and AV29S1 (5'-gggctcgagaagctgactggatattctgg-3'). The PCR products were incubated with Taq polymerase (Invitrogen Life Technologies) for 8 min at 72°C and subcloned directly into pCR2.1 using TOPO TA cloning. The purified plasmids were then digested with the appropriate endonucleases (New England Biolabs), and the gel-purified inserts were directionally cloned into pREP7 or pREP9 (Invitrogen Life Technologies) for the TCR{alpha} or TCR{beta} chains, respectively.

J.RT3-T.3 cells were transfected with the above cloned TCR pairs exactly as described (51), based on the method of Brawley and Concannon (52). Briefly, 12 x 107 cells and 20 µg of each expression plasmid in 0.3 ml of complete medium were incubated in a 0.4-cm gap eletroporation cuvette (Bio-Rad) for 10 min at 23°C, electroporated (250 V, 960 µF) with a Bio-Rad gene pulser, and incubated an additional 10 min at 23°C. The transfectants were cultured for 48 h at 37°C in complete medium and then transferred to complete medium containing 1 mg/ml G418 (Invitrogen Life Technologies) and 0.5 mg/ml hygromycin (Calbiochem). After 2–3 wk, TCR expression was confirmed by flow cytometry and transfectants were used for functional assays. For assays with monocyte-derived DCs, 1 x 105 transfectants were incubated with 5 x 104 monocyte-derived DCs in 200 µl of complete medium with and without the indicated Abs or Ags. PMA (10 nM) was included in all assays unless otherwise noted. Supernatants were removed after a 24-h incubation at 37°C and 25-µl aliquots were added to HT-2 cell cultures. IL-2 production, as measured by HT-2 proliferation, was assayed by adding 5000 HT-2 cells grown in 0.25 nM of IL-2 to 96 flat-well plates in 100 µl of complete medium. Supernatants were then added to a total volume of 125 µl and incubated for 18–24 h, before adding 1 µCi [3H]TdR (6.7 Ci/mMol) for the final 6 h of the culture. Cells were harvested and [3H]TdR incorporation was measured as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD1 self-reactive clones have diverse {alpha}{beta} TCRs

To obtain CD1-restricted T cell clones, we performed limiting dilution cloning from CD4-depleted T cells stimulated several times with microbial Ag preparations and CD1-expressing monocyte-derived DCs. In each initial culture, one of three preparations of hydrophobic microbial extracts derived by detergent phase separation (53) of microbial sonicates was included in stimulations, as indicated by the initials used in the designation of the resultant clones (Mt for M. tuberculosis, Ec for E. coli, and Ye for Y. enterocolitica (the previously designated clone KHO.8 was similarly derived by stimulation with an organic solvent extract of M. tuberculosis). Cloning efficiency ranged between 5 and 30% and was performed at densities between 0.3 and 3 cells per well. Screening large numbers of clones for CD1 restriction was accomplished by assessing Ab blocking of proliferation in response to DCs and the corresponding microbial detergent extract (in the remainder of the paper we will use the term "detergent extract" to refer to hydrophobic extracts derived by detergent phase separation). Systematic cloning followed by screening ~500 clones for proliferation inhibited by CD1 Abs yielded 15 T cell clones with reactivity to one of the group 1 CD1 isoforms in the absence of foreign Ags (Table I). We observed augmentation of CD1-restricted self-reactivity by the microbial detergent extracts initially used to stimulate the lines in most cases. No CD1-restricted clones had an absolute requirement for microbial stimulation. All the clones examined expressed the {alpha}{beta} TCR and either the CD8{alpha}{beta} or CD8{alpha}{alpha} accessory molecules. The expression of the CD8{alpha}{alpha} homodimer was a stable characteristic of those clones lacking the CD8{alpha}{beta} molecule, rather than simply an activation marker transiently expressed on in vitro stimulated cells (data not shown).


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  		Table I. Characteristics of the panel of self-reactive T cell clonesa

 
The TCR CDR regions of several clones were sequenced after inverse PCR amplification of cDNA derived from the clones (50, 51). This analysis revealed only one duplicate clone (Ec2.1) that was from the Ec-line and was identical in both TCR{alpha} and {beta} sequence to Ec2.55; otherwise, each clone was unique. The sequences indicate diverse TCR CDR3 regions, as well as heterogeneous V region gene usage (data not shown). Two common TCR{beta} V regions were used: BV7S1 for one CD1b- and two CD1c-restricted clones and BV9S1 for two CD1a-restricted clones. In contrast, no similarity was seen in the TCR{alpha} V regions described here and only one shared TCR{alpha} V gene is used, combining the CD1-restricted TCRs described here and a previous report (51). The current data suggest that the group 1 CD1 (CD1a, CD1b, and CD1c) self-reactive T cell subset uses diverse TCRs, although the possibility that some germline V genes are used preferentially cannot be excluded.

CD1-specific lysis of transformed cells and immature DCs

CD1 self-reactive T cell clones were potently cytolytic in 51Cr release assays of CD1-transfected target cells. Typical clones caused from 35 to 85% lysis of HeLa-CD1 transfectants (Fig. 1, A–D), contingent on expression of the cognate CD1 isoform and independent of exogenous Ag. Clones in the panel similarly lysed C1R-transfected cells with the same CD1 specificity (data not shown). The expression of group 1 CD1 molecules in vivo is prevalent on mature and immature DCs, with the particular isoforms expressed dependent on the tissue localization and degree of differentiation (54). We next confirmed that the cytolytic activity that the clones possessed toward CD1-transfected cell lines extended to nontransformed cells expressing CD1, such as monocyte-derived DCs. Indeed, the T cell clones lysed DCs, and CD1-specific Abs inhibited cytolytic activity by 60–75% (Fig. 1E).



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  		FIGURE 1. Foreign-Ag-independent CD1-restricted cytolysis. Standard 4-h 51Cr-release assays using HeLa-CD1 transfectants were performed as described in Materials and Methods and percent-specific lysis is shown for the indicated T cell clones with respect to the indicated CD1 transfectant. Error bars represent the means of triplicate determinations (A–D). Standard 4-h 51Cr-release assays using adherent monocytes grown for 1 day in GM-CSF and IL-4 as targets were performed as described in Materials and Methods and percent-specific lysis is shown for the indicated T cell clones in the presence and absence of the indicated mAb. Error bars represent the means of triplicate determinations (E).

 
Th1 and Th2 cytokine production without IL-4

We first used mitogenic stimulation to assess the capacity of CD1 self-reactive T cell clones to produce cytokines (Table II). IFN-{gamma} production was most pronounced for each clone, but significant quantities of the Th2 cytokine IL-13 were also produced. Unlike previously well-characterized human CD1d self-reactive T cells, the six clones tested did not produce detectable IL-4. Importantly, production of a range of cytokines occurred upon incubation of clones with monocyte-derived DCs, which was specifically inhibited by the relevant CD1 Abs (Fig. 2). The absence of IL-4 production in our panel was initially surprising in light of the considerable IL-13 and IL-5 secretion. However, several groups have described this discordance of Th2 cytokine production by CD8 T cells, which has been attributed to distinct activation pathways evident in the sensitivity of IL-4 production, but not IL-5 or IL-13 production, to cyclosporin (55, 56). More recent work has defined a DC population derived by stimulation with thymic stromal lymphopoietin and CD154 capable of polarizing naive CD8 cells toward a very similar IL-5, IL-13, and high IFN-{gamma} profile (57).


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  		Table II. Cytokine production by self-reactive T cell clones with mitogenic stimulationa

 


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  		FIGURE 2. Self-reactive CD1-restricted T cell clones produce diverse cytokines. Production of IFN-{gamma} (A), GM-CSF (B), IL-5 (C), and IL-13 (D) by clone Ec2.55 and clone Ye2.3 in the presence of a panel of CD1 blocking Abs. All values were determined by ELISA from triplicate cultures and are expressed in nanograms per milliliter. Error bars represent the means of triplicate determinations.

 
Self-reactive T cells also recognize foreign Ag

Because the T cells cloned were initially stimulated with microbial extracts, we also examined the response to these foreign Ag preparations. The response to microbial Ags far exceeded the self-reactive response seen in assays of T cell proliferation. Clone Mt2.21 exemplified this reactivity, demonstrating a stimulation index of 42 in 3-day proliferation assays using the original microbial detergent extract from M. tuberculosis as Ag (Fig. 3A). An anti-CD1b Ab inhibited this response by 90%. Mycobacterial sonicate provoked a somewhat weaker stimulation index of 16, but the response was also specifically inhibited by 90% with Ab blocking (Fig. 3B). These data suggested that the mycobacterial Ag served as a more potent T cell agonist for this clone than did CD1b with self-Ag. Consistent with this interpretation, the dose-response curve for proliferation of Mt2.21 to mycobacterial sonicate was markedly affected by the inclusion of exogenous IL-2 in the assay. Increasing concentrations of IL-2 enhanced T cell proliferation in response to self-CD1b such that, in 1 nM IL-2, the T cell clone showed essentially the same response as that induced by the mycobacterial sonicate in the absence of IL-2 (Fig. 3C). Added IL-2 also enhanced the proliferative response at every suboptimal Ag concentration. Confirming the CD1 dependence of this response, proliferation was inhibited by >95% by anti-CD1b Abs but was unaffected by an isotype control (data not shown). Thus, in changing the context of T cell activation by adding the cytokine IL-2, both self-CD1b and suboptimal microbial Ag concentration can result in identical plateau responses, suggesting weak rather than partial agonism (58).



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  		FIGURE 3. Microbial Ags are potent agonists for self-reactive T cell clones. The CD1b-restricted T cell clone Mt2.21 was cultured in the presence and absence of Mtb detergent extract (DE) or Mtb sonicate and the indicated mAb for 3 days. Proliferation was measured by [3H]TdR incorporation during the final 18 h of the culture (A and B). The Mt2.21 clone was incubated with a range of Mtb sonicate concentrations and the indicated concentration of IL-2. Proliferation was measured by [3H]TdR incorporation during the final 18 h of the culture (C). Mock or CD1b-transfected T2 cells were cultured overnight with 51Cr in the presence or absence of Mtb sonicate (10 µg/ml). The targets were washed and then cultured with the Mt2.21 clone at the indicated E:T ratio (D). Error bars represent the means of triplicate determinations in each panel.

 
Given that bacterial preparations can stimulate immature DCs, the above experiments do not exclude an indirect role for the mycobacterial preparation in activating the DC or the T cell clone itself. Such an indirect role appeared unlikely to account for the Ag reactivity because this preparation was ineffective at stimulating other T cell clones that recognized other foreign Ags (data not shown). To further support the role of the mycobacterial extract as a foreign Ag, CD1b-transfected cell lines were used as targets for cytolysis. The CD1b-transfected T2 cell line proved resistant to cytolysis in the absence of added Ag, but following overnight exposure to mycobacterial sonicate, increasing ratios of the Mt2.21 clone specifically lysed the CD1b transfectant by up to 35% at an E:T ratio of 25:1 (Fig. 3D). In contrast, the mock T2 transfectant with or without Ag exposure showed no dose-dependent increase in specific lysis. Finally, the recognition of foreign Ags by cloned transfected TCRs (below) provided direct evidence for TCR-mediated CD1-restricted foreign Ag recognition.

Self and foreign Ag reactivity are conferred by the same TCR

It was possible that the dual reactivity seen in these clones reflected multiple CD1-restricted T cells in what were in fact nonclonal cell lines. Subcloning of two of the clones (Ye2.3 and Mt2.33) yielded only T cells with the identical CD1 restriction and self-reactivity (data not shown), in contrast with previous studies of dual-reactive T cell lines, which found that subclones were either self or foreign Ag reactive (16). In addition, the clones expressing V{beta}7 or V{beta}9 TCRs stained homogeneously positive with the respective TCR V region specific mAb (data not shown), a finding consistent with the clonality of the T cells examined here. To further investigate the role of the TCR in CD1-restricted Ag recognition, we cloned full-length TCR cDNA. For each clone analyzed, only one intact TCR{alpha} and TCR{beta} sequence was isolated and at least five identical sequences were detected, suggesting that these T cells were in fact clonal.

Next, we transfected T cell receptors cloned from two index clones, Mt1.50 and Mt2.21, into the TCR-negative Jurkat line (J.RT3-T.3) for analysis (52). Both transfectants showed the predicted response to CD1-expressing cells as indicated by Ab-specific abrogation of IL-2 production in response to monocyte-derived DCs (Fig. 4). As controls, the previously described CD1a- and CD1b-restricted foreign Ag-specific J.RT3-T.3 tranfectants of the CD8-2 and DN1 TCR pairs (51), respectively, released no detectable IL-2 in the presence of immature DCs without added foreign Ag. The results confirm that the TCR transfer confers the self-reactivity to CD1. To examine the role of the TCR in foreign Ag reactivity, we examined the Mt1.50 TCR transfectant. The weak self-reactivity of the Mt1.50 transfectant was markedly augmented by the mycobacterial microbial detergent extract, whereas the sonicate induced only modest levels of IL-2 in a similar dose-response range (Fig. 5A). Analysis of a strictly foreign Ag reactive CD1a-restricted TCR transfectant as a control demonstrated the opposite pattern of reactivity (Fig. 5A). When these tranfectants were incubated with their optimal Ag in the presence of an anti-CD1a or anti-CD1b mAb, >95% inhibition was observed only with the CD1a Ab for each transfectant (Fig. 5B). Similar responses were obtained when comparing CD1a-transfected and CD1b-transfected C1R cells as APCs (data not shown). The transfectants were next tested against a panel of Ags. Potent stimulation of the CD8-2 transfectant was seen with the mycobacterial sonicate, the whole organic solvent extract, and the methanol fraction separated by silicic acid chromatography from the whole extract. This transfectant responded only weakly to the acetone-eluted fraction of the mycobacterial organic extract. In striking contrast, only the mycobacterial microbial detergent extract was capable of potently stimulating the Mt1.50 TCR transfectant (Fig. 5C). The fine specificity of Ag recognition by the Mt1.50 TCR transfectant, exemplified by the preferential response to the microbial detergent extract from M. tuberculosis over that from Y. enterocolitica, further argues against a nonspecific effect of the Ag preparation. Although we cannot exclude the possibility that an endogenous lipid present in higher concentrations results in the microbial extract’s potent agonism, previously identified group 1 CD1 self-ligands, such as phospholipids, sulfatides, and gangliosides (59, 60), did not augment the self-reactivity to immature DCs alone (data not shown).



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  		FIGURE 4. CD1 restriction is conferred by cloned TCRs. The J.RT3-T.5 transfectants were generated and reactivity was assessed as described in Materials and Methods. IL-2 release was measured by the HT-2 cell bioassay after incubation of the indicated TCR transfectant with monocyte-derived DCs in the presence or absence of the CD1a- and CD1b-specific Abs 10D12.2 and BCD1b3.1, respectively. Error bars represent the means of triplicate determinations in each case.

 


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  		FIGURE 5. Self and microbial recognition is conferred by the same TCR. The Mt1.50 and CD8-2 TCR transfectants were cultured overnight with monocyte-derived DCs and the indicated concentration of Mtb-DE or Mtb sonicate (A). The Mt1.50 and CD8-2 TCR transfectants were cultured overnight with monocyte-derived DCs and Mtb-DE and Mtb sonicate, respectively (30 µg/ml), in the presence or absence of CD1a- and CD1b-specific (10D12.2 and BCD1b3.1, respectively) Abs (B) or with monocyte-derived DCs and a panel of microbial Ag preparations (C). The Mt1.50 and CD8-2 TCR transfectants were cultured overnight with monocyte-derived DC and the indicated concentration of PMA (D). IL-2 release was measured by the HT-2 cell bioassay and error bars represent the means of triplicate determinations in each panel.

 
Self-Ag provides a weak TCR signal to dual-reactive T cells

The IL-2 released by J.RT3-T.3 transfectants in response to Ag stimulation is customarily measured in the presence of 10 nM PMA to provide an accessory stimulus in addition to signal 1 from the transfected TCR. One method to detect the strength of a TCR response is to examine the response to Ag stimulation in the presence of increasing costimulation (61). Dual-reactive TCRs should respond to increased accessory stimulation in the absence of foreign Ag because self-CD1 is itself a weak Ag. In contrast, foreign-Ag-dependent TCRs should not exhibit a response with increasing accessory stimulation in the absence of Ag. To assess the relationship between TCR and accessory signal strength, the concentration of PMA was varied between 0 and 10 nM in our transfectant assay. The self-reactive TCR (Mt1.50) showed dose-dependent increases in IL-2 release in the absence of exogenous Ag with increasing PMA concentrations (Fig. 5D). In contrast, the foreign-Ag-dependent transfectant (CD8-2) did not respond to any concentration of PMA in the absence of Ag but responded strongly to 30 mg/ml mycobacterial sonicate (36,000 cpm with 10 nM PMA; data not shown). These data are consistent with weak TCR recognition of CD1-restricted self Ags for the dual-reactive TCRs and strong recognition of foreign-Ag-loaded CD1 for both the foreign-Ag-dependent and the dual-reactive TCRs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The capacity for TCR self-recognition is inherent in the T cell repertoire as a consequence of the requirements for positive selection. Whereas thymocytes with an avidity for self above a certain threshold are deleted through negative selection, those with an avidity below a minimal threshold for self-recognition fail positive selection. At the far end of the avidity spectrum are T cells that exist at the margin of escape from negative selection. These cells display what we term "overt" self-reactivity in contrast with the "covert" self-reactivity resulting from the requirements for positive selection (62).

The evidence that group 1 CD1 molecules present microbial lipids is compelling (13, 14, 15, 16, 17, 18, 19, 20, 21, 22). Similarly, the evidence that each CD1 isoform thus far studied activates some T cells in the absence of added foreign Ag is equally sound (8, 10, 11, 16, 63, 64). It is noteworthy that in the initial descriptions of CD1 recognition, exogenous Ags were not added, implying a proclivity for self-reactivity in the T cell-CD1 recognition process. In this work, we identified a panel of 15 CD1-restricted self-reactive T cells from a screen of 500 clones (3%), which likely underestimates the precursor frequency of CD1a-, CD1b-, and CD1c-restricted self-reactive clones in circulation. Considering the predilection of effector T cells for migration through tissues (65, 66) and the prevalence of circulating and tissue-resident CD1d-restricted T cells, the total number of CD1-restricted cells with overt self-reactivity may be even more substantial than expected.

Despite the unequivocal self-reactivity specific to CD1a, CD1b, or CD1c exhibited by the panel of clones, analysis of transfected TCR specificities clearly demonstrates the capacity for dual reactivity to self and microbial lipid Ags. The greater potency of foreign microbial lipids to stimulate such T cells implicates the function of these T cells in microbial defense. Other overtly self-reactive T cells, such as the CD1d-restricted TCR{alpha} invariant NK T cells, are quiescent when unperturbed, but have immediate effector functions under inflammatory conditions (67) or in the face of infectious challenge (26, 27) in the absence of specific foreign lipid Ag recognition. Presumably, similar homeostatic mechanisms maintain the quiescent state of overtly self-reactive group 1 CD1-restricted T cells in the absence of inflammatory conditions.

Initially, the canonical nature of the NK T cell TCR{alpha}-chain appeared to distinguish T cells restricted by group 2 CD1 (CD1d) from those restricted by group 1 CD1, but it is now clear that an additional diverse population restricted by CD1d exists in the mouse (68, 69). Data demonstrating that a diverse CD1d-restricted TCR expressed transgenically gives rise to T cells with some characteristics of NK T cells (70) further supports the notion that CD1-restricted T cells are prone to overt self-reactivity. The finding of diverse TCR populations self-reactive to all of the CD1 isoforms implies that these T cells possess a broader repertoire of reactivities than previously appreciated, potentially explaining the foreign Ag recognition we observed. Efforts to demonstrate microbial Ag reactivity by murine TCR{alpha} invariant CD1d-restricted T cells have yielded conflicting data (71, 72) until very recently, with the characterization of several microbial lipids capable of activating CD1d-restricted T cells (36, 37, 38, 39). Consistent with our findings, the same invariant NKT cells from both mice and humans appear to react to both self and microbial lipids from {alpha}-Proteobacteria (36, 37, 40). The existing data also support the concept that Ag receptor promiscuity is a more common feature of T cells restricted by nonpolymorphic MHC molecules (73).

In transgenic models in which TCR peptide agonists are present during thymic selection, a population of T cells bearing the CD8{alpha}{alpha} homodimer escapes to the periphery with an activated phenotype and immediate effector capabilities in response to agonist peptide (74). Like the CD1-restricted T cells we describe here, the capacity to respond to foreign Ags remains despite the overt self-reactivity. Significantly, such cells fill mucosal compartments, at the front line of host defense where the need for immediate effector function would counterbalance the autoimmune hazard that self-reactive cells inherently pose. Consistent with this scenario, mucosal associated invariant T cells bearing a unique invariant TCR{alpha}-chain have recently been shown to be present in both human and murine mucosal sites and to display overt self-reactivity to the highly conserved MHC homologue MR1 (75).

Overtly self-reactive MHC-restricted T cells such as CD4+CD25+ regulatory T cells are clearly important for the maintenance of immune homeostasis (2, 76), but the vast majority of MHC-restricted T cells are covertly self-reactive and therefore available to participate in classical adaptive immune responses. In contrast, the majority of characterized CD1-restricted T cell responses manifests overt self-reactivity, which is associated with effector/memory T cell capabilities critical to rapid antimicrobial immune responses. We propose that the CD1-restricted diverse TCR population comprises both conventional naive T cells capable of adaptive responses to microbial lipids (20) and memory/effector T cells capable of both self-reactivity and foreign lipid reactivity (this work). The latter population might be unique to CD1-restricted T cells and impart the ability to respond more rapidly in an initial antimicrobial immune response, yet it still may allow for clonal expansion and subsequent long-standing cellular memory to foreign lipid Ags. Taken together, the data lead us to hypothesize that CD1-restricted T cells may be intrinsically subject to development toward a self-reactive memory/effector population.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Arthritis Foundation (to M.S.V.) and the National Institutes of Health (Grants K08AR01996 and R21AR48037 to M.S.V. and R01CA47724 and R37AI28973 to M.B.B.). Back

2 Address correspondence and reprint requests to Dr. Michael B. Brenner, Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham & Women’s Hospital and Harvard Medical School, One Jimmy Fund Way, Room 552, Boston, MA 02115; E-mail address: mbrenner{at}rics.bwh.harvard.edu or Dr. Michael S. Vincent at the current address: Amgen, One Amgen Center Drive, 38-3-A, Thousand Oaks, CA 91320; E-mail address: mvincent{at}amgen.com Back

3 Abbreviations used in this paper: DC, dendritic cell; DE, detergent extract. Back

Received for publication May 25, 2005. Accepted for publication July 26, 2005.


   References
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 Abstract
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 Materials and Methods
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 Discussion
 Disclosures
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