T-cell Receptor Chain Research Articles

404. Development of Combinational Cellular Immunotherapy to Treat Pediatric Acute Myeloid Leukemia Patients

Hematopoietic stem cell transplantation is the standard of care for pediatric acute myeloid leukemia (AML) patients, but relapse still occurs in approximately 40% of AML patients. Hence there is an unmet need to improve disease control with fewer adverse acute and late effects. We therefore aim to develop ‘HLA-matched’ modified cellular therapies based on a platform of cord blood transplantation (CBT) to treat pediatric AML patients. In the majority of AML patients the tumor oncogene Wilms tumor 1 (WT1) is overexpressed and can serve as a target for tumor specific therapy. In addition to our previously developed cord blood-derived dendritic cell vaccine (CB-DC) we are currently developing gene modified T lymphocytes from the same CB-unit to specifically target WT1 expressing AML to support and increase the anti-leukemic effects of CBT. Lentiviral vectors were developed to express recombinant T-cell receptors (TCR) specifically recognizing HLA-A2+ restricted WT1 peptide. TCR expression was improved by codon-optimization of the cDNA sequence including cysteines in the alpha and beta chain constant domains. These WT1-specific TCR α/β-chains were incorporated in a single lentiviral vector linked by a ‘self-cleaving’ T2A peptide to improve efficiency. The order of the alpha and beta TCR chains, and inclusion of a furin cleavage site in the expression cassette did not enhance TCR expression in Jurkat and primary T cells derived from either peripheral blood or cord blood. Primary T cells were stimulated with T2 cells and CB-DCs pulsed with the specific peptide, which resulted in T cell activation as measured by CD69 and CD137 upregulation, as well as IFNgamma production. To reduce the risk of off-target antigen recognition of gene-modified T cells, and to further improve efficacy, CRISPR/Cas9 can be used to eliminate endogenous TCR expression. Cas9 protein and guide RNA (gRNA) were delivered to GFP positive reporter Jurkat cells using plasmid and mRNA electroporation (EP), integrating and non-integrating lentiviral vectors and adenoviral vectors with the highest efficiency of knockout being achieved by EP. Two gRNAs targeting the constant domains of the alpha chain and three targeting those of the beta chain resulted in similar knockout efficiencies in Jurkat cells (approximately 25%). Of note, combining the gRNAs did not result in higher knockout efficiencies. Gene-editing at the molecular level was confirmed by next generation sequencing. We envision that addition of TCR-engineered T cells (introduction of tumor-specific TCR and knockdown of endogenous TCR), derived from the same cord blood as used for the transplantation, will more efficiently and with improved safety prevent early relapse after CBT by targeting WT1 AML.

Transplantational and specific antitumor immunity in retrospective view: new models based on transgenesis of individual chains of T-cell receptor

Findings in experimental oncology in beginning of last century and subsequent achievements of genetics of tissue compatibility resulted in divergence of transplantational immunology and oncoimmunology. However, central achievements of both scientific fields are based on unified phenomenon of interaction between T-cell receptor (TCR) and histocompatibility molecules. In this review we describe the history of ideas, achievements and unique experience of the team of the Laboratory of Regulatory Mechanisms in Immunity at Scientific Research Institute of Carcinogenesis, N.N. Blokhin Russian Cancer Research Center for all time of existence. This experience shows that efficiency of immunological defense including immunological surveillance are critically influenced by T-cell receptor repertoire. Transgenesis of individual chains of TCR is one of possible means to manage T-cell repertoire. Functional outcomes of transgenesis may be different due to diverse extent of dependence of α- and β-chains expression on the rules of allelic exclusion. Expression of transgenic β-chains results in the expansion of TCR repertoire diversity. Expression of β-chains is under strong control by allelic exclusion, resulting in formation of repertoire bearing mainly invariant transgenic β-chain pared with different α-chains and overall narrowing of repertoire. Earlier, we cloned genes encoding α- and β-chains of TCR of CD8+ memory cells specific to histocompatibility molecule H-2Kb . After introduction them in zigotes we have obtained transgenic mouse strains, which could be used for modeling of interactions between tumor cells and immune system of recipient. Normally, B10. D2 (R101) mice reject lymphoma EL4 cells in 12–14 days after transplantation, in spite of the fact, that allogeneic difference between B10. D2 (R101) (Kd Id Db ) mice and lymphoma EL4 (H-2b) cells is only in one product of MHC, the H-2Kb molecule. Transgenics carrying β-chains of TCR displayed compromised immunity to tumor cells resulting to their long persistence, tumor progression, the loss of H-2Kb molecule and death in 2–3 months after transplantation. This model allows to see all three phases of interaction between tumor and immune system of recipient – elimination, equilibrium and escape. Against, transgenics carrying α-chain reject tumor cells much more quickly, as in secondary immune response, in 3–6 days after transplantation. This rejection was mediated by intraepithelial T lymphocytes displaying features of resident memory cells – inability to recirculation, expression of CD103 and early activation antigen CD69 and intermediate density of T-cell markers CD3 and CD8. The capability to be located in nonlymphoid tissue and quickly destroy tumor cells makes them to be the most probable candidate to perform immunological surveillance functions.

MAPK1/ERK2 as novel target genes for pain in head and neck cancer patients.

BackgroundGenetic susceptibility plays an important role in the risk of developing pain in individuals with cancer. As a complex trait, multiple genes underlie this susceptibility. We used gene network analyses to identify novel target genes associated with pain in patients newly diagnosed with squamous cell carcinoma of the head and neck (HNSCC).ResultsWe first identified 36 cancer pain-related genes (i.e., focus genes) from 36 publications based on a literature search. The Ingenuity Pathway Analysis (IPA) analysis identified additional genes that are functionally related to the 36 focus genes through pathway relationships yielding a total of 82 genes. Subsequently, 800 SNPs within the 82 IPA-selected genes on the Illumina HumanOmniExpress-12v1 platform were selected from a large-scale genotyping effort. Association analyses between the 800 candidate SNPs (covering 82 genes) and pain in a patient cohort of 1368 patients with HNSCC (206 patients with severe pain vs. 1162 with non-severe pain) showed the highest significance for MAPK1/ERK2, a gene belonging to the MAP kinase family (rs8136867, p value = 8.92 × 10−4; odds ratio [OR] = 1.33, 95 % confidence interval [CI]: 1.13–1.58). Other top genes were PIK3C2G (a member of PI3K [complex], rs10770367, p value = 1.10 × 10−3; OR = 1.46, 95 % CI: 1.16–1.82), TCRA (the alpha chain of T-cell receptor, rs6572493, p value = 2.84 × 10−3; OR = 0.70, 95 % CI: 0.55–0.88), PDGFC (platelet-derived growth factor C, rs6845322, p value = 4.88 × 10−3; OR = 1.32, 95 % CI: 1.09–1.60), and CD247 (a member of CD3, rs2995082, p value = 7.79 × 10−3; OR = 0.76, 95 % CI: 0.62–0.93).ConclusionsOur findings provide novel candidate genes and biological pathways underlying pain in cancer patients. Further study of the variations of these candidate genes could inform clinical decision making when treating cancer pain.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0348-7) contains supplementary material, which is available to authorized users.

Hepatosplenic T-cell lymphoma: The problems of diagnosis and treatment

In the past decade, a notable advance has been made in the understanding of the pathogenesis of NK/T-cell lymphomas; however, their diagnosis remains difficult because of their rarity and clinical and morphological variabilities. The paper generalizes the ten-year experience of the Hematology Research Center, Ministry of Health of Russia, in diagnosing and treating hepatosplenic T-cell lymphoma (HSTL), considers the problems of differential diagnosis with other hematological diseases occurring with similar clinical and laboratory symptoms, and lays down current approaches to the diagnosis and treatment of this condition. A clinician's view of the problem of diagnosis and treatment of this disease is given. HSTL is shown to be a heterogeneous group of diseases differing in a T-cell receptor chain gene rearrangement, the clinical course of the disease, and overall survival (OS). According to our data, 3-year OS was 12%; the median survival was 26 months. Two-year OS for γδ and αβ HSTL was equal to 25 and 70%, respectively. The difference in OS for the variants of HSTL failed to reach statistical significance (because the sample might be insufficient).

Rapid cloning, expression, and functional characterization of paired αβ and γδ T-cell receptor chains from single-cell analysis

Transgenic expression of antigen-specific T-cell receptor (TCR) genes is a promising approach for immunotherapy against infectious diseases and cancers. A key to the efficient application of this approach is the rapid and specific isolation and cloning of TCRs. Current methods are often labor-intensive, nonspecific, and/or relatively slow. Here, we describe an efficient system for antigen-specific αβTCR cloning and CDR3 substitution. We demonstrate the capability of cloning influenza-specific TCRs within 10 days using single-cell polymerase chain reaction (PCR) and Gibson Assembly techniques. This process can be accelerated to 5 days by generating receptor libraries, requiring only the exchange of the antigen-specific CDR3 region into an existing backbone. We describe the construction of this library for human γδ TCRs and report the cloning and expression of a TRGV9/TRDV2 receptor that is activated by zoledronic acid. The functional activity of these αβ and γδ TCRs can be characterized in a novel reporter cell line (Nur77-GFP Jurkat 76 TCRα–β–) for screening of TCR specificity and avidity. In summary, we provide a rapid method for the cloning, expression, and functional characterization of human and mouse TCRs that can assist in the development of TCR-mediated therapeutics.

Chimeric Antigen Receptor Transgenic, T Cell Receptor/CD3 Negative Monospecific T Cells Generated from Cord Blood CD34 Positive Cells

Autologous T cells transduced to express chimeric antigen receptors (CAR) directed against CD19, a B cell antigen, are reported to induce complete remission in patients with leukemia or lymphoma of the B cell lineage. Although potentially very effective, this treatment strategy has major drawbacks. First, CAR therapy is based on autologous T cells and therefore dependent on the nature and quality of T cells present in the peripheral blood of these patients at the time of treatment. Poor quality of the T cells may cause treatment failure in some patients. In addition, therapy based on autologous cells is tailor-made i.e. CAR+ T cells have to be generated de novo for every patient. Finally, autologous cell therapy requires different, more complicated logistics than conventional therapy. We therefore investigate whether a general purpose, allogeneic CAR therapy based on HLA-matched cord blood obtained from cord blood banks can be devised. Here, we investigated whether functional CAR+ T cells can be generated in vitro that do not express an endogenous T cell receptor to avoid alloreactivity causing graft versus host reactions.We compared carcino-embryonic antigen (CEA)- specific CARs of the first generation (intracellular CD3ζ signaling chain), of the 2nd generation (intracellular CD3ζ and CD28 signaling chain) and of the 3rd generation (intracellular CD3ζ, CD28 and OX40 signaling chain). CD34+ progenitor cells were isolated from human cord blood or postnatal thymus and subsequently transduced with one of the three green fluorescent protein (GFP)-encoding CAR constructs. Transduced cells were subsequently co-cultured on OP9DL1 in the presence of stem cell factor, Flt3-ligand and interleukin-7. Unlike T cell receptor transduced precursors (1), expansion was not enhanced by transduction of the chimeric receptor. Expansion was highest with first generation CARs whereas second and third generation CARs displayed only restricted expansion. Similar to T cell receptor transduced progenitors, CAR transduced cells show an accelerated differentiation during co-culture compared to the non-transduced cells: first committed CD5+ CD7+ T precursors appear, then CD4+ CD8+ double positive cells (DP) and finally CD1- CD27- single positive or double negative (DN) mature T cells. In cultures transduced with 2nd and 3rd generation CARs, few transduced cells passed through the proliferative DP pathway but rather differentiated to mature CD1- CD27- non-proliferative DN cells without passing through the DP stage. This phenomenon is responsible for the limited expansion seen with precursors transgenic for 2nd or 3rd generation CARs. However, in all cultures CAR+ DP cells were generated and, as shown for TCR transgenic cells (1), we were able to induce CEA specific maturation after co-culturing these DP cells with a cell line expressing CEA or by antibody-induced cross-linking of the CAR, giving rise to CD1- CD27+ matured cells. The observations described above are compatible with data obtained in mice showing that strong T cell receptor (TCR) activation during thymocyte differentiation inhibits the generation of DP cells and induces maturation to DN cells. Both the spontaneously and induced mature CAR+ cells were TCR and CD3 negative, suggesting that the expression of a CAR in early T cell precursors shuts down rearrangements of the endogenous TCR chains. Moreover, these cells lack NK marker expression (CD56, NKG2D) and show expression of T cell markers (CD5, CD7, CD2), confirming their T cell nature. In conclusion, the CAR+ CD3/TCR negative cells are T cells as these are derived from T cell precursors (CD5+, DP cells) and express various membrane and nuclear T cell markers.Mature CD1- CD27- CAR+ cells can be expanded to large cell numbers using T cell expansion protocols. They displayed cytokine production specific for CEA+ tumor lines as well as specific cytotoxicity. Moreover, the 2nd and 3rd generation CAR expressing cells showed increased specific cytokine production when compared to the first generation CAR expressing cells. These results show that the cord blood-derived CAR+ cells have potent functional activity similar to peripheral blood derived CAR+ T cells.We believe that these in vitro generated CAR+ cells developed from HLA-matched cord blood progenitors may be ideal as an adjunct to cord blood transplantation.(1) Snauwaert et al, Leukemia, 2014 DisclosuresNo relevant conflicts of interest to declare.

Adoptive Transfer of WT1-Specific TCR Gene-Transduced Lymphocytes in Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia

Wilms' Tumor 1 (WT1) is expressed in a majority of MDS and AML cells and mRNA of WT1 in peripheral blood and bone marrow is monitored as a marker of minimal residual disease of AML and MDS. Several WT1 protein-derived epitopes that are recognized by cytotoxic T lymphocytes (CTLs) along with HLA molecules are determined. In vitro study and WT1 peptide vaccine trials have demonstrated that WT1-specfic CD8+ T cells with cytotoxic activity can be induced. Adoptive T cell therapy using ex vivo expanded WT1-specific CTLs or WT1-specific T-cell receptor (TCR)-gene transduced cells are potentially effective to refractory MDS and AML.Antigen-specific TCR-gene transfer may cause serious autoimmune disease by mispairing of introduced and endogenous TCR chains that recognize auto-antigens. We established a retroviral vector system encoding siRNAs for endogenous TCR genes to eliminate TCR-mispairing. Using the siRNA-encoding viral vector, we have conducted a first-in-man trial of WT1-specfic TCR-gene transduced T cell transfer. In the trial, we evaluate the safety of the TCR T cell transfer in patients with MDS and AML, and assess in vivo kinetics of the transferred cells. The study was designed as cell-dose escalation with three cohorts of 2x108, 1x109, and 5x109cells per infusion.Peripheral blood mononuclear cells were collected from each patient. Then, the cells were cultured with IL-2, anti-CD3 antibody, and RetroNectin®. Proliferating lymphocytes were infected with a retroviral vector, MS3-WT1-siTCR, which was constructed from DNA encoding WT1235-243/HLA*A24:02 specific TCR-α and -β chains and siRNAs for endogenous TCR genes. After 13-14 days in culture, the lymphocytes were harvested and frozen until infusion.Patients were enrolled to the clinical trial if they were refractory AML or MDS ineligible for allogeneic stem cell transplant, positive for HLA-A*24:02, had performance status of 0 to 2, and had normal organ function. WT1-TCR T cells were infused intravenously twice on days 0 and 28. Modified WT1235-243peptide (300μg) emulsified with Montanide, was given subcutaneously on day 2 and 16 after the second infusion.To date, 5 patients (4 MDS and 1 AML cases) with a median age of 69 years, received WT1-TCR T cells. Three received 2x108 cells (cohort 1) and 2 received 1x109 cells (cohort 2) per infusion, respectively. We did not see any severe adverse events related to the cell infusion or peptide vaccination. No renal or mesothelial damages were observed. We then assessed transduced TCR-gene copy numbers in peripheral blood samples collected at multiple pre-determined time points until day 58.TCR-gene marked cells were detected in all patients after the cell infusion. They appeared immediately after the infusion, reaching peak levels between 1 and 3 days. Then, the levels gradually declined. After the second infusion, which was followed by peptide vaccination, the cells appeared in the similar way to the first cycle. The peptide vaccine did not seem to affect the peripheral cell kinetics. Dose-dependent kinetics were shown between the cohort 1(2 x108 cells) and the cohort 2 (1x109cells). In two patients, transient decline of peripheral abnormal cells that were MDS-related erythroblasts, and decrease of bone marrow blasts were observed, respectively.Although the clinical trial is still ongoing, transfer of WT1-TCR-gene transduced lymphocyte to MDS and AML patients is safe and tolerable. TCR- T cells appeared in peripheral blood with cell-dose dependent manner. DisclosuresFujiwara:Celgene: Honoraria, Other: Travel, Acomodations, Expenses. Akatsuka:Takara Bio. Inc.: Other: Advisor to the CAR project. Tomura:TAKARO BIO INC.: Employment. Nukaya:TAKARA BIO INC.: Employment. Takesako:TAKARA BIO INC.: Employment.

Targeting Aurora Kinase with a Superior T-Cell Receptor Gene-Transfer Vector

[Background] Aurora Kinase A (AURKA) is a cancer-associated protein normally involved in the regulation of mitosis. It is over-expressed in a range of cancers, and has a 'cancer-testis' expression profile, making it a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor (TCR) sequences cognisant of HLA-A*0201-restricted AURKA antigen (aa207-215: YLILEYAPL) has previously been shown to transfer specific immunoreactivity against the target peptide in a HLA-restricted manner (Nagai K et al. Blood, 2012).While TCR gene-transfer has great potential in overcoming the difficulties of isolating and expanding tumor-reactive lymphocytes from a patient's own cells, one hurdle is potential mispairing and competition between exogenous and endogenous TCR chains. We have used a retroviral vector design bearing siRNAs that downregulates endogenous TCR chains, without affecting expression of the transgenic TCR sequences. The TCR expression cassette also includes a 2A self-cleaving peptide, resulting in equimolar expression of the TCR α and β chains, further enhancing formation of the desired TCR. Via a simple, modular cloning method, we have cloned the α and β chains of the anti-AURKA -reactive TCR into this 'siTCR' vector. In this study, we then compared the activity of this vector against the original, 'conventional' vector without siRNAs across a panel of assays.[Methods] After codon-optimization to be resistant to built-in siRNAs for endogenous TCRs, full-length of an HLA-A*0201-restricted and AURKA207-215 nonamer -specific TCR α/β genes (Vα3 /J20/Cα, Vβ10.3(12)/J1.1/Cβ1, respectively) obtained from our established epitope-specific CD8+ T cell clone (AUR2) linked using T2A peptide sequence were cloned into the siTCR vector carrying those siRNAs using an Infusion cloning method (Takara Bio). Either conventional retroviral AURKA-specific TCR expression vector without those siRNAs or this new retroviral AURKA-siTCR vector was transduced into CD8+ T cells from healthy volunteers using anti-CD3 antibody and RetronectinTM (Takara Bio) as previously reported. The epitope-specific and leukemia specific cytotoxicity and IFN-γ production of these transfectants were examined using 51Cr-releasing assay, CD107a assay and ELISA. Introduced viral vector copy numbers of these two types of transfectants were measured using qPCR kit (Bio Line) according to manufacturer's instruction, on an ABI Prism 7500 (Applied Biosystems/Life Technologies). To examine the potential to target leukemia stem cells mediated by these AURKA-siTCR transfectants, we employed the side population of the AURKA mRNA highly expressing HLA-A*0201+ leukemia cell line, GANMO-1, using the dye exclusion method with Hoechst 33342 (Sigma). Concurrently the on-target/off-tumor adverse effect against hematopoietic progenitor cells mediated by these transfectants was examined using HLA-A*0201+ cord blood CD34+ cells as a target.[Results] TCRs expressed from the AURKA-siTCR vector retained the cytotoxic functionality of the original vector, with evidence of reduced off-target reactivity. The rate of expression of correctly-formed TCRs demonstrated by AURKA/HLA-A*0201 tetramer labeling was superior using the siTCR design, and this was achieved at lower vector copy numbers. AURKA mRNA tended to be overexpressed by the side population of GANMO-1, and AURKA-siTCR gene-modified CD8+ T cells successfully recognized and killed this population, but not normal cord blood CD34+ cells.[Conclusion] Maintaining TCR efficacy with a reduced vector copy number reduces the risk of genotoxicity. The siTCR design also reduces the risk of mispairing and cross-reactivity, while increasing the functional titre. Such improvements in the safety of TCR gene-transfer will be crucial for clinical applications of this technology. DisclosuresFujiwara:Celgene: Honoraria, Other: Travel, Acomodations, Expenses. Okamoto:Takara Bio Inc.: Employment. Mineno:Takara Bio Inc.: Employment.

Cutaneous T-Cell Lymphoma: Deep Sequencing Offers a Diagnostic Boost

Diagnosis of cutaneous T-cell lymphoma (CTCL) rests for the most part on histopathologic evaluation with adjunctive immunohistochemistry. However, considerable overlap exists between forms of chronic inflammatory dermatitis and early mycosis fungoides. Even molecular tests to measure clonality of T-cell receptor (TCR) loci are limited by relatively low sensitivity and the fact that inflammatory reactions can be clonal. Each T cell possesses a unique combination of TCR chains formed …

T-cell receptor repertoire of human peripheral CD161hiTRAV1-2+ MAIT cells revealed by next generation sequencing and single cell analysis

Mucosal-associated invariant T (MAIT) cells are a T-cell subset that expresses a conserved TRAV1-2 (Vα7.2) T-cell receptor (TCR) chain and the surface marker CD161. They are involved in the defence against microbes as they recognise small organic molecules of microbial origin that are presented by the non-classical MHC molecule 1 (MR1). MAIT cells express a semi-restricted TCR α chain with TRAV1-2 preferentially linked to TRAJ33, TRAJ12, or TRAJ20 which pairs with a limited set of β chains. To investigate the TCR repertoire of human CD161hiTRAV1-2+ T cells in depth we analysed the α and β chains of this T-cell subset by next generation sequencing. Concomitantly we analysed 132 paired α and β chains from single cells to assess the αβ pairing preferences. We found that the CD161hiTRAV1-2+ TCR repertoire in addition to the typical MAIT TCRs further contains polyclonal elements reminiscent of classical αβ T cells.

Optimization of T-cell Reactivity by Exploiting TCR Chain Centricity for the Purpose of Safe and Effective Antitumor TCR Gene Therapy.

Adoptive transfer of T cells redirected by a high-affinity antitumor T-cell receptor (TCR) is a promising treatment modality for cancer patients. Safety and efficacy depend on the selection of a TCR that induces minimal toxicity and elicits sufficient antitumor reactivity. Many, if not all, TCRs possess cross-reactivity to unrelated MHC molecules in addition to reactivity to target self-MHC/peptide complexes. Some TCRs display chain centricity, in which recognition of MHC/peptide complexes is dominated by one of the TCR hemi-chains. In this study, we comprehensively studied how TCR chain centricity affects reactivity to target self-MHC/peptide complexes and alloreactivity using the TCR, clone TAK1, which is specific for human leukocyte antigen-A*24:02/Wilms tumor 1(235-243) (A24/WT1(235)) and cross-reactive with B*57:01 (B57). The TAK1β, but not the TAK1α, hemi-chain possessed chain centricity. When paired with multiple clonotypic TCRα counter-chains encoding TRAV12-2, 20, 36, or 38-2, the de novo TAK1β-containing TCRs showed enhanced, weakened, or absent reactivity to A24/WT1(235) and/or to B57. T cells reconstituted with these TCRα genes along with TAK1β possessed a very broad range (>3 log orders) of functional and structural avidities. These results suggest that TCR chain centricity can be exploited to enhance desired antitumor TCR reactivity and eliminate unwanted TCR cross-reactivity. TCR reactivity to target MHC/peptide complexes and cross-reactivity to unrelated MHC molecules are not inextricably linked and are separable at the TCR sequence level. However, it is still mandatory to carefully monitor for possible harmful toxicities caused by adoptive transfer of T cells redirected by thymically unselected TCRs.

T-cell receptor chain Vbeta subunit staining to quantify the malignant clone in adult T-cell leukemia

Adult T cell leukemia (ATL) is typically characterised by expansion of a single dominant malignantly transformed HTLV-1-infected clone. Clinically, the abundance of this clone can be estimated by flow cytometric assessment of CD4, CD25 and CD7 expression, and, more recently, by quantitative LM-PCR analysis of viral integration site. In addition to the unique proviral integration site, malignant clones arising from a single infection event also share an identical T cell receptor (TCR) sequence, utilising one of a potential 47 TCR Vbeta subunit genes. Using multiparametric flow cytometry, we quantified the frequency of peripheral T cells bearing each of 24 TCR Vbeta subunits in a cohort of individuals with adult T cell leukemia or lymphoma (n=26) at various stages of disease progression/remission. To establish an appropriate baseline, we also analysed T cells from asymptomatic carriers (n=11) and donors with HAM/TSP (n=13). Vbeta-specific antibodies stained 75% of CD3+ cells in donors without malignancy, and the median size of the largest single subset (CD4+Vbeta2+) was 7.6 % of CD3+ cells. We detected putative T cell clones which exceeded 20% of CD3+ T cells only in individuals with active chronic or acute leukemia (n=17), reaching frequencies of up to 96% of CD3+ T cells. With this method, we quantified the expression on clonally expanded populations of the putative ATL cell markers CD25, CCR4, TSLC1, CD7 and CD127. Almost 1% of expanded clonal populations in ATL patients were CCR4+TSLC1+CD7–. Surprisingly, in most donors only 60% of the dominant clonal population expressed high levels of CD25. This flow cytometric protocol is a potentially powerful method to both monitor disease progression and response to therapy, providing fast and accurate quantification of clonally expanded populations. We are currently employing this technique to assay lysis of expanded clones by autologous cytotoxic T lymphocytes in patients with ATL.

Identification of the Docking Site for CD3 on the T Cell Receptor β Chain by Solution NMR

The T cell receptor (TCR)-CD3 complex is composed of a genetically diverse αβ TCR heterodimer associated noncovalently with the invariant CD3 dimers CD3ϵγ, CD3ϵδ, and CD3ζζ. The TCR mediates peptide-MHC recognition, whereas the CD3 molecules transduce activation signals to the T cell. Although much is known about downstream T cell signaling pathways, the mechanism whereby TCR engagement by peptide-MHC initiates signaling is poorly understood. A key to solving this problem is defining the spatial organization of the TCR-CD3 complex and the interactions between its subunits. We have applied solution NMR methods to identify the docking site for CD3 on the β chain of a human autoimmune TCR. We demonstrate a low affinity but highly specific interaction between the extracellular domains of CD3 and the TCR constant β (Cβ) domain that requires both CD3ϵγ and CD3ϵδ subunits. The mainly hydrophilic docking site, comprising 9-11 solvent-accessible Cβ residues, is relatively small (∼400 Å(2)), consistent with the weak interaction between TCR and CD3 extracellular domains, and devoid of glycosylation sites. The docking site is centered on the αA and αB helices of Cβ, which are located at the base of the TCR. This positions CD3ϵγ and CD3ϵδ between the TCR and the T cell membrane, permitting us to distinguish among several possible models of TCR-CD3 association. We further correlate structural results from NMR with mutational data on TCR-CD3 interactions from cell-based assays.

Clinical applications of next-generation sequencing in histocompatibility and transplantation.

Next-generation sequencing (NGS) can overcome traditional methodological barriers to facilitate detailed studies of large genomes. Here, we summarize recent NGS-based developments in histocompatibility and transplantation, and highlight the dynamic range of clinical applications achievable on this platform. Multiple NGS-based protocols have been established to achieve unambiguous human leukocyte antigen genotyping. These methods are presently engaged to serve the high-throughput demand of large bone marrow registries; however, the scalable nature of NGS makes it an equally attractive technology for select applications within solid organ transplantation. Recently, the exquisite sensitivity of NGS has been leveraged to perform noninvasive allograft monitoring by tracking the dynamics of donor-derived cell-free DNA. Further, NGS-based T-cell receptor and immunoglobulin heavy chain repertoire profiling appear to be useful in clarifying disease-specific diagnoses in certain complex allograft pathology; detecting/quantifying minimal residual disease following allogeneic stem cell transplantation; and tracking donor-reactive T cells to understand the mechanism of tolerance in kidney transplant recipients. NGS is superior to classical Sanger sequencing in its throughput, sensitivity, and the ability to provide phase-defined sequence data. These unique properties allow its broad application to diverse areas in clinical transplantation.

Abstract 5011: generation of rejuvenated murine antigen-specific T cells by reprogramming to pluripotency and redifferentiation

Abstract Background: Adoptive cell therapy with autologous antigen-specific T cells is a promising approach for treating patients with chronic viral infections and various cancers. A major limitation of this approach is the difficulty of obtaining sufficient numbers of T cells with the characteristics necessary for in vivo effectiveness. Less-differentiated T cells with long telomeres would be an ideal T-cell subset for adoptive T cell therapy; however, generating large numbers of these “young” T cells is problematic. This limitation of adoptive T cell therapy can be overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, and provide an unlimited source of autologous T cells for immunotherapy. However, in vivo antitumor reactivity of iPSC-rejuvenated T cells remains unknown due to the lack of animal model. Methods: To establish preclinical model of evaluating iPSC-rejuvenated antigen-specific T cells, we reprogrammed OT-1 T cell receptor (TCR) transgenic CD8 T cells using Sendai virus vectors encoding Oct3/4, Sox2, Klf4 and c-Myc transcription factors. Pluripotency and differentiation capacity of generated iPSCs were evaluated. Results: Twenty days after infection with Sendai virus vectors, we obtained murine embryonic stem cell-like colonies from OT-1 CD8 T cells. Immunofluorescence staining and RT-PCR analyses revealed pluripotency of established colonies. The iPSCs formed embryoid bodies in vitro, and upregulation of marker genes for all three germ layers was detected. The iPSCs differentiated into teratomas in vivo comprising all three embryonic germ layers in immune-deficient NOD/SCID mice. The iPSCs retained the same rearranged configuration of TCR chain genes as the original OT-1 T cells. Furthermore, OT-1 T cell-derived iPSCs differentiated into T cells on delta-like ligand 1 expressing OP9 (OP9/DL1) murine bone marrow stromal cells. Conclusions: Generation of rejuvenated T cells by reprogramming to pluripotency and redifferentiation is feasible. This type of approach may be useful for adoptive T cell therapy. We acknowledge DNAVEC Corporation for kindly providing Sendai virus vectors. This study was supported by the University of Michigan Start-up fund. Citation Format: Fumito Ito, Noemi Fusaki, Hidehito Saito. generation of rejuvenated murine antigen-specific T cells by reprogramming to pluripotency and redifferentiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5011. doi:10.1158/1538-7445.AM2015-5011

Transfer of mRNA Encoding Invariant NKT Cell Receptors Imparts Glycolipid Specific Responses to T Cells and γδT Cells.

Cell-based therapies using genetically engineered lymphocytes expressing antigen-specific T cell receptors (TCRs) hold promise for the treatment of several types of cancers. Almost all studies using this modality have focused on transfer of TCR from CD8 cytotoxic T lymphocytes (CTLs). The transfer of TCR from innate lymphocytes to other lymphocytes has not been studied. In the current study, innate and adaptive lymphocytes were transfected with the human NKT cell-derived TCRα and β chain mRNA (the Vα24 and Vβ11 TCR chains). When primary T cells transfected with NKT cell-derived TCR were subsequently stimulated with the NKT ligand, α-galactosylceramide (α-GalCer), they secreted IFN-γ in a ligand-specific manner. Furthermore when γδT cells were transfected with NKT cell-derived TCR mRNA, they demonstrated enhanced proliferation, IFN-γ production and antitumor effects after α-GalCer stimulation as compared to parental γδT cells. Importantly, NKT cell TCR-transfected γδT cells responded to both NKT cell and γδT cell ligands, rendering them bi-potential innate lymphocytes. Because NKT cell receptors are unique and universal invariant receptors in humans, the TCR chains do not yield mispaired receptors with endogenous TCR α and β chains after the transfection. The transfection of NKT cell TCR has the potential to be a new approach to tumor immunotherapy in patients with various types of cancer.

αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell-related features.

Objectives:To characterize phenotypes of T cells that accumulated in multiple sclerosis (MS) lesions, to compare the lesional T-cell receptor (TCR) repertoire of T-cell subsets to peripheral blood, and to identify paired α and β chains from single CD8+ T cells from an index patient who we followed for 18 years.Methods:We combined immunohistochemistry, laser microdissection, and single-cell multiplex PCR to characterize T-cell subtypes and identify paired TCRα and TCRβ chains from individual brain-infiltrating T cells in frozen brain sections. The lesional and peripheral TCR repertoires were analyzed by pyrosequencing.Results:We found that a TCR Vβ1+ T-cell population that was strikingly expanded in active brain lesions at clinical onset comprises several subclones expressing distinct yet closely related Vα7.2+ α chains, including a canonical Vα7.2-Jα33 chain of mucosal-associated invariant T (MAIT) cells. Three other α chains bear striking similarities in their antigen-recognizing, hypervariable complementarity determining region 3. Longitudinal repertoire studies revealed that the TCR chains that were massively expanded in brain at onset persisted for several years in blood or CSF but subsequently disappeared except for the canonical Vα7.2+ MAIT cell and a few other TCR sequences that were still detectable in blood after 18 years.Conclusions:Our observation that a massively expanded TCR Vβ1-Jβ2.3 chain paired with distinct yet closely related canonical or atypical MAIT cell–related α chains strongly points to an antigen-driven process in early active MS brain lesions.

209. TCR Gene Editing in a Single Step of T Cell Activation To Redirect T Cell Specificity and Prevent GvHD

Transfer of T cell receptors (TCR) specific for tumor-associated antigens is a promising approach for cancer adoptive immunotherapy. Yet, TCR gene transfer into mature T cells results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted specificities. Thus, we developed a TCR gene editing procedure, based on the knockout of the endogenous TCR genes by transient exposure to α and β chain specific Zinc Finger Nucleases (ZFNs), followed by the introduction of tumor-specific TCR genes (Provasi et al, Nat. Med. 2012). While successful, the complete editing requires multiple manipulation steps involving repeated cell activation cycles and transductions. To reduce the duration and complexity of cell product generation, we recently developed a ‘single TCR editing’ (SE) procedure, based on the disruption of the endogenous TCR α chain only followed by the transfer of the tumor specific TCR genes. This SE method generates redirected T cells fully devoid of their natural TCR repertoire in a single round of cell activation. We validated the SE protocol exploiting an HLA-A2 restricted TCR specific for NY-ESO-1 (expressed by a considerable proportion of high risk multiple myeloma). The SE strategy allowed rapid production of high numbers of tumor specific T cells enriched for an early differentiation phenotype. When fucntionaly tested (co-culture, γ-IFN and 51Cr release) against the U266 myeloma cell line (NY-ESO-1+HLA-A2+), all NY-ESO-1 redirected T cells showed a strikingly high killing activity. However, when we assess the alloreactive potential of the different redirected T cells in mixed lymphocyte reactions, we observed that the allogeneic lysis by SE T cells was markedly lower (p=0.05) than that of conventional TCR transfer cells (TR). These results were validated in NSG mice, where the genetically modified T cells were infused after the engraftment of the U266 myeloma. All animals treated with tumor specific T cells were completely myeloma-free at the time of sacrifice, demonstrating the powerful anti-tumor potential of the NY-ESO-1 redirected T cells. However, the overall survival of mice treated with TR vs SE cells was 26% vs 100% respectively (p<0,001) corresponding with a significant difference in acute and chronic GvHD occurrence (71% vs 0%, p<0,001). Consistently, histopathological analysis of human T cell infiltration in the organs revealed a significantly higher score in mice treated with TR cells (p<0.001). The relative simplicity of the SE protocol enables rapid generation of highly performing tumor specific T cells, fully devoid of their endogenous TCR repertoire, and thus incapable of participating in GvHD. Such single TCR edited cells thus potentially represent a further advance in adoptive immunotherapy for cancer.

Paired TCRαβ analysis of virus-specific CD8(+) T cells exposes diversity in a previously defined 'narrow' repertoire.

T-cell receptor (TCR) usage has an important role in determining the outcome of CD8(+) cytotoxic T-lymphocyte responses to viruses and other pathogens. However, the characterization of TCR usage from which such conclusions are drawn is based on exclusive analysis of either the TCRα chain or, more commonly, the TCRβ chain. Here, we have used a multiplexed reverse transcription-PCR protocol to analyse the CDR3 regions of both TCRα and β chains from single naive or immune epitope-specific cells to provide a comprehensive picture of epitope-specific TCR usage and selection into the immune response. Analysis of TCR repertoires specific for three influenza-derived epitopes (D(b)NP(366), D(b)PA(224) and D(b)PB1-F2(62)) showed preferential usage of particular TCRαβ proteins in the immune repertoire relative to the naive repertoire, in some cases, resulting in a complete shift in TRBV preference or CDR3 length, and restricted repertoire diversity. The NP(366)-specific TCRαβ repertoire, previously defined as clonally restricted based on TCRβ analysis, was similarly diverse as the PA(224)- and PB1-F2(62)-specific repertoires. Intriguingly, preferred TCR characteristics (variable gene usage, CDR3 length and junctional gene usage) appeared to be able to confer specificity either independently or in concert with one another, depending on the epitope specificity. These data have implications for established correlations between the nature of the TCR repertoire and response outcomes after infection, and suggest that analysis of a subset of cells or a single TCR chain does not accurately depict the nature of the antigen-specific TCRαβ repertoire.

γδ T Lymphocytes in the Diagnosis of Human T Cell Receptor Immunodeficiencies.

Human T cell receptor (TCR) immunodeficiencies (TCRID) are rare autosomal recessive disorders caused by mutations affecting TCR, CD3, or CD247 chains, which share developmental, functional, and TCR expression defects (1). Their rapid diagnosis is fundamental for patient survival and early hematopoietic stem cell transplantation. Here, we propose that studying γδ T cells, which are often neglected, can be helpful for a timely diagnosis. We thus offer a diagnostic flowchart and some lab tricks based on published cases.