Surface Expression Of T-cell Receptor Research Articles

TMX1, a disulfide oxidoreductase, is necessary for T cell function through regulation of CD3ζ.

T cell-targeted therapies are commonly used to manage T cell hyperactivity in autoimmune disorders, graft-versus-host diseases (GVHD), and transplant rejections. However, many patients experience significant side effects or inadequate responses to current treatments, highlighting the urgent need for alternative strategies. In this study, we searched for regulators of T cells through proximity labeling with APEX2 to detect proteins interacting with CD8α, a coreceptor of the T-cell receptor (TCR). This screen revealed TMX1, an ER resident transmembrane disulfide oxidoreductase, is essential for T cell cytotoxicity and NFAT, NFκB, and AP1 signaling but not cell proliferation. TMX1 deletion decreases surface TCR expression and destabilizes CD3ζ, a subunit of TCR complex; however, overexpression of CD3ζ rescues the phenotype, suggesting that TMX1 is not required for CD3ζ function. Mechanistically, TMX1 was found to directly engage the CxxC motif of CD3δ, which has been reported to be essential for proper TCR assembly and function. We hypothesize that the loss of TMX1 interaction with CD3δ leads to impaired TCR assembly and subsequent CD3ζ destabilization. These findings identify TMX1 as a novel regulator of T-cell receptor assembly and a potential target for immunosuppressive therapy.

Modifications outside CDR1, 2 and 3 of the TCR variable β domain increase TCR expression and antigen-specific function.

T cell receptor (TCR) gene modified T cells are a promising form of adoptive cellular therapy against human malignancies and viral infections. Since the first human clinical trial was carried out in 2006, several strategies have been developed to improve the efficacy and safety of TCR engineered T cells by enhancing the surface expression of the introduced therapeutic TCRs whilst reducing the mis-pairing with endogenous TCR chains. In this study, we explored how modifications of framework residues in the TCR variable domains affect TCR expression and function. We used bioinformatic and protein structural analyses to identify candidate amino acid residues in the framework of the variable β domain predicted to drive high TCR surface expression. Changes of these residues in poorly expressed TCRs resulted in improved surface expression and boosted target cell specific killing by engineered T cells expressing the modified TCRs. Overall, these results indicate that small changes in the framework of the TCR variable domains can result in improved expression and functionality, while at the same time reducing the risk of toxicity associated with TCR mis-pairing.

Assessment of T Cell Receptor Complex Expression Kinetics in Natural Killer Cells.

Among the polypeptides that comprise the T cell receptor (TCR), only CD3ζ is found in Natural Killer (NK) cells, where it transmits signals from activating receptors such as CD16 and NKp46. NK cells are potent immune cells that recognize target cells through germline-encoded activating and inhibitory receptors. Genetic engineering of NK cells enables tumor-specific antigen recognition and, thus, has a significant promise in adoptive cell therapy. Ectopic expression of engineered TCR components in T cells leads to mispairing with the endogenous components, making a knockout of the endogenous TCR necessary. To circumvent the mispairing of TCRs or the need for knockout technologies, TCR complex expression has been studied in NK cells. In the current study, we explored the cellular processing of the TCR complex in NK cells. We observed that in the absence of CD3 subunits, the TCR was not expressed on the surface of NK cells and vice versa. Moreover, a progressive increase in surface expression of TCR between day three and day seven was observed after transduction. Interestingly, the TCR complex expression in NK92 cells was enhanced with a proteasome inhibitor (bortezomib) but not a lysosomal inhibitor (chloroquine). Additionally, we observed that the TCR complex was functional in NK92 cells as measured by estimating CD107a as a degranulation marker, IFNγ cytokine production, and killing assays. NK92 cells strongly degranulated when CD3ε was engaged in the presence of TCR, but not when only CD3 was overexpressed. Therefore, our findings encourage further investigation to unravel the mechanisms that prevent the surface expression of the TCR complex.

Abstract 5513: Chimeric CD3 fusion receptors expressed on iPSC-derived universal TCR-less CAR-T and -NK cells synergize with bispecific engagers to enhance antitumor activity and limit antigen escape

Abstract Despite success in hematologic malignancies, chimeric antigen receptor (CAR) T cells have been less effective in solid tumors, in part because of the heterogeneous expression of the CAR-specific target antigen (1° Ag) found within the tumor mass. In combination with a CAR, bispecific T cell engagers (BiTEs) can target additional tumor antigens (2° Ag) while engaging CD3 signaling molecules on T cells, providing a unique way to address tumor heterogeneity, mitigate antigen escape and further potentiate durable effector function. However, in generating off-the-shelf universal CAR-T and NK cells, a combination strategy is not feasible as neither modified cell product is compatible to specifically engage with a BiTE. In developing allogeneic CAR-T cells, the T cell receptor (TCR) surface expression must be eliminated to prevent graft versus host disease, but the absence of surface TCR expression leads to loss of surface CD3 expression and BiTE compatibility. Similarly, NK cells naturally lack TCR expression and have no surface CD3 molecules for BiTE engagement. Here we discuss the development of a novel chimeric CD3ε fusion receptor (CD3-CFR) with a modified transmembrane and endodomain enabling surface expression in TCR-less T or NK cells, allowing for a novel combinatorial solution between universal CAR-T or NK cells with BiTEs in an allogeneic setting. CD3-CFR signal transduction was initially investigated in TRAC knockout NFAT reporter (T-KO) Jurkat cells engineered with CD3-CFR and co-cultured with EpCAM+ target cells. When soluble EpCAM-BiTE was added to the co-culture, the observed 6.9-fold increase in NFAT activity indicated successful engagement associated with the interaction of CD3-CFR, target cells and the BiTE. CD3-CFR T-KO Jurkat cells were further modified to secrete the EpCAM-BiTE and showed increased NFAT activity, demonstrating the feasibility of self-secreting BiTE-mediated targeting through CD3-CFR signaling. We next engineered CD3-CFR expressing iPSC-derived CAR-T (iT) cells to show preserved T cell differentiation kinetics and maintained CAR activity against 1° Ag positive targets (>90% cytolysis). Importantly, while CAR-iT cells showed only background lysis of 1° Ag negative tumor cells, CD3-CFR+ CAR-iT cells were able to lyse ~99% of 1° Ag negative tumor cells with the addition of soluble EpCAM-BiTE targeting the 2° Ag. When CD3-CFR+ CAR-iT cells were further engineered to secrete the EpCAM-BiTE, CD3-CFR+ BiTE+ CAR-iT cells demonstrated superior killing of heterogenous tumors compared to CD3-CFR+ BiTE- CAR-iT cells (70% vs. 5% cytolysis). Taken together these data demonstrate for the first time that CD3 expression and BiTE engagement can be combined in a universal iPSC-derived CAR-iT cell product to overcome antigen heterogeneity and enhance efficacy in solid tumor settings. Citation Format: Eigen Peralta, Dan Lu, Mark Landon, Hui-Yi Chu, Amit Mehta, Philip Chu, Alec Witty, Tom Lee, Bahram Valamehr. Chimeric CD3 fusion receptors expressed on iPSC-derived universal TCR-less CAR-T and -NK cells synergize with bispecific engagers to enhance antitumor activity and limit antigen escape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5513.

Immunicy-1: Targeting BCMA with Cyad-211 to Establish Proof of Concept of an shRNA-Based Allogeneic CAR T Cell Therapy Platform

Off-the-shelf allogeneic CAR T cells derived from healthy donor cells have the potential to overcome many of the issues associated with the time-consuming manufacturing of autologous CAR T cells. However, adoptive transfer of allogeneic T cells carries the risk of graft-versus-host disease (GvHD). Most of the clinical experience with allogeneic CAR T cells is based on gene editing to eliminate T cell receptor (TCR) to mitigate the risk of GvHD. While clearly effective, the downsides of gene editing include multiple manufacturing steps requiring multiple clinical grade reagents, thus extending culture times, which can be associated with T cell exhaustion.As an alternative, we have explored short hairpin RNA (shRNA) as a means to knockdown TCR expression at the mRNA level. This shRNA is co-expressed along with the CAR in a single clinical grade vector, therefore requiring just one step of genetic modification.CYAD-211 is an allogeneic anti-BCMA CAR T that co-expresses a shRNA targeting CD3z which results in reduction of cell surface TCR expression. IMMUNICY-1 is an ongoing open-label Phase 1 trial (NCT04613557) designed to evaluate CYAD-211 in adult patients with refractory or relapsed multiple myeloma (MM) following at least two prior MM regimens. Patients receive non-myeloablative preconditioning (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, for 3 days) followed by a single CYAD-211 infusion in a 3+3 dose escalation design evaluating three dose-levels (DL): 30x10 6, 100x10 6 and 300x10 6 cells/infusion.As of July 29, 2021, nine patients were enrolled across the 3 DLs. Patients had received a median of four prior lines of treatment. Seventy-eight percent of patients were previously exposed to all three major MM drug classes (proteasome inhibitors, immunomodulatory drugs, and anti-CD38 antibody therapy). Eight patients had prior autologous stem cell transplantation.CYAD-211 was well tolerated. One patient developed grade 1 cytokine release syndrome. Two patients had Grade ≥ 3 hematologic toxicities possibly related to the experimental treatment. Two patients experienced infectious adverse events (1 grade 1 rhinitis and 1 grade 2 upper respiratory infection). There was no neurologic toxicity and no GvHD. There was no dose-limiting toxicity.Eight patients were evaluated for activity per IMWG criteria. Two patients achieved partial response at dose-levels 1 and 2 while 5 patients had stable disease (SD). One patient with an ongoing SD (3 months +) showed evidence of reduction in size of plasmacytomas.Analysis of peripheral blood samples by molecular methods confirmed the engraftment of CYAD-211. All patients had detectable CAR T cells. However, the engraftment was short lasting (3-4 weeks). There was a correlation between the depth of lymphodepletion and engraftment. There was also a dose-response in terms of CYAD-211 kinetics with a level neighboring 8,000 copies of CAR T per microgram of input DNA in patients at DL3.These early data indicate that CYAD-211 is well tolerated with a good safety profile. While further study is required to fully understand the anti-BCMA potency of the CAR used in this trial, the lack of observed GvHD despite engraftment of CYAD-211 provides proof of concept of the safe administration of CAR T using a shRNA-allogeneic platform. The lack of sustained engraftment of CYAD-211 can be explained by rejection of the allogeneic cells by the recovering immune system of the recipient and calls for exploring the role of augmented lymphodepletion. Furthermore, given the ability to include multiple shRNA within the single CAR vector, future strategies will also examine knocking down other molecules that are important in driving immune rejection. DisclosuresAl-Homsi: BMS: Other: Independent Medical Education Grant; Daichii Sankyo: Consultancy; Celyad Oncology: Other: Advisory Board. Deeren: Alexion: Consultancy; BMS: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Sanofi: Consultancy, Research Funding; Sobi: Consultancy; Takeda: Consultancy. Nishihori: Karyopharm: Research Funding; Novartis: Research Funding. Meuleman: iTeos Therapeutics: Consultancy. Abdul-Hay: Amgen: Membership on an entity's Board of Directors or advisory committees; Takeda: Speakers Bureau; Abbvie: Consultancy; Jazz: Other: Advisory Board, Speakers Bureau; Servier: Other: Advisory Board, Speakers Bureau. Braun: Celyad Oncology: Current Employment. Lonez: Celyad Oncology: Current Employment. Dheur: Celyad Oncology: Current Employment. Alcantar-Orozco: Celyad Oncology: Current Employment. Gilham: Celyad Oncology: Current Employment. Flament: Celyad Oncology: Current Employment. Lehmann: Celyad Oncology: Current Employment.

Abstract 332: Chimeric fusion receptors provide a CD3-mediated activation signal to off-the-shelf iPSC-derived TCR-less CAR-T cells for enhanced efficacy

Abstract T cells require a combination of signals to properly activate and exhibit their full potential; signal 1 from the T cell receptor (TCR), signal 2 from costimulatory receptors and signal 3 from cytokine receptors can shape the function of T cells. Second generation chimeric antigen receptor (CAR) T-cells targeting CD19 have shown success in hematologic malignancies, in part because signals 1 and 2 are provided by the CAR endodomain. As previously reported, we have developed an off-the-shelf iPSC-derived CAR19 T (CAR19-iT) cell product where the CAR is inserted into the T cell receptor alpha constant (TRAC) locus for enhanced efficacy and disruption of surface TCR expression. While the TCR-less CAR19-iT cells are ideal for allogeneic CAR-T cell therapies, as a consequence, they lack CD3 surface expression and the means by which to mediate CD3 signaling. To further improve CAR-iT cell performance and facilitate combinatorial strategies with T cell engagers such as bispecific T cell engagers (BiTEs), we designed chimeric fusion receptors (CFRs) comprising an ectodomain of CD3 or CD28, a transmembrane domain of CD28, and various endodomains that provide combinations of signals 1, 2, and 3. In addition to providing co-stimulatory signaling, the CFR designs can be controlled via agonistic antibodies or BiTEs to initiate signal transduction for precision control of the cell product. Furthermore, a series of CFRs with various CD3 endodomains were also developed to augment costimulatory signaling and the ER retention and endocytosis motifs found in CD3 endodomains were mutated to prevent downregulation of the CFRs from the cell surface of TCR-less cells. Selected CFRs were engineered into TRAC knockout Jurkat cells expressing a luciferase based NFAT reporter gene. After 48 hours, surface expression of the CFRs was confirmed by flow cytometry. Signal transduction via the CFRs was evaluated following exposure to agonistic antibodies or BiTEs. Measurement of the luciferase activity showed comparable levels to activated wildtype reporter cells. After construct validation, the CFRs were introduced into TCR-less CAR19-iT cells and co-cultured overnight with Nalm6 target cells in the presence of agonistic antibodies. Compared to control CAR19-iT cells, the armed CFR-CAR19 iT cells demonstrated superior killing of target cells. These results support the notion that enhancing signal 1 can increase CAR-T cell efficacy with current studies underway to investigate CFRs with endodomains that provide additional signal 2 or 3. We expect these constructs to further enhance CAR-iT cell persistence and engagement with BiTEs for activation and multi-antigen targeting. Collectively, the data suggest that TRC-less CAR-iT cells can be further armed with CFRs containing appropriate endodomains to provide superior antitumor function in a regulated manner. Citation Format: Eigen Peralta, Dan Lu, Mark Landon, Helen Chu, Shohreh Sikaroodi, Emily Carron, Natalie Navarrete, Alec Witty, Tom Lee, Anhco Nguyen, Bahram Valamehr. Chimeric fusion receptors provide a CD3-mediated activation signal to off-the-shelf iPSC-derived TCR-less CAR-T cells for enhanced efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 332.

CRISPR/Cas9-mediated knockout of clinically relevant alloantigenes in human primary T cells

BackgroundThe ability of CRISPR/Cas9 to mutate any desired genomic locus is being increasingly explored in the emerging area of cancer immunotherapy. In this respect, current efforts are mostly focused on the use of autologous (i.e. patient-derived) T cells. The autologous approach, however, has drawbacks in terms of manufacturing time, cost, feasibility and scalability that can affect therapeutic outcome or wider clinical application. The use of allogeneic T cells from healthy donors may overcome these limitations. For this strategy to work, the endogenous T cell receptor (TCR) needs to be knocked out in order to reduce off-tumor, graft-versus-host-disease (GvHD). Furthermore, CD52 may be knocked out in the donor T cells, since this leaves them resistant to the commonly used anti-CD52 monoclonal antibody lymphodepletion regimen aiming to suppress rejection of the infused T cells by the recipient. Despite the great prospect, genetic manipulation of human T cells remains challenging, in particular how to deliver the engineering reagents: virus-mediated delivery entails the inherent risk of altering cancer gene expression by the genomically integrated CRISPR/Cas9. This is avoided by delivery of CRISPR/Cas9 as ribonucleoproteins, which, however, are fragile and technically demanding to produce. Electroporation of CRISPR/Cas9 expression plasmids would bypass the above issues, as this approach is simple, the reagents are robust and easily produced and delivery is transient.ResultsHere, we tested knockout of either TCR or CD52 in human primary T cells, using electroporation of CRISPR/Cas9 plasmids. After validating the CRISPR/Cas9 constructs in human 293 T cells by Tracking of Indels by Decomposition (TIDE) and Indel Detection by Amplicon Analysis (IDAA) on-target genomic analysis, we evaluated their efficacy in primary T cells. Four days after electroporation with the constructs, genomic analysis revealed a knockout rate of 12–14% for the two genes, which translated into 7–8% of cells showing complete loss of surface expression of TCR and CD52 proteins, as determined by flow cytometry analysis.ConclusionOur results demonstrate that genomic knockout by electroporation of plasmids encoding CRISPR/Cas9 is technically feasible in human primary T cells, albeit at low efficiency.

Identification of T cells specific for DNAJB1-PRKACA fusion neoantigens in fibrolamellar carcinoma

Abstract Fibrolamellar carcinoma (FLC) is a rare liver tumor with poor outcomes and for which novel therapies are urgently needed. A potential therapeutic vulnerability in FLC is a gene fusion between DNAJB1 and PRKACA that is expressed in all FLC tumors and has been shown to drive FLC tumorigenesis. Genetic mutations that alter amino acid sequence, including gene fusions, can be presented as neoantigens by MHC class I molecules on tumor cells and thereby elicit anti-tumor CD8 T cell responses. We hypothesize that DNAJB1-PRKACA fusion neoantigens can elicit T cell responses in FLC patients and serve as targets for T cell-based immunotherapies for FLC. To test this hypothesis, T cells isolated and expanded from an FLC patient tumor were stimulated with DNAJB1-PRKACA fusion peptides, which reproducibly induced cytokine production measured by intracellular cytokine staining. Single cell gene expression and T cell receptor (TCR) repertoire analysis of patient tumors using the 10X Genomics platform identified clonally expanded TCR clonotypes that were associated with expression of cytokines and activation markers in response to fusion peptide stimulation. These TCRs were cloned and expressed in Jurkat 76 cells to test their fusion specificity and their ability to mediate in vitro functional responses to fusion neoantigens. When stimulated with their cognate fusion neoepitope, TCR-expressing cells produced cytokines, expressed the early activation marker CD69, and downregulated surface expression of TCR and CD3. These studies have therefore identified T cell responses against the DNAJB1-PRKACA fusion for the first time, as well as identified DNAJB1-PRKACA-specific TCR clonotypes that could be used to develop novel immunotherapies for FLC.

CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells

AbstractAdoptive transfer of T cells genetically modified to express a cancer-specific T-cell receptor (TCR) has shown significant therapeutic potential for both hematological and solid tumors. However, a major issue of transducing T cells with a transgenic TCR is the preexisting expression of TCRs in the recipient cells. These endogenous TCRs compete with the transgenic TCR for surface expression and allow mixed dimer formation. Mixed dimers, formed by mispairing between the endogenous and transgenic TCRs, may harbor autoreactive specificities. To circumvent these problems, we designed a system where the endogenous TCR-β is knocked out from the recipient cells using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) technology, simultaneously with transduction with a cancer-reactive receptor of choice. This TCR replacement strategy resulted in markedly increased surface expression of transgenic αβ and γδ TCRs, which in turn translated to a stronger, and more polyfunctional, response of engineered T cells to their target cancer cell lines. Additionally, the TCR-plus-CRISPR–modified T cells were up to a thousandfold more sensitive to antigen than standard TCR-transduced T cells or conventional model proxy systems used for studying TCR activity. Finally, transduction with a pan-cancer–reactive γδ TCR used in conjunction with CRISPR/Cas9 knockout of the endogenous αβ TCR resulted in more efficient redirection of CD4+ and CD8+ T cells against a panel of established blood cancers and primary, patient-derived B-cell acute lymphoblastic leukemia blasts compared with standard TCR transfer. Our results suggest that TCR transfer combined with genome editing could lead to new, improved generations of cancer immunotherapies.

Domain-swapped T cell receptors improve the safety of TCR gene therapy.

T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy.

Primary T-cell immunodeficiency with functional revertant somatic mosaicism in CD247

Primary T-cell immunodeficiency with functional revertant somatic mosaicism in CD247

646. Development of a Novel Chimeric Antigen Receptor as a Therapy Against Solid Tumors

The use of chimeric antigen receptor (CAR) T cells as a therapy for cancer is gaining traction as they potentially target tumors with high specificity and minimal off-target toxicities. Current CAR designs consist of an antigen-specific single chain antibody fragment (scFv) fused to the transmembrane and signaling domains of receptors capable of activating T cells. In CAR T cell therapy, patients’ T cells are transduced with cDNA encoding the CAR allowing CAR expressing T cells to target tumor cells. Clinical trials using anti-CD19 CAR T cells to target B cell malignancy have demonstrated the potential of this therapy with numerous patients achieving partial or full remission. In the treatment of solid tumors, however, CAR T cell therapy has not proven as effective. Evidence supporting T cell persistence is sparse, suggesting that currently used CARs may not provide optimal signals for memory cell generation. In addition, some patients have experienced life-threating side effects including cytokine release syndrome and on-target off-tumor activity, indicating a need for better modulation of CAR T cell activity. In line with these clinical observations, data from our laboratory and others demonstrate that currently used CARs can activate T cells in the absence of antigen, which promotes toxicity and T cell exhaustion while preventing differentiation into memory T cells. We propose that this antigen-independent activation is a result of high expression of CAR molecules that lack the regulatory context of the endogenous T cell receptor (TCR) complex. To more effectively mimic the temporal regulation of endogenous TCR signaling pathways, we developed a novel TCR-based CAR in which a scFv is fused to the constant region of TCRβ, thereby becoming incorporated into the native complex. We hypothesize that incorporation of our CAR into the TCR complex will mimic endogenous T cell activation and regulation by including contributions from all CD3 members (CD3Δ, CD3e, CD3γ, and CD3ζ). Furthermore, we propose that endogenous regulation of CAR signaling will prevent the constitutive T cell activation observed with currently used CAR constructs and allow for the formation of memory T cells. We have demonstrated that surface expression of our TCR CAR is modulated by endogenous CD3 expression levels and mirrors normal TCR surface expression. In contrast to both first and second generation CARs, T cells expressing our novel CAR lack constitutive activation, are specific for tumor cells expressing high levels of antigen, and retain higher proliferative potential in culture. Moreover, TCR CAR T cells are capable of signaling through the TCR complex and activating T cells in an antigen specific manner. Currently, we are examining the effector potential of our TCR CAR T cells compared to first and second generation CARs. Additionally, we are developing TCR CARs capable of signaling not only through the TCR complex but also through co-stimulatory receptors. In summary, we are designing the next generation of CARs by incorporating the tumor recognition component of a scFv into the endogenous TCR complex.

Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling

Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation. We identified ubiquitin-specific peptidase (Usp) 12 and Usp46, which had not been previously described in this pathway. Stimulation with anti-CD3 resulted in phosphorylation and time-dependent translocation of Usp12 from the nucleus to the cytosol. Usp12(-/-) Jurkat cells displayed defective NFκB, NFAT, and MAPK activities owing to attenuated surface expression of TCR, which were rescued on reconstitution of wild type Usp12. Proximity-based labeling with BirA-Usp12 revealed several TCR adaptor proteins acting as interactors in stimulated cells, of which LAT and Trat1 displayed reduced expression in Usp12(-/-) cells. We demonstrate that Usp12 deubiquitylates and prevents lysosomal degradation of LAT and Trat1 to maintain the proximal TCR complex for the duration of signaling. Our approach benefits from the use of activity-based probes in primary cells without any previous genome modification, and underscores the importance of ubiquitin-mediated regulation to refine signaling cascades.

Inmunodeficiencias congénitas del receptor de antígeno de los linfocitos T

T-cell receptor (TCR) immunodeficiencies of humans are low-prevalence autosomal recessive diseases characterized by impaired surface TCR expression and selective T lymphopenia (milder in CD3γ, TCRα or CD247 deficiency, and severe in individuals lacking CD3δ or CD3ɛ). The congenital absence of TCR components has a differential impact on T-cell development and function depending on the affected TCR chain and on the species, with human patients being, in some cases, rather different from mouse counterparts.The study of the immunophenotype by flow cytometry, along with molecular analyses, provides essential information for diagnosis and treatment, which is still to date the transplant of hematopoietic progenitors in severe immunodeficiency associated cases.

Enhancing the Efficacy of T Cell Receptor (TCR) Gene Therapy by Co-Transfer of TCR and Additional CD3 Molecules Into CD4+ T Cells

AbstractAbstract 2044 Introduction:The specificity of T cells can be redirected using retroviral T cell receptor (TCR) gene transfer. This has the potential to generate tumour specific T cells that can be adoptively transferred to target defined tumour antigens. The majority of TCR gene therapy studies have focused on the transfer of TCR genes into CD8 T cells. However the transfer of antigen specific CD8 T cells in the absence of antigen specific CD4 T cells leads to impaired anti-tumour responses and impaired memory development in vivo. Class I restricted TCR can be used to transduce CD4 T cells for use in adoptive transfer. The majority of class I restricted TCRs are CD8 dependent and thus require co-transduction of CD8 to be fully functional in CD4 T cells. CD4 T cells transduced with the class I restricted F5-TCR (specific for influenza peptide NP presented by H2-Dbclass I molecules) produce IL-2 and proliferate in vitro in response to class II negative tumour cells expressing NP peptide but these cells were not able to generate an IFN-γ response. In vivo, F5-TCR CD4 T cells could provide help for F5-TCR CD8 T cell mediated tumour eradication. These F5-TCR CD4 T cells persisted in vivo for up to 90 days post tumour regression and were able to re-expand following tumour challenge. In order to improve the function of class I restricted TCR expressing CD4 T cells, we co-transduced a vector containing all 4 chains of the CD3 complex. High surface expression of TCR has been shown to correlate with increased responsiveness to specific antigen. When additional TCR is introduced into a T cell, the introduced T cell must compete with the endogenous TCR for binding to CD3. The amount of CD3 within the cell will thus be rate limiting for the level of surface expression of the introduced TCR. Method:The retroviral vectors pMP71-F5α-2A-F5β (F5-TCR) and pMP71-CD3-ζ-2A-ε-2A-δ-2A-γ-IRES-GFP (CD3) were used for retroviral transduction. CD4 splenocytes obtained from C57BL/6 mice were activated with CD3/CD28 magnetic beads for 24 hours prior to transduction with either F5-TCR alone or F5-TCR and CD3. 5 days post transduction, transduced T cells were stimulated with C57BL/6 splenocytes loaded with NP (relevant) peptide or WT1 (irrelevant) peptide and cytokine production was measured by ELISA and intracellular cytokine staining and proliferation by [3H] thymidine incorporation. For in vivo tumour challenge, C57BL/6 recipient mice were irradiated with 5.5Gy and injected subcutaneously with 1 × 106 EL4-NP-luciferase cells (a lymphoma cell line stably transfected with NP peptide and luciferase) on day 0. On day 1, mice received 1 × 106 F5-TCR CD3 CD4 T cells or 1 × 106 F5-TCR CD4 T cells or 1 × 106 Mock Transduced T cells. Tumour area was measured by calipers and by bioluminescence imaging. For T cell trafficking experiments, the experimental set up was as above but transgenic CD4 luciferase T cells were used for adoptive transfer and EL4-NP luciferase negative cells were used for tumour challenge. Results:CD4 T cells transduced with F5-TCR and CD3 had a 5-fold higher expression of F5-TCR compared to cells transduced with F5-TCR alone. In vitro, F5-TCR CD3 CD4 T cells showed increased proliferation and increased production of IL-2 and IFN-γ in response to specific antigen compared to F5-TCR CD4 T cells. F5-TCR CD3 CD4 T cells responded to at a 2-fold lower concentration of specific peptide than F5-TCR CD4 T cells. Following adoptive transfer in murine models, F5-TCR CD3 CD4 T cells eradicated NP expressing EL4 tumours but transfer of equivalent doses of F5-TCR CD4 T cells did not lead to tumour regression. Using bioluminescence imaging, F5-TCR CD3 CD4 T cells trafficked to tumour site faster and accumulated in greater numbers than F5-TCR CD4 T cells. Following tumour challenge, there were higher numbers of F5-TCR CD3 CD4 T cells persisting in bone marrow, lymph node and peripheral blood than in mice that received F5-TCR CD4 T cells. Conclusion:Increased surface expression of class I restricted TCR in CD4 T cells leads to increased sensitivity to peptide in vitro and higher levels of proliferation and cytokine production in response to specific peptide. This translates in vivo to enhanced persistence of F5-TCR CD3 CD4 T cells and more efficient trafficking to tumour site and superior tumour protection. Therefore, the co-transduction of additional CD3 can improve the function of class I restricted TCR in CD4 T cells. Disclosures:Stauss:Cell Medica: Scientific Advisor Other.

High dose antigen stimulation leads to binding defects caused by persistent spatial segregation of TCR and CD8 in cytotoxic T cells (178.9)

Abstract The lateral organization of surface receptors controls ligand binding and activation. Our group has shown that activated T Cells exhibit persistent clustering of T Cell Receptor (TCR) several days after stimulation, leading to enhanced binding of soluble Major Histocompatibility Complex (MHC) dimers at low concentrations. In contrast, other groups have shown a complete loss of binding in activated T Cells during the same time period. Here, we resolve this contradiction by demonstrating that persistent binding behavior is controlled by the dose of antigen during primary activation. T Cells from TCR transgenic mice stimulated with high but not low dose of peptide are unable to specifically bind soluble Kb-SIY or Db-GP100 MHC dimer four days after stimulation. The behavior is not due to differences in the surface expression of TCR, CD8alpha, or CD8beta. Cytokine production mirrors the loss of binding. However, both high and low dose stimulated cells are able to bind the so-called ‘CD8 independent’ Ld-QL9 dimer, implicating the coreceptor in the binding defect. Using biophysical binding assays, k-space Image Correlation Spectroscopy, and Forster Resonance Energy Transfer between TCR and CD8, we demonstrate that TCR and CD8 are segregated, and TCR unclustered, on high dose activated T Cells, impairing CD8-dependent binding. Thus, membrane organization derived binding defects represent a mechanism for controlling T Cell responses to supraoptimal antigen.

Basal LAT-diacylglycerol-RasGRP1 signals in T cells maintain TCRα gene expression.

In contrast to the well-characterized T cell receptor (TCR) signaling pathways that induce genes that drive T cell development or polarization of naïve CD4 T cells into the diverse TH1, TH2, TH17 and Treg lineages, it is unclear what signals maintain specific gene expression in mature resting T cells. Resting T cells residing in peripheral lymphoid organs exhibit low-level constitutive signaling. Whereas tonic signals in B cells are known to be critical for survival, the roles of tonic signals in peripheral T cells are unknown. Here we demonstrate that constitutive signals in Jurkat T cell lines are transduced via the adapter molecule LAT and the Ras exchange factor RasGRP1 to maintain expression of TCRα mRNA and surface expression of the TCR/CD3 complex. Independent approaches of reducing basal activity through the LAT-diacylglycerol-RasGRP pathway led to reduced constitutive Ras-MEK-ERK signals and decreased TCRα mRNA and surface TCR expression in Jurkat cells. However, loss of TCR expression takes several days in these cell line experiments. In agreement with these in vitro approaches, inducible deletion of Lat in vivo results in reduced TCRα mRNA- and surface TCR- expression in a delayed temporal manner as well. Lastly, we demonstrate that loss of basal LAT-RasGRP signals appears to lead to silencing or repression of TCRα transcription. We postulate that basal LAT-diacylglycerol-RasGRP signals fulfill a regulatory function in peripheral T lymphocytes by maintaining proper gene expression programs.

The position of the AUG start codon in MFG‐based γ‐retroviral vectors has a dramatic effect on translation‐dependent protein expression

In the past three decades, much advancement has been made in γ-retroviral vector mediated gene transfer. One widely used vector design is based on the MFG vector, which uses the Moloney murine leukemia virus (MoMLV) transcriptional unit with extended packaging signals and insertion of the native MoMLV envelope splice acceptor region immediate 5' to the gene of interest inserted at an NcoI restriction site, which contains a translation start codon. Little is known about the impact of variations in start codon location within MFG-based vectors on protein expression. To evaluate variation in start condo placement, a gene encoding a T-cell receptor (TCR) was cloned into an MFG-based vector and site-directed mutagenesis was used to move the gene away from the splice acceptor, as well as alter the frame with respect to the upstream start codon. Kozak consensus sequences were also added to the gene in an attempt to improve translation. Protein expression as measured by TCR surface expression and biological activity was substantially reduced when the gene was placed downstream and out-of-frame with the NcoI start codon. Expression was reestablished by mutation of the upstream start site, although at a reduced level. These findings were repeated with two other genes, a dominant negative TGFβRII and the reporter protein dEGFP. These finding emphasize the scanning rule for translation initiation and stress the importance of cloning genes of interest into or near the native NcoI start site of MFG-based retroviral vectors.

Conditional deletion of SLP‐76 in mature T cells abrogates peripheral immune responses

The adaptor protein Src homology 2 domain-containing leukocyte-specific protein of 76 kDa (SLP-76) is central to the organization of intracellular signaling downstream of the T-cell receptor (TCR). Evaluation of its role in mature, primary T cells has been hampered by developmental defects that occur in the absence of WT SLP-76 protein in thymocytes. Here, we show that following tamoxifen-regulated conditional deletion of SLP-76, mature, antigen-inexperienced T cells maintain normal TCR surface expression but fail to transduce TCR-generated signals. Conditionally deficient T cells fail to proliferate in response to antigenic stimulation or a lymphopenic environment. Mice with induced deletion of SLP-76 are resistant to induction of the CD4+ T-cell-mediated autoimmune disease experimental autoimmune encephalomyelitis. Altogether, our findings demonstrate the critical role of SLP-76-mediated signaling in initiating T-cell-directed immune responses both in vitro and in vivo and highlight the ability to analyze signaling processes in mature T cells in the absence of developmental defects.

LKB1 regulates TCR-mediated PLCγ1 activation and thymocyte positive selection

The serine/threonine kinase LKB1 is a tumour suppressor that regulates cell growth, polarity, and proliferation in many different cell types. We previously demonstrated that LKB1 controls thymocyte survival via regulation of AMPK activation. In this study, we show that LKB1 was also involved in thymocyte positive selection through regulation of T cell receptor (TCR) signalling. Both Lck-Cre- and CD4-Cre-mediated deletion of LKB1 impaired the generation of mature CD4 and CD8 single positive (SP) thymocytes that might have resulted from the attenuated tyrosine phosphorylation of phospholipase C-γ 1 (PLCγ1) in the absence of LKB1. We found that LKB1 was directly phosphorylated by Lck at tyrosine residues 36, 261, and 365 and predominately interacted with LAT and PLCγ1 following TCR stimulation. Loss of LKB1 led to impaired recruitment of PLCγ1 to the LAT signalosome. Correlatively, LKB1-deficient thymocytes failed to upregulate lineage-specifying factors, and to differentiate into SP thymocytes even if their impaired survival was rescued. These observations indicated that LKB1 is a critical component involved in TCR signalling, and our studies provide novel insights into the mechanisms of LKB1-mediated thymocyte development.