The common HLA class I-restricted tumor-infiltrating T cell response in HPV16-induced cancer.

Immunotherapies targeting truly tumor-specific targets focus on the expansion and activation of T cells against neoantigens or oncogenic viruses. One target is the human papilloma virus type 16 (HPV16), responsible for several anogenital cancers and oropharyngeal carcinomas. Spontaneous and vaccine-induced HPV-specific T cells have been associated with better clinical outcome. However, the epitopes and restriction elements to which these T cells respond remained elusive. To identify CD8+ T cell epitopes in cultures of tumor infiltrating lymphocytes, we here used multimers and/or a functional screening platform exploiting single HLA class I allele-engineered antigen presenting cells. This resulted in the detection of 20 CD8+ T cell responses to 11 different endogenously processed HLA-peptide combinations within 12 HPV16-induced tumors. Specific HLA-peptide combinations dominated the response in patients expressing these HLA alleles. T cell receptors (TCRs) reactive to seven different HLA class I-restricted peptides could be isolated and analysis revealed tumor reactivity for five of the six TCRs analyzed. The tumor reactive TCRs to these dominant HLA class I peptide combinations can potentially be used to engineer tumor-specific T cells for adoptive cell transfer approaches to treat HPV16-induced cancers.

TCR repertoires of intratumoral T-cell subsets.

The infiltration of human tumors by T cells is a common phenomenon, and over the past decades, it has become increasingly clear that the nature of such intratumoral T-cell populations can predict disease course. Furthermore, intratumoral T cells have been utilized therapeutically in clinical studies of adoptive T-cell therapy. In this review, we describe how novel methods that are either based on T-cell receptor (TCR) sequencing or on cancer exome analysis allow the analysis of the tumor reactivity and antigen-specificity of the intratumoral TCR repertoire with unprecedented detail. Furthermore, we discuss studies that have started to utilize these techniques to probe the link between cancer exomes and the intratumoral TCR pool. Based on the observation that both the cancer epitope repertoire and intratumoral TCR repertoire appear highly individual, we outline strategies, such as 'autologous TCR gene therapy', that exploit the tumor-resident TCR repertoire for the development of personalized immunotherapy.

Towards error-free profiling of immune repertoires.

Deep profiling of antibody and T cell-receptor repertoires by means of high-throughput sequencing has become an attractive approach for adaptive immunity studies, but its power is substantially compromised by the accumulation of PCR and sequencing errors. Here we report MIGEC (molecular identifier groups-based error correction), a strategy for high-throughput sequencing data analysis. MIGEC allows for nearly absolute error correction while fully preserving the natural diversity of complex immune repertoires.

Peptide Splicing in the Proteasome Creates a Novel Type of Antigen with an Isopeptide Linkage.

The proteasome is able to create spliced Ags, in which two distant parts of a protein are excised and ligated together to form a novel peptide, for presentation by MHC class I molecules. These noncontiguous epitopes are generated via a transpeptidation reaction catalyzed by the proteasomal active sites. Transpeptidation reactions in the proteasome follow explicit rules and occur particularly efficiently when the C-terminal ligation partner contains a lysine or arginine residue at the site of ligation. Lysine contains two amino groups that theoretically may both participate in ligation reactions, implying that potentially not only peptide but also isopeptide linkages could be formed. Using nuclear magnetic resonance spectroscopy, we demonstrate in the present study that the proteasome can use the ε-amino group of an N-terminal lysine residue in transpeptidation reactions to create a novel type of posttranslationally modified epitopes. We show that the overall efficiency of ε ligation is only 10-fold lower as compared with α ligation, suggesting that the proteasome can produce sufficient isopeptide Ag to evoke a T cell response. Additionally, we show that isopeptides are more stable toward further proteasomal processing than are normal peptides, and we demonstrate that isopeptides can bind to HLA-A2.1 and HLA-A3 with high affinity. These properties likely increase the fraction of ε-ligated peptides presented on the cell surface for CD8(+) T cell surveillance. Finally, we show that isopeptide Ags are immunogenic in vivo. We postulate that ε ligation is a genuine posttranslational modification, suggesting that the proteasome can create a novel type of Ag that is likely to play a role in immunity.

Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules.

Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I-restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags.

HLA micropolymorphisms strongly affect peptide-MHC multimer-based monitoring of antigen-specific CD8+ T cell responses.

Peptide-MHC (pMHC) multimers have become one of the most widely used tools to measure Ag-specific T cell responses in humans. With the aim of understanding the requirements for pMHC-based personalized immunomonitoring, in which individuals expressing subtypes of the commonly studied HLA alleles are encountered, we assessed how the ability to detect Ag-specific T cells for a given peptide is affected by micropolymorphic differences between HLA subtypes. First, analysis of a set of 10 HLA-A*02:01-restricted T cell clones demonstrated that staining with pMHC multimers of seven distinct subtypes of the HLA-A*02 allele group was highly variable and not predicted by sequence homology. Second, to analyze the effect of minor sequence variation in a clinical setting, we screened tumor-infiltrating lymphocytes of an HLA-A*02:06 melanoma patient with either subtype-matched or HLA-A*02:01 multimers loaded with 145 different melanoma-associated Ags. This revealed that of the four HLA-A*02:06-restricted melanoma-associated T cell responses observed in this patient, two responses were underestimated and one was overlooked when using subtype-mismatched pMHC multimer collections. To our knowledge, these data provide the first demonstration of the strong effect of minor sequence variation on pMHC-based personalized immunomonitoring, and they provide tools to prevent this issue for common variants within the HLA-A*02 allele group.

Blockade of TGF-ß signaling greatly enhances the efficacy of TCR gene therapy of cancer.

TCR gene therapy is a promising approach for the treatment of various human malignancies. However, the tumoricidal activity of TCR-modified T cells may be limited by local immunosuppressive mechanisms within the tumor environment. In particular, many malignancies induce T cell suppression in their microenvironment by TGF-ß secretion. In this study, we evaluate whether blockade of TGF-ß signaling in TCR-modified T cells enhances TCR gene therapy efficacy in an autochthonous mouse tumor model. Treatment of mice with advanced prostate cancer with T cells genetically engineered to express a tumor-reactive TCR and a dominant-negative TGF-ß receptor II induces complete and sustained tumor regression, enhances survival, and leads to restored differentiation of prostate epithelium. These data demonstrate the potential to tailor the activity of TCR-modified T cells by additional genetic modification and provide a strong rationale for the clinical testing of TGF-ß signaling blockade to enhance TCR gene therapy against advanced cancers.

RNAi-mediated TCR knockdown prevents autoimmunity in mice caused by mixed TCR dimers following TCR gene transfer.

Genetically modified T cells that express a transduced T cell receptor (TCR) α/ß heterodimer in addition to their endogenous TCR are used in clinical studies to treat cancer. These cells express two TCR-α and two TCR-ß chains that do not only compete for CD3 proteins but also form potentially self-reactive mixed TCR dimers, composed of endogenous and transferred chains. To overcome these deficits, we developed an RNAi-TCR replacement vector that simultaneously silences the endogenous TCR and expresses an RNAi-resistant TCR. Transduction of the virus-specific P14 TCR without RNAi resulted in unequal P14 TCR-α and -ß chain surface levels, indicating heterodimerization with endogenous TCR chains. Such unequal expression was also observed following TCR gene optimization. Equal surface levels of the introduced TCR chains were however achieved by silencing the endogenous TCR. Importantly, all mice that received cells transduced with the native or optimized P14 TCR developed lethal TCR gene transfer-induced graft-versus-host-disease (TI-GVHD) due to formation of mixed TCR dimers. In contrast, TI-GVHD was almost completely prevented when using the RNAi-TCR replacement vector. Our data demonstrate that RNAi-assisted TCR replacement reduces the formation of mixed TCR dimers, and thereby significantly reduces the risk of TI-GVHD in TCR gene therapy.

Pairing of T-cell receptor chains via emulsion PCR.

Our ability to analyze adaptive immunity and engineer its activity has long been constrained by our limited ability to identify native pairs of heavy-light antibody chains and alpha-beta T-cell receptor (TCR) chains--both of which comprise coupled "halves of a key", collectively capable of recognizing specific antigens. Here, we report a cell-based emulsion RT-PCR approach that allows the selective fusion of the native pairs of amplified TCR alpha and beta chain genes for complex samples. A new type of PCR suppression technique was developed that makes it possible to amplify the fused library with minimal noise for subsequent analysis by high-throughput paired-end Illumina sequencing. With this technique, single analysis of a complex blood sample allows identification of multiple native TCR chain pairs. This approach may be extended to identify native antibody chain pairs and, more generally, pairs of mRNA molecules that are coexpressed in the same living cells.

Antiproliferative effects of selective adenosine receptor agonists and antagonists on human lymphocytes: evidence for receptor-independent mechanisms.

The effects of standard adenosine receptor (AR) agonists and antagonists on the proliferation of human T lymphocytes, unstimulated and phytohemagglutinin-stimulated human peripheral blood lymphocytes (PBL), and Jurkat T cells were investigated. Real-time PCR measurements confirmed the presence of all four AR subtypes on the investigated cells, although at different expression levels. A2A ARs were predominantly expressed in PBL and further upregulated upon stimulation, while malignant Jurkat T cells showed high expression levels of A1, A2A, and A2B ARs. Cell proliferation was measured by [(3)H]-thymidine incorporation assays. Several ligands, including the subtype-selective agonists CPA (A1), BAY60-6583 (A2B), and IB-MECA (A3), and the antagonists PSB-36 (A1), MSX-2 (A2A), and PSB-10 (A3) significantly inhibited cell proliferation at micromolar concentrations, which were about three orders of magnitude higher than their AR affinities. In contrast, further investigated AR ligands, including the agonists NECA (nonselective) and CGS21680 (A2A), and the antagonists preladenant (SCH-420814, A2A), PSB-1115 (A2B), and PSB-603 (A2B) showed no or only minor effects on lymphocyte proliferation. The anti-proliferative effects of the AR agonists could not be blocked by the corresponding antagonists. The non-selective AR antagonist caffeine stimulated phytohemagglutinin-activated PBL with an EC50 value of 104 µM. This is the first study to compare a complete set of commonly used AR ligands for all subtypes on lymphocyte proliferation. Our results strongly suggest that these compounds induce an inhibition of lymphocyte proliferation and cell death through AR-independent mechanisms.

Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7.

Major histocompatibility complex (MHC) class I multimer technology has become an indispensable immunological assay system to dissect antigen-specific cytotoxic CD8(+) T cell responses by flow cytometry. However, the development of high-throughput assay systems, in which T cell responses against a multitude of epitopes are analyzed, has been precluded by the fact that for each T cell epitope, a separate in vitro MHC refolding reaction is required. We have recently demonstrated that conditional ligands that disintegrate upon exposure to long-wavelength UV light can be designed for the human MHC molecule HLA-A2. To determine whether this peptide-exchange technology can be developed into a generally applicable approach for high throughput MHC based applications we set out to design conditional ligands for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Here, we describe the development and characterization of conditional ligands for this set of human MHC molecules and apply the peptide-exchange technology to identify melanoma-associated peptides that bind to HLA-A3 with high affinity. The conditional ligand technology developed here will allow high-throughput MHC-based analysis of cytotoxic T cell immunity in the vast majority of Western European individuals.

Adenosine regulates CD8 T-cell priming by inhibition of membrane-proximal T-cell receptor signalling.

Adenosine is a well-described anti-inflammatory modulator of immune responses within peripheral tissues. Extracellular adenosine accumulates in inflamed and damaged tissues and inhibits the effector functions of various immune cell populations, including CD8 T cells. However, it remains unclear whether extracellular adenosine also regulates the initial activation of naïve CD8 T cells by professional and semi-professional antigen-presenting cells, which determines their differentiation into effector or tolerant CD8 T cells, respectively. We show that adenosine inhibited the initial activation of murine naïve CD8 T cells after alphaCD3/CD28-mediated stimulation. Adenosine caused inhibition of activation, cytokine production, metabolic activity, proliferation and ultimately effector differentiation of naïve CD8 T cells. Remarkably, adenosine interfered efficiently with CD8 T-cell priming by professional antigen-presenting cells (dendritic cells) and semi-professional antigen-presenting cells (liver sinusoidal endothelial cells). Further analysis of the underlying mechanisms demonstrated that adenosine prevented rapid tyrosine phosphorylation of the key kinase ZAP-70 as well as Akt and ERK1/2 in naïve alphaCD3/CD28-stimulated CD8 cells. Consequently, alphaCD3/CD28-induced calcium-influx into CD8 cells was reduced by exposure to adenosine. Our results support the notion that extracellular adenosine controls membrane-proximal T-cell receptor signalling and thereby also differentiation of naïve CD8 T cells. These data raise the possibility that extracellular adenosine has a physiological role in the regulation of CD8 T-cell priming and differentiation in peripheral organs.

Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers.

Infiltration of human cancers by T cells is generally interpreted as a sign of immune recognition, and there is a growing effort to reactivate dysfunctional T cells at such tumor sites1. However, these efforts only have value if the intratumoral T cell receptor (TCR) repertoire of such cells is intrinsically tumor reactive, and this has not been established in an unbiased manner for most human cancers. To address this issue, we analyzed the intrinsic tumor reactivity of the intratumoral TCR repertoire of CD8+ T cells in ovarian and colorectal cancer-two tumor types for which T cell infiltrates form a positive prognostic marker2,3. Data obtained demonstrate that a capacity to recognize autologous tumor is limited to approximately 10% of intratumoral CD8+ T cells. Furthermore, in two of four patient samples tested, no tumor-reactive TCRs were identified, despite infiltration of their tumors by T cells. These data indicate that the intrinsic capacity of intratumoral T cells to recognize adjacent tumor tissue can be rare and variable, and suggest that clinical efforts to reactivate intratumoral T cells will benefit from approaches that simultaneously increase the quality of the intratumoral TCR repertoire.

Isolation of T cell receptors targeting recurrent neoantigens in hematological malignancies.

Mutation-derived neoantigens represent an important class of tumour-specific, tumour rejection antigens, and are attractive targets for TCR gene therapy of cancer. The majority of such mutations are patient-specific and targeting these requires a fully personalized approach. However, some mutations are found recurrently among cancer patients, and represent potential targets for neoantigen-specific TCR gene therapy that is more widely applicable. Therefore, we have investigated if some cancer mutations found recurrently in hematological malignancies encode immunogenic neoantigens presented by common European Caucasoid HLA class I alleles and can form targets for TCR gene therapy. We initially focused on identifying HLA class I neoepitopes derived from calreticulin (CALR) exon 9 mutations, found in ~ 80% of JAK2wt myeloproliferative neoplasms (MPN). Based on MHC class I peptide predictions, a number of peptides derived from mutant CALR (mCALR) were predicted to bind to HLA-A*03:01 and HLA-B*07:02. However, using mass spectrometry and ex vivo pMHC multimer staining of PBMC from MPN patients with CALR exon 9 mutations, we found no evidence that these peptides were naturally processed and presented on the surface of mCALR-expressing target cells. We next developed a protocol utilizing pMHC multimers to isolate CD8+ T cells from healthy human donor PBMC that are specific for mCALR and additional putative neoepitopes found recurrently in hematological malignancies. Using this approach, CD8+ T cells specific for HLA-A*03:01- and HLA-B*07:02-presented mCALR peptides and an HLA-A*11:01-presented mutant FBXW7 (mFBXW7) peptide were successfully isolated. TCRs isolated from mCALR-specific CD8+ T cell populations were not able to recognize target cells engineered to express mCALR. In contrast, mFBXW7-specific CD8+ T cells were able to recognize target cells engineered to express mFBXW7. In conclusion, while we found no evidence for mCALR derived neoepitope presentation in the context of the HLA class I alleles studied, our data suggests that the recurrent pR465H mutation in FBXW7 may encode an HLA-A*11:01 presented neoepitope, and warrants further investigation as a target for T cell based immunotherapy of cancer.

T cell receptor fingerprinting enables in-depth characterization of the interactions governing recognition of peptide-MHC complexes.

The promiscuous nature of T-cell receptors (TCRs) allows T cells to recognize a large variety of pathogens, but makes it challenging to understand and control T-cell recognition. Existing technologies provide limited information about the key requirements for T-cell recognition and the ability of TCRs to cross-recognize structurally related elements. Here we present a 'one-pot' strategy for determining the interactions that govern TCR recognition of peptide-major histocompatibility complex (pMHC). We measured the relative affinities of TCRs to libraries of barcoded peptide-MHC variants and applied this knowledge to understand the recognition motif, here termed the TCR fingerprint. The TCR fingerprints of 16 different TCRs were identified and used to predict and validate cross-recognized peptides from the human proteome. The identified fingerprints differed among TCRs recognizing the same epitope, demonstrating the value of this strategy for understanding T-cell interactions and assessing potential cross-recognition before selection of TCRs for clinical development.

High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma.

Tumor-specific neo-antigens that arise as a consequence of mutations are thought to be important for the therapeutic efficacy of cancer immunotherapies. Accumulating evidence suggests that neo-antigens may be commonly recognized by intratumoral CD8+ T cells, but it is unclear whether neo-antigen-specific CD4+ T cells also frequently reside within human tumors. In view of the accepted role of tumor-specific CD4+ T-cell responses in tumor control, we addressed whether neo-antigen-specific CD4+ T-cell reactivity is a common property in human melanoma.

Anti-CTLA-4 therapy broadens the melanoma-reactive CD8+ T cell response.

Anti-CTLA-4 treatment improves the survival of patients with advanced-stage melanoma. However, although the anti-CTLA-4 antibody ipilimumab is now an approved treatment for patients with metastatic disease, it remains unknown by which mechanism it boosts tumor-specific T cell activity. In particular, it is unclear whether treatment amplifies previously induced T cell responses or whether it induces new tumor-specific T cell reactivities. Using a combination ultraviolet (UV)-induced peptide exchange and peptide-major histocompatibility complex (pMHC) combinatorial coding, we monitored immune reactivity against a panel of 145 melanoma-associated epitopes in a cohort of patients receiving anti-CTLA-4 treatment. Comparison of pre- and posttreatment T cell reactivities in peripheral blood mononuclear cell samples of 40 melanoma patients demonstrated that anti-CTLA-4 treatment induces a significant increase in the number of detectable melanoma-specific CD8 T cell responses (P = 0.0009). In striking contrast, the magnitude of both virus-specific and melanoma-specific T cell responses that were already detected before start of therapy remained unaltered by treatment (P = 0.74). The observation that anti-CTLA-4 treatment induces a significant number of newly detected T cell responses-but only infrequently boosts preexisting immune responses-provides strong evidence for anti-CTLA-4 therapy-enhanced T cell priming as a component of the clinical mode of action.

High-throughput identification of antigen-specific TCRs by TCR gene capture.

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

TIL therapy broadens the tumor-reactive CD8(+) T cell compartment in melanoma patients.

There is strong evidence that both adoptive T cell transfer and T cell checkpoint blockade can lead to regression of human melanoma. However, little data are available on the effect of these cancer therapies on the tumor-reactive T cell compartment. To address this issue we have profiled therapy-induced T cell reactivity against a panel of 145 melanoma-associated CD8(+) T cell epitopes. Using this approach, we demonstrate that individual tumor-infiltrating lymphocyte cell products from melanoma patients contain unique patterns of reactivity against shared melanoma-associated antigens, and that the combined magnitude of these responses is surprisingly low. Importantly, TIL therapy increases the breadth of the tumor-reactive T cell compartment in vivo, and T cell reactivity observed post-therapy can almost in full be explained by the reactivity observed within the matched cell product. These results establish the value of high-throughput monitoring for the analysis of immuno-active therapeutics and suggest that the clinical efficacy of TIL therapy can be enhanced by the preparation of more defined tumor-reactive T cell products.

T-cell receptor gene therapy: critical parameters for clinical success.

T-cell receptor (TCR) gene therapy aims to induce immune reactivity against tumors by introducing genes encoding a tumor-reactive TCR into patient T cells. This approach has been extensively tested in preclinical mouse models, and initial clinical trials have demonstrated the feasibility and potential of TCR gene therapy as a cancer treatment. However, data obtained from preclinical and clinical studies suggest that both the therapeutic efficacy and the safety of TCR gene therapy can be and needs to be further enhanced. This review highlights those strategies that can be followed to develop TCR gene therapy into a clinically relevant treatment option for cancer patients.