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Molecular Immunology 53 (2013) 283– 294

Contents lists available at SciVerse ScienceDirect

Molecular Immunology

jo u rn al hom epa ge: www.elsev ier .com/ locate /mol imm

ubtle changes in TCR� CDR1 profoundly increase the sensitivity of CD4 T cells

ennifer N. Lyncha, David L. Donermeyera, K. Scott Webera, David M. Kranzb, Paul M. Allena,∗

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, United StatesDepartment of Biochemistry, University of Illinois, Urbana, IL 61801, United States

r t i c l e i n f o

rticle history:eceived 28 June 2012eceived in revised form 15 August 2012ccepted 18 August 2012

eywords:nergy

cellDR1

a b s t r a c t

Changes in the peptide and MHC molecules have been extensively examined for how they alter T cellactivation, but many fewer studies have examined the TCR. Structural studies of how TCR differencesalter T cell specificity have focused on broad variation in the CDR3 loops. However, changes in the CDR1and 2 loops can also alter TCR recognition of pMHC. In this study we focus on two mutations in theCDR1� loop of the TCR that increased the affinity of a TCR for agonist Hb(64-76)/I-Ek by increasing theon-rate of the reaction. These same mutations also conferred broader recognition of altered peptideligands. TCR transgenic mice expressing the CDR1� mutations had altered thymic selection, as most ofthe T cells were negatively selected compared to T cells expressing the wildtype TCR. The few T cells that

D4ffinityelection

escaped negative selection and were found in the periphery were rendered anergic, thereby avoidingautoimmunity. T cells with the CDR1� mutations were completely deleted in the presence of Hb(64-76)as an endogenous peptide. Interestingly, the wildtype T cells were not eliminated, identifying a thresholdaffinity for negative selection where a 3-fold increase in affinity is the difference between incomplete andcomplete deletion. Overall, these studies highlight how small changes in the TCR can increase the affinity

e con

of TCR:pMHC but with th

. Introduction

TCR:pMHC complex formation needs to exceed a specific bind-ng energy to achieve a T cell response (Davis-Harrison et al., 2007;akamsky et al., 2004; Garcia et al., 2001; Holler and Kranz, 2003;rogsgaard et al., 2003). Structural changes in the TCR are oftenequired to form a stable TCR:pMHC complex, but it is not clear ifuch conformational changes are necessary for productive signal-ng through the TCR (Borg et al., 2005; Burrows et al., 2010; Davist al., 1998; Holler and Kranz, 2004; Qi et al., 2006). It has long beennown that the variable CDR loops form the binding footprint forCR to contact the pMHC complex (Garcia et al., 1999; Huseby et al.,006). More recently it has been proposed that the TCR maintainsermline-encoded affinity for MHC by interactions between key

esidues on the MHC helices and CDRs 1 and 2 (Adams et al., 2011;arrack et al., 2008). CDRs 1 and 2 differ in the V gene segments,

nd it has been shown that introduction of mutations in these CDR

Abbreviations: APL, altered peptide ligand; CAb, clonotypic Ab for n3.L2 TCR; DN,ouble negative thymocytes; DP, double positive thymocytes; Hb, hemoglobin; koff,ff-rate constant; kon, on-rate constant; MHCII, MHC class II; pMHC, peptide MHComplex; scTCR, single chain TCR; SP, single positive thymocytes; t1/2, half-life; Treg,egulatory T cell.∗ Corresponding author at: Department of Pathology and Immunology, Washing-

on University in St Louis, 660 South Euclid, Campus Box 8118, St. Louis, MO 63110,nited States. Tel.: +1 314 362 8758; fax: +1 314 362 3939.

E-mail address: pallen@wustl.edu (P.M. Allen).

161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.molimm.2012.08.020

sequences of skewing selection and producing an unresponsive T cell.© 2012 Elsevier Ltd. All rights reserved.

loops can result in generation of high affinity TCRs (Chlewicki et al.,2005; Manning et al., 1999), potentially as a consequence of an opti-mal binding conformation or enhanced TCR:pMHC stability (Adamset al., 2011; Burrows et al., 2010; Dai et al., 2008; Holler and Kranz,2004; Willcox et al., 1999).

The TCR has inherent specificity for an agonist peptide but canretain binding to some variants of the agonist (Kersh and Allen,1996). The strength of recognition of an altered peptide ligand(APL) regulates the level of T cell response (Evavold et al., 1992).Some TCR flexibility is critical during T cell development in thethymus as it ensures recognition of foreign antigens during aninfection in addition to self peptides during selection. While flexiblerecognition of pMHC is advantageous, highly promiscuous T cellscan inappropriately recognize self-peptides and cause autoimmunedisease (Basu et al., 2001; Garcia et al., 2001). Therefore, it remainsimportant to understand the process by which a T cell discrimi-nates between peptides to generate a productive and appropriateimmune response.

There is an affinity continuum of a TCR for endogenous pMHCthat regulates selection of T cells in the thymus (Daniels et al., 2006).For T cells to be selected, they must have sufficient affinity forendogenous pMHC above the threshold for positive selection butbelow the threshold for negative selection (Hogquist and Bevan,

1996; Kosmrlj et al., 2008). A complex set of distinct signals reg-ulates positive and negative selection (Alberola-Ila et al., 1996;Gascoigne and Palmer, 2011), tuning T cell responsiveness in theperiphery. Negative selection results in apoptosis of T cells that

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re highly responsive to self pMHC in the process of central toler-nce (Klein et al., 2009; Williams et al., 1999). While the majorityf self-reactive T cells are deleted to generate central tolerance,t is thought that regulatory T cells can develop from these self-eactive, high affinity TCRs (Yu et al., 2008). Treg development isot merely a consequence of a high affinity TCR, as specific envi-onmental cues and TCR sequences may also be required (Bautistat al., 2009; Lathrop et al., 2008).

Negative selection is an incomplete process and some highffinity T cells escape to the periphery where they can causeutoimmune disease (Zehn and Bevan, 2006). While failure to elim-nate these cells breaks mechanisms of central tolerance, a back upystem is in place to prevent autoimmunity. Potentially autoreac-ive T cells can be rendered unresponsive to antigenic stimulationDe Boer et al., 2003) or deleted in the periphery to establish periph-ral tolerance (Williams et al., 1999). Anergy, a state of hypore-ponsiveness characterized by low IL-2 production and inhibitedroliferation (De Boer et al., 2003), has been induced in naive Tells through lack of costimulation (Schwartz, 1996), and exposureo APLs (Evavold et al., 1993; Klein et al., 2009; Sloan-Lancastert al., 1993). Anergized T cells downregulate TCR and costimula-ory receptors to maintain the hyporeactive state. Because of theual role of the TCR:pMHC interaction in selection and activation,he propensity for T cells to undergo tolerance may be set in thehymus by the affinity of the expressed TCR for self pMHC.

To understand how slight structural changes affect T cell devel-pment and sensitivity, we used a system that compares twoCRs recognizing the same cognate antigen, Hb(64-76)/I-Ek. In theouse, the Hb protein exists in two naturally occurring allelic

ariants, Hbd and Hbs, such that T cells can develop normally inbs strains and be reactive to cells from Hbd mice. The n3.L2CR was generated against the Hbd allele of the (64-76) peptideEvavold et al., 1992). We previously generated a series of mutantsf the n3.L2 TCR using a yeast display system (Weber et al., 2005).hese mutants were selected for increased surface stability oneast or increased binding to Hb(64-76)/I-Ek. Mutations selectedor increased affinity to Hb(64-76)/I-Ek were generated in the CDR3f the n3.L2 TCR. Surprisingly, one mutant, called M2, which hadwo mutations in the CDR1� loop (K25E and T28S), was selectednly for increased surface levels on yeast and not for increased affin-ty, yet it exhibited an increase in affinity. Here we verify that thewo CDR1� mutations in M2 resulted in a 3.7 stronger affinity dueo a faster on-rate, as measured by surface plasmon resonance. Weenerated and used hybridomas and transgenic mice expressinghe n3.L2 and M2 TCRs to determine how such a moderate changen the CDR1� loop could alter TCR specificity. The M2 TCR has aroader and more sensitive response to altered peptide ligands ofhe Hb(64-76) peptide. In addition, the M2 TCR has a stronger asso-iation with the I-Ek molecule, relying solely on contacts with theHCII� chain for pMHC recognition. As a consequence of these

hanges, T cell selection in the thymus was also altered. M2 T cellsere negatively selected at a higher rate and completely eliminatedhen exposed to Hbd(64-76) as self antigen. Interestingly, the feweripheral M2 T cells that escaped negative selection were ren-ered anergic. These studies show how subtle changes in the TCRtructure that modestly increase the TCR:pMHC affinity can have arofound effect on T cell development and activation.

. Materials and methods

.1. Mice and cells

In addition to the new mouse line described below, n3.L2/B6.KCR transgenic mice, B6.K, Rag1−/−, and Hbd congenic mice weresed in these studies. The n3.L2/B6.K mouse was previously gen-rated in the laboratory (Kersh et al., 1998) and was crossed to

nology 53 (2013) 283– 294

the Rag1−/− and separately to the Hbd mice for the studies pre-sented in this manuscript. All mice were bred and maintained ina pathogen-free barrier facility within Washington University inSt Louis following a protocol approved by and in accordance withguidelines from the Washington University Division of Compara-tive Medicine.

In addition to the new hybridoma cell lines described below,CH27 B cells were used to as antigen presentation cells insome of the hybridoma response experiments. CH27 cells weremaintained in RPMI + 10% FCS + 1% l-glutamine + 5 × 10−5 M �-2-mercaptoethanol + 0.5% gentamicin at 37 ◦C and 5% CO2.

2.2. Generation of the M2 TCR transgenic mouse line

The M2 mouse was generated using the method described forthe n3.L2 mouse (Kersh et al., 1998). The n3.L2 V-J� plasmid wasmutated by PCR to express the two amino acid changes in the M2CDR1� chain. The M2� chain was cloned into the TCR shuttle vec-tor. TCR� and � minigene constructs were coinjected into C57Bl/6pronuclei in the Washington University Department of Pathologyand Immunology’s Transgenic Core Facility. Transgenic mice wereidentified by PCR amplification of the M2� and � transgenes fromtail DNA. Expression of the M2� chain was confirmed by sequencingthe founders’ genomic DNA. One founder expressed both the M2�and � transgenes and was bred to the B6.K strain to provide theselecting MHC. Peripheral CD4 T cells in the M2/B6.K mouse stainedwith the clonotypic antibody, CAb. M2 and n3L.2 mice were furthercrossed to a Rag-1−/− background producing n3.L2/B.6K/Rag1−/−

and M2/B6.K/Rag1−/− mice. M2/B6.K mice were also crossed to Hbd

congenic mice. Mice were used at 4–8 weeks of age in these stud-ies, unless otherwise noted. All mice were bred and maintained ina pathogen-free barrier facility within Washington University inSt Louis following a protocol approved by and in accordance withguidelines from the Washington University Division of Compara-tive Medicine.

2.3. Flow cytometry

Antibodies used in flow cytometry are commercially availableexcept for the CAb antibody, which was previously generated inthe laboratory. CAb is a clonotypic antibody for the n3.L2 TCR. CAbwas conjugated with AlexaFluor-647 using an antibody conjugationkit (Invitrogen). FITC-, PE-, PE-Cy7-, PerCP-, APC-, APC-Cy7-, PacificBlue-, and Pacific Orange-labeled antibodies were used in vari-ous combinations. Intracellular labeling of FoxP3 was performedusing a kit from eBioscience. Cells were permeabilized and fixed for30 min followed by washing with permeabilization buffer and anti-body labeling with PE- anti-FoxP3. Data collection was performedusing a BD FACSCalibur, a FACSCanto, and a customized FACSLSR II.Data analysis was performed using FlowJo software.

2.4. Generation of n3.L2 and M2 hybridomas

n3.L2 and M2 TCR� and � chains were cloned into a p2A retro-viral vector with an IRES-GFP tag (pMIIG) developed by the Vignalilab (Holst et al., 2006), which places the � and � chains as a sin-gle polypeptide linked by the p2A peptide. No stability mutationswere added into the sequence. The 2A peptide (p2A) is cleaved post-translationally, ensuring equal expression of the � and � chains andresulting in efficient expression of transduced �� TCRs. PlatE cellswere transformed with lipofectamine + 30 �g plasmid DNA. Super-

natants from PlatE cells containing packaged retrovirus were usedto spinfect the 58 �−�− CD4+ hybridoma cell line. M2 and n3.L2expressing hybridomas were generated simultaneously, sorted forcomparable high GFP expression, and equal expression of the n3.L2

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nd M2 TCRs. Cells were sorted a second time to generate a pop-lation with stable TCR expression. Equivalent expression levelsf CD3, CD4, and TCR between n3.L2 and M2 hybridomas wereonfirmed by flow cytometry. Surface TCR levels were assessedith CAb, V�8.3, and H57. Hybridomas were cultured in RPMI + 10%

CS + 1% l-glutamine + 5 × 10−5M �-2-mercaptoethanol + 0.5% gen-amicin at 37 ◦C and 5% CO2.

.5. T cell IL-2 production and proliferation assays

To assess T cell hybridoma responses, 5 × 105 hybridomas wereultured overnight at 37 ◦C 5% CO2 in wells of a 96-well plateith 2 × 104 CH27 B cell APCs and varying concentrations of

he Hb(64-76) peptide or APLs from 0.0001 to 100 �M. Peptidesere synthesized in the lab and purified by HPLC prior to use.

8–20 h after culture, supernatants were assayed using an IL-2LISA. Briefly, IL-2 was captured using an anti-IL-2 antibody (BioX-ell) and detected with a second, biotinylated anti-IL-2 antibodyBioLegend). Biotin-labeled IL-2 was detected by a streptavidinonjugated horseradish peroxidase antibody (Southern Biotech)ollowed by TMB substrate for accurate detection of 5–500 pg/mLf IL-2 (1-step Ultra TMB ELISA, Thermo Scientific). After stop-ing the reaction with 2 M sulfuric acid, absorbance was read at50 nm. Absorbance levels were converted into amount of IL-2ased on a regression analysis of IL-2 standards using Graph-ad Prism (GraphPad Software). For the APL studies, the amountf peptide required for 50% maximal IL-2 production (EC50) wasalculated to determine relative levels of activation. For otherxperiments, the maximal IL-2 production and minimum stimu-atory dose were compared.

For isolated T cell stimulation, single cell suspensions wereade from pooled lymph nodes and spleen from transgenic mice.

D4+ cells were isolated by negative selection using Miltenyi CD4I isolation kit (Miltenyi Biotec). Non-CD4+ cells were depleted byinding to a Miltenyi AutoMACS column. Purity was tested by flowytometry analysis for percent CD4+ CAb+. To account for minorariations in the purity between n3.L2 and M2 isolation, cell num-ers were normalized to the percent CD4+ CAb+ cells to ensure theame number of specific CD4 cells were plated in each well of theL-2 or proliferation assays. 1 × 105 specific CD4 cells were platedn IMDM + 10% FCS with 5 × 105 irradiated B6.K splenocytes andarying concentrations of peptides. IL-2 production was measureds described. To measure proliferation, CD4 T cells were isolatednd cultured as in the IL-2 production assay. After 72 h in culturet 37 ◦C 5% CO2, each well was pulsed with 0.4 �Ci 3H thymidinend maintained in culture for an additional 24 h. Plates were thenarvested and measured for 3H thymidine incorporation.

.6. I-Ek Ig dimers

I-Ek dimers were produced in Drosophila S2 cells, as previ-usly described (Masteller et al., 2003). The I-Ek dimers have someeakly bound peptide that can easily be exchanged in vitro making

hem ideal for stimulation of T cells with specific pMHC com-lexes. Amino acid residues chosen for mutagenesis were locatedn the top of the MHCII � and � helices as potential TCR con-act residues. To generate mutant I-Ek dimers, mutations werentroduced into I-Ek constructs at one of 4 MHCII� and 3 MHCII�esidues chosen from a subset of mutants expressed in CHO cellsFelix et al., 2007). Wildtype and mutant I-Ek-Ig dimer constructsere transfected into Drosophila S2 cells. Dimers were isolated

rom culture supernatants by binding to Protein A. Dimers were

xposed to acidic pH to remove the endogenous, weakly bindingeptides and maintained in excess amounts of soluble peptide toubstitute the desired peptide into the peptide binding groove. Tossay the TCR:pMHC binding footprint, 96 well plates were coated

nology 53 (2013) 283– 294 285

overnight with Hb(64-76)-loaded I-Ek Ig dimers. After 20 h, plateswere washed to remove unbound dimer, hybridomas were added,and activation was assayed using IL-2 production as describedabove.

2.7. Surface plasmon resonance

We used established lab protocols to measure binding affinitiesfor n3.L2 and M2 single chain TCR (scTCR) to Hb(64-76)/I-Ek (Weberet al., 2005). 2500–3000 response units of Hb(64-76)-loaded I-Ek Igdimers were directly coupled to a CM5 sensor chip by amine cou-pling. Previously, refolded Hb(64-76)/I-Ek was generated from E.coli inclusion bodies for use in surface plasmon resonance studies.Both ligands were tested in this system and the affinity measure-ments were the same using either the refolded monomer or Igdimer and maintained a 1:1 TCR:MHC binding ratio ((Persaud et al.,2010), data not shown). Data presented are based on measurementsobtained using only peptide loaded I-Ek dimers. Refolded, solublesingle chain n3.L2 or M2 TCR (V�-linker-V�) (Holst et al., 2006;Shusta et al., 1999) was purified by fast protein liquid chromatog-raphy (FPLC), concentrated in PBS, and injected over flow cells withcoupled I-Ek at a rate of 30 �L/min. scTCR was injected in dupli-cate at increasing concentrations from 0 to 100 �M at 25 ◦C. Mothcytochrome C peptide loaded I-Ek was used as a negative controlfor binding. Moth cytochrome C sensograms were subtracted fromexperimental Hb/I-Ek sensograms to eliminate nonspecific bind-ing artifacts. Measurements were performed using a Biacore 2000.BiaEval version 4.1 software (Biacore AB) was used to generate1:1 Langmuir models of sensograms to determine KD, koff and kon.The Langmuir model was adjusted until a Chi2 value below 50 wasobtained, indicating the best approximation of data. KD values wereconfirmed by Scatchard analysis using GraphPad Prism (GraphPadSoftware). Statistical significance was measured by Student’s t testfor differences between n3.L2 and M2 parameters.

3. Results

3.1. CDR1 ̨ mutations increase the affinity of TCR:pMHC througha faster kon

The n3.L2 TCR is specific for the Hbd(64-76) peptide presentedon the I-Ek MHC class II molecule (Evavold et al., 1992). Previ-ously, the n3.L2 receptor was mutagenized using a yeast displaysystem to generate a series of higher affinity mutants (Weber et al.,2005). Mutants were isolated for enhanced stability measured byincreased surface levels on yeast. One TCR mutant, M2, containedtwo point mutations in the CDR1� chain (K25E and T28S). Althoughit had not been selected for higher affinity binding to Hb(64-76)/I-Ek, M2 was shown to have several fold improved affinity overthe n3.L2 TCR. Soluble single chain TCR molecules (V�-linker-V�;scTCR) were generated for the n3.L2 and M2 T cell receptors (Holstet al., 2006), containing several additional stabilizing mutationsin framework regions (Shusta et al., 1999). These scTCR stabiliz-ing mutations were needed to produce the soluble scTCR, but notfor expression of the mutants in T cells (Persaud et al., 2010). ThescTCRs were used previously to measure binding affinity of theseries of mutants to Hb(64-76)/I-Ek by surface plasmon resonance.To confirm and extend these studies, we performed similar sur-face plasmon resonance studies. n3.L2 had an affinity of 16.6 �Mfor Hb(64-76)/I-Ek (Fig. 1A and B). M2 had a 3.7-fold higher affin-ity for Hb(64-76)/I-Ek (4.3 �M) due to an equivalent change in kon,without a significant change in koff (Fig. 1A and B). Sensograms of

increasing concentrations of scTCR up to 100 �M were modeled fora 1:1 binding ratio to determine kinetic measurements of n3.L2 andM2 scTCR binding to Hb(64-76)/I-Ek dimers (Fig. 1C). The KD val-ues were confirmed by Scatchard analysis (Fig. 1D). These kinetic

286 J.N. Lynch et al. / Molecular Immunology 53 (2013) 283– 294

Fig. 1. M2 TCR has higher affinity for Hb(64-76) due to a change in kon. A and B. Kinetics of n3.L2, M2, K25E, and T28S were measured by surface plasmon resonance usingscTCRs binding to Hb(64-76)/I-Ek dimers. Values in arrows indicate fold change above n3.L2 for kon measurements. Measurements of n3.L2 and M2 kinetics are averages of5 independent experiments on separate CM5 chips. Measurements of K25E and T28S kinetics are averages of 3 independent experiments on separate CM5 chips. Statisticald re sho k

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ifferences were determined using Student’s t test. C. Representative sensograms ainetic constants were calculated by fitting sensograms to a 1:1 Langmuir bindincatchard plots of n3.L2 and M2 to confirm KD values predicted from 1:1 modeling.

alues were consistent with what was originally reported for n3.L2nd M2 (Weber et al., 2005). Therefore, the M2 TCR has an increasedffinity for Hb(64-76)/I-Ek as the result of two CDR1� mutations.

We next wanted to identify the individual contribution ofhe two amino acid differences between n3.L2 and M2. We werenterested to determine if one residue had a dominant effectn increasing n3.L2’s affinity for Hb(64-76)/I-Ek or if the twoutations were synergistic. Somewhat unexpectedly, the conser-

ative substitution of a threonine to a serine at position 28 (T28S)ncreased the affinity of the TCR by 1.9-fold over n3.L2, specifically

y changing the kon (Fig. 1A and B). The non-conservative singleubstitution of lysine to glutamic acid at position 25 (K25E) main-ained the wildtype n3.L2 affinity of the TCR for Hb(64-76)/I-Ek

Fig. 1A and B). Although the single substitution at position 25 did

wn for scTCR injections over bound I-E Ig dimer at the indicated concentrations.el. D. Equilibrium responses at each scTCR concentration were used to generate

esponse was highly linear, with r2 values of 0.8.

not have an effect on affinity, it must act cooperatively with thesubstitution at position 28 to achieve the 3.7-fold increase in affin-ity. The n3.L2 and M2 residues at position 28 are highly conservedin many TCRs with published crystal structures, suggesting theimportance of this residue in TCR stability and/or MHC interactions(Manning et al., 1999; Nalefski et al., 1990).

3.2. M2 and n3.L2 hybridomas have the same response toHb(64-76)/I-Ek

The M2 TCR had not been previously tested functionally.Because the M2 mutations altered the affinity of the TCR forpMHCII, we predicted M2 T cells would differ in their response(sensitivity or specificity) to the Hb(64-76) peptide in comparison

J.N. Lynch et al. / Molecular Immunology 53 (2013) 283– 294 287

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Fig. 3. Binding of n3.L2 and M2 TCRs to Hb(64-76)/I-Ek dimers. A. Plate bound Hb-loaded mutant I-Ek dimers were used to stimulate IL-2 production by n3.L2 andM2 hybridomas. We tested the response to 4 mutations in the I-Ek � chain and 3mutations in the I-Ek � chain. MHC residues were considered critical if mutationat that position inhibited >90% of IL-2 production (highlighted by the dashed line).Representative plot of mean + S.D. IL-2 production normalized to wildtype levels.B. Critical contact residues are highlighted by red circles on a surface model of thecrystal structure of Hb bound to I-Ek (Protein DataBank accession code, 1FNG) toidentify the TCR binding footprint. The MHCII� mutation at position 57 was notcritical for IL-2 production by n3.L2 or M2 and is not indicated on the model. Green

y n3.L2 (closed square) and M2 (open triangle) hybridomas in response to wild-ype Hb(64-76). Hybridomas were cultured in triplicate for 18–20 h with peptideoaded CH27 APCs. Mean + SEM is presented for triplicates at each concentration inhe dose curve, which is representative of 6 independent experiments.

ith n3.L2 T cells. To measure the sensitivity of n3.L2 and M2 TCRso Hb(64-76)/I-Ek, we generated T cell hybridomas expressingither the n3.L2 or M2 TCR. Sorted n3.L2 and M2 hybridomasxpressed equivalent levels of the n3.L2 or M2 TCR and CD4Supplemental Fig. 1). Hybridomas were stimulated by CH27 cellsulsed with varying doses of purified Hb(64-76) peptide. The n3.L2nd M2 response to agonist stimulation was measured by IL-2roduction. The response curves for the n3.L2 and M2 hybridomasere the same, with the same maximal response and sensitivity

o the Hb(64-76) peptide (Fig. 2). A similar result was obtained for related, higher affinity TCR, M15 (Weber et al., 2005). Therefore,he M2 TCR is fully functional and equivalent to the n3.L2 TCRn its ability to recognize and be stimulated by Hb(64-76)/I-Ek

ndependent of effects from development.

.3. CDR1 ̨ of M2 stabilizes the TCR to maintain activationespite mutations in the MHCII˛ chain

We wanted to begin to understand the structural basis for howubtle changes in the CDR1� loop could alter TCR recognition ofMHC. We used a set of mutant I-Ek dimers, each containing a singlemino acid substitution at a potential TCR contact site, to measurehanges in the TCR:pMHCII energetic footprint. Mutant I-Ek dimersere loaded with Hb(64-76) peptide and used to stimulate IL-2roduction by the n3.L2 and M2 hybridomas. Mutations that com-letely inhibited IL-2 production (> 90% reduction) were consideredritical in contributing to the energy required for TCR stimulation byMHCII. All 3 mutations in the MHCII� chain completely inhibited

L-2 secretion for both n3.L2 and M2 hybridomas (Fig. 3A and B)uggesting interactions with the MHCII� are highly specific andritical for stimulation through the TCR:pMHCII complex. The n3.L2ybridomas failed to recognize �53, �55, and �61 mutations, indi-ating these MHCII� residues are critical for the n3.L2 interactionFig. 3A and B). Interestingly, M2 hybridomas maintained stimula-ion by all MHCII� chain mutants except �61, which was criticalor both TCRs (Fig. 3A). The CDR1� loop may stabilize the M2 TCRnto an optimized conformation for MHCII recognition. It is unclear

hether the enhanced recognition of MHC dictated by CDR1� isirectly through generation of new contacts or through enhanced

nteractions with key amino acid residues.

.4. n3.L2 and M2 hybridomas differ in response to Hb(64-76)

PLs

Subtle changes in peptide and MHC sequence can alter a T cellesponse. We wanted to understand how moderate changes in the

ovals represent the general contact area for TCR CDR1� loops, where the two aminoacid differences between n3.L2 and M2 are located. Summarized results are basedon 3 independent experiments.

TCR could also affect specificity. We examined TCR specificity forpeptide by testing the ability of Hb APLs to stimulate the n3.L2 andM2 hybridomas. A panel of 76 peptides was generated by singlysubstituting each amino acid at the 4 TCR contact residues andeach APL was tested for the ability to stimulate IL-2 production.Of the 76 APLs, 13 APLs stimulated IL-2 production by the n3.L2hybridomas (Fig. 4A), whereas, 25 of the 76 peptides were agonistsfor M2 hybridomas. M2 recognized changes at each of the 4 TCRcontact residues. Additionally, M2 hybridomas were more sensi-tive, with a lower EC50 value for IL-2 production, to APLs that wereagonistic for both n3.L2 and M2 hybridomas (Fig. 4B). For exam-ple, the M69 APL stimulated both n3.L2 and M2 hybridomas. TheEC50 for M2 was 5 �M with a maximal IL-2 production at 350 pg/mL(Fig. 4B, left). M69 was able to stimulate n3.L2 hybridomas at con-centrations as low as 3.1 �M but did not produce maximal IL-2at 100 �M, the highest concentration testable. A similar differencewas seen with the D70 APL. M2 hybridomas produced more IL-2in response to D70 but the sensitivity of the two hybridomas wassimilar (Fig. 4B, right). Thus, the M2 CDR1� structural changes ledto enhanced recognition of a broader number of APLs.

3.5. Generation of M2/B6.K/Rag1−/− TCR transgenic mice

T cell hybridomas are immortalized cell lines and only requireTCR signals for activation. We wanted to determine how changes

in the TCR also affected T cell development and normal function.Therefore, we generated an M2 TCR transgenic mouse. The n3.L2TCR transgenic mouse has previously been described (Kersh et al.,1998). The M2 mouse was generated by coinjection of TCR � and �

288 J.N. Lynch et al. / Molecular Immunology 53 (2013) 283– 294

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cetetsm(

iwCiCh(tt

eptide (P2, P3, P5 and P8). Wildtype Hb(64-76) residues are noted under the labelriplicate up to 100 �M in at least 3 independent experiments. B. Stimulation of ILright) as examples of differences in APL response. Mean + SEM is presented for the

hains onto the B6 background. One founder was generated that co-xpressed the M2� and � chains. The M2�� founder was crossedo the B6.K/Rag1−/− background to provide the proper selectingnvironment for M2 T cells and eliminate any potential complica-ions from secondary TCR� rearrangements. The n3.L2 mouse wasimultaneously crossed to a B6.K/Rag1−/− background and the thy-ocytes and T cells of both mice were analyzed by flow cytometry

Fig. 5).Mature M2 Rag1−/− CD4+ T cells developed and were present

n the periphery of the M2 transgenic mouse (Fig. 5A). These cellsere completely mature and expressed high levels of TCR, CD3, andD4 (Fig. 5B). However, the CD4+ cell number was greatly reduced

n the M2 mouse in comparison to n3.L2 (Fig. 5A). The decreasedD4 population suggested a difference in T cell selection. M2 mice

ad dramatically reduced thymus cellularity in comparison to n3.L2Fig. 5C), also indicative of a difference in T cell selection. The M2hymus had an increased proportion of DN cells (Fig. 5C), arrested athe DN3 stage of development (Fig. 5D). While this block indicated

ch position. APLs that induced IL-2 production are shaded. Each APL was tested induction by n3.L2 (closed square) and M2 (open triangle) with M69 (left) and D70se curve which is representative of 4 independent experiments.

inefficient development of the M2 TCR, the transgenic V�8.3 washighly expressed on a small population at the same level as in then3.L2 mouse (Fig. 5E). The same TCR� chain was expressed on alarger population in the n3.L2 Rag1−/− mice (Fig. 5E), suggestingthe TCR� transgene was not efficiently expressed in the M2 Rag1−/−

mouse, potentially as a consequence of a transgene affect.Despite the inefficient development of the M2 T cells, the DN

population that did express V�8.3 developed into DP, SP, andperipheral T cells, permitting further analysis of those popula-tions. In the thymus, both the M2 and n3.L2 mice had a matureCD4 SP population (Fig. 5C). The CD4 SP populations expressed thesame levels of TCR, CD4, CD3 and maturation markers in the thy-mus and periphery (Fig. 5B and F). Very few Treg cells developedfrom the transgenic T cells either in the thymus or by peripheral

conversion (Supplemental Fig. 2A and B). Therefore, M2 CD4+ Tcells developed and persisted in the periphery in M2/B6.K/Rag1−/−

mice. While these cells appeared to be phenotypically nor-mal, we predicted the consequences of changes in selection in

J.N. Lynch et al. / Molecular Immunology 53 (2013) 283– 294 289

Fig. 5. Comparison of the n3.L2 and M2 Rag1−/− TCR transgenic mice. A. Flow cytometry analysis of the transgenic CD4 population in the spleens of age matched n3.L2 (left)and M2 (right) transgenic mice. Plots were gated on live lymphocytes. The percentage of specific transgenic CD4 (CAb+ CD4+) cells in the lymphocyte population is notedon each dot plot. Representative plots from 10 mice. Flow cytometry analysis was used to calculate the total number of CD4+ cells from the spleens of n3.L2 and M2 mice.n = 10 mice for each group. P values determined by Student’s t test. B. Histogram overlays showing TCR, CD3 and CD4 levels on the gated CAb+ CD4+ population in panel Afrom n3.L2 (black line) and M2 (filled histogram) TCR transgenic mice. C. Labeling of representative thymus from age matched n3.L2 (left) and M2 (right) transgenic mice forCD4 and CD8 expression. Percentage of DN, DP, CD4 and CD8 SP populations are listed. Bar graph indicates total thymus size for mice aged 4–6 weeks. P < 0.0001 measuredby Student’s t test. n = 10 mice for each group. D. Analysis of the DN population from the thymus plot and gating shown in panel C. E. Intracellular staining for the transgenicV�8.3 TCR � chain in n3.L2 (black) and M2 (filled) DN cells. Representative plots from 5 mice in 3 independent experiments. F. Overlays of TCR (CAb), CD3, and CD4 levelson gated n3.L2 (black line) and M2 (filled histogram) CD4 SP populations from panel C.

290 J.N. Lynch et al. / Molecular Immunology 53 (2013) 283– 294

Fig. 6. Selection of n3.L2 and M2 T cells by endogenous Hbd(64-76). A. n3.L2 and M2 Rag+/+ transgenic mice were crossed to congenic Hbd mice, which express the agonistH r presn 6 n3.Lc by del verla

ta

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bd(64-76) as a self peptide. Thymi and spleens from the crosses were assessed fo3.L2 × Hbd, M2 × Hbd, and transgenic negative Hbd littermate mice, aged 6 weeks.ells. B. n3.L2 × Hbd and M2 × Hbd mice were monitored for development of anemiaine) and M2 × Hbd (filled histogram) mice was labeled with a PE-pan Ig antibody. O

he thymus would alter CD4 function in response to cognatentigen.

.6. M2 T cells are deleted when exposed to Hb(64-76) as anndogenous peptide

The Hb model, involving two allelic forms of the antigen,fforded us the possibility to determine thymic selection changesn response to Hb(64-76) as an endogenous ligand. A strong signaluring T cell selection can result in deletion of T cells by negativeelection (Mirshahidi et al., 2004; Williams et al., 1999). We pro-osed that the slight increased affinity of the M2 TCR for Hb(64-76)ould result in enhanced negative selection, despite the identi-

al in vitro responses of n3.L2 and M2 to Hb(64-76). As a control,either T cell was reactive to the Hbs(64-76) peptide (data nothown). A B6.K/Hbd congenic line was bred to the Rag sufficient3.L2 and M2 TCR transgenic mice. Due to the high level of ago-ist antigen present in these mice, we predicted that n3.L2 and2 T cells would be deleted by negative selection. Surprisingly, a

mall number of n3.L2 CD4+ T cells were present in the peripheryf the n3.L2 × Hbd mice (Fig. 6A). While some specific T cells wereeleted due to recognition of Hbd(64-76) as self peptide, the n3.L2ffinity for Hbd(64-76) was apparently not above the threshold for

omplete negative selection. By 4 weeks of age, these mice devel-ped autoimmune hemolytic anemia as measured by the presencef anti RBC antibodies (Fig. 6B). In the n3.L2 × Hbd mice, we suggesthat the small population of Hb specific T cells induce production of

ence of CAb+ CD4+ cells. Percent CAb+ CD4+ is listed for representative n3.L2, M2,2 × Hbd and 6 M2 × Hbd mice were characterized for development of transgenic Tposition of antibodies on RBC. Peripheral blood from 5 week old n3.L2 × Hbd (blackys show levels of anti-Ig on gated RBCs for 3 n3.L2 × Hbd and 3 M2 × Hbd mice.

anti-Hbd antibodies by B cells in the spleen. The Rag1+/+ transgenicmice were used for Hbd crosses to preserve the B cell populationrequired to induce the autoimmune response against endogenousHbd(64-76). Anti-RBC antibodies are then deposited on red bloodcells resulting in their lysis and the mice develop autoimmunehemolytic anemia. We are then able to use antibody labeling ofRBCs as a readout for the onset of anemia.

In contrast to the n3.L2 × Hbd mice, the M2 × Hbd mice com-pletely lacked M2 specific cells in the periphery, though CAb+

cells were found in the thymus (Fig. 6A). M2 × Hbd mice failedto develop anemia due to complete elimination of the M2 pop-ulation (Fig. 6B), since the percentage of CD4 cells found in theperiphery of M2 × Hbd mice was not different from non-transgeniccontrols (Fig. 6A). The M2 × Hbd mice did not develop hemolyticanemia up to 10 weeks of age. The increased affinity of the M2 TCRfor Hbd leads to elimination of M2 cells by negative selection as aconsequence of stronger selection signals. These findings demon-strate the sensitivity of negative selection to minor changes in theTCR:pMHC interaction, and establish a threshold for complete neg-ative selection.

3.7. M2 Rag1−/− CD4 T cells are hyporesponsive to Hb(64-76) inthe periphery

The immune system has developed multiple mechanisms toensure self tolerance in the periphery in order to prevent autoim-munity. High affinity T cells that escape negative selection can

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Fig. 7. M2 CD4 T cells are hyporesponsive to Hb(64-76). A. Rag1−/− CAb+ CD4+ cellsisolated from n3.L2 (closed squares) and M2 (open triangle) mice were cultured intriplicate overnight with irradiated B6.K splenocytes pulsed with indicated concen-trations of purified Hb(64-76) peptide. After 18–20 h, IL-2 production was assayedby ELISA. Dose curves are mean + SEM of triplicate samples from 3 independentexperiments. B. The same cells assayed for IL-2 production in panel A were main-tained in culture for an additional 72 h to measure proliferation of isolated n3.L2(closed squares) and M2 (open triangle) cells by 3H incorporation. Dose curvesare mean + SEM of triplicate samples from 3 independent experiments. C. Levelsof costimulatory and inhibitory molecules on the surface of naive CAb+ CD4+ cellsfR

belssTnps

2008), we cannot determine if changes in M2’s peptide fine speci-

rom the spleen of n3.L2 (black line) and M2 (filled histogram) TCR transgenic mice.epresentative plots from 5 mice in 3 independent experiments.

ecome unresponsive in the periphery (Kirberg et al., 1993; Maciant al., 2004) with decreased cytokine production and inhibited pro-iferation. Isolated M2 and n3.L2 Rag1−/− CD4+ T cells had the sameensitivity in producing IL-2 in response to Hb(64-76)-I-Ek pre-ented on irradiated B6.K splenocytes (Fig. 7A). However, the M2

cells produced significantly lower levels of IL-2 in comparison to

3.L2 T cells (Fig. 7A). Interestingly, isolated M2 T cells failed toroliferate in response to Hb(64-76) stimulation in contrast to thetrong proliferation of n3.L2 T cells (Fig. 7B). The reduced ability to

nology 53 (2013) 283– 294 291

produce IL-2 and inhibition of proliferation is a classic pattern of Tcell anergy (Sloan-Lancaster et al., 1993).

An anergic state is often accompanied by downregulation of theTCR or costimulatory molecules on the T cell surface. M2 and n3.L2T cells retained high TCR levels in the periphery of the Rag1−/−

transgenic mice (Fig. 5B). CD3 and CD4 levels were the same onn3.L2 and M2 cells (Fig. 5B), suggesting the unresponsive natureof M2 T cells is not due to an inability to receive activating sig-nals. Levels of CD25, CD28, PD-1, and CTLA-4 were also similaron n3.L2 and M2 CD4 cells (Fig. 7C). As the induction of anergydid not appear to be due to decreased costimulation or increasedinhibitory signals, M2’s hyporesponsive phenotype may be set inthe thymus as a result of different T cell selection in comparison ton3.L2. Alternatively, the anergic state could be set in the peripheryas a consequence of altered activation. In either case, the TCR struc-ture is fine tuned for T cell activation. Slight changes in the TCR thatimprove TCR:pMHC affinity, such as the CDR1� mutations resultingin the M2 TCR, do not necessarily result in enhanced T cell function.Selection in the thymus determines productive pMHC engagementand what structural changes improve peptide recognition by a Tcell.

4. Discussion

The affinity of a TCR for specific pMHC is determined by thestrength of contacts between the TCR and pMHC. We have gen-erated a TCR, M2, that has a stronger affinity for Hb(64-76)/I-Ek

than the wildtype TCR, n.3L2. The same mutations in the CDR1�loop that result in M2s increased affinity alter the TCR:pMHCbinding footprint. The M2 TCR has increased sensitivity to andbroader recognition of APLs. The increased breadth and strength ofresponse to APLs may contribute to the high level of negative selec-tion observed in the M2 TCR transgenic mice. M2 T cells undergocomplete negative selection when exposed to agonist Hbd(64-76)endogenously as a consequence of the stronger affinity. Instead ofimproving the T cell response, M2 T cells that escape negative selec-tion are anergic. Therefore, subtle changes in the TCR that enhancebinding to pMHC can lead to generation of unresponsive T cells.

What is unique about our system is that the M2 TCR has ahigher affinity due to two amino acid changes in the CDR1� loop.In the traditional view of TCR:pMHC binding, the CDR3 loops arethe primary regions that interact with the peptide, conferring thecross-reactive property of T cells, and therefore have been the mainfocus in determining what makes a T cell potentially autoreactive(Reiser et al., 2003; Yin et al., 2011). A few additional studies havedirectly mutagenized CDR 1, 2, and 3 loops and seen large changesin antigen specificity (Jones et al., 2008; Zhao et al., 2007) and selec-tion (Sim et al., 1996). With M2, mutations in the CDR1� loop notonly generate a higher affinity TCR (Chlewicki et al., 2005) but alsoregulate activation through the TCR. The stability of the TCR or theTCR:pMHC complex conferred by the CDR1� mutations may main-tain an affinity closer to the threshold for T cell activation. As aresult, M2 T cells are stimulated to produce IL-2 with a broad rangeof APLs and can potentially recognize a larger number of selectingpeptides in the thymus. Previously the two amino acids at positions26 and 28 were shown to affect binding pMHC for arsenic specificTCRs (Nalefski et al., 1990). These same residues were individu-ally important in contributing to stronger affinity and enhancedTCR:pMHC energetics (Pierce et al., 2010), though these residuesmay not be important for all TCR:pMHC complexes. While CDR1�loops in general have a flexible conformation (Armstrong et al.,

ficity are due to greater flexibility or merely the result of strongeraffinity interactions through alterations in peptide or MHC contactby CDR1� or changes in the structure of other CDRs.

2 Immu

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92 J.N. Lynch et al. / Molecular

The increased level of negative selection of M2 cells maye a consequence of broader recognition of selecting peptidesr stronger interactions with self peptides that select n3.L2. Byncreasing the affinity of the TCR for self pMHC complexes, pep-ides which were low affinity, inducing positive selection, wouldave high enough affinity to overcome the threshold for negativeelection. Therefore, the M2 CDR1� mutations may in fact be gener-ting a TCR with stronger recognition of MHCII in the thymus. Whilee were unable to identify a positively selecting peptide, this doesot disprove M2s stronger interaction with endogenous pMHCII, asositively selecting peptides are highly specific and do not reflecthe structural features of agonists (Dao et al., 2003; Hogquist et al.,997; Lo et al., 2009).

The threshold between thymocyte survival and death by neg-tive selection is very narrow. Previous work using n3.L2 T cellselected in the presence of Hb(64-76) APLs identified that inter-ctions longer than 2 s (t1/2) led to complete negative selectionWilliams et al., 1999). Based on our new findings using n3.L2 × Hbd

nd M2 × Hbd T cell development, this selection threshold for CD4 cells needs to be adjusted. Both n3.L2 and M2 exceeded thewo second half life for binding Hb/I-Ek but differed in the levelf negative selection by Hbd. N3.L2 T cells bound Hb(64-76)/I-k with an affinity of 16.6 �M. While developing n3.L2 T cellsere subjected to low levels of negative selection in the pres-

nce of endogenous Hbd(64-76), a large proportion of the n3.L2D4 T cells did persist in the periphery of the n3.L2 × Hbd mice. By

ncreasing the TCR:pMHC affinity 3.7-fold to 4 �M, M2 thymocytesere completely eliminated by negative selection in the M2 × Hbd

ice. This value is consistent with the threshold measured forD8 T cells (Naeher et al., 2007). The difference between n3.L2nd M2 clearly defines the negative selection threshold for CD4

cells and shows that the overall affinity, not the t1/2, dictates thishreshold.

Despite evidence supporting an affinity model for selection,tudies have implicated signaling differences regulate positiveersus negative selection. Even so, the quality and quantity ofMHC-specific signaling received by a thymocyte during devel-pment must still be regulated by the affinity of the TCR:pMHComplex. Low affinity interactions have maintained both selec-ion and activation of specific 2C CD8 T cells to induce target lysisSykulev et al., 1994; Udaka et al., 1992). This same functionalityas based on the number of pMHC complexes available to TCRs

n the cell surface (Delaney et al., 1998). The ability to form mul-iple TCR:pMHC interactions, either with neighboring moleculesr through rebinding, can determine the level of phosphorylationf downstream signaling molecules and the level of T cell func-ion. Rebinding is dependent upon the on-rate (Huang et al., 2010;uppa et al., 2010) and therefore could be a determining param-ter for the difference between n3.L2 and M2 T cells. It would benteresting to identify signaling differences between n3.L2 and M2hymocytes that could explain the skewed selection phenotypebserved in vivo. The affect is not the same as was described byim et al. (1996) where mutation of CDR1 skewed CD4 develop-ent to CD8, however the increased level of negative selection

f M2 thymocytes could also be a consequence of altered MHCecognition. Levels of costimulatory and inhibitory molecules canodulate thymus and peripheral T cell reactivity to cytokines and

nduction of apoptosis resulting in negative selection and anergyXing and Hogquist, 2012). While surface expression of costimula-ory and inhibitory molecules was identical between the two TCRs,he functionality of these receptors was not tested and could con-ribute to the skewed selection profile and induction of anergy for

2 T cells. Further dissection of signaling pathways downstreamf the TCR may uncover a mechanism for how altering CDR1� andncreasing TCR affinity for pMHC changes the T cell response toelecting peptides and peripheral antigens.

nology 53 (2013) 283– 294

As a mechanism to prevent autoimmune disease, high affinityTCRs can be tolerized, leaving a population of autoreactive T cellsunresponsive to agonist stimuli. M2 T cells develop in an envi-ronment lacking the Hbd agonist peptide and do not respond toHbs stimulation, both indicators that M2 (and n3.L2) T cells areunlikely to become autoreactive in the transgenic mouse. Even so,M2 T cells are anergized in the periphery. Weak agonist TCR:pMHCinteractions, such as is measured with some APLs, often result inincomplete T cell activation and induction of anergy (Evavold et al.,1993; Garbi et al., 2010; Lyons et al., 1996; Rabinowitz et al., 1996;Sloan-Lancaster et al., 1993; Zehn et al., 2009). Rechallenge withan agonist after anergy induction by APL stimulation leads to aninability to proliferate but restored IL-2 production both in vitro andin vivo (Edwards and Evavold, 2010). Because of broadened peptiderecognition due to the CDR1� mutations, M2 T cells may recognizeweakly stimulatory peptides that are null for n3.L2. M2 T cells thatescape negative selection following selection by a weakly stimula-tory peptide would be hyporesponsive when exposed to Hb(64-76)agonist. Early signaling events downstream of the hyporesponsiveM2 TCR may be insufficient to trigger full IL-2 production and pro-liferation, as has been seen with cytolytic activity of CD8 T cells(Beal et al., 2009). The developmental programming imposed onthese cells in the thymus leads to an attenuated response, despiteenhanced contact with agonist pMHC for full activation.

The optimized M2 TCR structure has a faster on-rate for bindingHb(64-76) I-Ek

. The CDR1� region stabilizes the TCR with pMHC bycontributing energetically to the overall complex as a result of con-tact with MHC or by directly influencing the CDR3� contact withpeptide (Armstrong et al., 2008; Borg et al., 2005; Burrows et al.,2010). Interestingly, in one study position 28 of the TCR� chainwas important in increasing the association rate of TCRs with addi-tional mutations through enhancing interaction between residueson CDR1� and CDR3� loops (Pierce et al., 2010). Since mutationson the I-Ek � chain maintained sufficient interaction with M2for IL-2 production but not n3.L2, the M2 CDR1� mutations mayresult in new or stronger contacts with the MHCII, decreasing therequirement for others. An altered binding orientation is unlikely,as structures of high affinity variants in the 2C system do not showaltered footprints (Varani et al., 2007). Instead the faster kon of M2binding pMHC may reflect a more optimal conformation for associ-ation with MHC, maintaining affinity above the threshold for T cellactivation with a broad range of peptides. The M2 CDR1� loop maybe more flexible, which would contribute to an altered ability to bein a “ready” conformation for faster pMHC binding. Alternatively,the CDR1� mutations may change pMHC contact directly or indi-rectly by altering the orientation of the CDR1� or other CDR loops ofthe n3.L2 TCR, as has been seen for other systems (Krogsgaard et al.,2003; Manning et al., 1999; Pierce et al., 2010; Reiser et al., 2003).A crystal structure of the M2: Hb/I-Ek co-complex would identifythe exact structural changes induced by the M2 CDR1� mutations.What is clear, however, is that a TCR with enhanced binding topMHC does not always produce a better overall T cell.

Acknowledgments

We thank Darren Kreamalmeyer for maintenance and breedingof all mice used in this manuscript; Terri Sherlinski, and the Wash-ington University Transgenic Mouse Core for generation of the M2TCR transgenic mouse; Wan-Lin Lo for generating the mutant I-Ek

Ig dimers; Stephen Horvath, Kazuo Omi, and Stephen Persaud forassistance with various aspects of the experimental studies in thismanuscript. We thank Ted Hansen, Chyi Hseih, and Yina Huang for

review of early versions of this manuscript and constructive advice.These studies were supported by the NIH grants AI21457 (P.M.A.),and CA097296 (D.M.K.) and The Rheumatic Diseases Core Centergrant (AR48335).

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ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/.molimm.2012.08.020.

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