Synthetic peptides with inadvertent chemical modifications can activate potentially autoreactive T cells

Abstract

The human CD8⁺ T cell clone 6C5 has previously been shown to recognize the tert-butyl-modified Bax_161–170 peptide LLSY(3-tBu)FGTPT presented by HLA-A*0201. This non-natural epitope was likely created as a by-product of fluorenylmethoxycarbonyl protecting group (Fmoc) peptide synthesis and bound poorly to HLA-A*0201. In this study, we used a systematic approach to identify and characterize natural ligands for the 6C5 TCR. Functional analyses revealed that 6C5 T cells only recognized the LLSYFGTPT peptide when tBu was added to the tyrosine (Y) residue and did not recognize the LLSYFGTPT peptide modified with larger (di-tBu) or smaller (Me) chemical groups. Combinatorial peptide library screening further showed that 6C5 T cells recognized a series of self-derived peptides with dissimilar amino acid sequences to LLSY(3-tBu)FGTPT. Structural studies of LLSY(3-tBu)FGTPT and two other activating nonamers (IIGWMWIPV and LLGWVFAQV) in complex with HLA-A*0201 demonstrated similar overall peptide conformations and highlighted the importance of the position 4 (P4) residue for T cell recognition, particularly the capacity of the bulky amino acid tryptophan (W) to substitute for the tBu-modified Y residue in conjunction with other changes at P5 and P6. Collectively, these results indicated that chemical modifications directly altered the immunogenicity of a synthetic peptide via molecular mimicry, leading to the inadvertent activation of a T cell clone with unexpected and potentially autoreactive specificities.

Introduction

Characterization of the peptide epitopes recognized by CD8⁺ T cells is important for understanding the immunobiology of cancer and infectious diseases and for the development of related immunotherapeutics. The use of synthetic peptides has been instrumental in this endeavor (1). T cell recognition is both highly specific, such that single amino acid substitutions in a given cognate epitope can abrogate functional responses, and highly cross-reactive, such that more than a million different peptides can be engaged productively by a single degenerate TCR (2). Epitope mapping is further complicated by the need to define individual restriction elements and the fact that antigenic peptides can be generated by splicing discontinuous segments of the same protein (3, 4).

TCRs can accommodate naturally occurring post-translational modifications to agonist MHC class I-bound peptide structures arising from deamidation (5, 6), phosphorylation (7), nitration (8), and glycosylation (9). Similar peptide modifications, including citrullination (10) and nitration (11), have been described in the context of MHC class II. T cells can also recognize chemically modified peptides that do not occur in nature (12, 13). We recently described a CD8⁺ T cell clone that recognized a peptide (LLSYFGTPT) modified via the addition of tBu to the tyrosine (Y) residue at P4 (14). This modification was likely introduced during peptide synthesis as a low-frequency contaminant (~1%). We further showed that this non-natural peptide bound poorly to HLA-A*0201, and computer modeling suggested that the 3-tBu-modified Y residue was a likely contact point for the TCR (14). However, it was not clear why LLSY(3-tBu)FGTPT was immunogenic in the face of competition from unmodified peptides that were more abundant in the initial screening mixture and bound more strongly to HLA-A*0201.

In this study, we used a variety of approaches to investigate the influence of chemical groups on T cell specificity. Our data revealed that molecular mimicry between chemically modified and naturally occurring peptides enabled the activation of potentially autoreactive T cells, exemplified by clone 6C5.

Materials and Methods

T cell culture

The CD8⁺ T cell clone 6C5 was originally derived and expanded from a patient with chronic lymphocytic leukemia (CLL) (14). Aliquots of cryopreserved T cells were thawed and rested overnight prior to use in functional assays as described previously (14). The clonality of 6C5 was confirmed by derivation of a single TCRα sequence (TRAV12-13*01/02/CAMSYYNNNDMR/TRAJ43*01) and a single TCRβ sequence (TRBV6-5*01/CASSSSYEQY/TRBJ2-7*01) from extracted mRNA (15).

Combinatorial peptide library screens

6C5 T cells were prescreened using sizing scan peptide mixtures (16) and showed a preference for nonamer peptides (data not shown). Subsequent screens were performed using a nonamer combinatorial peptide library (CPL) synthesized in a positional scanning format (PepScan Ltd.) as described previously (2, 16–18). Briefly, 6 × 10⁴ C1R-HLA-A*0201 cells were preincubated for 2 hr at 37°C with each peptide mixture (100 μM) in RPMI 1640 medium containing 100 U/ml penicillin, 100 mg/ml streptomycin, 2mM L-glutamine, and 2% heat-inactivated FCS (Thermo Fisher Scientific). After the peptide pulsing phase, 3 × 10⁴ 6C5 T cells were added to each well, and the plates were incubated for 16–18 hr at 37°C. All assays were performed in duplicate. Negative control wells lacked exogenous peptides, and positive control wells contained PHA (10 μg/ml; Sigma-Aldrich). Cell-free supernatants were collected after incubation and assayed for MIP-1β content by ELISA (17).

Identification of agonist peptides

Data were processed as described previously to identify preferred amino acid residues at each position across the peptide backbone (2) and generate a predicted optimal epitope sequence (16, 18). Sequence-driven database searches were then conducted using the PI CPL webtool hosted by the Warwick Systems Biology Centre (18). Peptides were ranked in order of likelihood of recognition according to the agonist likelihood score generated by the PI CPL webtool search, and potential agonists were further stratified according to origin and predicted binding affinity to HLA-A*0201 (www.iedb.org). The final list of candidate agonists included 11 peptides derived from human viruses and 26 peptides derived from human proteins (self). Three modified versions of LLSYFGTPT were also included as representatives of substitutions predicted to alter the TCR interaction (4Y > 4W), substitutions predicted to have no effect (4Y > 4F), or substitutions predicted to improve peptide binding to HLA-A*0201 (9T > 9V). These individual peptides (n = 40) were synthesized commercially at ≥95% purity (Mimotopes). All peptides were dissolved in DMSO at 10 mg/ml stock and stored in aliquots at −80°C. Fresh aliquots were used for each experiment, and the final DMSO concentration in culture wells never exceeded 0.4%.

Initial screens were performed at a peptide dose of 4 μg/ml (approximately 4 μM). Briefly, 5 × 10⁴ peptide-pulsed T2 cells, which express HLA-A*0201, were pulsed with peptide for 1 hr at 37°C, washed, and cocultured with 6C5 T cells at a ratio of 1:1 for 18 hr at 37°C. Negative control wells contained unpulsed T2 cells with DMSO at a concentration of 0.4%, and positive control wells contained T2 cells pulsed with LLSY(3-tBu)FGTPT. Cell-free supernatants were collected after incubation and assayed for MIP-1β content by ELISA (17). Agonist peptides were tested similarly in dose-response experiments at concentrations ranging from 10¹ μg/ml to 10⁻⁶ μg/ml, stratified according to maximal production of MIP-1β. Dose-response curves were generated in Prism version 8 (GraphPad).

Flow cytometry

T cell functions were assessed using flow cytometry as described previously (14). Briefly, 6C5 T cells were incubated with T2 cells ± peptide (10 μg/ml) in the presence of GolgiStop (0.7 μl/ml; BD Biosciences) and anti-CD107a–FITC (clone H4A3; BD Biosciences). Negative control tubes contained equivalent DMSO or no T2 cells, and positive control tubes contained PMA (10 ng/ml; Sigma-Aldrich) and ionomycin (1.5 μg/ml; Sigma-Aldrich). Cells were incubated for 6 hr at 37°C, washed with FACS buffer (Thermo Fisher Scientific), and stained for 15 min at 4°C with anti-CD8a–PerCP-Cy5.5 (clone SK1), anti-CD19–BV510 (clone HIB19), and Zombie NIR (all from BioLegend). After a further wash, cells were fixed/permeabilized using a FIX & PERM Cell Fixation & Permeabilization Kit (Thermo Fisher Scientific), washed again, and stained for 20 min at 4°C with anti-IFN-γ–V450 (clone B27; BD Biosciences), anti-IL-2–APC (clone MQ1-17H12; BioLegend), anti-MIP-1β–PE (clone D21-1351; BD Biosciences), and anti-TNF-α–PE-Cy7 (clone MAb11; BioLegend). Data were acquired immediately using a FACSCanto flow cytometer (BD Biosciences) and analyzed using FlowJo software version 9.9.4 (FlowJo LLC). The gating strategy is shown in Supplemental Fig. 1.

Peptide binding assay

Peptide binding was quantified via by the upregulation of HLA-A*0201 on the surface of T2 cells (19). Briefly, 2 × 10⁵ T2 cells were incubated for 18 hr at 37°C with each test peptide at a concentration of 50 μg/ml in serum-free RPMI 1640. Negative control tubes contained equivalent DMSO, and positive control tubes contained the Bax_136–144 peptide (IMGWTLDFL). Cells were then stained with anti-HLA-A*0201 FITC (clone BB7.2; BioLegend). Data were acquired using an Accuri C6 flow cytometer (BD Biosciences) and analyzed using CFlow software (BD Biosciences). Upregulation of HLA-A*0201 was calculated using the following formula: % increase = [(mean fluorescence with peptide − mean fluorescence without peptide)/(mean fluorescence without peptide) × 100].

Chemically modified peptides and mass spectrometry

Chemically modified peptides were synthesized at ≥95% purity: LLSY(3-Me)FGTPT (Mimotopes, Wirral, UK) and LLSY(3,5-di-tBu)FGTPT and LLSY(3-tBu)FGTPT (both made by PolyPeptide Group, Strasbourg, France). Peptide modifications and sequences were verified by mass spectrometry with collision-induced dissociation using an amaZon SL Ion Trap (Bruker) (Supporting data, Fig. 1). All peptides were dissolved in DMSO at 10 mg/ml stock and stored in aliquots at −80°C. Fresh aliquots were used for each experiment, and the final DMSO concentration in culture wells never exceeded 0.4%.

Figure 1. Side-chain specificity of clone 6C5.

(A) From left to right: structures of Y, 3-tBu-Y, W, 3-Me-Y, and 3,5-di-tBu-Y. (B) Functional recognition of LLSYFGTPT (LLSY), LLSY(3-tBu)FGTPT (LLSY-tBu), LLSWFGTPT (LLSW), LLSY(3-Me)FGTPT (LLSY-Me), and LLSY(3,5-di-tBu)FGTPT (LLSY-di-tBu). T2 cells were mixed with saturating concentrations of the indicated peptides in duplicate and incubated for 6 hr with clonal 6C5 T cells. Negative controls: equivalent DMSO or no T2 cells (T cells alone). Positive control: PMA/ionomycin. Individual functions were measured by flow cytometry (key). Representative of two independent experiments. Data are shown as mean ± SD.

Crystallization and structure determination

Soluble peptide–HLA-A*0201 complexes were generated and purified as described previously (20, 21). Purified proteins were set up for crystallization trials in a sitting-drop vapor-diffusion arrangement. Crystallization screen and protein solutions were dispensed in a standard SB-format 96-well plate using an ARI Phoenix Robot (Alpha Biotech Ltd.). Screens were performed using PACT Premier (Molecular Dimensions) and TOPS (22). The best crystals were obtained with TOPS condition A12 for LLSY(3-tBu)FGTPT–HLA-A*0201, PACT Premier condition G06 for IIGWMWIPV–HLA-A*0201, and PACT Premier condition D02 for LLGWVFAQV–HLA-A*0201 (Supplemental Table 1)

Crystals were harvested into thin plastic loops and cryocooled in liquid nitrogen for transfer to the Diamond Light Source (Didcot, UK). Diffraction data were collected on beamlines I02 and I03, and data reduction was completed automatically through XDS called from within XIA2 (23, 24). Data were scaled and merged using AIMLESS and TRUNCATE in the CCP4 suite (25). The structures were solved using molecular replacement in PHASER (26), with PDB entry 5EU5 as the starting model (27), and subsequently refined using REFMAC5 (28). Data collection and refinement statistics are summarized in Supplemental Table 1. The final model was adjusted to match density maps in COOT (29). Additional figures depicting all structures with associated electron density are available upon request. The final model co-ordinates were deposited in the protein database (PDB) and assigned accession codes; 6Z9V (IIGWMWIPV–HLA-A*0201), 6Z9W (LLGWVFAQV–HLA-A*0201) and 6Z9X (LLSY(3-tBu)FGTPT–HLA-A*0201).

Statistics

Specific tests are indicated in the relevant figure legends. All statistical comparisons were performed using Prism version 8 (GraphPad).

Results

Side-chain specificity of CD8⁺ T cell clone 6C5

The human CD8⁺ T cell clone 6C5 has been shown to recognize the synthetic peptide LLSY(3-tBu)FGTPT but not the unmodified peptide LLSYFGTPT (14). To investigate the fine specificity of this phenomenon, we tested the agonist properties of peptides modified with larger (di-tBu) or smaller (Me) chemical groups attached to the Y residue at P4 (Fig. 1A). As expected, the tBu-modified peptide (LLSY-tBu) induced multiple effector functions, namely degranulation, measured via the surface mobilization of CD107a, and the production of IL-2, IFN-γ, MIP-1β, and TNF-α (Fig. 1B). In contrast, the di-tBu-modified (LLSY-di-tBu) and Me-modified peptides (LLSY-Me) were largely inert, although the latter induced a small increase in the production of MIP-1β (Fig. 1B). Earlier modeling of the LLSY(3-tBu)FGTPT–HLA-A*0201 complex suggested that the tBu-modified Y residue pointed up toward the expected position of the TCR (14). This model also predicted that a bulky amino acid might be able to mimic the structure of 3-tBu-Y. In our assays, however, a peptide containing tryptophan (W) in place of Y at P4 (LLSWFGTPT) failed to elicit any effector functions (Fig. 1B). Collectively, these data suggested that the 6C5 TCR was able to discriminate between different chemical modifications at P4.

Cross-reactivity profile of CD8⁺ T cell clone 6C5

Although clone 6C5 was originally derived from a patient with CLL, no reactivity was detected against autologous or HLA-A*0201-matched CLL cells (14). In an attempt to discover the natural epitope, we screened clone 6C5 against a nonamer CPL presented by CIR-A2 cells, which express HLA-A*0201 (2). As expected for peptides that bind HLA-A*0201, there was a preference for hydrophobic residues at P2 and P9 (Fig. 2A). There also appeared to be constraints on the number of amino acids recognized in the middle portion of the peptide (P3–P6). This was particularly evident at P3, where alanine (A) and glycine (G) were the preferred residues, and at P4, where there was a clear preference for W (Fig. 2A).

Figure 2. Nonamer CPL screen of clone 6C5.

C1R-HLA-A*0201 cells were pulsed with 180 different peptide mixtures in duplicate and incubated overnight with clonal 6C5 T cells. Cell-free supernatants were collected after incubation and assayed for MIP-1β content by ELISA. (A) Bar graphs depicting the amount of MIP-1β secreted in response to each fixed amino acid at each position across the peptide backbone (P1–P9). Data are shown as mean ± SD. (B) Graphical summary of the results shown in A. Responses are ordered by color and size (key). The optimal peptide sequence was identified as IIGWMWIPV.

The predicted optimal agonist sequence based on preferred residues across the entire peptide backbone (P1–P9) was IIGWMWIPV (Fig. 2B). No direct matches for this sequence were identified in a database search of human viral pathogens and self-proteins (18). However, a list of >400 peptide sequences ranked in order of likelihood of recognition was generated using this approach (data not shown), and these potential agonists were further refined to a list of 37 candidate peptides based on predicted binding affinity to HLA-A*0201. These candidate peptides included 11 nonamer sequences derived from common human viruses and 26 nonamer sequences derived from self-proteins (Table 1). Three modified versions of LLSYFGTPT were also included for downstream testing as representatives of substitutions predicted to alter the TCR interaction (4Y > 4W), substitutions predicted to have no effect (4Y > 4F), or substitutions predicted to improve peptide binding to HLA-A*0201 (9T > 9V) (Table 1).

Table 1. Characteristics of candidate agonist peptides.

Type	Sequence	Source	Predicted IC50 (nM)*
Index	LLSY(tBu)FGTPT	N/A	1459**
Optimal	IIGWMWIPV	N/A	24
Viral	LLGYVLART	Cytomegalovirus US8	1458
	FLARFWTRA	Herpes simplex virus 1 ENV	31
	FTSYGFSNV	Coronavirus spike glycoprotein	42
	ITAYGLVLV	Herpes simplex virus 1 ENV	65
	LTAYMLLAI	Human cytomegalovirus UL147	65
	SLGWVLTSA	Human herpes virus 8 G protein coupled receptor	81
	VLGYIGATV	Coronavirus replicase	81
	IVAWFLLLI	Vaccinia virus uncharacterized protein	95
	FIGYMVKNV	Vaccinia virus rifampicin target	147
	ILGHCWVTA	Human herpes virus 8 ENV	228
	LLFYMWSGT	Human herpes virus 6 UL32	393
Modified	LLSW	Wildtype peptide (4Y > 4W)	597
	LLSFFGTPT	Wildtype peptide (4Y > 4F)	1596
	LLSYFGTPV	Wildtype peptide (9T > 9V)	9
Self	ILAYVFPGV	Speriolin-like protein	4
	FLAYFLVSI	Galactosylxylosylprotein 3-beta-glucuronosyltransferase 3	5
	FLAWGGVPL	Centrosomal protein	6
	VLAWGLLNV	Transmembrane protein 209	9
	IMAWGLATL	G-protein coupled receptor 143	15
	FMAYFGVSA	Low affinity cationic amino acid transporter 2	17
	YTAYVFIPI	Serine palmitoyltransferase small	24
	LLGWMLSQV	Isoform 2 of Protein NLRC3	34
	LLGYGWAAA	Protein LYRIC	461
	MLAYVLLPL	Probable G-protein coupled receptor 157	7
	MLAWIFLPI	Sodium/myo-inositol cotransporter 2	8
	YMAYMFLTL	Sodium- and chloride-dependent GABA transporter 1	8
	LLAWHFVAV	Vacuolar protein sorting-associated protein 8 homolog	9
	MTAWILLPV	Transmembrane protein 150A	9
	LLGWLFAPV	Sodium/glucose cotransporter 2	12
	ALGWVFVPV	Sodium/glucose cotransporter 4	13
	FLAYSGIPA	Transferrin receptor protein 1 Homo sapiens	14
	IIWYYFPSA	Pro-neuregulin-3, membrane-bound precursor	14
	ALAWVFVPI	Sodium/glucose cotransporter 5	17
	LMGYSFAAV	Sodium/myo-inositol cotransporter 2	18
	YVAWFLVFA	Polycystic kidney disease and receptor for egg jelly-related protein precursor	18
	LIGWGLPTV	Vasoactive intestinal polypeptide receptor 2	21
	LLGWVFAQV	Tissue factor precursor	27
	LLGWVFIPI	Sodium/myo-inositol cotransporter	27
	FASYYWLTV	Protocadherin fat 2 precursor	28
	ILGWIFVPI	Low affinity sodium-glucose cotransporter	28

^*Predicted binding affinity for HLA-A*0201 (iedb.org).

^**Assumed value for peptide binding based on the prediction for LLSYFGTPT.

In functional assays, 6 of the 37 natural peptides activated clone 6C5 more potently than LLSY(3-tBu)FGTPT, and all of these agonists were derived from self-proteins (Fig. 3). None of the variant peptides with substitutions at P4 (LLSWFGTPT and LLSFFGTPT) or P9 (LLSYFGTPV) were recognized in parallel assays (Fig. 3), although as predicted empirically, the 9T > 9V substitution enhanced binding to HLA-A*0201 (Supplemental Fig. 2A).

Figure 3. Validation of candidate ligands for clone 6C5.

T2 cells were pulsed with saturating concentrations of the indicated peptides in duplicate and incubated overnight with clonal 6C5 T cells. Cell-free supernatants were collected after incubation and assayed for MIP-1β content by ELISA. Negative control: equivalent DMSO. Representative of three independent experiments. Data are shown as mean ± SD.

Of note, two peptides that elicited functional responses in the initial screens (LTAYMLLAI and FMAYFGVSA) were subsequently found to be inert using high purity (>95%) preparations (Supplemental Fig. 2B). Mass spectrometric analysis of the false-positive LTAYMLLAI peptide (initial purity 35%) further indicated artefactual tBu modification of the Y residue at P4 (Supporting data Fig. 2). These findings suggested that tBu modification enabled the functional recognition of largely unrelated peptides via the 6C5 TCR.

It was also notable that peptide immunogenicity did not correlate with surface stabilization of HLA-A*0201 (Supplemental Fig. 2C). For example, some peptides that bound strongly to HLA-A*0201, such as ILAYVFPGV and SLGWVLTA (Supplemental Fig. 2A), failed to elicit a T cell response (Fig. 3). As a prerequisite for antigenicity, however, all 6 of the agonist peptides that activated clone 6C5 more potently than LLSY(3-tBu)FGTPT (Fig. 3) were able to stabilize HLA-A*0201 (Fig. 4A and Supplemental Fig. 2A).

Figure 4. Functional potency of candidate ligands for clone 6C5.

(A) T2 cells were pulsed with the indicated peptides across a range of concentrations in duplicate and incubated overnight with clonal 6C5 T cells. Cell-free supernatants were collected after incubation and assayed for MIP-1β content by ELISA. Nonlinear curves were fitted to the corresponding mean values. (B) T2 cells were mixed with saturating concentrations of the indicated peptides in duplicate and incubated for 6 hr with clonal 6C5 T cells. Negative controls: equivalent DMSO or no T2 cells (T only). Positive control: PMA/ionomycin (PMA/IONO). Individual functions were measured by flow cytometry. The heatmap shows mean percent function⁺ cells normalized to the maximal response (key). Representative of three independent experiments. (C) T2 cells were incubated with saturating concentrations of the indicated peptides in duplicate for 18 hr. Negative control: equivalent DMSO. Positive control: Bax_136–144 (Bax IMG). Upregulation of HLA-A*0201 was measured by flow cytometry relative to DMSO. Representative of two independent experiments. Data are shown as mean ± SD.

To confirm our initial results, we tested a smaller panel of 14 peptides across a wider range of concentrations (Fig. 4B). This panel included peptides identified as more potent (e.g. LLGWVFAQV and MLAWIFLPI), equivalently potent (e.g. ILGWIFIVPI and FLAYFLVSI), or less potent (e.g. YMAYMFLTL) than LLSY(3-tBu)FGTPT. In line with the initial screens, we found that seven of these peptides, including the predicted optimal agonist (IIGWMWIPV), activated clone 6C5 to a greater extent than LLSY(3-tBu)FGTPT. The six most potent peptides had half-maximal effective concentration (EC₅₀) values up to 600-fold higher than LLSY(3-tBu)FGTPT based on the production of MIP-1β (Supplemental Fig. 3).

In further experiments, we used flow cytometry to measure other effector functions elicited by the agonist and non-agonist peptides originally defined on the basis of MIP-1β (Fig. 4C). Similar response patterns were observed for the seven most potent agonists, with minor variations in the production of IL-2 and TNF-α (Fig. 4C). The predicted optimal agonist (IIGWMWIPV) elicited the strongest responses (Fig. 4C). In contrast, peptides that elicited suboptimal levels of MIP-1β, namely LLGWMLSQV, YVAWFLVFA, and ILGWIFVPI, largely failed to induce degranulation or the production of IL-2, IFN-γ, or TNF-α (Fig. 4C).

Collectively, these experiments identified six agonist ligands that activated clone 6C5 more potently than LLSY(3-tBu)FGTPT. Shared features of these ligands included an A or a G residue at P3, a W residue at P4, and a phenylalanine (F) residue at P6. Otherwise, these agonist peptides were largely dissimilar, with minimal linear sequence homology to LLSY(3-tBu)FGTPT (Fig. 7).

Figure 7. Peptide specificity of clone 6C5.

Top: non-agonist ligands. Bottom: agonist ligands. Red font indicates peptide modifications. Orange highlights indicate peptide similarities.

Structural analysis of agonist ligands for CD8⁺ T cell clone 6C5

In an attempt to understand how ligands with disparate linear sequences could trigger the same TCR, we solved the binary structures of LLSY(3-tBu)FGTPT and two more potent agonists, namely the predicted optimal peptide (IIGWMWIPV) and a self-peptide derived from tissue factor precursor (LLGWVFAQV), in complex with HLA-A*0201. All three peptides adopted a typical bulged-out conformation, and two conformations were observed for each peptide–HLA-A*0201 complex (Fig. 5A–C, Supporting data Fig. 3). The main chain trace of all six peptide copies superimposed on each other in bound form revealed similar central conformations (Fig. 6A). In particular, the amino acid residues at P4 all pointed upward away from the peptide-binding groove of HLA-A*0201, consistent with the earlier in silico model (14). The bulk of the tBu-modified Y residue appeared to be similar to that of the W residue at P4. A preference for flexible nonpolar side-chains, namely phenylalanine (F), methionine (M), or valine (V), suggested that P5 was also a potential contact residue for the 6C5 TCR (Fig. 5A–C). In contrast, the orientation of residues at P6 was more consistent with a secondary anchor role, potentially stabilizing peptide binding to HLA-A*0201 (Fig. 6B). Previous studies have shown that the P6 residue can associate with the P3 side-chain to support the central bulge of nonamer peptides bound to HLA-A*0201 (21, 27). This support role could not be achieved by the short side-chain of serine (S) in conjunction with G at P6 in the bound conformation of LLSY(3-tBu)FGTPT, allowing the central bulge to subside slightly in the binary structure compared with IIGWMWIPV–HLA-A*0201 and LLGWVFAQV–HLA-A*0201.

Figure 5. Superposition of the two peptide copies observed for each agonist ligand in complex with HLA-A*0201.

Peptides are shown as sticks. (A) LLSY(3-tBu)FGTPT (yellow and gray). (B) IIGWMWIPV (green and cyan). (C) LLGWVFAQV (blue and orange).

Figure 6. Overall conformation of each agonist ligand in complex with HLA-A*0201.

Peptide colors match those shown in Fig. 5. (A) Superposition of the main chain traces. All rms values were below 1.0 Å. (B) Superposition of the peptide chains. (C) Least-squares superposition of the three side-chains at P4. Green, Y; orange, 3-tBu-Y; blue, W.

Although similar in terms of overall conformation, some differences were apparent between the two copies of each bound peptide, most notably with respect to the side-chains at P4, P5, and P6. The largest deviation was observed for IIGWMWIPV. In this structure, the W at P4, the M at P5, and the W at P6 adopted opposing conformations in complex with HLA-A*0201 (Fig. 5A).

Collectively, these data suggested a common mode of immunogenicity involving the central bulged region of each peptide, likely focused on P4 and P5, and further indicated that the tBu-modified Y residue mimicked the bulky nature of W at P4 (Fig. 6C and Fig. 7).

Discussion

In this study, we used functional assays, CPL screens, and X-ray crystallography to identify and characterize natural ligands for the orphan 6C5 TCR, which was previously shown to recognize the chemically modified Bax_161–170 peptide LLSY(3-tBu)FGTPT (14). We identified 6 peptides that activated clone 6C5 more potently than LLSY(3-tBu)FGTPT. All of these peptides were derived from self-proteins and bound HLA-A*0201. Structural analyses further revealed that the tBu-modified Y residue in the LLSY(3-tBu)FGTPT mimicked the unmodified W residue in two more potent agonist complexes, namely IIGWMWIPV–HLA-A*0201 and LLGWVFAQV–HLA-A*0201.

Multiple rounds of in vitro stimulation with a peptide mixture containing LLSY(3-tBu)FGTPT were required to generate clone 6C5 (14, 30). It therefore seemed likely that the parent clonotype was relatively infrequent in the peripheral circulation and that recognition of the chemically modified LLSY(3-tBu)FGTPT epitope, which does not occur in nature, was a consequence of cross-reactivity at the level of the TCR (12). To identify the primary specificity of clone 6C5, we used functional screens in combination with a nonamer CPL (2, 16–18). This approach has been used previously with advanced bioinformatics to identify candidate peptide ligands for orphan TCRs (18, 31, 32).

The predicted optimal agonist ligand based on residue preferences across the peptide backbone was IIGWMWIPV. Although the corresponding synthetic peptide was a potent activator of clone 6C5, no direct match for this sequence was found in a database search of human viral pathogens and self-proteins (18). CPL-driven database searches nonetheless identified several candidate ligands with peptide sequences derived mostly from intracellular proteins. All of these proteins were encoded by ubiquitously expressed genes with no known links to carcinogenesis. Moreover, peptides derived from some of these proteins, including sodium/myo-inositol cotransporter 2, vacuolar protein sorting-associated protein 8 homolog, and galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase, have been identified in previous studies of the global immunopeptidome, albeit not in the context of HLA-A*0201 (33). It is also important to note that clone 6C5 did not respond functionally to CD40L-activated HLA-A*0201⁺ CLL cells (14). Further work is therefore required to determine the extent to which these agonist peptides are naturally presented on the cell surface bound to HLA-A*0201.

All of the candidate ligands were more potent agonists than the tBu-modified peptide, likely reflecting greater antigen density as a consequence of enhanced binding to HLA-A*0201 and a better fit with the 6C5 TCR. Despite some conserved features at the linear sequence level, these naturally occurring peptides were largely dissimilar to LLSY(3-tBu)FGTPT, highlighting the value of unbiased ligand identification using CPLs (2). However, the binary structures of two of these agonist ligands (IIGWMWIPV and LLGWVFAQV) revealed conformational similarities with LLSY(3-tBu)FGTPT–HLA-A*0201, providing initial insights into the specificity of the 6C5 TCR.

Our data showed that neither LLSY(3-Me)FGTPT nor LLSY(3,5-di-tBu)FGTPT were able to activate clone 6C5, suggesting that the size of the chemical modification was an important determinant of recognition via the TCR. Moreover, the unmodified peptide could not be converted into an agonist ligand via a 4Y > 4W substitution, despite a strong preference for W at P4 in the CPL screens and the similarity in size between W and 3-tBu-Y. This observation suggested that neighboring peptide residues were key determinants of activation. In line with these results, the structural data indicated that the 3-Me moiety would likely be too small and the 3,5-di-tBu moeity would likely be too big to trigger a functional response, assuming a necessity for overall shape complementarity between P4 and the 6C5 TCR. Similarly, the LLSWFGTPT peptide likely failed to activate clone 6C5 as a consequence of the F residue at P5, which presumably breached the clearance needed after the W side-chain to make appropriate contacts with the TCR. In contrast, the LLGWVFAQV peptide was likely a potent activator of clone 6C5 as a consequence of the V residue at P5, the side-chain of which tucked in underneath the central peptide bulge toward the TCR, whereas the IIGWMWIPV peptide was likely a potent activator of clone 6C5 as a consequence of the M residue at P5, the flexible side-chain of which was free to swivel out of the way, preserving the central peptide bulge toward the TCR (21).

In summary, we have demonstrated that synthetic peptides with inadvertent chemical modifications can activate potentially autoreactive T cells as a consequence of molecular mimicry. Although the original isolation of clone 6C5 was serendipitous, our results will likely have important implications for peptide-based immunotherapies and vaccines, specifically emphasizing the need for rigorous manufacturing processes and quality controls to mitigate the risk of adverse effects in vivo.