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The Journal of Immunology, 2002, 168: 1018-1027.
Copyright © 2002 by The American Association of Immunologists

CD4 T Cells Selected by Antigen Under Th2 Polarizing Conditions Favor an Elongated TCR{alpha} Chain Complementarity-Determining Region 3

Rosemary J. Boyton*, Nathan Zaccai{ddagger}, E. Yvonne Jones{ddagger} and Daniel M. Altmann2,*,{dagger}

* Transplantation Biology Group, Medical Research Council Clinical Sciences Centre, and {dagger} Human Disease Immunogenetics Group, Department of Infectious Diseases, Imperial College School of Science, Technology, and Medicine, Hammersmith Hospital, London, United Kingdom; and {ddagger} Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The affinity of the MHC/peptide/TCR interaction is thought to be one factor determining the differentiation of CD4+ T cells into Th1 or Th2 phenotypes. To study whether CD4+ cells generated under conditions favoring Th1 or Th2 responses select structurally different TCRs, Th1 and Th2 clones and lines were generated from nonobese diabetic and nonobese diabetic H2-E transgenic mice against the peptides proteolipoprotein 56–70, glutamic acid decarboxylase65 524–543, and heat shock protein-60 peptides 168–186 and 248–264. Th1/Th2 polarization allowed the generation of clones and lines with fixed peptide specificity and class II restriction but differing in Th1/Th2 phenotype in which the impact on TCR selection and structure could be studied. The Th2 clones tended to use longer TCR complementarity-determining region (CDR)3{alpha} loops than their Th1 counterparts. This trend was confirmed by analyzing TCR{alpha} transcripts from Th1 and Th2 polarized, bulk populations. Molecular modeling of Th1- and Th2-derived TCRs demonstrated that Th2 CDR3{alpha} comprised larger side chain residues than Th1 TCRs. The elongated, bulky Th2 CDR3{alpha} loops may be accommodated at the expense of less optimal interactions between the MHC class II/peptide and other CDR loops of the TCR. We propose that CD4+ T cells selected from the available repertoire under Th2 polarizing conditions tend to have elongated TCR CDR3{alpha} loops predicted to alter TCR binding, reducing contact at other interfaces and potentially leading to impeded TCR triggering.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Responses of CD4 T cells can be divided on the basis of their differential cytokine responses into Th1 and Th2 (1, 2). The contribution of Th1 and Th2 responses to autoimmune disease in humans and mice has been widely examined, leading to a view that pathology is often associated with Th1 cytokine responses and protection or recovery with Th2 responses. Much attention has focused on the factors influencing the development of T cells into Th1 or Th2 populations (2, 3). The local cytokine environment at the time of priming is critical, both in terms of effects on the responding T cells and on the APC (3). The type of APC is important, as are the costimulatory molecules expressed by the APC (4). Attempts to find phenotypic markers that distinguish Th1 and Th2 populations have revealed several differences. Many of these reflect differential transcriptional control leading to the activation of Th1 or Th2 cytokine genes, including the differential expression of c-MAF, GATA-3, NF-ATc1, and T-bet (5, 6). Other differences are in the expression of cytokine receptors and of signaling molecules downstream of these receptors such as STAT-4 and STAT-6 (7, 8, 9, 10).

The transcriptional choice to produce Th1 or Th2 cytokines cannot be hardwired to the TCR sequence because TCR-transgenic T cells originally derived from a given Th1 or Th2 T cell clone can be experimentally driven to differentiate into both Th1 and Th2 effector cells (11, 12, 13, 14). Nevertheless, a number of studies suggest a relationship between structural features of the TCR interaction with peptide/MHC and the resulting cytokine response. Studies using altered peptide ligands (APL)3 have shown that peptide/MHC-TCR affinity is a factor influencing whether a CD4 T cell produces Th1 or Th2 cytokines (15, 16). APL/MHC complexes showing a low-affinity interaction with TCR are often associated with Th2 responses. Proteolipoprotein (PLP) 139–151-specific clones selected under Th2-favoring conditions show shifted peptide-TCR primary contact residues compared with Th1 clones (17). In another system, a point mutation in the TCR{alpha} complementarity-determining region (CDR)2 is associated with a shift in the phenotype of TCR-transgenic T cells from Th1 to Th2 (18). T cell populations undergoing expansion during successive restimulation in vivo or in vitro have been shown to undergo selective changes in epitope specificity, TCR usage, and TCR affinity at the population level (19, 20). A prediction from these studies is that T cell populations against a given peptide/MHC complex, when polarized to develop as Th1 or Th2 cultures, may preferentially expand clones using different TCRs and of different affinities.

The experimental system used in this study to investigate this prediction is based in two autoimmune diseases in the nonobese diabetic (NOD) mouse. NOD mice spontaneously develop type I diabetes, whereas H2-E transgenic NOD mice (NOD.E) are protected (21). This situation is reversed in experimental autoimmune encephalomyelitis of NOD mice because after immunization with PLP 56–70 NOD mice suffer very mild disease, whereas H2-E transgenics develop severe disease with demyelination (22). In our hands, the diabetes susceptibility in NOD and NOD.E mice correlates with the magnitude of response to the glutamic acid decarboxylase (GAD)65 524–543 epitope, although other laboratories have also described other candidate disease-related peptides (23, 24). In the course of the present study, we also defined two epitopes from another self-Ag, heat shock protein (Hsp)60, and we describe the Th1 and Th2 responses to these epitopes. The NOD and NOD.E models do not involve an exclusive association of Th1 cytokines with disease and Th2 with protection (22, 23). Rather, disease is associated with a relative shift toward a more Th1 phenotype. T cell clones were generated with fixed peptide specificity and class II restriction but differing Th1/Th2 phenotype in which the impact on TCR selection and structure could be studied. The resulting clones and lines demonstrated that the selection of clones making either Th1 or Th2 cytokines correlates with TCR{alpha} CDR3 usage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Immunization

PLP 56–70 DYEYLINVIHAFQYV was used for priming T cell responses and the substituted analog carrying lysine for tyrosine at position 57 and 59 for in vitro restimulation of cells. GAD65-specific responses were generated by immunization with GAD 524–543, SRLSKVAPVIKA (synthesized by Haemeostasis Group, Medical Research Council Clinical Sciences Centre, London, U.K.). Two newly identified murine Hsp60 class II epitopes in the NOD mouse, 168–186, AQVATISANGDKDIGN, and 248–264, KKISSVQSIVPALEIA (S. G. Newton, O. Birk, I. R. Cohen, and D. M. Altmann, manuscript in preparation) were also used to generate polarized lines and clones. NOD and NOD.E transgenic mice (21) were bred and housed in the Centre for Biological Services at the MRC Clinical Sciences Center.

T cell proliferation assays

Mice were immunized with 50 µg of GAD, PLP, or Hsp60 peptide in CFA. After 10 days, popliteal lymph nodes were removed and single cell suspensions were prepared in HL-1 medium (Hycor Biomedical, Irvine, CA). Cells were cultured in triplicate in 96-well plates in the presence of peptide for 3 days. [3H]Thymidine was added 18 h before termination, and cultures were harvested for beta scintillation counting.

T cell lines and clones

T cell lines from immunized lymph nodes were initially set up in the presence of GAD, Hsp60, or PLP peptide. After 48 h, recombinant cytokines were added to the medium and the cultures were incubated for an additional 7 days. Cells were then resuspended in RPMI 1640 medium (Life Technologies, Paisley, U.K.) and 5% FCS, and were restimulated with 50 µg/ml of the appropriate peptide in the presence of irradiated splenocytes. The 10-day cycle was repeated at least four times. Recombinant cytokines (R&D Systems, Abingdon, U.K.) were added to the culture medium to promote the growth of Th1 and Th2 cells. Th1 lines and clones were maintained in IL-2 (330 IU/ml) and IL-12 (100 IU/ml), and Th2 lines and clones were maintained in IL-2 (330 IU/ml) and IL-4 (100 IU/ml). The short-term lines described in Table IIIGo and Figs. 3Go and 4Go were additionally polarized by supplementing Th1 medium throughout with neutralizing anti-IL-4 at 10 µg/ml (R&D Systems and BioSource International, U.K) and Th2 medium with neutralizing anti-IFN-{gamma} at the same concentration. Flow cytometric analysis of lines at the time of making cDNA (after three restimulations) showed them to contain >85% CD4 cells, this population being equivalent between peptides and between Th1 and Th2 polarization (data not shown).


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  		Table III. CDR3{alpha} sequences from cloning of TCR{alpha}-amplified cDNA from short-term Th1 or Th2 polarized NOD.E T cell lines against PLP 56–70 and NOD lines specific for Hsp60 168–186 and 248–2641

 


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  		FIGURE 3. Cytokine analysis of short-term, polarized T cell lines made from NOD mice against Hsp60 248–264 and 168–184. After three restimulations and at the time of making cDNA (see Table IIIGo and Fig. 4Go) Th1 and Th2 polarized lines were analyzed for T cell proliferation and for synthesis of IFN-{gamma} and IL-4. Triplicate samples were analyzed. •, Results for the Th1 line; {triangleup}, results for the Th2 line.

 


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  		FIGURE 4. Use of common Th1-derived 12-mer CDR3{alpha} motif and Th2-derived 14-mer CDR3{alpha} motif in cDNA from short-term Th1 and Th2 polarized bulk cultures generated from NOD.E mice immunized with PLP 56–70. A, RT-PCR of cDNA from ex vivo draining lymph node cells, used at day 10 after immunization and not polarized in vitro; the RT-PCR uses oligonucleotides from the common 12- or 14-mer CDR3{alpha} (see Table IIIGo), used together with the C{alpha} oligonucleotide NJ108 (see Ref. 25 ). Hypoxanthine phosphoribosyltransferase amplification is shown as a cDNA loading control. B, RT-PCR of Th1 and Th2 polarized line cDNA using oligonucleotides from the common 12- or 14-mer CDR3{alpha} used together with the C{alpha} oligonucleotide NJ108. C, Representative membranes showing hybridization of 32P-labeled 12- or 14-mer CDR3{alpha} probes to screen bacterial colonies containing TCR{alpha} PCR-amplified sequences from Th1 and Th2 cDNA. D, Frequency (out of total colonies) of bacterial colonies hybridizing to each probe. Paired, filled bars indicate results from separate repeat experiments.

 
T cell cytokine assays

T cell proliferation assays of immunized lymph node cells were set up as described above. Cells were stimulated with GAD, PLP, or Hsp60 peptides. After 66 h of stimulation, 50 µl of supernatant was removed from each well to determine cytokine production. The remainder was pulsed with [3H]thymidine and cultured for an additional 18 h, and then incorporated radioactivity was counted. The IL-4, IL-5, IL-6, IL-10, and IFN-{gamma} content of the supernatants was measured by specific ELISA, measuring against linear standard curves (Endogen (Cambridge, U.K.) for IL-5 and IFN-{gamma}, and R&D Systems for IFN-{gamma}, IL-4, IL-6, and IL-10).

TCR subcloning and sequencing

Total RNA was extracted from T cell clones at 28 days or more after the last restimulation with APC and peptide using the acid phenol method (RNAzol B; Biogenesis, Bournemouth, U.K.). cDNA was synthesized using random hexamers and superscript reverse transcriptase (Life Technologies). TCR{alpha} and TCR{beta} transcripts were amplified as described (25) and ligated into the pCR2.1 TA cloning vector. Six independent colonies for each TCR{alpha} and TCR{beta} product were sequenced using M13 forward and reverse primers (Cambridge Biosciences, Cambridge, U.K.).

For analysis of TCR{alpha} usage in short-term polarized Th1 and Th2 bulk lines, cells were restimulated ex vivo with PLP 56–70, Hsp60 168–186, or Hsp60 248–264 peptide and APC as described above. Cells were maintained under polarizing conditions (IL-2 (330 IU/ml), IL-12 (100 IU/ml), and anti-IL-4 (10 µg/ml) for Th1; IL-2 (330 IU/ml), IL-4 (100 IU/ml), and anti-IFN-{gamma} (10 µg/ml) for Th2). After three rounds of restimulation, cDNA was generated and TCR{alpha} transcripts were amplified as described above. Following TA cloning, these were either sequenced or plated on Luria-Bertani agar plates and hybridized to Hybond-N membranes (Amersham, Little Chalfont, Buckinghamshire, U.K.) for screening with [32P]{gamma}ATP-labeled CDR3 sequence oligonucleotide probes (see below).

Modeling of the H2-Ag7-PLP/TCR complexes

The refined-structure MHC class I/TCR complex of Garboczi and coworkers (Ref. 26 ; Brookhaven Protein Database accession number 1AO7) was used to provide a template for the MHC/TCR docking geometry. The structure of an MHC class II-restricted TCR was superimposed onto the model complex. A second template was subsequently created from the MHC class II/TCR complex determined by Reinherz and coworkers (27). This structure was superimposed onto the first template using as a basis the {alpha} variable domain of the TCR. In each template, the structure of the murine MHC class II molecule H2-Ag7 (Ref. 28 ; Brookhaven Protein Database accession number 1ESO) was superimposed onto the MHC positions, and the sequence of the PLP peptide was substituted using the interactive graphics Program O (29). The appropriate TCR CDR3{alpha} and CDR3{beta} loops were modeled manually in Program O on the basis of the available structural database of TCRs and TCR-MHC-peptide complexes (26, 27, 30, 31, 32, 33, 34, 35). Each superposition was performed with the program SHP (36). Fig. 5Go was produced using Bobscript and was rendered with Raster3D (University of Washington, Seattle, WA) (37).



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  		FIGURE 5. Molecular models of Th1- and Th2-derived TCRs (A10 (2 ) and D3 (3 ), respectively), recognizing the MHC class II H2-Ag7/PLP complex. A, View of the side of the peptide-binding groove/TCR interface. The MHC class II H2-Ag7 main chain is shown schematically ({alpha}-chain, light blue; {beta}-chain, light green) and the peptide, PLP 56–70, is depicted in brown ball-and-stick representation (with the leucine, valine, and alanine residues most exposed to putative TCR interactions highlighted in pink). A single representative scaffold for the TCR{alpha} and TCR{beta} variable domain main chain is shown schematically in gray, with the modeled CDR3{alpha} and CDR3{beta} loops for a Th1 profile TCR colored in blue, and for a Th2 profile colored in red. B, A close-up view of the PLP peptide-TCR interface with the MHC class II {alpha}- and {beta}-chains omitted. Structural elements are colored as in A. The side chains at the putative apices of the CDR3 loops are depicted in ball-and-stick representation. C, View onto the peptide-binding groove/TCR interface. Only the apices of the TCR CDR loops are shown. Structural elements are colored as in the previous panels. The modeled main chains for the CDR3{beta} loop in the Th1 profile TCR and in the Th2 profile TCR are superimposed in this view, appearing as a single blue and red interlaced loop.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of Th1 and Th2 clones specific for PLP 56–70, GAD65 524–543, and Hsp60 248–264

Several long-term T cell lines were generated from NOD or NOD.E transgenic mice, maintained by restimulation and expansion for several months, and then cloned by limiting dilution. The clones were obtained from separate cloning sessions of lines, each line being obtained from the pooled cells of four immunized mice. T cell clones were analyzed for MHC class II restriction (by presentation using H2-E-positive or -negative APC) and for cytokine production by ELISA and intracellular FACS staining. The clones selected for further analysis of TCR usage are illustrated in Fig. 1Go. All of the Th1 clones generated in response to PLP peptide from NOD.E mice were H2-Ag7 restricted. They are classified as Th1 because of their strong IFN-{gamma} production at low peptide concentration and lack of IL-4, -5, or -10 production. These clones produced IL-6 at higher peptide concentrations (Fig. 1Go, upper panel). Th2 clones were generated against either PLP 56–70 or GAD65 524–543 and were restricted either by H2-Ag7 or by H2-E (Fig. 1Go, lower panel). The cytokine profiles of the Th2 clones were more diverse. The NOD anti-GAD clones, C11 (3) and G6 (3), both produced IL-4, -5, -6, and -10, but no IFN-{gamma}, whereas the NOD.E anti-GAD H2-E-restricted clone, B3 (1), produced IL-4, -5, -6, -10, and, in addition, IFN-{gamma}. The IFN-{gamma} was produced at higher peptide concentrations than in the Th1 clones. Two additional clones generated against the Hsp60 epitope 248–264 presented by H2-Ag7 in NOD mice were also analyzed. TLC C10 is a Th1 clone that recognizes Hsp60 248–264 and responds to peptide at 0.04 µg/ml with a high IFN-{gamma} response and no IL-4 (data not shown). TLC E2 is specific for the same peptide/MHC complex but responds by making a high IL-4 response in the absence of IFN-{gamma}. Using such clones, comparisons can be made of TCR usage in T cells with different cytokine profiles: clones A10 (2) and D3 (3) are a useful pair in this respect, having completely polarized cytokine profiles but recognizing the same peptide/MHC complex.



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  		FIGURE 1. Characteristics of Th1 (upper panel) and Th2 (lower panel) T cell clones. T cell clones were analyzed for proliferation and cytokine production in response to peptide. Th1 clones were defined as those producing IFN-{gamma} in the absence of IL-4. Th2 clones produced IL-4 and/or IL-5. Note that the Th2 panel contains two clones, E4(2) and B3(1), which produce IFN-{gamma}, albeit in response to higher peptide doses than Th1 clones, and can therefore be considered Th0.

 
Analysis of TCR V{alpha} and V{beta} usage in Th1 and Th2 clones: TCR from Th2-differentiated populations have elongated CDR3{alpha} loops

Clones isolated from the Th1 NOD.E anti-PLP lines were all V{beta}13+J{beta}2.6+ and used an identical CDR3 sequence, ASSPLDWGDEQY (Table IGo). Interestingly, most of the H2-Ag7-restricted clones used J{beta}2.6, irrespective of their V{alpha} and V{beta} usage, peptide ligand, or Th1/Th2 phenotype. Two reports of TCR sequences from NOD islet T cell infiltrates describe over-representation of J{beta}2.6 (38, 39), although diabetogenic clones using other J{beta} segments have been described (25). In view of the over-representation of J{beta}2.6 cells among our H2-Ag7-restricted cells against PLP and GAD peptides and among the reported infiltrating cells, it seems likely that this J segment is simply a favored one in NOD mice for interactions with H2-Ag7-bound peptides. Although the Th2 clones showed more diversity than the Th1 clones, no clear trend differentiated their TCR{beta} chains and both had CDR3 lengths of 11–13 amino acids. Analysis of the TCR{alpha} chains in the Th1 clones showed that there were two types of V{beta}13+ clones against the PLP peptide: two-thirds carried only a V{alpha}9/J{alpha}11 TCR{alpha} chain, whereas one-third reproducibly gave the same two in-frame TCR{alpha} chains from repeated bacterial colonies (Table IIGo). Thus, three of the clones carried both V{alpha}9/J{alpha}11 and V{alpha}1/J{alpha}A10 TCR{alpha} chains. The pair of T cell clones generated against Hsp60 248–264 follows the same trend as those against PLP and GAD65: C10 is a Th1 clone that recognizes H2-Ag7/284–265 using a 12-residue CDR3{alpha} sequence. The equivalent Th2 clone, E2, does so using a CDR3{alpha} that incorporates two additional amino acids.


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  		Table I. TCR{beta} sequences of T cell clones

 

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  		Table II. TCR{alpha} sequences of T cell clones

 
The most noteworthy difference identified between the Th1 and Th2 clones was in the length of CDR3{alpha} loops and in the characteristics of amino acids predominating in the loops (see below). Overall, the mean CDR3{alpha} length was significantly longer in Th2 (mean = 13 ± 0.3) compared with Th1 clones (mean = 10.8 ± 0.4; p < 0.0008; Table IIGo and Fig. 2Go). The structural importance of this difference in length of CDR3{alpha} was studied by TCR/peptide/MHC modeling (see below).



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  		FIGURE 2. CDR3{alpha} length of Th1 (filled bars) and Th2 (open bars) clones. Upper and lower horizontal bars indicate the mean lengths of Th2 and Th1 CDR3{alpha} loops, respectively.

 
TCR{alpha} analysis of short-term Th1 or Th2 polarized T cell lines

In an attempt to confirm the association between CDR3{alpha} length and selection of Th2 polarized cells, TCR{alpha} transcripts were further analyzed using cDNA from bulk populations of independently derived, polarized, short-term T cell lines (Table IIIGo and Fig. 3Go). To achieve this, bulk populations of ex vivo PLP 56–70- (in NOD.E mice), Hsp60 168–186-, or Hsp60 248–264 (both in NOD mice)-primed lymph node cells derived from four mice were divided into two plates in each case for culture under either Th1 or Th2 conditions. Two trends seen in the analysis of Th1 and Th2 clones were again evident. First, even in these short-term cultures, there is rapid selection of dominant clones, which are repeatedly isolated from TCR{alpha}-amplified cDNA. Second, the choice of these dominant receptors from the same initial pool differs under Th1 and Th2 conditions: for the NOD.E PLP responses, the 12-aa CDR3{alpha} loop, AVSWDNYAQGLT, dominated the receptors amplified from Th1 cDNA, and the 14-aa CDR3{alpha} loop, ALEGIASSSFSKLV, was favored by Th2s. Taking into account the dominant TCR{alpha} as well as the others identified, the CDR3{alpha} loops differed between Th2 and Th1 PLP bulk cultures, the former showing a mean CDR3{alpha} length of 13 ± 0.3 and the latter showing a mean length of 11.4 ± 0.2. This analysis does not take into account the multiple isolations of the dominant Th1, 12-mer sequence and the dominant Th2, 14-mer sequence, but rather treats each as if it were one event. Thus, this analysis, if anything, underestimates the differential skewing of CDR3 lengths at a functional level. Nevertheless, comparison of CDR3{alpha} lengths in the two PLP lines gives a p value of <0.005. In summary, the selection of a short-term line against PLP 56–70 under Th2 compared with Th1 polarizing conditions has favored the expansion of receptors using one to two additional amino acids.

Similar analysis was conducted on polarized NOD lines against Hsp60 168–186 (Table IIIGo and Fig. 3Go). The mean CDR3{alpha} length of Th1 receptors was 9.5 ± 1.5 and of Th2 receptors, was 12.0 ± 0, making the populations significantly different (p < 0.05). Comparison between lines against the same peptide/MHC complex is more meaningful than predicting favored generic lengths of the TCR{alpha} for Th1 or Th2 responses: the favored "short" loop of Th1 receptors against PLP 56–70 is the same length (12 amino acids) as the loop that, in context of the response to Hsp60, is considered "long" and is favored by Th2 polarization. This is presumably due to specific differences in the conformation and molecular interactions with different MHC/peptide complexes. Comparison of Th1 and Th2 sequences for a third pair of polarized NOD T cell lines against Hsp60 248–264 (Table IIIGo and Fig. 3Go) does not reach statistical significance (although Th1 receptors show a mean length of 10.6 and Th2 receptors show a mean of 11.3), mainly because some Th2 cells were identified that, like the Th1 cells, were able to use CDR3{alpha} loops of 10–11 amino acids. This demonstrates that although the use of the longer CDR3{alpha} loop may be one of the factors predisposing to selection of a clone in a Th2 environment, it is clearly not the only way in which this can be achieved; Th2 clones may also use "short" CDR3{alpha} loops like Th1 cells (see Fig. 4GoB). Th1 cells, in contrast, rarely use elongated CDR3{alpha} loops.

Notwithstanding the above caveats as to comparison between different MHC/peptide combinations, when all Th1-Th2 sequences are compared for the polarized lines, the mean Th1 CDR3{alpha} length is 10.8 ± 0.3 and the mean Th2 length is 12.4 ± 0.3 (p < 0.0009).

To obtain a broader picture of the relationship between the predominant 12- and 14-mer CDR3{alpha} sequences and Th1/Th2 conditions, we conducted RT-PCRs to probe for the presence of these transcripts in either Th1 or Th2 cDNA (Fig. 4Go). We initially conducted PCR amplification of cDNA from nonpolarized, ex vivo draining lymph node cells from NOD.E mice immunized 10 days earlier with PLP 56–70 (Fig. 4GoA). As expected, the initial pool of cells from which lines would subsequently be polarized contains, at this stage, transcripts for both 12- and 14-mer CDR3{alpha} receptors. Indeed, looking at the short-term polarized lines (Fig. 4GoB) we found that, though not identified as such during sequencing of individual colonies, the dominant 12-mer CDR3{alpha} transcript was present in both Th1 and Th2 cDNA (Fig. 4GoB). However, the Th2-derived 14-mer CDR3{alpha} could only be amplified from Th2 cDNA and was not present in Th1. That is, during the three rounds of restimulation and cytokine polarization between the starting population shown in Fig. 4GoA and the lines shown in Fig. 4GoB, the cells bearing the longer, 14-mer CDR3{alpha} loops have, under Th1 conditions, been lost in preference to others. A more quantitative measure of the dominance of these common 12- and 14-mer CDR3{alpha} sequences in the Th1 and Th2 populations was obtained by screening bacterial colonies containing TCR{alpha}-amplified PCR inserts from the short-term bulk lines (Fig. 4Go, C and D). Libraries representing total, PCR-amplified, and subcloned TCR{alpha} inserts were plated on Luria-Bertani agar and transferred to duplicate colony lifts that were probed with oligonucleotides representing the previously identified, common Th1 CDR3 AVSWDNYAQGLT, or the longer, Th2 CDR3, ALEGIASSSFSKLV. Representative data from one of these experiments are shown in Fig. 4GoC and summarized in Fig. 4GoD. The 12-mer Th1-derived CDR3 motif is present in ~50% of all Th1 TCR{alpha} sequences and in ~10% of Th2 TCR{alpha} sequences. Twenty percent of the Th2 TCR{alpha} sequences use the 14-mer Th2-derived CDR3. However, use of this receptor seems to be incompatible with polarization to a Th1 phenotype because, among this population, <0.1% have the 14-mer CDR3. These results strongly support the view that selection for particular TCR sequences is one of the ways in which bulk populations may become skewed to polarized cytokine programs.

Structural analysis of H2-Ag7/TCR interactions in Th1 and Th2 clones with different CDR3{alpha} length

Recent structural analyses of MHC/TCR complexes have provided detailed insights into the architecture of this recognition interface (26, 27, 30, 34). A clear theme has emerged that suggests a common geometry for binding defined by the conserved positioning of the TCR{alpha} domain CDR loops and, in particular, the CDR{alpha}1 and -2 loops (32). Structural information for the MHC class II/TCR complexes is at present still scarce (Ref. 27 and J. Hennecke and D. Wiley, unpublished observations). However, functional data (40), coupled with the similarity in molecular topology, provide strong evidence that the MHC class I/TCR docking mode is also conserved in the MHC class II/TCR interactions (D. Wiley, unpublished observations). Thus, analysis of the available TCR/MHC class II structure and a simple modeling exercise to substitute an MHC class II structure into MHC class I/TCR complexes can immediately provide a useful context in which to assess the structural relevance of the observed differences between CDR3{alpha} loops in the Th1 and Th2 clones A10 (2) and D3(3) (Fig. 1Go and Table IIGo).

An MHC class II-bound peptide is constrained by conserved hydrogen bonds to adopt an extended polyproline-II-like conformation deep within the binding groove (41). Thus, the H2-Ag7/PLP complex may be modeled with a reasonable level of accuracy based on the H2-Ag7 structure (28) and knowledge of the peptide anchor residues (42, 43). The surface presented for TCR recognition primarily comprises the flanking {alpha} helices of H2-Ag7 and tyrosine, asparagine, and histidine side chains from the PLP peptide (Fig. 5Go, A and B); however, the exposure to TCR of the PLP residues is markedly reduced in comparison with that of the central portion of a standard MHC class I peptide. This difference may be expected to have significant impact on the length of TCR{alpha} and TCR{beta} CDR3 loops required to make direct contact with MHC class II-presented peptide.

The lengths of {alpha} and {beta} CDR loops for the TCRs sequenced in this study, with the exception of the {alpha} CDR3s, lie in the range observed in MHC/TCR complex structures. The 10- to 11-residue length of the CDR3{alpha} loop for the Th1 TCRs in Table IGo also corresponds to the standard examples in MHC/TCR complexes, whereas the 12- to 14-residue Th2 TCR CDR3{alpha} loops are longer than any of the published TCR structures. This difference in Th1 and Th2 TCR CDR3{alpha} structures is exacerbated by the nature of the residues at the loop apex, the Th2 loop sequences generally comprising larger side chain residues (for example, valine, aspartic acid, asparagine, and tyrosine; Fig. 5GoB) than the equivalent sequences of Th1 TCRs (for example, glutamic acid, glycine, and glycine) raised against the same PLP peptide. When this observation is combined with the differences in peptide position between MHC class I and class II structures, the Th1 TCR CDR3{alpha} loop appears poorly fitted to mediated extensive interactions with the PLP peptide in H2-Ag7 (Fig. 5Go). The minimal contribution of a short, glycine-rich CDR3{beta} loop to the surface shape complementarity at the interface between a TCR and MHC class I molecule has been directly observed by Garcia et al. (32). For the MHC class I complex, the large cavities at the interface, in part arising from the short CDR3{beta} loop length, correlate with the weak affinity of this TCR. The current modeling study cannot provide sufficiently accurate coordinates to merit a detailed calculation of the shape complementarity at the interface, but it does highlight the possibility that the Th1 CDR3{alpha} loop may only make a minimal contribution to peptide recognition. The situation for the Th2 CDR3{alpha} loop is, in contrast, one in which the extra loop length risks steric clashes of its apical aspartic acid and asparagine residues with the asparagine of the PLP peptide and the MHC class II {alpha}1 helix (Fig. 5Go, A and B). Contributions to the interface by the CDR1 and CDR2 define the optimal docking geometry. Modeling clearly implies that the additional bulk of the Th2 CDR3{alpha} loop can only be accommodated at the expense of less optimal interactions between the MHC class II/PLP and the other CDR loops of the TCR. Indeed, comparison of two MHC class I-TCR crystal structures has illustrated that the presence of a particularly bulky CDR3{beta} loop substantially reduces the contact between TCR and MHC/peptide complex (33).

Although it is known from comparative analyses of MHC class I/TCR complex and isolated TCR structures that CDR3{alpha} loops can undergo large physical rearrangements to optimize their binding to the MHC molecule (30, 32), the current modeling clearly indicates significant differences in the contributions of the CDR3{alpha} loop for Th1 and Th2 TCR binding. The mechanism is most probably indirect through, for example, an altered positioning of either the CDR3{alpha} loop itself and/or of the TCR{beta} domain.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TCR-transgenic cells expressing a single receptor derived from a Th1 clone can be differentiated into either Th1 or Th2 cells. Therefore, the cytokine response of the cell cannot be an inherent property of the TCR sequence. Furthermore, Th1 cells can be "reversed" into a Th2 profile by retroviral infection with the Th2 transcription factor GATA3, further arguing against any hardwired relationship between features of the TCR sequence and cytokine response (44). The downstream, Th2 transcriptional boost of GATA3 expression presumably bypasses cell surface receptor signaling differences associated with Th1 or Th2 preference. However, single amino acid-substituted APL or antagonists can induce immune deviation in T cell populations, indicating that cytokine profiles can be altered by stimulation with ligands of reduced affinity (16). This difference in behavior between agonists and antagonists can be explained using the kinetic proofreading model (45). That is, because a certain amount of contact time is required to achieve recruitment and activation of ZAP-70 and full commitment to T cell activation, ligands that bind the receptor with low affinities and fast dissociation rates may not fully trigger T cell activation. It has been proposed that TCR engagement with APLs that are associated with the production of Th2 cytokines involves incomplete triggering such that ZAP 70 recruitment and Lck phosphorylation do not proceed (46). Differential strength of TCR engagement has been linked to differential cytokine transcription: extracellular signal-regulated kinase and c-Jun N-terminal kinase activation is a gradual phenomenon depending on the intensity of TCR stimulation and extracellular signal-regulated kinase- and p38 mitogen-activated protein kinase-dependent pathways are required for IFN-{gamma} transcription (47). If, as seems to be the case, TCR/peptide/MHC affinity is one of the factors determining Th1 and Th2 cytokine responses, it may be proposed that T cells selected for activation in Th1- or Th2-favoring environments will show preferential use of TCRs that facilitate this affinity difference.

We initially noted that selection of T cell lines under conditions favoring Th1 or Th2 responses can result in the evolution of lines with different receptor usage in studies with TCR mAbs (data not shown). With respect to TCR V{beta} families, the differences were minor. Indeed, analysis of TCR V{beta} sequence from Th1 and Th2 clones failed to identify differences in V{beta} family, CDR3 length, or amino acid motifs. Therefore, attention was focused on TCR{alpha} differences between the two populations, which were more apparent.

In several experiments using separate populations of Th1 and Th2 polarized cells and using a range of experimental approaches, we demonstrated that TCR{alpha} sequences, selected out of lines, differ under the two sets of conditions. In our panel of established Th1 and Th2 clones, the latter group tended to have longer CDR3{alpha} loops. This trend was confirmed by analyzing cells that had been selected in vitro but without a long-term program of restimulation and cloning. A single, bulk population of primed lymph node cells was polarized into Th1 or Th2 and was then made into cDNA and TCR{alpha} transcripts and compared. It was again found that the Th2 conditions tended to select for T cells with elongated CDR3{alpha} loops. Although there was a tendency for cultures to become rapidly dominated by particular clones, it is informative that the favored clones differed with the Th1/Th2 polarizing conditions. The 14-mer CDR3{alpha} motif that was identified as a frequent transcript among anti-PLP Th2 receptors was found on RT-PCR to be undetectable in Th1 cDNA. However, the predominant 12-mer CDR3{alpha} motif that was isolated from Th1 material was also identified in RT-PCR of Th2 cDNA. Thus, it is possible for Th2 clones to develop either with long or short CDR3{alpha} loops. If low-avidity T cell activation is a prerequisite for Th2 activation, Th2 cells may achieve this by other means, such as changes in accessory molecule interactions. However, no Th1 receptors were ever observed to use CDR3{alpha} loops longer than those found in the equivalent Th2 population. The expression of a TCR{alpha} chain with an elongated and sterically obstructive CDR3 loop on a Th1 cell may be incompatible with the quantitative and qualitative nature of signaling (for example mitogen-activated protein kinase activation) required to initiate transcription of Th1 cytokines. It is well established that restimulation of lines with Ag in vitro is similar to chronic stimulation in vivo, favoring the competitive outgrowth of clones of focused Ag specificity and TCR usage. Multiple clones carrying the same receptor have been viewed either as evidence of immunologically pertinent clonal expansion (48, 49) or of the fortuitous, repeated isolation of sister clones (25). The fact that the "sister clones," which thrive and predominate, have different features when a single pool is separated into Th1 and Th2 culture wells suggests that these preferential expansions have a structural relevance in the Th1/Th1 context.

Of the Th1, V{beta}13 clones, one-third carried both V{alpha}9J{alpha}11 and V{alpha}1J{alpha}A10 TCR{alpha} chains. The existence of dual TCR{alpha} T cells and their potential relevance to autoimmunity have previously been described (50). It is not possible to distinguish between TCR{alpha} sequences that encode the dominant, functional TCR expressed at the cell surface and a TCR{alpha} sequence that may be a subsidiary, "leaky" receptor, or indeed one that may be rearranged but never paired and expressed at the cell surface. However, because it is clear that we reproducibly observe a trend of different TCR{alpha} usage between Th1 and Th2 populations, this most likely reflects the behavior of the functional cell surface receptors.

Our data emphasize a role for TCR{alpha} sequence in the nature of the interaction with peptide/MHC and the determination of cytokine profile. There is a precedent for this, albeit with respect to CDR2{alpha} rather than CDR3{alpha} differences, in recent work from Janeway’s laboratory (18). It was demonstrated in D10 transgenic mice that mutation of a leucine to a serine at TCR{alpha} residue 51, which in transfection studies was associated with a 100-fold reduction in response, resulted in transgenic mice with skewing to Th2 responses among naive CD4 T cells. It was argued that a single peptide/MHC complex may be recognized by two different CD4 T cell clones, which, due to differences in avidity and outcome of activation, may differ in their potential to differentiate into either Th1 or Th2.

The TCR crystal structures that have been determined indicate an interaction between peptide/MHC and CDR3{alpha}, although it is perhaps hard to generalize about the relative contribution of V{alpha} and V{beta} CDR3s as all the crystal structures published to date are of relatively short V{alpha} CDR3s. Structural analyses of MHC class I/TCR recognition have indeed indicated the dominant role of the TCR{alpha} subunit in defining the basic docking geometry (51). Even minor changes within this part of the binding footprint appear more likely to have a dramatic effect on function than changes within the TCR{beta} binding region (52). Overall, our modeling highlights a potentially very significant impact on MHC class II binding arising from the difference in CDR3{alpha} length for Th1 and Th2 TCRs. The shorter Th1 loop appears unlikely to make extensive contact with the peptide, implying that other CDR loops must provide the interactions conferring the specificity to a particular MHC class II-restricted epitope. In contrast, the bulky Th2 loop may act as a wedge at the interface, imposing a shift in the MHC-TCR docking geometry.

It is notoriously difficult to predict the relative binding affinities of MHC-TCR complexes, even given detailed crystal structures (34). The energetic contributions of total buried surface, detailed shape, and charge complementarity are all of importance. Recent studies on the binding kinetics and thermodynamics of MHC-TCR recognition have also highlighted the particular importance of entropic penalties in this system (53), which are apparently attributable to the flexibility of the CDR3 loops in the unbound TCR (35). From the modeling, it appears likely that a long CDR3{alpha} loop in the Th2 TCRs precludes the other CDRs contributing optimally to the interface in the TCR-MHC complex, while the potentially greater flexibility of a long loop in the uncomplexed state would incur a higher entropic penalty on binding. Although such arguments cannot definitively point to a lower MHC class II binding affinity for Th2 verses Th1 TCRs, the structural analyses underscore the potential importance of loop length in modulating the characteristics of the functional binding.

Several groups have investigated the relationship between the strength of the peptide/MHC/TCR signal and the cytokine profile of the T cell response. Hosken and coworkers (12) showed that stimulation of naive TCR-transgenic cells with Ag at very high or very low concentrations favored the development of Th2 responses, with intermediate doses leading to a Th1 response. Where APL have been used to stimulate T cell responses, peptides with reduced affinity for the MHC class II molecule have generally been associated with Th2 responses (16, 46, 54). An exception to this is the model of experimental autoimmune encephalomyelitis induction by myelin basic protein Ac1–11 peptide in which the encephalitogenic peptide is of exceptionally low affinity for class II and induces a Th1 response, whereas the Ac1–11[4Y] substituted peptide binds with high affinity and induces a Th2 response (55). The apparent requirement in most models for a low-affinity interaction to generate Th2 responses does not necessarily extend to other ligand-receptor interactions between the T cell and APC. Th2 responses appear to have a greater CD4 dependency than Th1 as well as a greater dependence on a CD28/B7 interaction (56, 57). However, in both these cases, the differential requirement from the accessory molecule interaction is likely to be for costimulatory signaling rather than simply enhanced avidity (57).

TCR signaling studies show that interactions leading to Th2 cytokine production show features of incomplete triggering, including reduced ZAP 70 recruitment. We favor the hypothesis that under conditions where the extracellular environment is dictating the appropriateness of mounting a Th2 response, clones for which the avidity of the T cell/APC interaction is minimized are preferred. In the present case, this may be inferred from modeling to be by selection for a bulky CDR3{alpha} wedge, precluding optimal contact at other CDR/peptide-MHC interfaces. Reduced expression of accessory molecules could also contribute to this effect. The data are compatible with a model in which Th2 responses preferentially ensue from interactions involving impeded serial triggering.


   Acknowledgments
 
We thank L. Wedderburn for advice.


   Footnotes
 
1 Part of this work was supported by the Multiple Sclerosis Society of Great Britain and Northern Ireland. R.J.B. is funded by the Wellcome Trust. Back

2 Address correspondence and reprint requests to Dr. Daniel M. Altmann, Human Disease Immunogenetics Group, Department of Infectious Diseases, Imperial College of Science, Technology, and Medicine, Hammersmith Hospital, London, U.K. E-mail address: d.altmann{at}ic.ac.uk Back

3 Abbreviations used in this paper: APL, altered peptide ligand; CDR, complementarity-determining region; NOD, nonobese diabetic; NOD.E, H2-E transgenic NOD; Hsp, heat shock protein; PLP, proteolipoprotein; GAD, glutamic acid decarboxylase. Back

Received for publication December 12, 2000. Accepted for publication November 13, 2001.


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