The immunogenicity of a T cell Ag is correlated with the ability of its antigenic epitope to bind HLA and be stably presented to T cells. This presents a challenge for the development of effective cancer immunotherapies, as many self-derived tumor-associated epitopes elicit weak T cell responses, in part due to weak binding affinity to HLA. Traditional methods to increase peptide–HLA binding affinity involve modifying the peptide to reflect HLA allele binding preferences. Using a different approach, we sought to analyze whether the immunogenicity of wild-type peptides could be altered through modification of the HLA binding pocket. After analyzing HLA class I peptide binding pocket alignments, we identified an alanine 81 to leucine (A81L) modification within the F binding pocket of HLA-A*24:02 that was found to heighten the ability of artificial APCs to retain and present HLA-A*24:02–restricted peptides, resulting in increased T cell responses while retaining Ag specificity. This modification led to increased peptide exchange efficiencies for enhanced detection of low-avidity T cells and, when expressed on artificial APCs, resulted in greater expansion of Ag-specific T cells from melanoma-derived tumor-infiltrating lymphocytes. Our study provides an example of how modifications to the HLA binding pocket can enhance wild-type cognate peptide presentation to heighten T cell activation.

The TCR governs T cell–mediated immunity through recognition of peptide–MHCs (pMHCs) displayed on the surface of most nucleated cells. The formation of stable pMHC class I molecules is dependent on the binding affinity between peptide and MHC. Peptide binding to MHC class I is accomplished through interactions between peptide amino acid side chains and discrete pockets located in the α1/α2 domains of the MHC class I H chain, called the peptide-binding region. Typically, for human MHC class I (called HLA class I), binding energy is predominantly provided by residue interactions between those at position 2 and the C terminus of the peptide epitope with those at the B and F binding pockets of the HLA class I molecule, respectively (1, 2). Crystallographic studies of peptide–HLA class I (pHLA) complexes have revealed that HLA residues 9, 45, 63, 66, 67, 70, and 99 are considered the key residues of the B pocket that engage with residue 2 of the peptide, whereas residues 77, 80, 81, and 116 are key residues within the F pocket that interact with the C terminus of the peptide (2).

The generation of strong immunogenic responses against tumor Ags remains an important facet of cancer immunotherapy. Due to the observation that natural nonmutated tumor-associated epitopes, the majority of which are derived from self-antigens, elicit relatively weak T cell responses (3, 4), new methods to enhance epitope immunogenicity are needed. At least one factor contributing to weak responses is the presence of low-affinity peptide Ags generated from tumor cells, which result in reduced surface expression of pMHC complexes (3, 4). In attempts to increase peptide vaccine efficiency, researchers have used anchor-modified “heteroclitic” peptides, which usually contain modifications to position 2 or to the C terminus of peptides to match preferred HLA peptide-binding motifs, thus increasing peptide affinity toward HLA or that of the pHLA complex toward TCR (410). Though modified peptides generate heightened T cell responses toward vaccine candidates in vitro and in vivo (410), their use has failed to show success in clinical trials, owing partly to differences in the T cell repertoires primed against modified and unmodified peptides. Using a commonly described and highly immunogenic human tumor-related MHC class I–restricted epitope, the HLA-A*02:01–restricted Melan-A/MART-126–35 (EAAGIGILTV) Ag, research has found that patients with melanoma vaccinated with anchor-modified Melan-A/MART-126–35 (ELAGIGILTV) develop subtle differences in their TCR repertoire compared with those vaccinated with wild-type peptide (11, 12). Though anchor-modified Melan-A/MART-126–35 induces heightened T cell responses (13, 14), T cells primed with the natural epitope were shown to have stronger tumor reactivity (15), indicating that T cells generated against heteroclitic peptides may not be identical to those induced against natural epitopes and may fail to recognize natural epitopes present in tumors in vivo.

Balancing the need to induce stable peptide complexes through strong pHLA affinity with a need to preserve TCR responses toward wild-type Ags, we explored T cell activity resulting from modifications to the HLA binding pocket. We found that a single amino acid substitution within the F binding pocket of HLA-A*24:02 heightens cognate T cell responses against naturally occurring HLA-A*24:02–restricted epitopes. We show that this modified HLA can have practical applications, such as improved detection of TCRs using pHLA multimers and enhanced expansion of HLA-A*24:02–restricted tumor Ag-specific T cells without the requirement of modified peptides.

Peripheral blood samples were obtained from healthy donors after Institutional Review Board approval. Mononuclear cells were obtained via density gradient centrifugation (Ficoll-Paque PLUS; GE Healthcare). K562 is an erythroleukemic cell line with defective HLA expression. T2 is a T cell leukemia/B-lymphoblastoid cell line hybrid cell line defective in its ability to load endogenously derived cytosolic class I peptides (16, 17). Jurkat 76 is a T cell leukemic cell line lacking TCR and CD8 expression (a gift from Dr. M. Heemskerk, Leiden University Medical Center, Leiden, the Netherlands) (18). The K562, T2, and HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA). The K562, T2, and Jurkat 76 cell lines were grown in RPMI 1640 supplemented with 10% FBS and 50 μg/ml gentamicin (Thermo Fisher Scientific). The HEK293T cell line was grown in DMEM supplemented with 10% FBS and 50 μg/ml gentamicin. Tumor-infiltrating lymphocytes (TILs) isolated from a patient with metastatic melanoma were grown in vitro as reported previously (19). High-resolution HLA DNA typing (American Red Cross) was performed on the TIL sample. Melanoma specimens were obtained from the UHN Biospecimen Program (Toronto, ON, Canada). This study was conducted in accordance with the Declaration of Helsinki and approved by the Research Ethics Board of the University Health Network (Toronto, ON, Canada; REB numbers 11-0343 and 11-0348). Written informed consent was obtained from all healthy donors who provided peripheral blood samples.

Synthetic peptides were purchased from GenScript (Piscataway, NJ) and dissolved at 50 mg/ml in DMSO. Peptides used were A2-restricted heteroclitic NY-ESO-1157–165 (SLLMWITQV), gp100154–162 (KTWGQYWQV), HIV pol476–484 (ILKEPVHGV), HLA-A24:02–restricted gp100-intron4170–178 (VYFFLPDHL), gp100-intron4161–180 (PSQPIIHTCVYFFLPDHLSF), gp100-itnron4166–185 (IHTCVYFFLPDHLSFGRPFH), wild-type WT1235–243 (CMTWNQMNL), heteroclitic WT1235–243 (CYTWNQMNL), HTLV-1 tax301–309 (SFHSLHLLF), and HIV env584–592 (RYLRDQQLL) peptides. HIV pol476–484, HTLV-1 tax301–309, and HIV env584–592 peptides were used as negative controls. The peptide sequences tested for the pHLA binding assay and measurement of peptide exchange efficiency are listed in Supplemental Tables I and II, respectively.

To allow for indirect tracking of gene expression, all HLA-A*24:02 genes were linked to a truncated nerve growth factor receptor (ΔNGFR) gene using a Furin-SGSG-F2A sequence and cloned into the pMX retrovirus plasmid (2025). The full-length gp100 gene was purchased from Dharmacon (Lafayette, CO). Genomic DNA of gp100 was isolated using the PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific). All genes were cloned into the pMX retrovirus vector and transduced using the 293GPG cell-based retrovirus system (26).

Jurkat 76/CD8 cells were transduced to stably express CD8α/β with individual TCRα and TCRβ genes as reported previously (21). The Jurkat 76/CD8-derived TCR transfectants were purified (>95% purity) using CD3 Microbeads (Miltenyi Biotec). The K562-based artificial APCs (aAPCs) expressing HLA-A*24:02 (wild-type or A81L) in conjunction with CD80 and CD83 have been reported previously (27). PG13-derived retrovirus supernatants were used to transduce TCR genes into human primary T cells. T2 cells were retrovirally transduced with HLA-A*24:02 (wild-type) or A*24:02 (A81L, L82R, and R83G) to generate T2-A*24:02 or T2-A*24:02 (A81L, L82R, R83G), respectively. To generate β2-microglobulin–knockout T2 (T2/β2mKO) cells, β2m single guide RNA plasmid (Origene) was electroporated into T2 cells using Gene Pulser Xcell (Bio-Rad Laboratories) (24). T2/β2mKO cells were obtained by negative selection using biotin-conjugated anti-β2m Ab (clone 2M2; BioLegend) and anti-biotin microbeads (Miltenyi Biotec). To generate T2/β2mKO-expressing single-chain β2m-linked HLA-A*02:01 (T2/β2mKO/β2m-A*02:01), T2/β2mKO cells were retrovirally transduced with a construct containing full-length β2m linked to the α1 domain of HLA-A*02:01 (wild-type or L81A) via (GGGGS)3 linker. The β2m-positive cells were purified (>95% purity) and used in subsequent experiments. All of the HLA-A*02 genes were tagged with the ΔNGFR gene as described above, and the ΔNGFR+ cells were purified (>95% purity) and used in subsequent experiments.

Cell surface molecules were stained with a PC5-conjugated anti-CD8 mAb (clone B9.11; Beckman Coulter), FITC-conjugated anti-NGFR (clone ME20.4; BioLegend), and APC/Cy7-conjugated anti-CD3 (clone UCHT1; BioLegend). Dead cells were discriminated with the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies). For intracellular staining, cells were fixed and permeabilized by using a Cytofix/Cytoperm kit (BD Biosciences). Stained cells were collected using an FACSCanto II (BD Biosciences), and data analysis was performed using FlowJo version 10.8 (BD Biosciences).

T2-A24 (wild-type) cells or T2-A24 cells with a single amino acid substitution at position 81, 82, or 83 were pulsed with 50 μg/ml of biotinylated peptide overnight at 37°C. After intensive washing, the cells were stained by PE-conjugated streptavidin (SA) and washed, and fluorescence intensity was measured by flow cytometry analysis.

For the IL-2 ELISPOT assay, polyvinylidene difluoride plates (Millipore, Bedford, MA) were coated with capture mAb (SEL002; R&D Systems, Minneapolis, MN). T cells were incubated with 2 × 104 target cells/well in the presence or absence of peptide for 20–24 h at 37°C for 20–24 h at 37°C. The plates were washed and incubated with biotin-conjugated detection mAb (SEL002; R&D Systems). After washing, alkaline phosphatase–conjugated SA (Jackson ImmunoResearch Laboratories) was added. The plates were washed and incubated with NBT/5-bromo-4-chloro-3-indolyl phosphate (Promega), and IL-2 spots were developed. For IFN-γ ELISPOT analysis, polyvinylidene difluoride plates (Millipore, Bedford, MA) were coated with the capture mAb (1-D1K; Mabtech, Mariemont, OH), and T cells were incubated with 2 × 104 target cells/well for 20–24 h at 37°C. The plates were subsequently washed and incubated with a biotin-conjugated detection mAb (7-B6-1; Mabtech). HRP-conjugated SA (Jackson ImmunoResearch Laboratories) was then added, and IFN-γ spots were developed. The reaction was stopped by rinsing thoroughly with cold tap water. ELISPOT plates were scanned and counted using an ImmunoSpot plate reader and ImmunoSpot version 5.0 software (Cellular Technology Limited, Shaker Heights, OH).

CD8+ TILs were purified through negative magnetic selection using a CD8+ T Cell Isolation Kit (Miltenyi Biotec). HLA-A*24:02 aAPCs were pulsed with 10 μg/ml class I–restricted peptides of interest for 6 h. The aAPCs were then irradiated at 200 Gy, washed, and added to the TILs at an E:T ratio of 20:1. After 48 h, 10 IU/ml IL-2 (Novartis), 10 ng/ml IL-15 (PeproTech), and 30 ng/ml IL-21 (PeproTech) were added to the cultures every 3 d.

CD3+ T cells were purified through negative magnetic selection using a Pan T Cell Isolation Kit (Miltenyi Biotec). Purified T cells were stimulated with aAPC/mOKT3 irradiated with 200 Gy at an E:T ratio of 20:1. After overnight incubation, activated T cells were retrovirally transduced with cloned TCR genes via centrifugation for 1 h at 1000 × g at 32°C for 3 consecutive days. After 48 h, 100 IU/ml IL-2 and 10 ng/ml IL-15 were added to the TCR-transduced T cells. The culture medium was replenished every 2 to 3 d.

The affinity-matured HLA-A*24:02 gene was engineered to carry a Glu (E) residue in lieu of the Gln (Q) residue at position 115 of the α2 domain and a mouse Kb gene-derived α3 domain instead of the HLA class I α3 domain (28). By fusing the extracellular domain of the affinity-matured HLA-A*24:02 gene with a Gly-Ser flexible linker followed by a 6× His tag, we generated the soluble A*24:02Q115E-Kb gene. HEK293T cells were individually transduced with the soluble A*24:02Q115E-Kb gene using the 293GPG cell–based retrovirus system (26). Stable HEK293T cells expressing the soluble affinity-matured A*24:02Q115E-Kb gene were grown until confluent, and the medium was changed. Forty-eight hours later, conditioned medium was harvested and used immediately or frozen at −80°C for later use. The soluble A*24:02Q115E-Kb–containing supernatant produced by HEK293T transfectants was mixed with 100 μg/ml of A*24:02-restricted peptide of interest and incubated overnight at 37°C for in vitro peptide exchange. Soluble monomeric A*24:02Q115E-Kb loaded with peptide was dimerized using an anti-His mAb (clone AD1.1.10; Abcam) conjugated to a fluorochrome such as PE at a 2:1 molar ratio for 2 h at room temperature or overnight at 4°C. The concentration of functional soluble A*24:02Q115E-Kb molecules was measured by specific ELISA using an anti–HLA class I mAb (clone W6/32) and an anti-His tag biotinylated mAb (clone AD1.1.10; R&D Systems) as capture and detection Abs, respectively.

T cells (2 × 105) were incubated for 30 min at 37°C in the presence of 50 nM dasatinib (LC Laboratories) (29). The cells were then washed and incubated with 5–10 μg/ml of pHLA multimer for 30 min at room temperature, and R-PE–conjugated AffiniPure Fab fragment goat anti-mouse IgG1 (Jackson ImmunoResearch Laboratories) was added for 15 min at 4°C. Next, the cells were washed three times and costained with an anti-CD8 mAb for 15 min at 4°C. Dead cells were discriminated using the LIVE/DEAD Fixable Dead Cell Stain Kit.

Efficiency of peptide exchange in monomer was assessed using a competition binding assay and ELISA. Monomer was loaded with 100 μg/ml biotinylated peptide with sequence 37TYFSLNNK(-biotin)F45 derived from Adenovirus 5 Hexon and incubated overnight at 37°C. Biotinylated pHLA was purified, exchanged into PBS using Amicon Ultra filters (molecular mass cutoff of 10 kDa) (Millipore Sigma, Burlington, MA), mixed with 1 mg/ml peptide of interest, and incubated overnight at 37°C. ELISA plates were coated with anti–HLA class I mAb (clone W6/32) at 10 μg/ml in PBS overnight at 4°C. The plates were washed and blocked with 10% nonfat dry milk in PBS for 30 min at room temperature. pHLA monomer was added and incubated for 2 h at room temperature. After washing, the plates were incubated with SA-conjugated alkaline phosphatase for 30 min at room temperature. Finally, the plates were washed and incubated with p-nitrophenyl phosphate substrate (Pierce, Rockford, IL) at room temperature. The reaction was terminated by adding 1 M/l NaOH. The OD (405 nm) was read (Spectramax 190 Microplate Reader; Molecular Devices, Sunnyvale, CA). The OD values from the control wells containing nonbiotinylated peptide were subtracted from the OD values in test wells containing biotinylated peptide. The efficiency of peptide exchange for each monomer was calculated as follows: peptide exchange efficiency = (1 − [OD value with peptide/OD value with DMSO]) × 100. Every sample was assayed in triplicate wells.

Statistical analysis was performed using GraphPad Prism 9. To determine whether two groups were significantly different for a given variable, we conducted an analysis using Welch t test (two-sided). The p values <0.05 were considered significant.

During assessments of peptide immunogenicity, we searched for allelic variations between HLA class I alleles. We noticed that among key residues forming the HLA class I peptide binding pockets, position 81 within the F pocket shows two polymorphisms: HLA-A and -B alleles express either an Ala or a Leu residue, whereas HLA-C alleles only express a Leu residue (30). Moreover, with the exception of most members of the A24 supertype and several other alleles, Leu was the residue most frequently found in position 81 among all HLA class I alleles (2). Further, alignment of the most prevalent HLA class I alleles found within the general population showed that the α1 domain of HLA-A*24:02 differs from those of other HLA alleles at positions 81 to 83 (Fig. 1), suggesting that these residues may uniquely define HLA-A*24:02 and influence its peptide binding.

FIGURE 1.

HLA-A*24:02 differs from other HLA class I alleles at positions 81–83. Amino acid sequence alignment of the α1 domain of the most prevalent HLA class I alleles expressed within the world population. The rectangle indicates amino acid residues at positions 81–83.

FIGURE 1.

HLA-A*24:02 differs from other HLA class I alleles at positions 81–83. Amino acid sequence alignment of the α1 domain of the most prevalent HLA class I alleles expressed within the world population. The rectangle indicates amino acid residues at positions 81–83.

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Although the HLA-A24 supertype is represented in all ethnic groups, it is the second most frequent HLA-A allele in the world (31, 32). Thus, in attempts to understand how HLA-A*24:02 presents Ags, we investigated the biological effects of amino acid substitutions within the HLA-A*24:02 peptide binding pocket. Using TAP-deficient T2 cells, we transduced HLA-A*24:02 constructs expressing single amino acid substitutions at positions 81–83, substituting each position with the most common residues found in other HLA class I alleles (Fig. 1). T2 lines expressing HLA-A*24:02 with an Ala to Leu substitution at position 81 (A81L), Leu to Arg at position 82 (L82R), or Arg to glycine at position 83 (R83G) were all successfully transduced. Consistent with observations that HLA-A24 surface expression is less dependent on TAP expression than that of other HLA alleles (33), we were able to detect transduction of some mutant alleles by flow cytometry; however, stark differences in anti-HLA Ab binding were observed (Fig. 2A). Whereas the pan-anti–HLA class I Ab (clone W6/32) was able to similarly bind all HLA-A*24:02–transduced T2 cells with greater mean fluorescence intensity (MFI) than untransduced T2 cells, we found that anti–HLA-A*24 (clone 22E1) lost the ability to detect the mutant HLA-A*24:02 (L82R).

FIGURE 2.

HLA-A*24:02 (A81L) improves pHLA complex surface formation. (A) Surface expression of ΔNGFR, HLA class I, and HLA-A*24:02 on T2 cells transduced with the full-length HLA-A*24:02 genes tagged with ΔNGFR and analyzed by flow cytometry following staining with indicated Abs (open curve) or isotype control (filled curve). (B) MFI of biotinylated peptides with SA-PE on the T2–HLA-A*24:02 (wild-type or A81L) cells. Peptides used are shown in Supplemental Table I. The B7-restricted HPV E75–13 and C7-restricted MART151–61 peptides were used as irrelevant controls. For data analysis, the MFI of the samples to the nonbiotinylated peptide was subtracted from the MFI of the samples to the biotinylated peptide. p values were determined by repeated-measures one-way ANOVA with Tukey multiple-comparisons test for each peptide (F = 1128 [Adenovirus 5 Hexon], 45.78 [CMV-IE1], 453.3 [CMV pp65], 484.2 [HIV env], 20.08 [EpCAM], and 430.2 [KM–HN–1]; df = 11). **p < 0.01, ***p < 0.001.

FIGURE 2.

HLA-A*24:02 (A81L) improves pHLA complex surface formation. (A) Surface expression of ΔNGFR, HLA class I, and HLA-A*24:02 on T2 cells transduced with the full-length HLA-A*24:02 genes tagged with ΔNGFR and analyzed by flow cytometry following staining with indicated Abs (open curve) or isotype control (filled curve). (B) MFI of biotinylated peptides with SA-PE on the T2–HLA-A*24:02 (wild-type or A81L) cells. Peptides used are shown in Supplemental Table I. The B7-restricted HPV E75–13 and C7-restricted MART151–61 peptides were used as irrelevant controls. For data analysis, the MFI of the samples to the nonbiotinylated peptide was subtracted from the MFI of the samples to the biotinylated peptide. p values were determined by repeated-measures one-way ANOVA with Tukey multiple-comparisons test for each peptide (F = 1128 [Adenovirus 5 Hexon], 45.78 [CMV-IE1], 453.3 [CMV pp65], 484.2 [HIV env], 20.08 [EpCAM], and 430.2 [KM–HN–1]; df = 11). **p < 0.01, ***p < 0.001.

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Because such alterations occur within a fundamental region of the HLA peptide binding cleft, we assessed whether T2 transfectants expressing HLA-A*24:02 constructs were able to present wild-type Ags. T2 cells were pulsed with an array of known HLA-A*24:02–restricted peptides derived from viral and tumor-associated Ags, and cell surface presentation of pHLA complexes was analyzed by flow cytometry. Consistent with increased pan–HLA class I expression observed in T2 transfectants, we found that all HLA-A*24:02 constructs, including the HLA-A*24:02 (L82R) construct, were capable of presenting peptides (Fig. 2B), suggesting that substitutions at positions 81–83 did not alter surface expression or folding of HLA-A*24:02 but rather affected epitope recognition by anti–HLA-A*24 (clone 22E1) Ab. However, our assays found that T2–HLA-A*24:02 (A81L) had a significantly greater ability to present nonmodified HLA-A*24:02–restricted peptides compared with wild-type or other HLA-A*24:02 mutants. These results highlight the importance of this substitution in peptide anchoring and suggest that the HLA-A*24:02 (A81L) construct can better sustain the presentation of HLA-A*24:02 Ags on the cell surface of APCs.

A Leu residue at position 81 renders HLA-A*02:01 more similar to other common HLA-B and C alleles than to HLA-A24 supertype members (Fig. 1). Knowing the A81L substitution in HLA-A*24:02 has a profound effect on the surface expression of pMHC complexes, we examined whether such a substitution would alter Ag presentation involving HLA-A*02:01. To control for endogenous expression of HLA-A*02:01 in T2 cells (16), a β2m-linked HLA-A*02:01 (β2m–HLA-A*02:01) with an L81A substitution expressed as a single chain was generated and transduced into T2/β2mKO. Consistent with previous observations, endogenous HLA expression could be detected on the surface of wild-type T2 cells (16) but was absent in T2/β2mKO, as detected using anti-β2m or pan–anti-HLA Abs (Fig. 3A). Unlike β2m–HLA-A*02:01 (wild-type), β2m–HLA-A*02:01 containing the L81A substitution resulted in significantly reduced expression, staining only slightly above that of T2/β2mKO (Fig. 3A, left). To determine if the L81A substitution only disrupted surface expression of β2m–HLA-A*02:01, we stained, fixed, and permeabilized T2 cells. After fix/permeabilization, we found that although intracellular β2m expression remained intact, intracellular staining using conformation-dependent pan–HLA class I Abs W6/32 or B9.12.1 did not detect β2m–HLA-A*02:01 (L81A) at any greater degree than that of surface staining (Fig. 3A, right).

FIGURE 3.

Position 81 is critical for the surface expression of HLA-A*02:01. (A) Expression of endogenous β2m and HLA class I molecules in T2 cells and β2m and HLA class I derived from transduced β2m–HLA-A*02:01 (wild-type or L81A) in T2/β2mKO cells was analyzed via flow cytometry following staining with an anti-β2m and HLA class I mAbs (open curve) and an isotype control (filled curve) expressed on the surface (left panel) or after fixation and permeabilization (right panel). (B) Jurkat 76/CD8 cells transduced with A2/NY-ESO-1157–165 TCR (clone 1G4LY) or A2/gp100154–162 TCR were used as responder cells in IFN-γ ELISPOT analysis. The indicated T2 cells pulsed with 10 μg/ml heteroclitic NY-ESO-1157–165, gp100154–162, or HIV pol476–484 control peptide were used as stimulator cells. The data shown represent the mean ± SD of experiments performed in triplicate. **p < 0.01, ***p < 0.001 (two-tailed Welch t tests).

FIGURE 3.

Position 81 is critical for the surface expression of HLA-A*02:01. (A) Expression of endogenous β2m and HLA class I molecules in T2 cells and β2m and HLA class I derived from transduced β2m–HLA-A*02:01 (wild-type or L81A) in T2/β2mKO cells was analyzed via flow cytometry following staining with an anti-β2m and HLA class I mAbs (open curve) and an isotype control (filled curve) expressed on the surface (left panel) or after fixation and permeabilization (right panel). (B) Jurkat 76/CD8 cells transduced with A2/NY-ESO-1157–165 TCR (clone 1G4LY) or A2/gp100154–162 TCR were used as responder cells in IFN-γ ELISPOT analysis. The indicated T2 cells pulsed with 10 μg/ml heteroclitic NY-ESO-1157–165, gp100154–162, or HIV pol476–484 control peptide were used as stimulator cells. The data shown represent the mean ± SD of experiments performed in triplicate. **p < 0.01, ***p < 0.001 (two-tailed Welch t tests).

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To confirm that the reduced surface expression of β2m–HLA-A*02:01 (L81A) translated to biological activity, we performed functional assays using Jurkat 76/CD8 cells expressing HLA-A*02:01–restricted NY-ESO-1157–165-specific TCR (clone 1G4LY) or gp100154–162-specific TCR (clone gp100154). Whereas cognate peptide-pulsed T2-β2m–HLA-A*02:01 (wild-type) was able to induce IFN-γ from TCR-Jurkat 76/CD8 cells in ELISPOT assays, coculture with T2-β2m–HLA-A*02:01 (L81A) resulted in significantly reduced IFN-γ production (Fig. 3B). These results suggest that the reduced surface expression of β2m–HLA-A*02:01 (L81A) was not due to altered epitope recognition by pan–anti-HLA Ab, but rather was likely due to the L81A substitution disrupting the formation of stable HLA-A*02:01 pMHC complexes.

We next investigated whether the increased surface expression of modified HLA-A*24:02 led to increased T cell activation. Using Jurkat 76/CD8 cells transduced with TCRs of varying avidities toward the HLA-A*24:02/WT1235–243 epitope (21), we found that T2–HLA-A*24:02 (A81L) displayed a greater ability to induce T cell secretion of IL-2, as compared with T2–HLA-A*24:02 (wild-type). This occurred in conditions in which WT1235–243 peptide was continuously present within culture media (Fig. 4A, top row) or pulsed (Fig. 4a, bottom row) onto cells prior to T cell assays. Observed for all WT1235–243-specific TCRs irrespective of their avidity toward pMHC, these results suggest that the A81L substitution may enhance the ability of HLA-A*24:02 to retain surface expression. As short peptides readily bind HLA on the cell surface, thereby bypassing the deficient endosomal processing pathway of T2 cells (16, 17), we tested the extent of peptide presentation of T2–HLA-A*24:02 (A81L) by pulsing long HLA-A*24:02–restricted peptides. Using T2 transfectants, we pulsed 20-mer–long peptides containing HLA-A*24:02–restricted gp100int4170–178 epitopes and found results consistent with its ability to enhance T cell activation toward short peptides: 20-mer–long peptide-pulsed T2–HLA-A*24:02 (A81L) resulted in a >4-fold increase in the number of IFN-γ–secreting spots as compared with HLA-A*24:02 (wild-type) (Fig. 4B).

FIGURE 4.

HLA-A*24:02 (A81L) enhances the presentation of Ags to T cells. (A) Jurkat 76/CD8 cells individually transduced with the indicated HLA-A*24:02–restricted WT1235–243 TCR were used as responder cells in IL-2 ELISPOT analysis. T2 cells transduced with HLA-A*24:02 (wild-type or A81L) pulsed with graded concentrations of A24/heteroclitic WT1235–243 peptide were cocultured with T cells in the presence of peptide (top panel) or T2 cells pulsed with peptide (bottom panel). Secretion of IL-2 relative to the maximal response was analyzed. (B) Primary T cells transduced with HLA-A*24/gp100-intron4170–178 TCR were used as responder cells in IFN-γ ELISPOT analysis. T2 or T2 transduced with HLA-A*24:02 (wild-type or A81L) cells were pulsed with the indicated gp100-intron4 peptide in serum-free media and used as stimulator cells. The HIV env584–592 peptide and untransduced primary T cells were employed as negative controls. *p < 0.05, **p < 0.01 (two-tailed Welch t tests).

FIGURE 4.

HLA-A*24:02 (A81L) enhances the presentation of Ags to T cells. (A) Jurkat 76/CD8 cells individually transduced with the indicated HLA-A*24:02–restricted WT1235–243 TCR were used as responder cells in IL-2 ELISPOT analysis. T2 cells transduced with HLA-A*24:02 (wild-type or A81L) pulsed with graded concentrations of A24/heteroclitic WT1235–243 peptide were cocultured with T cells in the presence of peptide (top panel) or T2 cells pulsed with peptide (bottom panel). Secretion of IL-2 relative to the maximal response was analyzed. (B) Primary T cells transduced with HLA-A*24/gp100-intron4170–178 TCR were used as responder cells in IFN-γ ELISPOT analysis. T2 or T2 transduced with HLA-A*24:02 (wild-type or A81L) cells were pulsed with the indicated gp100-intron4 peptide in serum-free media and used as stimulator cells. The HIV env584–592 peptide and untransduced primary T cells were employed as negative controls. *p < 0.05, **p < 0.01 (two-tailed Welch t tests).

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A major limitation of TCR-based cancer immunotherapies is the difficulty of identifying TCRs recognizing low-affinity epitopes. Designing multimers that can reliably stain such TCRs can lead to more promising cancer treatment options. Having found the A81L substitution enhanced Ag presentation of naturally occurring HLA-A*24:02 peptides to T cells, we tested whether modified HLA could be used to improve the study of HLA-A*24:02–restricted T cells. Previously, we developed novel peptide exchangeable affinity-matured HLA class I multimers, which were used to detect, sort, and clone tumor Ag-specific TCRs from tumor infiltrates (23). Using this platform, we generated affinity-matured HLA-A*24:02 multimers expressing the A81L substitution to determine whether this substitution could further enhance the utility of these multimers in detecting low-affinity TCRs. Using a cell-free assay, we tested peptide exchange efficiency with HLA-A*24:02–restricted peptides derived from an array of viral and tumor-associated Ags (see Supplemental Table II for peptide list). When compared with wild-type HLA-A*24:02 monomers, the A81L substitution significantly enhanced the efficiency of peptide exchange for all peptides having low exchange efficiency in wild-type HLA-A*24:02 affinity-matured monomers, increasing exchange efficiency by at least 2-fold or more (Fig. 5A). Notably, for the TSPAN1076–84 peptide, the affinity-matured HLA-A*24:02 (A81L) monomer resulted in detectable peptide exchange, which was not the case with the wild-type monomer (Fig. 5A, peptide 32). The A81L substitution did not compromise the specificity of peptides, as HLA-B*18–restricted peptide MAGE-A3167–176, HLA-B*27–restricted peptide VEGF (untranslated region), and HLA-C*06–restricted peptide GAGE1/2/89–16 did not lead to any appreciable exchange into affinity-matured HLA-A*24:02 (A81L) monomer (Fig. 5A, peptides 65–67). Having found the HLA-A*24:02 (A81L) monomer capable of more efficient peptide exchange, we stained Jurkat 76/CD8 cells expressing cognate TCRs of varying avidities with wild-type and A81L HLA-A*24:02 WT1235–243 multimers. Consistent with observed exchange efficiency, the HLA-A*24:02 (A81L) multimers showed a greater ability to stain low-avidity TCRs as compared with HLA-A*24:02 wild-type multimers. For TCRs A133 and A186, HLA-A*24:02 (A81L) multimers resulted in staining of T cells that otherwise would not have been detected under the experimental conditions used (Fig. 5B).

FIGURE 5.

Peptide-loaded HLA-A*24:02 (A81L) multimers can be used to stain low-affinity TCRs. (A) The peptide exchange efficiency in soluble monomeric HLA-A*24:02 wild-type (top panel) or with A81L substitution (bottom panel) was measured by peptide competition binding assay and ELISA. The HLA-A*24:02–restricted peptides used are shown in Supplemental Table II. HLA-B*18–restricted MAGE-A3167–176 (no. 65), HLA-B*27–restricted VEGF (no. 66), and HLA-C*16–restricted MAGE-A4293–301 (no. 67) peptides were used as irrelevant controls. The data shown represent the mean ± SD of experiments performed in triplicate. (B) HLA-A*24:02 multimers with A81L substitution efficiently stain low-affinity cognate TCRs. Jurkat 76/CD8 cells transduced with three different A24/WT1 TCRs or A24/gp100-intron4170–178 TCR as control were stained with the HLA-A*24:02Q115E-Kb/wild-type WT1 (top panel) or HLA-A*24:02Q115E/A81L-Kb/wild-type WT1 (bottom panel) multimers. The percentage of multimer+ cells in CD8+ T cells is shown. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5.

Peptide-loaded HLA-A*24:02 (A81L) multimers can be used to stain low-affinity TCRs. (A) The peptide exchange efficiency in soluble monomeric HLA-A*24:02 wild-type (top panel) or with A81L substitution (bottom panel) was measured by peptide competition binding assay and ELISA. The HLA-A*24:02–restricted peptides used are shown in Supplemental Table II. HLA-B*18–restricted MAGE-A3167–176 (no. 65), HLA-B*27–restricted VEGF (no. 66), and HLA-C*16–restricted MAGE-A4293–301 (no. 67) peptides were used as irrelevant controls. The data shown represent the mean ± SD of experiments performed in triplicate. (B) HLA-A*24:02 multimers with A81L substitution efficiently stain low-affinity cognate TCRs. Jurkat 76/CD8 cells transduced with three different A24/WT1 TCRs or A24/gp100-intron4170–178 TCR as control were stained with the HLA-A*24:02Q115E-Kb/wild-type WT1 (top panel) or HLA-A*24:02Q115E/A81L-Kb/wild-type WT1 (bottom panel) multimers. The percentage of multimer+ cells in CD8+ T cells is shown. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

In addition to enhancing detection of low-avidity T cells, we tested whether the A81L substitution would be useful in the expansion of T cells from tumor infiltrates. Using our previously developed aAPC platform (27, 34), we performed a short-term expansion of melanoma patient-derived TILs using aAPCs expressing HLA-A*24:02 (A81L) or wild-type, pulsed with gp100int4170–178 or control HTLV-1tax301–309 peptide. After 2 wk of coculture, aAPC–HLA-A*24:02 (A81L) pulsed with gp100int4170–178 resulted in a >2-fold expansion of Ag-specific CD8+ T cells as compared with aAPC expressing wild-type HLA-A*24:02 (Fig. 6A). This ability to enhance expansion of Ag-specific T cells was consistent across three replicates (Fig. 6B) and resulted in a ∼6-fold expansion from baseline TILs (Figs. 6C and 7).

FIGURE 6.

aAPC/HLA-A*24:02 (A81L) enhances expansion of tumor Ag–specific T cells. CD8+ T cells isolated from the melanoma TILs were stimulated with the aAPC/HLA-A*24:02 (wild-type or A81L) pulsed with 10 µg/ml gp100-intron4170–178 or HTLV-1 tax301–309 peptide. (A) Staining with the indicated multimers before stimulation (day 0) and after 14-d stimulation. Gates indicate percentage multimer+ CD8+ T cells. (B) Cumulative frequency of gp100-intron4170–178-multimer+ CD8+ T cells after 14 d of expansion cultures. (C) Cumulative fold change of gp100-intron4170–178-multimer+ CD8+ T cells after 14 d of expansion cultures. The data shown represent the mean ± SD. *p < 0.05 (paired test).

FIGURE 6.

aAPC/HLA-A*24:02 (A81L) enhances expansion of tumor Ag–specific T cells. CD8+ T cells isolated from the melanoma TILs were stimulated with the aAPC/HLA-A*24:02 (wild-type or A81L) pulsed with 10 µg/ml gp100-intron4170–178 or HTLV-1 tax301–309 peptide. (A) Staining with the indicated multimers before stimulation (day 0) and after 14-d stimulation. Gates indicate percentage multimer+ CD8+ T cells. (B) Cumulative frequency of gp100-intron4170–178-multimer+ CD8+ T cells after 14 d of expansion cultures. (C) Cumulative fold change of gp100-intron4170–178-multimer+ CD8+ T cells after 14 d of expansion cultures. The data shown represent the mean ± SD. *p < 0.05 (paired test).

Close modal

Collectively, our results show that the generation of a modified HLA-A*24:02–presenting cognate Ag can heighten the activation of Ag-specific T cells, resulting in increased cytokine secretion and T cell expansion without the need to modify the recognized peptide epitope. In cell-free assays, HLA-A*24:02 (A81L) significantly increased the efficiency of peptide exchange toward peptides and, importantly, did not alter the restriction of those peptides, as no increase in presentation by T2 cells or in cell-free peptide exchange assays were observed toward non-HLA-A*24:02–restricted peptides. We show that a modified HLA-A*24:02 leads to increased expansion and detection of low-avidity T cells.

Given the importance of residue 81 in forming part of the F binding pocket, it was surprising that the A81L substitution did not adversely alter the binding of HLA-A*24:02–restricted peptides, but in fact led to increased surface availability of pHLA-A*24:02 complexes and heightened T cell responses. This was particularly true for HLA-A*24:02–restricted peptides that poorly bind wild-type HLA-A*24:02. The overall structure of HLA-A*24:02 is similar to other HLA I alleles, adopting the well-described α1 and α2 domains formed by an antiparallel β sheet and two long α helices that make up the peptide binding interface presented to TCRs (3537). However, unique to HLA-A*24:02 are the unusually deep B and F peptide binding pockets that can accommodate bulky aromatic and large hydrophobic side chains, such as anchor residues Y or F (at position 2) and F, L, I, or W (at the C termini) of peptide ligands (2, 35). Whether the A81L substitution (see (Fig. 7 for in silico modeling) alters this preference is not clear; however, we did not observe any bias toward the presentation of particular HLA-A*24:02–restricted peptides, but rather an ability to enhance peptide presentation and T cell activation of all tested peptides. This observation likely places emphasis on the importance of secondary peptide anchors known to strongly influence the binding capacity of peptide ligands toward HLA-A*24:02 (38). Indeed, the A81L substitution may affect how secondary peptide anchors influence the unique conformation that peptides adopt within HLA-A*24:02. Several crystal structures have shown that peptide ligands bound to HLA-A*24:02 adopt a moderately large “A” or “M” shape (in which position 5 provides an anchor) at central residues relative to other HLA I alleles (3537), providing a unique peptide “bulge” for TCR docking and T cell recognition. Interestingly, as with the unique effects secondary anchors have on different HLA I alleles (38, 39), modification of position 81 contributes significantly to pHLA complex formation; this was evident as a substitution of position 81 within HLA-A*02:01 rendered this allele incapable of activating T cells, highlighting the importance of this residue in surface expression of pHLA complexes.

FIGURE 7.

Model showing A81L substitution in HLA-A*24:02 peptide complex. Top view of the peptide-binding groove of HLA-A*24:02 complexed with Flu PB1498–505 peptide, including a position 81 side chain (RCSB Protein Data Bank: 4F7T). The models with alanine at position 81 (A81) and leucine at position 81 (81L) are shown.

FIGURE 7.

Model showing A81L substitution in HLA-A*24:02 peptide complex. Top view of the peptide-binding groove of HLA-A*24:02 complexed with Flu PB1498–505 peptide, including a position 81 side chain (RCSB Protein Data Bank: 4F7T). The models with alanine at position 81 (A81) and leucine at position 81 (81L) are shown.

Close modal

As the affinity between peptide and HLA, and thus surface expression, is an important factor in determining the immunogenicity of pMHC complexes, we suggest that the A81L modification to the F binding pocket likely increases the binding affinity of peptides to HLA-A*24:02 or modifies the conformation of peptide binding, resulting in heightened T cell activation. Given the TAP-deficient nature of T2 cells, the ability of HLA-A*24:02 (A81L) to also enhance the presentation of long peptides containing cognate T cell epitopes may fit with this interpretation. Rather than cross-presentation, another potential mechanism may be that HLA-A*24:02 (A81L) has an increased affinity for cognate T cell epitopes, forcing their conformation into a standard presentation motif while allowing the noncognate peptide region of the long peptide to protrude from the core binding groove, as has been observed with Toxoplasma gondii long peptides eluted from HLA of infected cells (40). Alternatively, HLA-A*24:02 (A81L) may induce an altered peptide binding motif with long peptides, similar to observations that unusually long viral epitopes adopt a “super bulge” within the HLA (41, 42).

An important aspect not explored in our study is how this modification may influence the presentation of naturally processed Ags, a possibility exemplified by the observations of drug hypersensitivity reactions toward the guanosine-related HIV reverse transcriptase inhibitor abacavir and other HLA-linked drugs (4346). Abacavir has been shown to bind within the F pocket of HLA-B*57:01, changing the shape and chemistry of the Ag-binding cleft, thus altering the endogenous repertoire of presented peptides by HLA-B*57:01 (45, 46). This example highlights the possibility that minor modifications to the HLA binding cleft can lead to changes in T cell reactivity. Nonetheless, a modified HLA-A*24:02 revealed practical aspects when applying this substitution toward various immunotherapeutic applications. We found that TCRs deemed low affinity, which were not detected by wild-type monomers, could be readily detected using binding cleft–modified HLA multimers. In line with this observation, we also show that aAPCs expressing HLA-A*24:02 (A81L) resulted in increased expansion of Ag-specific T cells from TILs. The ability of HLA-A*24:02 (A81L) multimers to detect low-affinity TCRs coupled with the ability of aAPCs to expand Ag-specific T cells might allow for a more robust scrutiny of both high- and low-affinity T cell repertoires against any given epitope derived from tumor Ags.

One of the outstanding questions is whether TCR repertoires generated through wild-type or HLA-A*24:02 (A81L) may be identical. However, as no highly immunogenic HLA-A*24:02 epitope has been identified that can induce Ag-specific T cells responses with such diverse/heterogeneous TCR repertoires as that of HLA-A*02:01 Melan-A/MART-126–35 peptide, this would be difficult to address. Nonetheless, we provide an example of an HLA binding pocket modification that increases pHLA immunogenicity and heightens cognate T cell activation and expansion toward a broad breadth of naturally occurring peptides. Our data suggest that the specific A81L substitution could be directly applied to HLA class I alleles containing the F binding pocket motif RI/TALR79–83, in which Ala is expressed at position 81; namely, the majority of A24-supertype members (HLA-A*23 and HLA-A*24 alleles) as well as various members of the A01 (HLA-A*25, HLA-A*31, and HLA-A*32 alleles), B07 (HLA-B*51 allele), B44 (HLA-B*44 allele), and B58 (HLA-B*58 allele) supertypes, all of which share F pocket peptide specificity with HLA-A*24:02 (2). Given the results of our study, we envision that crystal structure–guided modifications beyond position 81 could be applied to other HLA alleles and thereby alleviate the reliance on heteroclitic peptides in the development of vaccines and immunotherapies.

We thank the members of our laboratory for their helpful discussions.

This work was supported by the Ontario Institute for Cancer Research Clinical Investigator Award IA-039 (to N.H.), the Ira Schneider Memorial Cancer Research Foundation (to N.H.), the Princess Margaret Cancer Foundation (to N.H. and M.O.B.), the Mitacs internship (to K.M.), the Uehara Memorial Foundation Research Fellowship Program (to K.Su.), the Longo Family Cancer Immunotherapy Fellowship (to Y.M.), and the Frederick Banting and Charles Best Canada Graduate Scholarship (to C.-H.W.).

The online version of this article contains supplemental material.

K.M. and N.H. designed the project; K.M., D.L., H.S., Y.M., K.Su., F.I., D.O., Y.O., K.Sa., C.-H.W., E.Y.F.Z., and B.D.B. performed the experimental work; M.O.B. provided clinical samples; K.M., D.L., H.S., Y.M., and N.H. analyzed the results and wrote the manuscript.

Abbreviations used in this article:

     
  • aAPC

    artificial APC

  •  
  • β2m–HLA-A*02:01

    β2-microglobulin–linked HLA-A*02:01

  •  
  • MFI

    mean florescence intensity

  •  
  • pHLA

    peptide–HLA class I

  •  
  • pMHC

    peptide–MHC

  •  
  • SA

    streptavidin

  •  
  • T2/β2mKO

    β2-microglobulin–knockout T2

  •  
  • TIL

    tumor-infiltrating lymphocyte

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M.O.B. has served on advisory boards for Merck, Bristol-Myers Squibb, Novartis, GlaxoSmithKline, Immunocore, Immunovaccine Inc., Sanofi, and EMD Serono and received research funding for investigator-initiated clinical trials from Merck and Takara Bio. N.H. has received research funding from and served as a consultant for Takara Bio, is a cofounder of TCRyption, and has equity in Treadwell Therapeutics. The University Health Network has filed a patent application related to this study on which K.M., D.L., H.S., Y.M., and N.H. are named as inventors.

Supplementary data