Neoantigen T-Cell Receptor Gene Therapy in Pancreatic Cancer.

A patient with progressive metastatic pancreatic cancer was treated with a single infusion of 16.2×109 autologous T cells that had been genetically engineered to clonally express two allogeneic HLA-C*08:02-restricted T-cell receptors (TCRs) targeting mutant KRAS G12D expressed by the tumors. The patient had regression of visceral metastases (overall partial response of 72% according to the Response Evaluation Criteria in Solid Tumors, version 1.1); the response was ongoing at 6 months. The engineered T cells constituted more than 2% of all the circulating peripheral-blood T cells 6 months after the cell transfer. In this patient, TCR gene therapy targeting the KRAS G12D driver mutation mediated the objective regression of metastatic pancreatic cancer. (Funded by the Providence Portland Medical Foundation.).

One-Step Next-Generation Sequencing of Immunoglobulin and T-Cell Receptor Gene Recombinations for MRD Marker Identification in Acute Lymphoblastic Leukemia.

Within the EuroClonality-NGS group, immune repertoire analysis for target identification in lymphoid malignancies was initially developed using two-stage amplicon approaches, essentially as a progressive modification of preceding methods developed for Sanger sequencing. This approach has, however, limitations with respect to sample handling, adaptation to automation, and risk of contamination by amplicon products. We therefore developed one-step PCR amplicon methods with individual barcoding for batched analysis for IGH, IGK, TRD, TRG, and TRB rearrangements, followed by Vidjil-based data analysis.

Immunoglobulin/T-Cell Receptor Gene Rearrangement Analysis Using RNA-Seq.

Identification of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements in acute lymphoblastic leukemia (ALL) patients at initial presentation are crucial for monitoring of minimal residual disease (MRD) during subsequent follow-up and thereby for appropriate risk-group stratification. Here we describe how RNA-Seq data can be generated and subsequently analyzed with ARResT/Interrogate to identify possible MRD markers. In addition to the procedures, possible pitfalls will be discussed. Similar strategies can be employed for other lymphoid malignancies, such as lymphoma and myeloma.

Minimal Residual Disease Analysis by Monitoring Immunoglobulin and T-Cell Receptor Gene Rearrangements by Quantitative PCR and Droplet Digital PCR.

Analysis of immunoglobulin and T-cell receptor gene rearrangements by real-time quantitative polymerase chain reaction (RQ-PCR) is the gold standard for sensitive and accurate minimal residual disease (MRD) monitoring; it has been extensively standardized and guidelines have been developed within the EuroMRD consortium ( www.euromrd.org ). However, new generations of PCR-based methods are standing out as potential alternatives to RQ-PCR, such as digital PCR technology (dPCR), the third-generation implementation of conventional PCR, which has the potential to overcome some of the limitations of RQ-PCR such as allowing the absolute quantification of nucleic acid targets without the need for a calibration curve. During the last years, droplet digital PCR (ddPCR) technology has been compared to RQ-PCR in several hematologic malignancies showing its proficiency for MRD analysis. So far, no established guidelines for ddPCR MRD analysis and data interpretation have been defined and its potential is still under investigation. However, a major standardization effort is underway within the EuroMRD consortium ( www.euromrd.org ) for future application of ddPCR in standard clinical practice.

Profiling the T Cell Receptor Alpha/Delta Locus in Salmonids.

In jawed vertebrates, two major T cell populations have been characterized. They are defined as α/ß or γ/δ T cells, based on the expressed T cell receptor. Salmonids (family Salmonidae) include two key teleost species for aquaculture, rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) which constitute important models for fish immunology and important targets for vaccine development. The growing interest to decipher the dynamics of adaptive immune responses against pathogens or vaccines has resulted in recent efforts to sequence the immunoglobulin (IG) or antibodies and T cell receptor (TR) repertoire in these species. In this context, establishing a comprehensive and coherent locus annotation is the fundamental basis for the analysis of high-throughput repertoire sequencing data. We therefore decided to revisit the description and annotation of TRA/TRD locus in Atlantic salmon and two strains of rainbow trout (Swanson and Arlee) using the now available high-quality genome assemblies. Phylogenetic analysis of functional TRA/TRD V genes from these three genomes led to the definition of 25 subgroups shared by both species, some with particular feature. A total of 128 TRAJ genes were identified in Salmo, the majority with a close counterpart in Oncorhynchus. Analysis of expressed TRA repertoire indicates that most TRAV gene subgroups are expressed at mucosal and systemic level. The present work on TRA/TRD locus annotation along with the analysis of TRA repertoire sequencing data show the feasibility and advantages of a common salmonid TRA/TRD nomenclature that allows an accurate annotation and analysis of high-throughput sequencing results, across salmonid T cell subsets.

Subpopulations of swine γδ T cells defined by TCRγ and WC1 gene expression.

γδ T cells constitute a major portion of lymphocytes in the blood of both ruminants and swine. Subpopulations of swine γδ T cells have been distinguished by CD2 and CD8α expression. However, it was not clear if they have distinct expression profiles of their T-cell receptor (TCR) or WC1 genes. Identifying receptor expression will contribute to understanding the functional differences between these subpopulations and their contributions to immune protection. Here, we annotated three genomic assemblies of the swine TCRγ gene locus finding four gene cassettes containing C, J and V genes, although some haplotypes carried a null TRGC gene (TRGC4). Genes in the TRGC1 cassette were homologs of bovine TRGC5 cassette while the others were not homologous to bovine genes. Here we evaluated three principal populations of γδ T cells (CD2+/SWC5-, CD2-/SWC5+, and CD2-/SWC5-). Both CD2- subpopulations transcribed WC1 co-receptor genes, albeit with different patterns of gene expression but CD2+ cells did not. All subpopulations transcribed TCR genes from all four cassettes, although there were differences in expression levels. Finally, the CD2+ and CD2- γδ T-cell populations differed in their representation in various organs and tissues, presumably at least partially reflective of different ligand specificities for their receptors.

Search for MHC/TCR-Like Systems in Living Organisms.

Highly polymorphic loci evolved many times over the history of species. These polymorphic loci are involved in three types of functions: kind recognition, self-incompatibility, and the jawed vertebrate adaptive immune system (AIS). In the first part of this perspective, we reanalyzed and described some cases of polymorphic loci reported in the literature. There is a convergent evolution within each functional category and between functional categories, suggesting that the emergence of these self/non-self recognition loci has occurred multiple times throughout the evolutionary history. Most of the highly polymorphic loci are coding for proteins that have a homophilic interaction or heterophilic interaction between linked loci, leading to self or non-self-recognition. The highly polymorphic MHCs, which are involved in the AIS have a different functional mechanism, as they interact through presented self or non-self-peptides with T cell receptors, whose diversity is generated by somatic recombination. Here we propose a mechanism called "the capacity of recognition competition mechanism" that might contribute to the evolution of MHC polymorphism. We propose that the published cases corresponding to these three biological categories represent a small part of what can be found throughout the tree of life, and that similar mechanisms will be found many times, including the one where polymorphic loci interact with somatically generated loci.

Immunophenotypic switch in cutaneous T-cell lymphoma: A series of three cases and review of the literature.

Primary cutaneous T-cell lymphoma (CTCL) comprises a heterogeneous group of neoplasms with variable clinical behavior. Immunophenotypic switch (IS) is a phenomenon that occurs during lymphoma progression and is defined by an alteration in the immunophenotypic expression of a tumor with retention of its genotypic signature. This has been well-recognized in hematopoietic neoplasms; however, it has been rarely reported in CTCL and its clinical implications are not well understood. We present the clinical, histopathologic, immunophenotypic, and genetic findings of three cases of CTCL that demonstrated IS post treatment with variable outcomes. We add our cases to the small number previously reported to increase awareness of this phenomenon and its diagnostic challenge.

The clinicopathological relevance of uniform CD56 expression in anaplastic large cell lymphoma: a retrospective analysis of 18 cases.

BACKGROUND: Anaplastic large cell lymphoma (ALCL) with uniform CD56 expression is a rare condition, that has been described in limited literature, and its clinicopathological features have not yet been well illustrated. The aim of our study was to fully investigate the clinical, histological, immunohistochemical and molecular features of CD56+ ALCL. METHODS: The clinical and histological characteristics of CD56+ ALCL cases were retrospectively evaluated. The immunohistochemical phenotype, status of Epstein-Barr virus (EBV) and T-cell receptor (TCR) gene rearrangement were examined. Overall survival was also analyzed. RESULTS: Eighteen (5.8%) cases with diffuse CD56 expression were identified out of 313 archived ALCL cases with CD56 test. CD56 expression was significantly higher in ALK+ systemic ALCLs (sALCLs) (13/64, 20.3%) than in ALK- sALCLs (3/101, 3.0%) (p < 0.001) as well as primary cutaneous ALCLs (2/148, 1.4%) (p < 0.001). Regarding the CD56+ ALK+ sALCLs, the median age was 20 years (range, 8-60 years) with a male-to-female ratio of 2.3:1, and these cases more frequently affected extranodal sites (11/38, 28.9%) rather than lymph nodes (2/26, 7.7%) (p = 0.038). Eleven (84.6%) cases presented with stage I-II diseases, which was significantly more than their CD56- ALK+ counterparts (45.5%) (p = 0.015). Histologically, 2 ALK+ cases were of small cell variant and all the others displayed characteristic morphology of classic ALCL. Regarding the immunophenotype, both CD30 and CD56 were diffusely positive in all cases. CD3, CD43, anaplastic lymphoma kinase-1 (ALK1), TIA-1, EMA expression was observed in 30.8% (4/13), 90.9% (10/11), 100% (13/13), 100% (9/9), and 80.0% (8/10) cases, respectively. EBV infection was consistently absent. Monoclonal TCR gene rearrangement was found in 100% (5/5) of investigated ALK+ cases. Chemotherapy with a CHOP regimen was most frequently employed. The 3-year overall survival (OS) rate for CD56+ ALK+ patients was 92.0%, compared with 73.0% for their CD56- counterparts, but there was no significant difference in OS between the two groups (p = 0.264). CONCLUSIONS: Uniform CD56 expression is an unexpected condition in ALCL. Of ALK+ ALCLs, CD56 expression correlated with a high frequency of early stage and an extranodal predominance. It is of great importance to raise awareness of this condition and familiarity with its characteristic features to avoid diagnostic and therapeutic pitfalls. Further investigations are warranted for a better understanding of this unusual phenotype and the significance of CD56 expression in ALCL.

Regulation of T-cell Receptor Gene Expression by Three-Dimensional Locus Conformation and Enhancer Function.

The adaptive immune response in vertebrates depends on the expression of antigen-specific receptors in lymphocytes. T-cell receptor (TCR) gene expression is exquisitely regulated during thymocyte development to drive the generation of αß and γδ T lymphocytes. The TCRα, TCRß, TCRγ, and TCRδ genes exist in two different configurations, unrearranged and rearranged. A correctly rearranged configuration is required for expression of a functional TCR chain. TCRs can take the form of one of three possible heterodimers, pre-TCR, TCRαß, or TCRγδ which drive thymocyte maturation into αß or γδ T lymphocytes. To pass from an unrearranged to a rearranged configuration, global and local three dimensional (3D) chromatin changes must occur during thymocyte development to regulate gene segment accessibility for V(D)J recombination. During this process, enhancers play a critical role by modifying the chromatin conformation and triggering noncoding germline transcription that promotes the recruitment of the recombination machinery. The different signaling that thymocytes receive during their development controls enhancer activity. Here, we summarize the dynamics of long-distance interactions established through chromatin regulatory elements that drive transcription and V(D)J recombination and how different signaling pathways are orchestrated to regulate the activity of enhancers to precisely control TCR gene expression during T-cell maturation.

A Potential Mechanism of Anticancer Immune Response Coincident With Immune-related Adverse Events in Patients With Renal Cell Carcinoma.

BACKGROUND/AIM: Some reports showed encouraging efficacy of immune checkpoint inhibitors among patients who experienced immune-related adverse events (irAEs). Thus, characterization of T-cell repertoire and immune signatures in peripheral blood mononuclear cells (PBMCs) and tumors before and after immune checkpoint inhibitors treatment should contribute to better understanding of irAE-provoked anticancer immune responses. MATERIALS AND METHODS: We applied expression analysis of immune-related genes and T-cell receptor sequencing in tumor and PBMCs from five patients with renal cell carcinoma before combined immunotherapy and at the onset of severe irAEs. RESULTS: We found that the cluster of differentiation 8 (CD8)/forkhead box P3(FOXP3), granzyme B(GZMB)/CD3, perforin 1(PRF1)/CD3 and programmed cell death 1(PD1)/CD8 expression ratios were significantly elevated in PBMCs at the onset of irAEs. In addition, we found expansion of certain T-cell clones in metastatic tissue after irAEs, which were already increased in peripheral blood at the onset of irAEs. CONCLUSION: irAE-provoked T-cells may also circulate and attack distant tumors, leading to durable response in patients with irAEs.

Characterization and Monitoring of Antigen-Responsive T Cell Clones Using T Cell Receptor Gene Expression Analysis.

High-throughput T-cell receptor repertoire sequencing constitutes a powerful tool to study T cell responses at the clonal level. However, it does not give information on the functional phenotype of the responding clones and lacks a statistical framework for quantitative evaluation. To overcome this, we combined datasets from different experiments, all starting from the same blood samples. We used a novel, sensitive, UMI-based protocol to perform repertoire analysis on experimental replicates. Applying established bioinformatic routines for transcriptomic expression analysis we explored the dynamics of antigen-induced clonal expansion after in vitro stimulation, identified antigen-responsive clones, and confirmed their activation status using the expression of activation markers upon antigen re-challenge. We demonstrate that the addition of IL-4 after antigen stimulation drives the expansion of T cell clones encoding unique receptor sequences. We show that our approach represents a scalable, high-throughput immunological tool, which can be used to identify and characterize antigen-responsive T cells at clonal level.

Past and Future of the Molecular Characterization of the T Cell Repertoire: Some Highlights of Eli Sercarz's Contributions.

The contribution of Eli E. Sercarz to immunology and immunopathology has been remarkable and achieved many milestones in the understanding of the processes of the mechanisms fine-tuning immune responses. A part of his work was dedicated to the study of the deep complexity of the lymphocyte T cell repertoire and its importance during the physiologic development and disease, such as clonal heterogeneity of T cell responses. Starting from these studies, under his mentoring, we had the opportunity to implement the spectratyping method and apply it to human and experimental autoimmune diseases, obtaining intriguing results. The open question of this brief review is the possible role of this fine and complex technique, the immunoscope analysis, in the era of the big data and omics.

qPCR Applications for the Determination of the Biological Age.

Individual age is a phenotypic trait that provides useful information not only in forensic investigations but also in the aging research which is becoming an urgent call due to the dramatic growth of the aging population worldwide.TaqMan quantification PCR (qPCR) can be successfully applied to biological age estimation, using method defined in Zubakov et al. (Curr Biol 20:R970-R971, 2010). Since levels of signal joint T-cell receptor rearrangement excision circle (sjTREC) in human lymphocytes are known to decrease with age increasing, the qPCR of sjTREC represents a simple and relatively reproducible technique which offers highly accurate age estimation results.

Understanding TCR affinity, antigen specificity, and cross-reactivity to improve TCR gene-modified T cells for cancer immunotherapy.

Adoptive cell transfer (ACT) using T cell receptor (TCR) gene-modified T cells is an exciting and rapidly evolving field. Numerous preclinical and clinical studies have demonstrated various levels of feasibility, safety, and efficacy using TCR-engineered T cells to treat cancer and viral infections. Although evidence suggests their use can be effective, to what extent and how to improve these therapeutics are still matters of investigation. As TCR affinity has been generally accepted as the central role in defining T cell specificity and sensitivity, selection for and generation of high affinity TCRs has remained a fundamental approach to design more potent T cells. However, traditional methods for affinity-enhancement by random mutagenesis can induce undesirable cross-reactivity causing on- and off-target adverse events, generate exhausted effectors by overstimulation, and ignore other kinetic and cellular parameters that have been shown to impact antigen specificity. In this Focussed Research Review, we comment on the preclinical and clinical potential of TCR gene-modified T cells, summarize our contributions challenging the role TCR affinity plays in antigen recognition, and explore how structure-guided design can be used to manipulate antigen specificity and TCR cross-reactivity to improve the safety and efficacy of TCR gene-modified T cells used in ACT.

Framework engineering to produce dominant T cell receptors with enhanced antigen-specific function.

TCR-gene-transfer is an efficient strategy to produce therapeutic T cells of defined antigen specificity. However, there are substantial variations in the cell surface expression levels of human TCRs, which can impair the function of engineered T cells. Here we demonstrate that substitutions of 3 amino acid residues in the framework of the TCR variable domains consistently increase the expression of human TCRs on the surface of engineered T cells.The modified TCRs mediate enhanced T cell proliferation, cytokine production and cytotoxicity, while reducing the peptide concentration required for triggering effector function up to 3000-fold. Adoptive transfer experiments in mice show that modified TCRs control tumor growth more efficiently than wild-type TCRs. Our data indicate that simple variable domain modifications at a distance from the antigen-binding loops lead to increased TCR expression and improved effector function. This finding provides a generic platform to optimize the efficacy of TCR gene therapy in humans.

Cancer targeting by TCR gene-engineered T cells directed against Kita-Kyushu Lung Cancer Antigen-1.

T cell receptor (TCR) gene-engineered T cells have shown promise in the treatment of melanoma and synovial cell sarcoma, but their application to epithelial cancers has been limited. The identification of novel therapeutic TCRs for the targeting of these tumors is important for the development of new treatments. Here, we describe the preclinical characterization of a TCR directed against Kita-Kyushu Lung Cancer Antigen-1 (KK-LC-1, encoded by CT83), a cancer germline antigen with frequent expression in human epithelial malignancies including gastric cancer, breast cancer, and lung cancer. Gene-engineered T cells expressing the KK-LC-1 TCR (KK-LC-1 TCR-Ts) demonstrated recognition of CT83+ tumor lines in vitro and mediated regression of established CT83+ xenograft tumors in immunodeficient mouse models. Cross-reactivity studies based on experimental determination of the recognition motifs for the target epitope did not demonstrate cross-reactivity against other human proteins. CT83 gene expression studies in 51 non-neural tissues and 24 neural tissues showed expression restricted exclusively to germ cells. CT83 was however expressed by a range of epithelial cancers, with the highest expression noted in gastric cancer. Collectively, these findings support the further investigation and clinical testing of KK-LC-1 TCR-Ts for gastric cancer and possibly other malignancies.

Orthotopic replacement of T-cell receptor α- and ß-chains with preservation of near-physiological T-cell function.

Therapeutic T cells with desired specificity can be engineered by introducing T-cell receptors (TCRs) specific for antigens of interest, such as those from pathogens or tumour cells. However, TCR engineering is challenging, owing to the complex heterodimeric structure of the receptor and to competition and mispairing between endogenous and transgenic receptors. Additionally, conventional TCR insertion disrupts the regulation of TCR dynamics, with consequences for T-cell function. Here, we report the outcomes and validation, using five different TCRs, of the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) with non-virally delivered template DNA for the elimination of endogenous TCR chains and for the orthotopic placement of TCRs in human T cells. We show that, whereas the editing of a single receptor chain results in chain mispairing, simultaneous editing of α- and ß-chains combined with orthotopic TCR placement leads to accurate αß-pairing and results in TCR regulation similar to that of physiological T cells.

Standardized next-generation sequencing of immunoglobulin and T-cell receptor gene recombinations for MRD marker identification in acute lymphoblastic leukaemia; a EuroClonality-NGS validation study.

Amplicon-based next-generation sequencing (NGS) of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements for clonality assessment, marker identification and quantification of minimal residual disease (MRD) in lymphoid neoplasms has been the focus of intense research, development and application. However, standardization and validation in a scientifically controlled multicentre setting is still lacking. Therefore, IG/TR assay development and design, including bioinformatics, was performed within the EuroClonality-NGS working group and validated for MRD marker identification in acute lymphoblastic leukaemia (ALL). Five EuroMRD ALL reference laboratories performed IG/TR NGS in 50 diagnostic ALL samples, and compared results with those generated through routine IG/TR Sanger sequencing. A central polytarget quality control (cPT-QC) was used to monitor primer performance, and a central in-tube quality control (cIT-QC) was spiked into each sample as a library-specific quality control and calibrator. NGS identified 259 (average 5.2/sample, range 0-14) clonal sequences vs. Sanger-sequencing 248 (average 5.0/sample, range 0-14). NGS primers covered possible IG/TR rearrangement types more completely compared with local multiplex PCR sets and enabled sequencing of bi-allelic rearrangements and weak PCR products. The cPT-QC showed high reproducibility across all laboratories. These validated and reproducible quality-controlled EuroClonality-NGS assays can be used for standardized NGS-based identification of IG/TR markers in lymphoid malignancies.

Clonotypic heterogeneity in cutaneous T-cell lymphoma (mycosis fungoides) revealed by comprehensive whole-exome sequencing.

Mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma, is believed to represent a clonal expansion of a transformed skin-resident memory T cell. T-cell receptor (TCR) clonality (ie, identical sequences of rearranged TCRα, TCRß, and TCRγ), the key premise of this hypothesis, has been difficult to document conclusively because malignant cells are not readily distinguishable from the tumor-infiltrating reactive lymphocytes that contribute to the TCR clonotypic repertoire of MF. Here, we have successfully adopted targeted whole-exome sequencing (WES) to identify the repertoire of rearranged TCR genes in tumor-enriched samples from patients with MF. Although some of the investigated MF biopsies had the expected frequency of monoclonal rearrangements of TCRγ corresponding to that of tumor cells, the majority of the samples presented multiple TCRγ, TCRα, and TCRß clonotypes by WES. Our findings are compatible with the model in which the initial malignant transformation in MF does not occur in mature memory T cells but rather at the level of T-lymphocyte progenitors before TCRß or TCRα rearrangements. We have also shown that WES can be combined with whole-transcriptome sequencing in the same sample, which enables comprehensive characterization of the TCR repertoire in relation to tumor content. WES/whole-transcriptome sequencing might be applicable to other types of T-cell lymphomas to determine clonal dominance and clonotypic heterogeneity in these malignancies.