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Tumor-infiltrating regulatory T cells (Tregs) contribute to an immunosuppressive tumor microenvironment. Despite extensive studies, the prognostic impact of tumor-infiltrating Tregs in B-cell non-Hodgkin lymphomas (B-NHLs) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotypes and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Here, we applied single-cell RNA sequencing and T-cell receptor sequencing combined with high-dimensional cytometry to decipher the heterogeneity of intratumoral Tregs in diffuse large B-cell lymphoma and follicular lymphoma (FL), compared with that in nonmalignant tonsillar tissue. We identified 3 distinct transcriptional states of Tregs: resting, activated, and unconventional LAG3+FOXP3- Tregs. Activated Tregs were enriched in B-NHL tumors, coexpressed several checkpoint receptors, and had stronger immunosuppressive activity compared with resting Tregs. In FL, activated Tregs were found in closer proximity to CD4+ and CD8+ T cells than other cell types. Furthermore, we used a computational approach to develop unique gene signature matrices, which were used to enumerate each Treg subset in cohorts with bulk gene expression data. In 2 independent FL cohorts, activated Tregs was the major subset, and high abundance was associated with adverse outcome. This study demonstrates that Tregs infiltrating B-NHL tumors are transcriptionally and functionally diverse. Highly immunosuppressive activated Tregs were enriched in tumor tissue but absent in the peripheral blood. Our data suggest that a deeper understanding of Treg heterogeneity in B-NHL could open new paths for rational drug design, facilitating selective targeting to improve antitumor immunity., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Acute myeloid leukemia (AML), the most frequent leukemia in adults, is driven by recurrent somatically acquired genetic lesions in a restricted number of genes. Treatment with tyrosine kinase inhibitors has demonstrated that targeting of prevalent FMS-related receptor tyrosine kinase 3 (FLT3) gain-of-function mutations can provide significant survival benefits for patients, although the efficacy of FLT3 inhibitors in eliminating FLT3-mutated clones is variable. We identified a T cell receptor (TCR) reactive to the recurrent D835Y driver mutation in the FLT3 tyrosine kinase domain (TCR FLT3D/Y ). TCR FLT3D/Y -redirected T cells selectively eliminated primary human AML cells harboring the FLT3 D835Y mutation in vitro and in vivo. TCR FLT3D/Y cells rejected both CD34 + and CD34 - AML in mice engrafted with primary leukemia from patients, reaching minimal residual disease-negative levels, and eliminated primary CD34 + AML leukemia-propagating cells in vivo. Thus, T cells targeting a single shared mutation can provide efficient immunotherapy toward selective elimination of clonally involved primary AML cells in vivo., (© 2023. The Author(s).)

Cancer immunotherapy using T cell receptor-engineered T cells (TCR-Ts) represents a promising treatment option. However, technologies for pre-clinical safety assessment are incomplete or inaccessible to most laboratories. Here, TCR-T off-target reactivity was assessed in five steps: (1) Mapping target amino acids necessary for TCR-T recognition, followed by (2) a computational search for, and (3) reactivity screening against, candidate cross-reactive peptides in the human proteome. Natural processing and presentation of recognized peptides was evaluated using (4) short mRNAs, and (5) full-length proteins. TCR-Ts were screened for recognition of unintended HLA alleles, and as proxy for off-target reactivity in vivo, a syngeneic, HLA-A*02:01-transgenic mouse model was used. Validation demonstrated importance of studying recognition of full-length candidate off-targets, and that the clinically applied 1G4 TCR has a hitherto unknown reactivity to unintended HLA alleles, relevant for patient selection. This widely applicable strategy should facilitate evaluation of candidate therapeutic TCRs and inform clinical decision-making., (© 2023. Springer Nature Limited.)

The emergence of SARS-CoV-2 variants of concern (VOC) is driven by mutations that mediate escape from neutralizing antibodies. There is also evidence that mutations can cause loss of T cell epitopes. However, studies on viral escape from T cell immunity have been hampered by uncertain estimates of epitope prevalence. Here, we map and quantify CD8 T cell responses to SARS-CoV-2-specific minimal epitopes in blood drawn from April to June 2020 from 83 COVID-19 convalescents. Among 37 HLA ligands eluted from five prevalent alleles and an additional 86 predicted binders, we identify 29 epitopes with an immunoprevalence ranging from 3% to 100% among individuals expressing the relevant HLA allele. Mutations in VOC are reported in 10.3% of the epitopes, while 20.6% of the non-immunogenic peptides are mutated in VOC. The nine most prevalent epitopes are conserved in VOC. Thus, comprehensive mapping of epitope prevalence does not provide evidence that mutations in VOC are driven by escape of T cell immunity., Competing Interests: Declaration of interests J.O. is the author on a patent protecting a method for identification of T cell receptors and on patent applications protecting T cell receptor sequences for potential use in cancer immunotherapy. J.O. is a member of the Scientific Advisory Board of Asgard Therapeutics. The other authors declare no competing interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Immunotherapy in cancer takes advantage of the exquisite specificity, potency, and flexibility of the immune system to eliminate alien tumor cells. It involves strategies to activate the entire immune defense, by unlocking mechanisms developed by tumor cells to escape from surrounding immune cells, as well as engineered antibody and cellular therapies. What is important to note is that these are therapeutics with curative potential. The earliest example of immune therapy is allogeneic stem cell transplantation, introduced in 1957, which is still an important modality in hematology, most notably in myeloid malignancies. In this review, we discuss developmental trends of immunotherapy in hematological malignancies, focusing on some of the strategies that we believe will have the most impact on future clinical practice in this field. In particular, we delineate novel developments for therapies that have already been introduced into the clinic, such as immune checkpoint inhibition and chimeric antigen receptor T-cell therapies. Finally, we discuss the therapeutic potential of emerging strategies based on T-cell receptors and adoptive transfer of allogeneic natural killer cells., (© 2021 The Authors. Journal of Internal Medicine published by John Wiley & Sons Ltd on behalf of Association for Publication of The Journal of Internal Medicine.)

B-cell depletion induced by anti-cluster of differentiation 20 (CD20) monoclonal antibody (mAb) therapy of patients with lymphoma is expected to impair humoral responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination, but effects on CD8 T-cell responses are unknown. Here, we investigated humoral and CD8 T-cell responses following two vaccinations in patients with lymphoma undergoing anti-CD20-mAb therapy as single agent or in combination with chemotherapy or other anti-neoplastic agents during the last 9 months prior to inclusion, and in healthy age-matched blood donors. Antibody measurements showed that seven of 110 patients had antibodies to the receptor-binding domain of the SARS-CoV-2 Spike protein 3-6 weeks after the second dose of vaccination. Peripheral blood CD8 T-cell responses against prevalent human leucocyte antigen (HLA) class I SARS-CoV-2 epitopes were determined by peptide-HLA multimer analysis. Strong CD8 T-cell responses were observed in samples from 20/29 patients (69%) and 12/16 (75%) controls, with similar median response magnitudes in the groups and some of the strongest responses observed in patients. We conclude that despite the absence of humoral immune responses in fully SARS-CoV-2-vaccinated, anti-CD20-treated patients with lymphoma, their CD8 T-cell responses reach similar frequencies and magnitudes as for controls. Patients with lymphoma on B-cell depleting therapies are thus likely to benefit from current coronavirus disease 2019 (COVID-19) vaccines, and development of vaccines aimed at eliciting T-cell responses to non-Spike epitopes might provide improved protection., (© 2022 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Neoantigens are commonly defined as HLA-bound peptides that are altered as a consequence of DNA damage and recognized by T cells. Current efforts to target neoantigens in therapy rely on algorithms that predict HLA-binding and immunogenicity from DNA sequence data. Datasets obtained by mass spectrometry of peptides eluted from mono-allelic cell lines have greatly improved our ability to predict HLA-binding. The main challenge lies in selecting those that are likely to be immunogenic. Here we argue that the current approach of searching for antigens that have evoked T-cell responses in untreated patients may underestimate immunogenicity. Results from clinical trials show that cancer vaccines often primarily engage the naïve T-cell repertoire. We therefore propose a new pipeline where HLA-binding is detected directly by mass spectrometry and immunogenicity is determined as the ability to prime naïve T cells from healthy donors., (Copyright © 2022. Published by Elsevier Ltd.)

Unlike chimeric antigen receptors, T-cell receptors (TCRs) can recognize intracellular targets presented on human leukocyte antigen (HLA) molecules. Here we demonstrate that T cells expressing TCRs specific for peptides from the intracellular lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT), presented in the context of HLA-A*02:01, specifically eliminate primary acute lymphoblastic leukemia (ALL) cells of T- and B-cell origin in vitro and in three mouse models of disseminated B-ALL. By contrast, the treatment spares normal peripheral T- and B-cell repertoires and normal myeloid cells in vitro, and in vivo in humanized mice. TdT is an attractive cancer target as it is highly and homogeneously expressed in 80-94% of B- and T-ALLs, but only transiently expressed during normal lymphoid differentiation, limiting on-target toxicity of TdT-specific T cells. TCR-modified T cells targeting TdT may be a promising immunotherapy for B-ALL and T-ALL that preserves normal lymphocytes., (© 2021. The Author(s).)

Activated T cells secrete interferon-γ, which triggers intracellular tryptophan shortage by upregulating the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme 1-4 . Here we show that despite tryptophan depletion, in-frame protein synthesis continues across tryptophan codons. We identified tryptophan-to-phenylalanine codon reassignment (W>F) as the major event facilitating this process, and pinpointed tryptophanyl-tRNA synthetase (WARS1) as its source. We call these W>F peptides 'substitutants' to distinguish them from genetically encoded mutants. Using large-scale proteomics analyses, we demonstrate W>F substitutants to be highly abundant in multiple cancer types. W>F substitutants were enriched in tumours relative to matching adjacent normal tissues, and were associated with increased IDO1 expression, oncogenic signalling and the tumour-immune microenvironment. Functionally, W>F substitutants can impair protein activity, but also expand the landscape of antigens presented at the cell surface to activate T cell responses. Thus, substitutants are generated by an alternative decoding mechanism with potential effects on gene function and tumour immunoreactivity., (© 2022. The Author(s).)

Background: Current prognostic variables can only partly explain the large outcome heterogeneity in diffuse large B cell lymphoma (DLBCL). We aimed to investigate the utility of systems-level protein and immune repertoire profiling for outcome prognostication in DLBCL., Methods: In this retrospective study, we used proximity extension assay technology to quantify 81 immune-related proteins in serum or plasma in 2 independent cohorts in a total 111 DLBCL patients. Protein levels were assessed before and after treatment with rituximab and chemotherapy, and the patients were compared with 19 age- and sex-matched healthy blood donors. In a subset of the patients, we performed a broad mass cytometric characterization of immune cell repertoires in peripheral blood., Findings: Patients displayed large deviations in protein profiles compared with healthy controls. Development of a systemic protein deviation (SPD) score provided a 4-protein-based metric that reflected the overall degree of protein deviations compared with age- and sex-matched healthy blood donors. The SPD score identified patients with very poor overall survival in both cohorts and correlated with increased frequencies of peripheral blood PD-1 + CD8 + T cells, and expansion of myeloid-derived suppressor cells., Conclusions: Our results show that a simple metric based on measurement of a small set of serum or plasma proteins can be used to probe systemic immune changes associated with poor survival in DLBCL. This finding warrants further investigation in larger, prospective studies to establish a clinical prognostic biomarker., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Extensive tumour inflammation, which is reflected by high levels of infiltrating T cells and interferon-γ (IFNγ) signalling, improves the response of patients with melanoma to checkpoint immunotherapy 1,2 . Many tumours, however, escape by activating cellular pathways that lead to immunosuppression. One such mechanism is the production of tryptophan metabolites along the kynurenine pathway by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which is induced by IFNγ 3-5 . However, clinical trials using inhibition of IDO1 in combination with blockade of the PD1 pathway in patients with melanoma did not improve the efficacy of treatment compared to PD1 pathway blockade alone 6,7 , pointing to an incomplete understanding of the role of IDO1 and the consequent degradation of tryptophan in mRNA translation and cancer progression. Here we used ribosome profiling in melanoma cells to investigate the effects of prolonged IFNγ treatment on mRNA translation. Notably, we observed accumulations of ribosomes downstream of tryptophan codons, along with their expected stalling at the tryptophan codon. This suggested that ribosomes bypass tryptophan codons in the absence of tryptophan. A detailed examination of these tryptophan-associated accumulations of ribosomes-which we term 'W-bumps'-showed that they were characterized by ribosomal frameshifting events. Consistently, reporter assays combined with proteomic and immunopeptidomic analyses demonstrated the induction of ribosomal frameshifting, and the generation and presentation of aberrant trans-frame peptides at the cell surface after treatment with IFNγ. Priming of naive T cells from healthy donors with aberrant peptides induced peptide-specific T cells. Together, our results suggest that IDO1-mediated depletion of tryptophan, which is induced by IFNγ, has a role in the immune recognition of melanoma cells by contributing to diversification of the peptidome landscape.

Competing Interests: Conflict-of-interest disclosure: J.O. has a research collaboration with Kite Pharma and is a member of the Scientific Advisory Board of Intellia Therapeutics. F.L.-J. declares no competing financial interests.

The identification of immunogenic neoantigens and their cognate T cells represents the most crucial and rate-limiting steps in the development of personalized cancer immunotherapies that are based on vaccination or on infusion of T cell receptor (TCR)-engineered T cells. Recent advances in deep-sequencing technologies and in silico prediction algorithms have allowed rapid identification of candidate neoepitopes. However, large-scale validation of putative neoepitopes and the isolation of reactive T cells are challenging because of the limited availablity of patient material and the low frequencies of neoepitope-specific T cells. Here we describe a standardized protocol for the induction of neoepitope-reactive T cells from healthy donor T cell repertoires, unaffected by the potentially immunosuppressive environment of the tumor-bearing host. Monocyte-derived dendritic cells (DCs) transfected with mRNA encoding candidate neoepitopes are used to prime autologous naive CD8 + T cells. Antigen-specific T cells that recognize endogenously processed and presented epitopes are detected using peptide-MHC (pMHC) multimers. Single multimer-positive T cells are sorted for the identification of TCR sequences, after an optional step that includes clonal expansion and functional characterization. The time required to identify neoepitope-specific T cells is 15 d, with an additional 2-4 weeks required for clonal expansion and downstream functional characterization. Identified neoepitopes and corresponding TCRs provide candidates for use in vaccination and TCR-based cancer immunotherapies, and datasets generated by this technology should be useful for improving algorithms to predict immunogenic neoantigens.

Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19 + targets, and maintained their intrinsic degranulation response toward CD19 - K562 cells. The response of redirected NK-cell subsets against CD19 + targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA-specific KIR(s), displayed superior ability to kill CD19 + , HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells. Cancer Immunol Res; 6(4); 467-80. ©2018 AACR ., (©2018 American Association for Cancer Research.)

T cells can recognize peptides encoded by mutated genes, but analysis of tumor-infiltrating lymphocytes suggests that very few neoantigens spontaneously elicit T-cell responses. This may be an important reason why immune checkpoint inhibitors are mainly effective in tumors with a high mutational burden. Reasons for clinically insufficient responses to neoantigens might be inefficient priming, inhibition, or deletion of the cognate T cells. Responses can be dramatically improved by cancer immunotherapy such as checkpoint inhibition, but often with temporary effects. By contrast, T cells from human leukocyte antigen (HLA)-matched donors can cure diseases such as chronic myeloid leukemia. The therapeutic effect is mediated by donor T cells recognizing polymorphic peptides for which the donor and patient are disparate, presented on self-HLA. Donor T-cell repertoires are unbiased by the immunosuppressive environment of the tumor. A recent study demonstrated that T cells from healthy individuals are able to respond to neoantigens that are ignored by tumor-infiltrating T cells of melanoma patients. In this review, we discuss possible reasons why neoantigens escape host T cells and how these limitations may be overcome by utilization of donor-derived T-cell repertoires to facilitate rational design of neoantigen-targeted immunotherapy.

Common variable immunodeficiency (CVID) is defined by hypogammaglobulinemia and B-cell dysfunction, with significant clinical and immunological heterogeneity. Severe non-infectious complications, such as autoimmunity, granulomatous disease and splenomegaly, constitute a major cause of morbidity in CVID patients. T cells are generally regarded important for development of these clinical features. However, while T-cell abnormalities have been found in CVID patients, functional characteristics of T cells corresponding to well-defined clinical subtypes have not been identified. As common γ-chain cytokines play important roles in survival and differentiation of T cells, characterization of their signaling pathways could reveal functional differences of clinical relevance. We characterized CVID T cells functionally by studies of cytokine-induced signaling, and correlated the findings to defined clinical subtypes. Peripheral blood T cells from 29 CVID patients and 19 healthy donors were analyzed for i) phenotype, ii) cytokine-induced (interleukin (IL)-2, IL-4, IL-7 and IL-21) phosphorylation of signal transducer and activator of transcription (STAT) 3, STAT5 and STAT6, and iii) T-helper (Th)1/Th2 polarization. Expression of IL-4 receptor and downstream signaling molecules was measured. A subgroup of CVID patients (n = 7) was identified by impaired IL-4-induced p-STAT6 in naive and memory CD4 and CD8 T cells. This corresponded to patients with the largest accumulation of severe (non-infectious) complications. The signaling defect persisted over years and was not due to constitutively activated p-STAT6. The CD4 T cells were strongly Th1-skewed, but IL-4 signaling was impaired independently of Th status. However, IL-4Rα and Janus kinase (JAK) 1 mRNA levels were significantly lower than in normal donors, providing a likely mechanism for the defective IL-4-induced p-STAT6 and Th1-bias. In conclusion, we identified a subgroup of CVID patients with defective IL-4 signaling in T cells, with severe clinical features of inflammation and autoimmunity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Patients with common variable immunodeficiency (CVID) constitute a clinically and immunologically heterogeneous group characterized by B-cell dysfunction with hypogammaglobulinemia and defective immunoglobulin class switch of unknown etiology. Current classification systems are insufficient to achieve precise disease management. Characterization of signaling pathways essential for B-cell differentiation and class switch could provide new means to stratify patients. We evaluated constitutive and induced signaling by phospho-specific flow cytometry in 26 CVID patients and 18 healthy blood donors. Strong responses were induced both in CVID and healthy donor B cells upon activation. In contrast, constitutive phosphorylation levels of STAT3,-5,-6, Erk, PLC-γ and Syk were significantly increased in CVID B cells only. Hierarchical clustering revealed a subgroup of CVID patients with elevated constitutive phosphorylation of Syk and PLC-γ. All these patients had non-infectious complications, indicating that a distinct phosphorylation pattern of kinases in B cells identifies a clinically important subgroup of CVID patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background & Aims: Recruitment of gut-derived memory T-cells to the liver is believed to drive hepatic inflammation in primary sclerosing cholangitis (PSC). However, whether gut-infiltrating and liver-infiltrating T-cells share T cell receptors (TCRs) and antigenic specificities is unknown. We used paired gut and liver samples from PSC patients with concurrent inflammatory bowel disease (PSC-IBD), and normal tissue samples from colon cancer controls, to assess potential T cell clonotype overlap between the two compartments., Methods: High-throughput sequencing of TCRβ repertoires was applied on matched colon, liver and blood samples from patients with PSC-IBD (n=10), and on paired tumor-adjacent normal gut and liver tissue samples from colon cancer patients (n=10)., Results: An average of 9.7% (range: 4.7-19.9%) memory T cell clonotypes overlapped in paired PSC-IBD affected gut and liver samples, after excluding clonotypes present at similar frequencies in blood. Shared clonotypes constituted on average 16.0% (range: 8.7-32.6%) and 15.0% (range: 5.9-26.3%) of the liver and gut memory T-cells, respectively. A significantly higher overlap was observed between paired PSC-IBD affected samples (8.7%, p=0.0007) compared to paired normal gut and liver samples (3.6%), after downsampling to equal number of reads., Conclusion: Memory T-cells of common clonal origin were detected in paired gut and liver samples of patients with PSC-IBD. Our data indicate that this is related to PSC-IBD pathogenesis, suggesting that memory T-cells driven by shared antigens are present in the gut and liver of PSC-IBD patients. Our findings support efforts to therapeutically target memory T cell recruitment in PSC-IBD., Lay Summary: Primary sclerosing cholangitis (PSC) is a devastating liver disease strongly associated with inflammatory bowel disease (IBD). The cause of PSC is unknown, but it has been suggested that the immune reactions in the gut and the liver are connected. Our data demonstrate for the first time that a proportion of the T-cells in the gut and the liver react to similar triggers, and that this proportion is particularly high in patients with PSC and IBD., (Copyright © 2016 European Association for the Study of the Liver. All rights reserved.)

Accumulating evidence suggests that clinically efficacious cancer immunotherapies are driven by T cell reactivity against DNA mutation-derived neoantigens. However, among the large number of predicted neoantigens, only a minority is recognized by autologous patient T cells, and strategies to broaden neoantigen-specific T cell responses are therefore attractive. We found that naïve T cell repertoires of healthy blood donors provide a source of neoantigen-specific T cells, responding to 11 of 57 predicted human leukocyte antigen (HLA)-A*02:01-binding epitopes from three patients. Many of the T cell reactivities involved epitopes that in vivo were neglected by patient autologous tumor-infiltrating lymphocytes. Finally, T cells redirected with T cell receptors identified from donor-derived T cells efficiently recognized patient-derived melanoma cells harboring the relevant mutations, providing a rationale for the use of such "outsourced" immune responses in cancer immunotherapy., (Copyright © 2016, American Association for the Advancement of Science.)

Unlabelled: Hepatic T-cell infiltrates and a strong genetic human leukocyte antigen association represent characteristic features of various immune-mediated liver diseases. Conceptually the presence of disease-associated antigens is predicted to be reflected in T-cell receptor (TCR) repertoires. Here, we aimed to determine if disease-associated TCRs could be identified in the nonviral chronic liver diseases primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and alcoholic liver disease (ALD). We performed high-throughput sequencing of the TCRβ chain complementarity-determining region 3 of liver-infiltrating T cells from PSC (n = 20), PBC (n = 10), and ALD (n = 10) patients, alongside genomic human leukocyte antigen typing. The frequency of TCRβ nucleotide sequences was significantly higher in PSC samples (2.53 ± 0.80, mean ± standard error of the mean) compared to PBC samples (1.13 ± 0.17, P < 0.0001) and ALD samples (0.62 ± 0.10, P < 0.0001). An average clonotype overlap of 0.85% was detected among PSC samples, significantly higher compared to the average overlap of 0.77% seen within the PBC (P = 0.024) and ALD groups (0.40%, P < 0.0001). From eight to 42 clonotypes were uniquely detected in each of the three disease groups (≥30% of the respective patient samples). Multiple, unique sequences using different variable family genes encoded the same amino acid clonotypes, providing additional support for antigen-driven selection. In PSC and PBC, disease-associated clonotypes were detected among patients with human leukocyte antigen susceptibility alleles., Conclusion: We demonstrate liver-infiltrating disease-associated clonotypes in all three diseases evaluated, and evidence for antigen-driven clonal expansions. Our findings indicate that differential TCR signatures, as determined by high-throughput sequencing, may represent an imprint of distinctive antigenic repertoires present in the different chronic liver diseases; this thereby opens up the prospect of studying disease-relevant T cells in order to better understand and treat liver disease., (© 2015 by the American Association for the Study of Liver Diseases.)

T cells engineered to express chimeric antigen receptors (CARs) targeted to CD19 are effective in treatment of B-lymphoid malignancies. However, CARs recognize all CD19 positive (pos) cells, and durable responses are linked to profound depletion of normal B cells. Here, we designed a strategy to specifically target patient B cells by utilizing the fact that T-cell receptors (TCRs), in contrast to CARs, are restricted by HLA. Two TCRs recognizing a peptide from CD20 (SLFLGILSV) in the context of foreign HLA-A*02:01 (CD20p/HLA-A2) were expressed as 2A-bicistronic constructs. T cells re-directed with the A23 and A94 TCR constructs efficiently recognized malignant HLA-A2(pos) B cells endogenously expressing CD20, including patient-derived follicular lymphoma and chronic lymphocytic leukemia (CLL) cells. In contrast, a wide range of HLA-A2(pos)CD20(neg) cells representing different tissue origins, and HLA-A2(neg)CD20(pos) cells, were not recognized. Cytotoxic T cells re-directed with CD20p/HLA-A2-specific TCRs or CD19 CARs responded with similar potencies to cells endogenously expressing comparable levels of CD20 and CD19. The CD20p/HLA-A2-specific TCRs recognized CD20p bound to HLA-A2 with high functional avidity. The results show that T cells expressing CD20p/HLA-A2-specific TCRs efficiently and specifically target B cells. When used in context of an HLA-haploidentical allogeneic stem cell transplantation where the donor is HLA-A2(neg) and the patient HLA-A2(pos), these T cells would selectively kill patient-derived B cells and allow reconstitution of the B-cell compartment with HLA-A2(neg) donor cells. These results should pave the way for clinical testing of T cells genetically engineered to target malignant B cells without permanent depletion of normal B cells.

T lymphocytes can be redirected to recognize a tumor target and harnessed to combat cancer by genetic introduction of T-cell receptors of a defined specificity. This approach has recently mediated encouraging clinical responses in patients with cancers previously regarded as incurable. However, despite the great promise, T-cell receptor gene therapy still faces a multitude of obstacles. Identification of epitopes that enable effective targeting of all the cells in a heterogeneous tumor while sparing normal tissues remains perhaps the most demanding challenge. Experience from clinical trials has revealed the dangers associated with T-cell receptor gene therapy and highlighted the need for reliable preclinical methods to identify potentially hazardous recognition of both intended and unintended epitopes in healthy tissues. Procedures for manufacturing large and highly potent T-cell populations can be optimized to enhance their antitumor efficacy. Here, we review the current knowledge gained from preclinical models and clinical trials using adoptive transfer of T-cell receptor-engineered T lymphocytes, discuss the major challenges involved and highlight potential strategies to increase the safety and efficacy to make T-cell receptor gene therapy a standard-of-care for large patient groups., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

When a T-cell Receptor (TcR) interacts with its cognate peptide-MHC (pMHC), it triggers activation of a signaling cascade that results in the elicitation of a T cell effector function. Different models have been proposed to understand which parameters are needed to obtain an optimal activation of the signaling. It was speculated that improving the binding of a TcR could bring a stronger pMHC recognition, hence a stronger stimulation of the T cell. However, it was recently shown that an increase in affinity does not seem to be sufficient to guarantee improved functionality. A combination of factors is necessary to place the modified TcR in an optimal functional window. We here compared the binding parameters of two mutants of the melanoma antigen peptide MART-127-35 specific TcR DMF5. The first mutant was previously isolated by others in a screen for improved TcR. It was reported to have an increased CD8-independent activity. We confirmed these data and showed that the enhancement was neither due to change in half life (t1/2) nor Kd of the pMHC-TcR complex. The second mutant was designed based on a previous report claiming that a particular polymorphic residue in the TRAV12-2 chain was stabilizing the TcR. We created a DMF5 mutant for this residue and showed that, unexpectedly, this TcR had acquired a reduced overall activity although the TcR-pMHC complex was more stable when compared to the TcR wild type complex (increased t1/2). In addition, the soluble TcR form of this mutant bound target cells less efficiently. From this we concluded that kinetic parameters do not always predict the superior functionality of mutant TcRs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Recently, technology has become available to generate soluble T-cell receptors (sTCRs) that contain the antigen recognition part. In contrast to antibodies, sTCRs recognize intracellular in addition to extracellular epitopes, potentially increasing the number of applications as reagents for target detection and immunotherapy. Moreover, recent data show that they can be used for identification of their natural peptide ligands in disease. Here we describe a new and simplified expression method for sTCRs in human cells and show that these sTCRs can be used for antigen-specific labeling and elimination of human target cells. Four different TCRs were solubilized by expression of constructs encoding the TCR alpha (α) and beta (β) chains lacking the transmembrane and intracellular domains, linked by a ribosomal skipping 2A sequence that facilitates equimolar production of the chains. Cell supernatants containing sTCRs labeled target cells directly in a peptide (p)-human leukocyte antigen (HLA)-specific manner. We demonstrated that a MART-1p/HLA-A*02:01-specific sTCR fused to a fluorescent protein, or multimerized onto magnetic nanoparticles, could be internalized. Moreover, we showed that this sTCR and two sTCRs recognizing CD20p/HLA-A*02:01 could mediate selective elimination of target cells expressing the relevant pHLA complex when tetramerized to streptavidin-conjugated toxin, demonstrating the potential for specific delivery of cargo. This simple and efficient method can be utilized to generate a wide range of minimally modified sTCRs from the naturally occurring TCR repertoire for antigen-specific detection and targeting.

Therapeutic vaccines for follicular lymphoma have had limited success. A novel in situ immunotherapeutic strategy combining 3 different treatment modalities induced regression of disseminated follicular lymphoma, which correlated with systemic antitumor T-cell immunity. These results should renew interest in the development of local combined radio- and immunotherapies to achieve abscopal effects.

Advanced stage follicular lymphoma (FL) is incurable by conventional therapies. In the present pilot clinical trial, we explored the efficacy and immunogenicity of a novel in situ immunotherapeutic strategy. Fourteen patients with untreated or relapsed stage III/IV FL were included and received local radiotherapy to solitary lymphoma nodes and intranodal injections of low-dose rituximab (5 mg), immature autologous dendritic cells, and granulocyte-macrophage colony-stimulating factor at the same site. The treatment was repeated 3 times targeting different lymphoma nodes. Primary end points were clinical responses and induction of systemic immunity. Five out of 14 patients (36%) displayed objective clinical responses, including 1 patient with cutaneous FL who showed regression of skin lesions. Two of the patients had durable complete remissions. Notably, the magnitude of vaccination-induced systemic CD8 T-cell-mediated responses correlated closely with reduction in total tumor area (r = 0.71, P = .006), and immune responders showed prolonged time to next treatment. Clinical responders did not have a lower tumor burden than nonresponders pretreatment, suggesting that the T cells could eliminate large tumor masses once immune responses were induced. In conclusion, the combined use of 3 treatment modalities, and in situ administration in single lymphoma nodes, mediated systemic T-cell immunity accompanied by regression of disseminated FL. The trial was registered at www.clinicaltrials.gov as #NCT01926639., (© 2015 by The American Society of Hematology.)