TCR hypervariable regions expressed by T cells that respond to effective tumor vaccines
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A major goal of immunotherapy for cancer is the activation of T cell responses against tumor-associated antigens (TAAs). One important strategy for improving antitumor immunity is vaccination with peptide variants of TAAs. Understanding the mechanisms underlying the expansion of T cells that respond to the native tumor antigen is an important step in developing effective peptide-variant vaccines. Using an immunogenic mouse colon cancer model, we compare the binding properties and the TCR genes expressed by T cells elicited by peptide variants that elicit variable antitumor immunity directly ex vivo. The steady-state affinity of the natural tumor antigen for the T cells responding to effective peptide vaccines was higher relative to ineffective peptides, consistent with their improved function. Ex vivo analysis showed that T cells responding to the effective peptides expressed a CDR3β motif, which was also shared by T cells responding to the natural antigen and not those responding to the less effective peptide vaccines. Importantly, these data demonstrate that peptide vaccines can expand T cells that naturally respond to tumor antigens, resulting in more effective antitumor immunity. Future immunotherapies may require similar stringent analysis of the responding T cells to select optimal peptides as vaccine candidates.
KeywordsPeptide vaccines T cell repertoire Tumor antigens Peptide variants
Increased frequencies of cytotoxic T lymphocytes (CTLs), which recognize antigenic peptides from tumors presented by major histocompatibility (MHC) class I molecules, correlate with improved cancer patient survival . CTLs and the effector molecules they produce are critical in the elimination of cancer cells [2, 3]; however, they are also subject to regulatory mechanisms. Tumor-specific CTLs often recognize peptides derived from tumor-associated antigens (TAAs). One important strategy for improving antitumor immunity and overcoming these self-tolerance mechanisms is vaccination with peptide variants of TAAs (also known as mimotopes, heteroclitic peptides, peptide analogues, or altered peptide ligands) that improve binding to either MHC molecules  or tumor-specific T cell receptor (TCR) molecules [5, 6]. These vaccines stimulate the expansion of the often low-affinity TAA-specific T cells that escape negative selection during development [7, 8] and are suboptimally activated by native tumor antigens [9, 10]. Clinical trials have shown increased frequencies of TAA-specific T cells following vaccination with peptide variants, demonstrating the potential of antigen-specific immunotherapy .
Peptide variants are most often selected as vaccine candidates because they bind with stronger affinity or stimulate TAA-specific T cells more effectively than the native tumor antigen in vitro. Paradoxically, vaccination with these variants often elicits T cells with diminished antitumor activity relative to T cells that naturally respond to native tumor antigens [12, 13, 14, 15]. Using the CT26 transplantable tumor model [7, 16], we previously showed that vaccination with variants of the immunodominant peptide, gp70423–431 (AH1), elicited variable antitumor immunity [17, 18]. These peptides robustly stimulated the tumor-specific T cell clone used to identify the variants and elicited more tumor-specific T cells from the endogenous repertoire of BALB/c mice than the native AH1 peptide [17, 18]. However, vaccination with these peptides elicited a range of antitumor immunity. The ineffective peptides elicited T cells that failed to kill AH1-loaded target cells or produce IFNγ after stimulation with the native tumor antigen . Surprisingly, these functionally deficient T cells produced IFNγ after stimulation with the variant peptide used in the vaccine, suggesting that the ineffective peptide variants were not antagonists or partial agonists, as in other systems .
Understanding the mechanisms underlying the expansion of T cells that respond poorly to native tumor antigens is an important step in developing effective peptide-variant vaccines. The repertoire of T cells responding to peptide variants is often different than the repertoire of T cells responding to native tumor antigens [10, 12, 20, 21]. As addressed in this study, we hypothesized that the effective peptide variants likely elicited a different repertoire of tumor-specific T cells that expressed TCRs with higher affinity for the native tumor antigen. Since in vitro expansion of tumor antigen-specific T cells can dramatically skew T cell cultures in favor of higher-affinity T cell clones , we compared the binding properties and the TCR genes expressed by T cells elicited by peptide variants directly ex vivo. The T cells elicited by an effective peptide-variant bound fluorescent MHC-tetramers containing the native tumor antigen with a higher staining intensity. These T cells did not cross-react with an ineffective peptide variant, suggesting that the repertoires of T cells responding to these variant vaccines were different and did not overlap. In addition, the effective peptide variants elicited a repertoire of T cells that was closely related in sequence to the T cells elicited by the native TAA, expressing a common Jβ sequence and a common CDR3β motif, and the frequency of sequences containing this CDR3β motif correlated with tumor protection. Thus, the effective peptide-variant vaccines enhanced the expansion of T cells that respond to the native TAA, rather than activating a new subset of T cells. Importantly, these results demonstrate that T cells responding to native tumor antigens can be effectively stimulated to prevent tumor growth, provided they are activated through the TCR by the appropriate signal. The implication of these results is that the most effective peptide vaccines may be identified by measuring the responses of tumor antigen-specific T cells found in patients, rather than isolated T cell clones.
Materials and methods
Six- to eight-week-old female BALB/cAnNCr mice were purchased from the National Cancer Institute/Charles River Laboratories. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of National Jewish Health.
Sf9 and High Five insect cells (, Invitrogen) were cultured and infected as described [2, 17]. T cell clones were cultured as described . The T cell lines were cloned by limiting dilution and expanded with 5 × 105/ml irradiated CT26 tumor cells expressing the costimulatory molecule B7 for 2 weeks prior to flow cytometry and TCR sequence analysis.
Antibodies and staining reagents
Preparation of the fluorescent Ld tetramer linked to the AH1 peptide using a disulfide trap or loaded with exogenous AH1 peptide has been described [17, 18]. Soluble TCRs containing CDR3β motifs were prepared by inserting Vβ8.3 and Vα6 gene segments expressed by the A5-4E11 or 39-1D4 T cell clones into a BV expression vector . Purified TCR molecules were conjugated to fluorescent streptavidin molecules as described and used to stain insect cells expressing membrane-bound peptide-MHC molecules . The median fluorescent intensity (MFI) of Ld high cells was determined. Antibodies specific for the Ld molecule (28.14.8S), CD8 (53–6.7, Southern Biotech), IFNγ (XMG1.2, eBioscience), B220 (RA3-6B2, BD Pharmigen), CD4 (RM4-5, BD Pharmigen), and MHC-II (M5/114.15.2, BD Pharmigen) were used for flow cytometric analyses. The B220, CD4, and MHC-II antibodies were analyzed in the “dump gate” for the tetramer, IFNγ, and Vβ staining experiments. For costaining experiments, splenocytes from vaccinated mice were stained with Ld-tet linked to the AH1 peptide with a disulfide trap and conjugated to steptavidin-PE for 2 h at 37°C to facilitate internalization and limit competition between tetramers and Ld-tet linked to the F1A5 or WMF peptides conjugated to streptavidin-AF647 were added and stained for an additional 2 h at 4°C. All samples were run on a BD FACSCaliber flow cytometer.
Intracellular cytokine staining
Splenocytes were stimulated with the indicated peptide for 5 h in the presence of monensin, a protein transport inhibitor (GolgiStop, BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit, BD Pharmingen), fixed, permeabilized, and stained as described .
Tetramer decay and titration
Normalized fluorescence (Nf) was calculated by dividing the Tf at each time point by the Tf at time 0. The natural log (ln) of the Nf was plotted versus time and the data for each mouse were fit to a one-phase exponential decay curve [(y = y0*e(−kt) where y = ln Nf, k = decay constant, and t = time)] using Prism version 4.0, Graphpad software. The half-lives were calculated for each mouse [(half-life = −ln(2)/k)] and compared using an unpaired two-tailed t test.
For the tetramer titration assay, splenocytes were stained as above for 2 h at 4°C. The MFI was calculated using the following formula : MFI = (Tf)/(% of AH1-tet+ of CD8+ cells). The MFI was plotted versus the concentration of tetramer and fit to a one-site binding curve [Y = (Bmax*X)/(KD + X)] using Prism version 4.0, Graphpad software. The KD values for each mouse were compared using an unpaired two-tailed t test.
Approximately 1 × 105 CD8+ AH1-tet+ splenocytes were separated with a MoFlo® High-Performance Cell Sorter. RNA was extracted using the RNeasy Mini Kit (Qiagen), and cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions. For the plasmid sequencing method, the cDNA was PCR-amplified using the primer sets described in Supplemental Table 1 [27, 28, 29]. Amplified DNA was cloned into the pCR2.1 vector using the TOPO TA Cloning Kit (Invitrogen), and sequences were determined using an internal Cβ primer (Supplemental Table 1) and capillary DNA sequencing instruments (ABI 3730s). For the high-throughput method, the cDNA was PCR-amplified for 15 cycles using a forward primer specific for all Vβ8 family members containing an adaptor sequence (in bold) and an internal multiplex identifier (MID) sequence (in italics, Supplemental Table 1) and a reverse primer specific for Cβ containing a different adaptor sequence (in bold, Supplemental Table 1). PCR fragments were separated by gel electrophoresis, purified using the Gel Extraction Kit (Qiagen), and further amplified 30 cycles using primers specific for the adaptor sequences. After gel extraction and quantification, the PCR products were combined and subjected to high-throughput sequencing as previously described . Sequences from both methods were analyzed using a computer software program developed by our laboratory that uses a BLAST-type algorithm to identify Vβ and Jβ gene sequence information [28, 31]. This program translated and aligned the sequences, identified the MID sequences and sorted the results according to each vaccine, distinguished the germline-encoded and randomized CDR3β region of each sequence, calculated the length of each CDR3β region, and determined the number of sequences containing the shared CDR3β motif. The sequences of the TCRs expressed by the T cell clones (Fig. 6a) were determined by directly sequencing PCR products amplified from their cDNA using the Vβ8.3 and Cβ primers or Vα6 primers.
Vaccination with the F1A5 peptide elicits T cells with higher affinity for the AH1 peptide
Previously identified peptide variants effectively stimulated a tumor antigen-specific T cell clone both in vitro and in vivo, but elicited variable antitumor responses from the endogenous T cell repertoire . Vaccination with the F1A5 peptide variant protected 90% of mice from tumor growth, while tumors grew in all of the mice vaccinated with the WMF peptide [2, 17]. The increased tumor protection afforded by the F1A5 peptide was attributed to the expansion of more tumor-specific T cells that exhibited effector function after stimulation with the AH1 peptide, although the mechanism for this differential expansion of functional T cells was not determined .
Since tetramer-dissociation rates correlate with peptide-MHC/TCR affinities in some systems , we measured the half-life (t1/2) of tetramer binding to the responding T cell repertoire (Fig. 1b). The average t1/2 of T cells from F1A5- and WMF-vaccinated mice was not statistically different (Fig. 1biii). Although many factors contribute to Kd values calculated using multivalent interactions , these observed avidity differences may be due to faster on-rates rather than longer half-lives . In summary, the F1A5-elicited T cells bound to the AH1 antigen with increased affinity, which may explain the previously described improved effector function exhibited by T cells responding to this peptide vaccine .
Few tumor-specific T cells cross-react with both the F1A5 and WMF peptides
In some systems, functional measurements more accurately assess the number of antigen-specific T cells . To ensure the tetramer-binding experiments did not underestimate the number of T cells that cross-react with both peptide variants, we analyzed intracellular IFNγ production by splenocytes from vaccinated mice stimulated with either the F1A5 or the WMF peptides (Fig. 2c, d). Similar to the tetramer staining results, few T cells cross-reacted with both variant peptides. In addition, as previously reported , few WMF-elicited T cells produced IFNγ after stimulation with the AH1 peptide (Fig. 2d). Together, these data suggest that vaccination with the F1A5 and WMF peptides elicited different portions of the endogenous AH1-specific T cell repertoire.
F1A5 and WMF peptides elicit different repertoires of T cells
To directly characterize the TCR gene usage of the AH1-specific T cells elicited by the peptide variants, we co-stained splenocytes from vaccinated mice with the AH1-tetramer and a panel of antibodies to the mouse TCR Vβ genes (Supplementary Fig. 2a). In addition to the F1A5 and WMF peptides, we also analyzed the response to the native tumor antigen and to three other previously characterized peptide variants, 15 (ineffective), and A5 and 39 (effective) [9, 17, 18]. All of these peptide variants were also selected using the Vβ8.3+ CT T cell clone and, not surprisingly, they elicited AH1-specific T cells that predominantly expressed the Vβ8 family of TCRs (Supplementary Fig. 2b).
Effective and ineffective peptides elicit different repertoires of AH1-specific T cells
Surprisingly, although these junctional sequences were similar to that expressed by the CT T cell clone (Fig. 6a), we did not find the exact CT-TCR sequence in these mice or in the mice vaccinated with the peptide variants. The absence of the CT T cell clone in this study suggests that this clone is not representative of the endogenous repertoire of T cells responding to these vaccines and explains the lack of cross-reactivity between the F1A5- and WMF-elicited repertoires (Fig. 2). Perhaps the different mode of immunization (whole cell irradiated CT26-GM ) and the in vitro expansion used to generate the CT clone accounts for its dominance in those studies, and absence here.
TCRs containing the CDR3β motif bind to the WMF peptide with lower affinity
To study the binding properties of the motif-containing T cells described above, we required both the β- and α-chain sequences of the TCRs identified in Figs. 3 and 4. We expanded and cloned AH1-specific T cells from vaccinated mice as described . The TCR genes expressed by these T cell clones did not reflect the sequencing results we obtained from fresh uncultured splenocytes, particularly for the T cell clones from mice vaccinated with the ineffective peptide variants (Fig. 6a). However, we obtained several antigen-specific T cell clones that expressed TCRs containing the CDR3β motif and that had identical Vβ chains as some of those identified ex vivo. These T cell clones expressed a Vα6 TCR, distinct from the Vα4.11 TCR expressed by the CT T cell clone (Fig. 6a).
To determine the binding properties of TCRs containing the CDR3β motif, we subcloned the TCR molecules expressed by the A5-4E11 and the 39-1D4 T cell clones (Fig. 6a) into a BV expression vector. The sequences of these TCRs were highly selected in mice vaccinated with AH1, A5, and F1A5 peptides and not identified in mice vaccinated with the ineffective 15 and WMF peptides. We previously showed that the relative binding of multimeric-soluble TCRs to peptide–MHC complex expressed on BV-infected insect cells correlates with the affinity of monomeric TCR for the peptide–MHC complex determined by surface plasmon resonance [17, 18, 33, 39, 40]. Therefore, we stained insect cells expressing each of the peptide variant–Ld complexes with fluorescent multimeric TCRs derived from the A5-4E11 and 39-1D4 clones. The soluble TCRs bound to insect cells expressing the native tumor antigen, but not the irrelevant βgal peptide, demonstrating that these TCRs are specific for the AH1 peptide (Fig. 6b, c). The staining intensity of insect cells expressing the WMF peptide was significantly lower, indicating that this CDR3β motif-containing TCR does not bind efficiently to the WMF peptide (Fig. 6b). The binding of the 39-1D4 TCR more closely correlated with its representation in the ex vivo repertoire analysis, binding to the A5 and F1A5 peptides and not to the WMF peptide (Fig. 6c). The low-affinity interaction between the CDR3β motif-containing TCRs and the WMF peptide provides a simple mechanistic explanation for the absence of these cells following vaccination with the WMF peptide.
Although the peptide variants used in our study effectively stimulated a tumor-specific T cell clone in vitro, they elicited variable responses from the endogenous T cell population. In this study, we found that effective cancer peptide vaccines stimulate T cells with hypervariable regions similar to those that respond to the natural tumor antigen, not a de novo T cell repertoire. These results suggest that the heterogeneity in the repertoire of AH1-specific T cells contributes to the ineffectiveness of some of the peptide-variant vaccines.
Similar to our results, a study of the T cell response to the Melan-A/MART-126–35 TAA showed that the TCRβ gene usage of the T cells responding to the native peptide was broader than that responding to the peptide variant . They also identified a CDR3β motif that was shared among several patients, a so-called public TCR, although these motif-containing T cells were not among the dominant clones identified in each patient and did not have a higher functional avidity . They concluded that the repertoires responding to the peptide variants and the native tumor antigen were subtly different but over-lapping, and that the observed functional differences were due to subtle structural changes in the TCR. In contrast, the T cells responding to the ineffective peptide variants in our study express a unique repertoire of TCRs that do not overlap with the repertoire responding to the native tumor antigen and that do not contain a significant fraction of the shared CDR3β motif. Therefore, in our study, only the effective peptide variants elicited a repertoire of T cells that over-lapped with those responding to the native tumor antigen.
The most effective peptide variants (A5 and F1A5) have minimal amino acid sequence changes relative to the AH1 peptide. It is, therefore, not surprising that these vaccines raise similar T cell repertoires. These data suggest that peptides with conservative amino acid changes may be more effective in stimulating antitumor immunity by eliciting a repertoire of T cells that mimics the response to the native tumor antigen. In agreement with this premise, another study concluded that substitutions at MHC-binding positions create immunogenic peptide variants with an overall similar structure to the native peptide . However, the opposite has also been shown in these studies. Peptides specifically designed to encode conservative amino acid changes were ineffective vaccines and raised a repertoire of T cells different from that of the native tumor antigen [10, 20, 41]. These discrepancies suggest that even subtle amino acid substitutions may cause unpredictable changes in the repertoire and binding kinetics of the responding endogenous T cells.
The T cells responding to the F1A5 peptide express higher-affinity T cell receptors that functionally recognize the AH1 TAA better than the T cells responding to the ineffective peptide, WMF. Furthermore, soluble TCRs encoding the common CDR3β motif do not bind efficiently to the WMF peptide, providing a mechanistic explanation for the lack of these TCRs in the responding repertoire. We predict from these data that the structure of the WMF peptide prevents productive interactions with TCRs containing the common CDR3β motif. Specifically, the tryptophan residue at position 7 of the WMF peptide may affect the position of the adjacent tyrosine residue at position 6, precluding its predicted interaction with the tyrosine residue encoded by the Jβ2.6 gene segment of the motif-containing TCRs. Important for peptide vaccine development, these results imply that T cells responding to the ineffective variants (like WMF) cannot be “rescued” or forced to respond to the tumor antigen with better adjuvants; they are not anergic or improperly activated, they simply express T cell receptors that interact poorly with the native tumor antigen.
A clinically relevant strategy in the design of effective peptide vaccines is to optimize the T cells used in the identification of peptide variants. We selected our peptide variants based on improved binding to and activation of the CT clone, an AH1-specific T cell clone that was in vitro expanded and is not similar to those analyzed ex vivo (Fig. 6). The T cell clones that were found in the endogenous repertoire bound to the peptides that elicit better tumor-specific responses (Fig. 6). Therefore, we predict that peptide variants selected with an ex vivo tumor-specific repertoire, rather than an in vitro selected T cell clone, may be the most effective peptides for cancer vaccines.
Due to the unpredictable nature of the responding T cell repertoire, even if optimal T cells are used for peptide selection, ineffective peptides may be unavoidable in peptide-variant vaccine development. Therefore, a general strategy to improve the response to peptide variants such as the WMF peptide should be considered. In this particular case, rather than providing additional adjuvants or cytokines during T cell activation, the repertoire of the T cell response needs to be re-focused to those with high affinity for the native tumor antigen. Although strategies to engineer T cells with receptors that have high affinity for tumor antigens have been developed and employed in the clinical treatment for cancer [42, 43], effective antitumor immunity may be more easily achieved by “boosting” the peptide-variant responses with native tumor antigens [14, 44]. This strategy may increase the number of high-affinity T cells that cross-react with native tumor antigens and reduce competition with T cells that only bind to peptide variants.
In summary, peptide-variant vaccines improve the proliferation and activation of T cells that respond to native tumor antigens. Because the T cell response is unpredictable and different for every antigen, optimal peptide variants may be most successfully identified using a representative TAA-specific T cell population to select preferred residue changes from a large and random set of peptides.
This work was supported by NCI CA109560 and ACS RSG-08-184-01-LIB. K.R.J. and J.D.B. were supported in part by the Cancer Research Institute Predoctoral Emphasis Pathway in Tumor Immunology Fellowship.
Conflict of interest
The authors have no conflicting financial interests.
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