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TCR Ligand Discovery via T-Scan
Please cite this article in press as: Wang et al., TCR Ligand Discovery via T-Scan, Trends in Immunology (2019), https://doi.org/10.1016/ j.it.2019.10.003
Trends in Immunology
Spotlight
TCR Ligand Discovery via T-Scan Zhe Wang,1,2 Genhong Cheng,3,* and Guideng Li1,2,* T cell receptor (TCR) ligand discovery is crucial to monitoring T cell responses to antigen and to identifying antigens reactive against orphan TCRs of interest. In a recent article, Elledge and colleagues describe a functional T cell ligand screening platform for unbiased TCR ligand discovery. T cells are central mediators of immunity and are required to maintain the genetic homeostasis of host tissues. TCRs on the surface of a T cell can recognize a specific antigen fragment (peptide and/or epitope) presented on MHC molecules. The most common antigenic peptides are derived from pathogen proteins, viral genes, or aberrant gene products from somatic nonsynonymous mutations. Mounting evidence indicates that the interaction of TCR with the peptide–MHC complex (pMHC) is complex and even promiscuous. A single TCR can bind to different antigenic peptides and vice versa. This inevitably leads to off-target reactivity or cross-reactivity of therapeutic TCRs. In the clinic, unexpected immune responses against self-antigens constitute one of the main reasons for autoimmunity and off-target toxicities caused by TCR-T cell therapy. Therefore, an increased understanding of TCR– pMHC recognition is fundamental to the development of clinically beneficial and safe immunotherapies in cancer. Several methods have been established to identify ligands reactive against orphan TCRs of interest. Functional T cell expansion assays and pMHC multimer-based screening are the two most common antigen-directed approaches
[1,2]. However, these methods require foreknowledge of those antigenic targets and are low throughput, therefore precluding unbiased screening. Recently, scientists have also made considerable progress in TCR-guided antigen discovery [3]. An unbiased method was developed to screen a yeast cell surface-displayed pMHC library by using soluble, fluorescently labeled TCR reagents [3]. However, this system requires a laborious process to make soluble TCR reagents. Two innovative approaches that utilize different biological processes to label the target cells in a co-culture system were established to overcome these disadvantages [4,5]. One approach utilizes a phenomenon called ‘trogocytosis’, a biological process by which cells share membrane and membrane-associated proteins while conjugated [5]. TCR–pMHC interactions lead to specific transfer of surface proteins (e.g., TCRs) to cognate antigen-presenting cells, which can be measured by flow cytometry. This platform was successfully used to identify the cognate neoepitope for a subject-derived neoantigen-specific TCR [5]. The other approach takes advantage of signaling and antigen-presenting bifunctional receptors (SABRs), in which an intracellular CD3z signaling domain with a CD28 co-stimulatory domain is linked to the pMHC complex [4]. Following pMHC–TCR interactions, the intracellular signal results in the expression of GFP in target cells, which can be isolated and sequenced to identify the specific peptide recognized by the TCR [4]. A similar strategy has been developed for screening the antigenic epitope presented by MHC-II molecules [6]. These methods make TCRdirected antigen discovery more simple, scalable, and flexible. Additionally, a single-cell droplet microfluidicsbased TCR T cell pairing method was established to monitor the kinetics of
TCR–antigen recognition in live cells within droplets using a NFAT-eGFP reporter [7]. Most recently, Elledge and colleagues developed a state-of-the-art method called ‘T-Scan’ (Figure 1), which can identify functional T cell targets from genome-wide antigen libraries with high complexity [8]. Upon TCR–pMHC engagement, serine protease granzyme B (GzB) can be delivered into target cells and trigger apoptosis. The authors exploited the activity of the GzB reporter as the functional readout of physiological TCR–pMHC engagement. They first generated an infrared fluorescent protein (IFP)-based GzB reporter (IFPGZB) that contains a GzB-specific cleavage sequence. Upon T cell recognition, GzB-mediated cleavage results in the activation of the IFPGZB reporter. To ensure screening efficiency, the authors further made improvements to reduce background noise and preserve signal. They engineered MHC-I null epitope discovery cells (EDCs), which expressed both the IFPGZB reporter and a mutated inhibitor of caspase-activated DNase (ICAD), to prevent caspase activation-induced genomic DNA fragmentation [8]. Cytomegalovirus (CMV)-specific TCRs were first used to validate the productivity of T-Scan. The authors established two synthesized oligonucleotides libraries comprising peptides across either the entire CMV genome or the entire human virome. After co-culturing EDCs expressing these peptide libraries and CD8+ T cells expressing two different CMV-specific TCRs, the most enriched peptides expressed in the cell-sorted IFP+ cells represented the known target peptide NLVPMVATV (NLV) derived from the CMV-pp65 protein. Additionally, when co-culturing these EDCs with NLV-reactive T cells (CD8+ T cells from CMV+ donors that had been expanded
Trends in Immunology, -- 2019, Vol. --, No. --
1
Please cite this article in press as: Wang et al., TCR Ligand Discovery via T-Scan, Trends in Immunology (2019), https://doi.org/10.1016/ j.it.2019.10.003
Trends in Immunology
Trends in Immunology
Figure 1. Schematic Illustration of the Genome-Wide T-Scan Screening Platform. Epitope discovery cells (EDCs) are genetically engineered with a granzyme B (GzB) cleavage-activating infrared fluorescent protein (IFPGzB), which works as a reporter to monitor GzB activity. A mutated inhibitor of caspase-activated DNase (ICAD CR ) is also introduced to EDCs to prevent GzBinduced genomic DNA fragmentation, therefore facilitating the recovery of antigen information from EDCs. Upon co-culture of EDCs expressing a library of candidate antigens with T cells of interest, GzB-mediated cleavage leads to the activation of IFP GZB reporter in the target cells. IFP + EDCs are sorted by fluorescence-activated cell sorting (FACS), followed by genomic DNA extraction, PCR, and next-generation sequencing (NGS) to uncover information relating to the targeting epitopes [8]. Abbreviations: CMV, cytomegalovirus; pMHC, peptide–MHC complex; TCR, T cell receptor.
against the NLV peptide), the authors unexpectedly identified a new epitope from the IE1 gene; this suggested that this platform has the potential for the discovery of novel targets in subclonal populations of T cells. After demonstrating the capacity of T-Scan in genome-wide screening, the authors also applied T-Scan to T cell library-on-peptide library screening using bulk memory CD8+ T cells from patients who were CMV seropositive and identified a set of both known and new CMV-specific epitopes [8]. These results are relevant because they highlight the value of T-scan in the unbiased discovery of novel antigens. The authors further demonstrated the feasibility of T-Scan in understanding the molecular basis of TCR cross-reactivity. They successfully used a comprehensive single mutant library of the NLV epitope to map the critical residues and to identify specific mimotopes for NLV TCR recognition. Finally, they applied TScan to understand the specificity of tumor-reactive TCR in a human-genome 2
wide library using a TCR-targeting HLAA1-restricted epitope of tumor antigen MAGE-A3. The top two enriched epitopes were confirmed as either known MAGE-A3 epitopes or epitopes derived from the related protein MAGE-A6. In addition, the authors identified two additional cross-reactive epitopes derived from PLD5 and FAT2. These epitopes shared a three-amino acid core sequence with the MAGE-A3 peptide, but differed at the remaining six positions [8]. These findings highlight the challenge of bioinformatically predicting TCR cross-reactivity based on sequence similarity, demonstrating the value of unbiased experimental approaches to finding TCR cross-reactivities. T-Scan enables the rapid identification of antigens targeted by an immune response, a key goal in the fields of infectious disease, tumor immunology, autoimmunity, and immunotherapy. It has several advantages over existing technologies, including: (i) the T-Scan
Trends in Immunology, -- 2019, Vol. --, No. --
screening platform utilizes the natural processing and/or presentation of large-scale antigens (260 000 candidate antigens) and the physiological engagement of TCR–pMHC, which enables the interrogation of significantly larger antigen space than was previously possible. This feature also allows unbiased proteome-wide screening and permits the identification of biological relevant targets in the human genome, which differentiates this platform from other approaches; (ii) T-Scan breaks down the bottlenecks of TCR-T based immunotherapy by its library-to-library screening ability to rapidly identify tumor-specific antigens (TSAs) and tumor-reactive therapeutic TCRs without self-target toxicities; and (iii) T-Scan can simultaneously screen for target epitopes presented by the full set of MHC-I alleles from individuals. However, it is not clear whether T-Scan can also be efficiently applied to TCRs mediating MHC-II-restricted responses, which represents another
Please cite this article in press as: Wang et al., TCR Ligand Discovery via T-Scan, Trends in Immunology (2019), https://doi.org/10.1016/ j.it.2019.10.003
Trends in Immunology
important player when considering autoimmune diseases and antitumor responses. Thus, it will be fascinating to follow the development of further applications of T-Scan in the future.
Acknowledgments This work was supported by the National Natural Science Foundation (81972875) and The CAMS Initiative for Innovative Medicine (2016-I2M-1-005). 1Center
of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China
2Suzhou
Institute of Systems Medicine, Suzhou 215123, China
3Department
of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA *Correspondence: [email protected], [email protected] https://doi.org/10.1016/j.it.2019.10.003 ª 2019 Elsevier Ltd. All rights reserved.
References 1. Gros, A. et al. (2016) Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients. Nat. Med. 22, 433–438
2. Stronen, E. et al. (2016) Targeting of cancer neoantigens with donor-derived T cell receptor repertoires. Science 352, 1337–1341 3. Gee, M.H. et al. (2018) Antigen identification for orphan T cell receptors expressed on tumor-infiltrating lymphocytes. Cell 172, 549–563 4. Joglekar, A.V. et al. (2019) T cell antigen discovery via signaling and antigenpresenting bifunctional receptors. Nat. Methods 16, 191–198 5. Li, G. et al. (2019) T cell antigen discovery via trogocytosis. Nat. Methods 16, 183–190 6. Kisielow, J. et al. (2019) Deciphering CD4(+) T cell specificity using novel MHC-TCR chimeric receptors. Nat. Immunol. 20, 652–662 7. Segaliny, A.I. et al. (2018) Functional TCR T cell screening using single-cell droplet microfluidics. Lab Chip 18, 3733–3749 8. Kula, T. et al. (2019) T-Scan: a genome-wide method for the systematic discovery of T cell epitopes. Cell 178, 1016–1028
Trends in Immunology, -- 2019, Vol. --, No. --
3
Trends in Immunology
Spotlight
TCR Ligand Discovery via T-Scan Zhe Wang,1,2 Genhong Cheng,3,* and Guideng Li1,2,* T cell receptor (TCR) ligand discovery is crucial to monitoring T cell responses to antigen and to identifying antigens reactive against orphan TCRs of interest. In a recent article, Elledge and colleagues describe a functional T cell ligand screening platform for unbiased TCR ligand discovery. T cells are central mediators of immunity and are required to maintain the genetic homeostasis of host tissues. TCRs on the surface of a T cell can recognize a specific antigen fragment (peptide and/or epitope) presented on MHC molecules. The most common antigenic peptides are derived from pathogen proteins, viral genes, or aberrant gene products from somatic nonsynonymous mutations. Mounting evidence indicates that the interaction of TCR with the peptide–MHC complex (pMHC) is complex and even promiscuous. A single TCR can bind to different antigenic peptides and vice versa. This inevitably leads to off-target reactivity or cross-reactivity of therapeutic TCRs. In the clinic, unexpected immune responses against self-antigens constitute one of the main reasons for autoimmunity and off-target toxicities caused by TCR-T cell therapy. Therefore, an increased understanding of TCR– pMHC recognition is fundamental to the development of clinically beneficial and safe immunotherapies in cancer. Several methods have been established to identify ligands reactive against orphan TCRs of interest. Functional T cell expansion assays and pMHC multimer-based screening are the two most common antigen-directed approaches
[1,2]. However, these methods require foreknowledge of those antigenic targets and are low throughput, therefore precluding unbiased screening. Recently, scientists have also made considerable progress in TCR-guided antigen discovery [3]. An unbiased method was developed to screen a yeast cell surface-displayed pMHC library by using soluble, fluorescently labeled TCR reagents [3]. However, this system requires a laborious process to make soluble TCR reagents. Two innovative approaches that utilize different biological processes to label the target cells in a co-culture system were established to overcome these disadvantages [4,5]. One approach utilizes a phenomenon called ‘trogocytosis’, a biological process by which cells share membrane and membrane-associated proteins while conjugated [5]. TCR–pMHC interactions lead to specific transfer of surface proteins (e.g., TCRs) to cognate antigen-presenting cells, which can be measured by flow cytometry. This platform was successfully used to identify the cognate neoepitope for a subject-derived neoantigen-specific TCR [5]. The other approach takes advantage of signaling and antigen-presenting bifunctional receptors (SABRs), in which an intracellular CD3z signaling domain with a CD28 co-stimulatory domain is linked to the pMHC complex [4]. Following pMHC–TCR interactions, the intracellular signal results in the expression of GFP in target cells, which can be isolated and sequenced to identify the specific peptide recognized by the TCR [4]. A similar strategy has been developed for screening the antigenic epitope presented by MHC-II molecules [6]. These methods make TCRdirected antigen discovery more simple, scalable, and flexible. Additionally, a single-cell droplet microfluidicsbased TCR T cell pairing method was established to monitor the kinetics of
TCR–antigen recognition in live cells within droplets using a NFAT-eGFP reporter [7]. Most recently, Elledge and colleagues developed a state-of-the-art method called ‘T-Scan’ (Figure 1), which can identify functional T cell targets from genome-wide antigen libraries with high complexity [8]. Upon TCR–pMHC engagement, serine protease granzyme B (GzB) can be delivered into target cells and trigger apoptosis. The authors exploited the activity of the GzB reporter as the functional readout of physiological TCR–pMHC engagement. They first generated an infrared fluorescent protein (IFP)-based GzB reporter (IFPGZB) that contains a GzB-specific cleavage sequence. Upon T cell recognition, GzB-mediated cleavage results in the activation of the IFPGZB reporter. To ensure screening efficiency, the authors further made improvements to reduce background noise and preserve signal. They engineered MHC-I null epitope discovery cells (EDCs), which expressed both the IFPGZB reporter and a mutated inhibitor of caspase-activated DNase (ICAD), to prevent caspase activation-induced genomic DNA fragmentation [8]. Cytomegalovirus (CMV)-specific TCRs were first used to validate the productivity of T-Scan. The authors established two synthesized oligonucleotides libraries comprising peptides across either the entire CMV genome or the entire human virome. After co-culturing EDCs expressing these peptide libraries and CD8+ T cells expressing two different CMV-specific TCRs, the most enriched peptides expressed in the cell-sorted IFP+ cells represented the known target peptide NLVPMVATV (NLV) derived from the CMV-pp65 protein. Additionally, when co-culturing these EDCs with NLV-reactive T cells (CD8+ T cells from CMV+ donors that had been expanded
Trends in Immunology, -- 2019, Vol. --, No. --
1
Please cite this article in press as: Wang et al., TCR Ligand Discovery via T-Scan, Trends in Immunology (2019), https://doi.org/10.1016/ j.it.2019.10.003
Trends in Immunology
Trends in Immunology
Figure 1. Schematic Illustration of the Genome-Wide T-Scan Screening Platform. Epitope discovery cells (EDCs) are genetically engineered with a granzyme B (GzB) cleavage-activating infrared fluorescent protein (IFPGzB), which works as a reporter to monitor GzB activity. A mutated inhibitor of caspase-activated DNase (ICAD CR ) is also introduced to EDCs to prevent GzBinduced genomic DNA fragmentation, therefore facilitating the recovery of antigen information from EDCs. Upon co-culture of EDCs expressing a library of candidate antigens with T cells of interest, GzB-mediated cleavage leads to the activation of IFP GZB reporter in the target cells. IFP + EDCs are sorted by fluorescence-activated cell sorting (FACS), followed by genomic DNA extraction, PCR, and next-generation sequencing (NGS) to uncover information relating to the targeting epitopes [8]. Abbreviations: CMV, cytomegalovirus; pMHC, peptide–MHC complex; TCR, T cell receptor.
against the NLV peptide), the authors unexpectedly identified a new epitope from the IE1 gene; this suggested that this platform has the potential for the discovery of novel targets in subclonal populations of T cells. After demonstrating the capacity of T-Scan in genome-wide screening, the authors also applied T-Scan to T cell library-on-peptide library screening using bulk memory CD8+ T cells from patients who were CMV seropositive and identified a set of both known and new CMV-specific epitopes [8]. These results are relevant because they highlight the value of T-scan in the unbiased discovery of novel antigens. The authors further demonstrated the feasibility of T-Scan in understanding the molecular basis of TCR cross-reactivity. They successfully used a comprehensive single mutant library of the NLV epitope to map the critical residues and to identify specific mimotopes for NLV TCR recognition. Finally, they applied TScan to understand the specificity of tumor-reactive TCR in a human-genome 2
wide library using a TCR-targeting HLAA1-restricted epitope of tumor antigen MAGE-A3. The top two enriched epitopes were confirmed as either known MAGE-A3 epitopes or epitopes derived from the related protein MAGE-A6. In addition, the authors identified two additional cross-reactive epitopes derived from PLD5 and FAT2. These epitopes shared a three-amino acid core sequence with the MAGE-A3 peptide, but differed at the remaining six positions [8]. These findings highlight the challenge of bioinformatically predicting TCR cross-reactivity based on sequence similarity, demonstrating the value of unbiased experimental approaches to finding TCR cross-reactivities. T-Scan enables the rapid identification of antigens targeted by an immune response, a key goal in the fields of infectious disease, tumor immunology, autoimmunity, and immunotherapy. It has several advantages over existing technologies, including: (i) the T-Scan
Trends in Immunology, -- 2019, Vol. --, No. --
screening platform utilizes the natural processing and/or presentation of large-scale antigens (260 000 candidate antigens) and the physiological engagement of TCR–pMHC, which enables the interrogation of significantly larger antigen space than was previously possible. This feature also allows unbiased proteome-wide screening and permits the identification of biological relevant targets in the human genome, which differentiates this platform from other approaches; (ii) T-Scan breaks down the bottlenecks of TCR-T based immunotherapy by its library-to-library screening ability to rapidly identify tumor-specific antigens (TSAs) and tumor-reactive therapeutic TCRs without self-target toxicities; and (iii) T-Scan can simultaneously screen for target epitopes presented by the full set of MHC-I alleles from individuals. However, it is not clear whether T-Scan can also be efficiently applied to TCRs mediating MHC-II-restricted responses, which represents another
Please cite this article in press as: Wang et al., TCR Ligand Discovery via T-Scan, Trends in Immunology (2019), https://doi.org/10.1016/ j.it.2019.10.003
Trends in Immunology
important player when considering autoimmune diseases and antitumor responses. Thus, it will be fascinating to follow the development of further applications of T-Scan in the future.
Acknowledgments This work was supported by the National Natural Science Foundation (81972875) and The CAMS Initiative for Innovative Medicine (2016-I2M-1-005). 1Center
of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China
2Suzhou
Institute of Systems Medicine, Suzhou 215123, China
3Department
of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA *Correspondence: [email protected], [email protected] https://doi.org/10.1016/j.it.2019.10.003 ª 2019 Elsevier Ltd. All rights reserved.
References 1. Gros, A. et al. (2016) Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients. Nat. Med. 22, 433–438
2. Stronen, E. et al. (2016) Targeting of cancer neoantigens with donor-derived T cell receptor repertoires. Science 352, 1337–1341 3. Gee, M.H. et al. (2018) Antigen identification for orphan T cell receptors expressed on tumor-infiltrating lymphocytes. Cell 172, 549–563 4. Joglekar, A.V. et al. (2019) T cell antigen discovery via signaling and antigenpresenting bifunctional receptors. Nat. Methods 16, 191–198 5. Li, G. et al. (2019) T cell antigen discovery via trogocytosis. Nat. Methods 16, 183–190 6. Kisielow, J. et al. (2019) Deciphering CD4(+) T cell specificity using novel MHC-TCR chimeric receptors. Nat. Immunol. 20, 652–662 7. Segaliny, A.I. et al. (2018) Functional TCR T cell screening using single-cell droplet microfluidics. Lab Chip 18, 3733–3749 8. Kula, T. et al. (2019) T-Scan: a genome-wide method for the systematic discovery of T cell epitopes. Cell 178, 1016–1028
Trends in Immunology, -- 2019, Vol. --, No. --
3