Transgenic T-cell Receptor Research Articles

A Transgenic Dual-Luciferase Reporter Mouse for Longitudinal and Functional Monitoring of T Cells In Vivo.

Adoptive T-cell therapy (ATT) efficacy is limited when targeting large solid tumors. The evaluation of ATT outcomes using accessory treatment would greatly benefit from an in vivo monitoring tool, allowing the detection of functional parameters of transferred T cells. Here, we generated transgenic bioluminescence imaging of T cells (BLITC) mice expressing an NFAT-dependent click-beetle luciferase and a constitutive Renilla luciferase, which supports concomitant in vivo analysis of migration and activation of T cells. Rapid transferability of our system to preestablished tumor models was demonstrated in the SV40-large T antigen model via both crossbreeding of BLITC mice into a T-cell receptor (TCR)-transgenic background and TCR transduction of BLITC T cells. We observed rapid tumor infiltration of BLITC CD8+ T cells followed by a burst-like activation that mirrored rejection kinetics. Using the BLITC reporter in the clinically relevant H-Y model, we performed female to male transfers and detected H-Y-specific alloreactivity (graft-versus-host disease) in vivo In an H-Y solid tumor model, we found migration of adoptively transferred H-Y TCR-transgenic CD4+ T cells into the tumor, marked by transient activation. This suggests a rapid inactivation of infiltrating T cells by the tumor microenvironment, as confirmed by their expression of inhibitory receptors. In summary, the BLITC reporter system facilitates analysis of therapeutic parameters for ATT, is rapidly transferable to models of interest not restricted to tumor research, and is suitable for rapid screening of TCR clones for tumor rejection kinetics, as well as off-target effects. Cancer Immunol Res; 6(1); 110-20. ©2018 AACR.

T helper cells with specificity for an antigen in cardiomyocytes promote pressure overload-induced progression from hypertrophy to heart failure

We investigated whether CD4+-T cells with specificity for an antigen in cardiomyocytes promote the progression from hypertrophy to heart failure in mice with increased pressure load due to transverse aortic constriction (TAC). OT-II mice expressing a transgenic T cell receptor (TCR) with specificity for ovalbumin (OVA) on CD4+-T cells and cMy-mOVA mice expressing OVA on cardiomyocytes were crossed. The resulting cMy-mOVA-OT-II mice did not display signs of spontaneous autoimmunity despite the fact that their OVA-specific CD4+-T cells were not anergic. After TAC, progression to heart failure was significantly accelerated in cMy-mOVA-OT-II compared to cMy-mOVA mice. No OVA-specific antibodies were induced in response to TAC in cMy-mOVA-OT-II mice, yet more CD3+ T cells infiltrated their myocardium when compared with TAC-operated cMy-mOVA mice. Systemically, the proportion of activated CD4+-T cells with a Th1 and Th17 cytokine profile was increased in cMy-mOVA-OT-II mice after TAC. Thus, T helper cells with specificity for an antigen in cardiomyocytes can directly promote the progression of heart failure in response to pressure overload independently of autoantibodies.

Hematologic Malignancies: Plasma Cell Disorders.

Multiple myeloma (MM) is a plasma cell malignancy characterized by the growth of tumor cells in the bone marrow. Properties of the tumor microenvironment provide both potential tumor-promoting and tumor-restricting properties. Targeting underlying immune triggers for evolution of tumors as well as direct attack of malignant plasma cells is an emerging focus of therapy for MM. The monoclonal antibodies daratumumab and elotuzumab, which target the plasma cell surface proteins CD38 and SLAMF7/CS1, respectively, particularly when used in combination with immunomodulatory agents and proteasome inhibitors, have resulted in high response rates and improved survival for patients with relapsed and refractory MM. A number of other monoclonal antibodies are in various stages of clinical development, including those targeting MM cell surface antigens, the bone marrow microenvironment, and immune effector T cells such as antiprogrammed cell death protein 1 antibodies. Bispecific preparations seek to simultaneously target MM cells and activate endogenous T cells to enhance efficacy. Cellular immunotherapy seeks to overcome the limitations of the endogenous antimyeloma immune response through adoptive transfer of immune effector cells with MM specificity. Allogeneic donor lymphocyte infusion can be effective but can cause graft-versus-host disease. The most promising approach appears to be genetically modified cellular therapy, in which T cells are given novel antigen specificity through expression of transgenic T-cell receptors (TCRs) or chimeric antigen receptors (CARs). CAR T cells against several different targets are under investigation in MM. Infusion of CD19-targeted CAR T cells following salvage autologous stem cell transplantation (SCT) was safe and extended remission duration in a subset of patients with relapsed/refractory MM. CAR T cells targeting B-cell maturation antigen (BCMA) appear most promising, with dramatic remissions seen in patients with highly refractory disease in three ongoing trials. Responses are associated with degree of CAR T-cell expansion/persistence and often toxicity, including cytokine release syndrome (CRS) and neurotoxicity. Ongoing and future studies are exploring correlates of response, ways to mitigate toxicity, and "universal" CAR T cells.

Epicutaneous sensitization to house dust mite allergen requires interferon regulatory factor 4–dependent dermal dendritic cells

Exposure to allergens, such as house dust mite (HDM), through the skin often precedes allergic inflammation in the lung. It was proposed that TH2 sensitization through the skin occurs when skin barrier function is disrupted by, for example, genetic predisposition, mechanical damage, or the enzymatic activity of allergens. We sought to study how HDM applied to unmanipulated skin leads to TH2 sensitization and to study which antigen-presenting cells mediate this process. HDM was applied epicutaneously by painting HDM on unmanipulated ear skin or under an occlusive tape. HDM challenge was through the nose. Mouse strains lacking different dendritic cell (DC) populations were used, and 1-DER T cells carrying a transgenic T-cell receptor reactive to Der p 1 allergen were used as a readout for antigen presentation. The TH2-inducing capacity of sorted skin-derived DC subsets was determined by means of adoptive transfer to naive mice. Epicutaneous HDM application led to TH2 sensitization and eosinophilic airway inflammation upon intranasal HDM challenge. Skin sensitization did not require prior skin damage or enzymatic activity within HDM extract, yet was facilitated by applying the allergen under an occlusive tape. Primary proliferation of 1-DER T cells occurred only in the regional skin-draining lymph nodes. Epicutaneous sensitization was found to be driven by 2 variants of interferon regulatory factor 4-dependent dermal type 2 conventional DC subsets and not by epidermal Langerhans cells. These findings identify skin type 2 conventional DCs as crucial players in TH2 sensitization to common inhaled allergens that enter the body through the skin and can provoke features of allergic asthma.

Activation induced cell death (AICD) of human melanoma antigen-specific TCR engineered CD8 T cells involves JNK, Bim and p53

ABSTRACTObjectives: Adoptive cancer immunotherapy (ACT) with transgenic T cell receptor (TCR) engineered (TCReng) anti-tumor T cells has produced encouraging results, however, efficacy of these approaches need improvement. Since premature activation induced cell death (AICD) of adoptively administered T cells could be a major impediment, we examined the mechanism(s) underlying AICD in TCReng CD8+ cytolytic T lymphocytes (CTL).Methods: AICD in human tumor antigen-specific MHC class I restricted TCR engineered CD8+ CTL was induced by exposing them to cognate peptide epitope.Results: We show that TCReng CD8+ human primary CTL undergo AICD even upon encountering their cognate peptide epitope for the very first time. AICD in TCReng CTL is a death-receptor-independent, JNK activation-driven intrinsic processes, in which p53-mediated mitochondria-centric, non-transcription-dependent pathway plays an essential role. Activated JNK modulates mitochondrial membrane integrity in CTL undergoing AICD by directly interacting with Bcl family protein, Bim, and the mitochondrial membrane pore complex, voltage dependent anion channel (VDAC), leading to the release of caspase-independent death executioner, apoptosis inducing factor (AIF), accumulation of single strand DNA breaks and eventually to cell death.Conclusions: Our findings offer opportunities to interfere with AICD in TCReng CD8+ anti-tumor CTL for sustaining them longer for producing better clinical outcomes.

Towards 'Off-the-Shelf ' Universal Chimeric Antigen Receptor (CAR) T Cells: Mouse Anti-3rd Party Central Memory CD8 Veto Cells Prolong Functional Engraftment of Allogeneic Genetically Modified T Cells

We recently demonstrated that mouse anti-3rd party central memory CD8 veto cells (Tcm) enable engraftment of fully mis-matched T cell depleted bone marrow (TDBM) in sublethally irradiated (5.5 Gy TBI) recipients (Eran et al. Blood 2013). Here we found that Tcm generated from (host x donor)F1 or fully allogeneic donors were able to engraft and sustain their presence in such sub-lethally irradiated mice in the absence of TDBM transplants. Limiting dilution analysis revealed that the reactivity of host T cells (HTC) against donor spleen cells was significantly diminished in the chimeric mice treated with Tcm, whereas reactivity against 3rd party was comparable betweenTcm treated and non-treated mice (Fig. 1). This specific immune tolerance indicated that vetoTcm could potentially enable the use of 'off-the-shelf' allogeneic CAR T cells or any other genetically modified T cells from the same donor.To further investigate this possibility, we tested the ability of Tcmveto cells to enable engraftment of CD8 T cells from OT1 mice, expressing a transgenic T cell receptor (TCR) directed againstovalbumin (OVA) residues 257-264 in the context of H2Kb MHC-I. To that end, 1x106 OT1 CD8 T cells (H2b) were infused into Balb/c (H2Dd) recipients sublethally irradiated with 5.25 Gy TBI in the presence or absence of different doses of veto Tcm. As shown in Fig. 2, when tested at 2 months post-transplant, OT1 cells could not be detected in the peripheral blood of untreated recipient mice, while they were readily detectable in mice receiving 2x106 Tcm (0.78±0.36), reaching a higher level (1.18± 0.61) upon infusion of 5x106Tcm.To evaluate the functionality of the engrafted OT1 T cells in the chimeric mice, we developed and calibrated a new murine model, using a melanoma B16-cell line of C57BL background that expresses the ovalbumin peptide and the tdTomatomarker (B16-OVA-tdTomato) (Fig 3A). In this model, we tried to mimic a state of minimal residual disease by injecting a small number of B16-OVAtdTomato melanoma cells (0.25x106) into syngeneic C57BL mice; we then treated the mice with sublethal irradiation (6Gy TBI) to simulate treatment of the tumor, but also to clear out space for the T-cell adoptive transfer. Finally, we injected 1x106 naïve OT-1 cells generated on a (Balb x-OT1-CD45.1+RAG2-) F1 background in the presence or absence of Balb/c veto Tcm (5x106), and followed the development of the tumor. We found thatBalb/c vetoTcm prolonged the engraftment of the transgenic donor-derived F1 OT1 CD8 T cells inBalb/c recipients, thereby enabling significantly improved control of tumor growth when tested at day 20 post-transplant ( p<0.05). In contrast non OVA-expressing tumor cells grew at the same rate in all treatment groups, thereby demonstrating the specificity of the anti-tumor effect (Fig.3B).Previous studies demonstrated that Tcmare depleted of GVH reactivity by virtue of their culture with 3rd party stimulators under cytokine starvation. Our present results demonstrate that suchTcm veto cells can pave the way for functional engraftment of CD8 T cells with genetically modified specificity. Taken together, these results provide a proof of concept for the potential clinical use of such veto cells to enable therapy with 'off-the-shelf ' allogeneic CAR T cells. The relative ease of growing humanTcm in large numbers over a short period of time (9-12 days) suggests that it could be possible to harvest byleukapheresis from a single donor, a sufficient number of cells, to support CAR therapy for more than 50 allogeneic recipients.* N.O.G and R.G equally contributed [Display omitted] [Display omitted] [Display omitted] DisclosuresOr Geva:Yeda LTD: Patents & Royalties. Gidron:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Exploring RhoA G17V-Mediated T-Cell Dysfunction

The RhoA G17V mutation occurs in 60% of tumors from patients with Angioimmunoblastic T-cell Lymphomas (AITL) as well as in a subset of Peripheral T-cell Lymphoma, not otherwise specified (PTCL-NOS). The non-malignant counterparts of AITL cells are posited to be follicular-helper T-cells (TFH), and RhoA G17V mutation is strongly associated with a TFH gene expression program in both AITL and PTCL-NOS cases. The G17V substitution is predicted to lock the small GTPase RhoA in an inactive conformation, generating a protein that can bind guanine exchange factors more avidly than unmutated RhoA, likely producing neomorphic effects by inhibiting other small GTPases and G-coupled protein receptors.We have generated mice that express RhoA G17V under the control of regulatory elements from the murine CD4 locus, including two separate founder lines with essentially identical developmental and functional phenotypes. These mice demonstrate a severe reduction in thymic and splenic T cells beginning at the thymic CD4/CD8 double-positive stage, consistent with effects reported from loss-of-function (LOF) RhoA models. RhoA G17V mice also show distinctive effects in peripheral T-cell compartments, including a marked reduction in splenic naïve T-cells with a relative increase in the proportion of activated and/or memory T-cells among those that remain. Notably, there is an increase in the proportion of TFHcells among RhoA G17V mice, suggesting that the mutant protein skews differentiation towards this lineage. Also, unlike reports from RhoA LOF models, we observe increased activation of RhoA G17V T cells when sub-optimal T-cell receptor (TCR)-pathway stimulation is performed with CD3 crosslinking, consistent with a neomorphic activity.We have also observed a striking inflammatory tail fibrosis that occurs with essentially complete penetrance by 8-10 weeks of age in RhoA G17V mice but not in littermate controls. Development of fibrosis is preceded by a cellular infiltrate of hematopoietic cells. including a prominent population of CD4+cells, suggesting that CD4-specific RhoA G17V expression promotes autoimmunity.Finally, we crossed RhoA G17V mice with mice lacking expression of TET2 in the hematopoietic compartment (via Vav-Cre-mediated deletion) and the ovalbumin-specific OT-II transgenic TCR. These mice developed spontaneous T-cell lymphomagenesis comprised of Bcl6-expressing CD4 cells and involving the liver, spleen, and peripheral lymph nodes. Further immunization of these mice with NP40-ovalbumin and alum resulted in Bcl-6-expressing T cell lymphomas within 1-4 months after initial immunization. Thus, the same genotype observed in >50% of AITLs (i.e., RhoA G17V/TET2 loss), combined with TCR stimulation rapidly produces T cell lymphomas with TFHphenotypes.In conclusion, T-cells with RhoA G17V mutations may overcome regulatory pathways before and after malignant transformation, including those that control activation and prevent autoreactivity. These phenotypes also provide a platform from which to determine the specific biochemical effects mediated by RhoA G17V that contribute to T-cell hyperactivation and transformation. DisclosuresWeinstock:Novartis: Consultancy, Research Funding.

Antigen exposure shapes the ratio between antigen-specific Tregs and conventional T cells in human peripheral blood

The T-cell receptor (TCR) is required for maturation and function of regulatory T cells (Tregs), but the ligand specificities of Tregs outside the context of transgenic TCRs are largely unknown. Using peptide-MHC tetramers, we isolated rare specific Foxp3+ cells directly ex vivo from adult peripheral blood and defined their frequency and phenotype. We find that a proportion of circulating Tregs recognize foreign antigens and the frequency of these cells are similar to that of self-reactive Tregs in the absence of cognate infection. In contrast, the frequencies of Tregs that recognize some common microbial antigens are significantly reduced in the blood of most adults. Exposure to peripheral antigens likely has a major influence on the balance between Tregs and conventional T-cell subsets because a larger proportion of flu-specific T cells has a regulatory cell phenotype in the cord blood. Consistent with this finding, we show that lymphocytic choriomeningitis virus infection can directly modulate the ratio of virus-specific effectors and Tregs in mice. The resulting change in the balance within an antigen-specific T-cell population further correlates with the magnitude of effector response and the chronicity of infection. Taken together, our data highlight the importance of antigen specificity in the functional dynamics of the T-cell repertoire. Each specific population of CD4+ T cells in human peripheral blood contains a subset of Tregs at birth, but the balance between regulatory and effector subsets changes in response to peripheral antigen exposure and this could impact the robustness of antipathogen immunity.

Abstract 671: Assessing the origins of anaplastic large cell lymphoma in murine models via forced lineage specificity of NPM-ALK expression in T cells

Abstract Background: Anaplastic large cell lymphoma (ALCL) is a T cell lymphoma representing 10-15% of all childhood non Hodgkin lymphomas, whereby those positive for aberrant expression of Anaplastic Lymphoma kinase (ALK) account for 50-80% of cases. The oncogene Nucleophosmin 1 (NPM)-ALK is considered the main driver of pediatric ALCL identified in 83% of ALK+ cases. ALCL cells rarely express a T cell receptor (TCR), CD4 nor CD8 despite displaying an activated cytotoxic T cell phenotype (production of perforin and Granzyme B). Expression of NPM-ALK in mice from the T-cell specific CD4 promoter (which gives rise to expression throughout thymic development) gives rise to thymic lymphomas not mimicking human ALCL suggesting other events are required for peripheral T cell lymphoma development and/or expression of NPM-ALK at different stages of T cell progression: some theories have revolved around the possibility of an infectious aetiology, or subversion of the development of T cells within the thymus in the pathogenesis of ALCL. Objective: To assess the potential cell of origin of ALCL through lineage restriction of NPM-ALK to defined T cell lineages (CD4 or CD8) using TCR transgenic mice. Experimental procedure: Disease presentation following backcrossing of the CD4/NPM-ALK mouse to a clonal transgenic TCR OT1 (CD4/NPM-ALK/OTI) or OTII (CD4/NPM-ALK/OTII) genetic background in the presence and absence of RAG was monitored. The OTI and OTII transgenic mice express a transgenic TCR which is MHC class I restricted, with a CD8 or MHC class II with a CD4 developmental skew respectively. The TCR in these mice has been engineered to recognise ovalbumin peptides (ova). Results: CD4/NPM-ALK/OTI mice develop peripheral disease histopathologically mimicking human ALCL. Conversely, CD4/NPM-ALK/OTII mice develop cortical thymic lymphomas similar to the parental CD4/NPM-ALK strain and mostly of a CD4 single positive T cell phenotype (64%). Intriguingly, the peripheral T cell tumours that develop in the CD4/NPM-ALK/OTI mice do not express the transgenic OTI TCR (or endogenously rearranged TCR). Additionally, haemopoietic tumors only occur on a RAG competent genetic background or in the absence of ova stimulation even though TCR transgene expressing T cells are detected in the periphery of these mice (CD4/NPM-ALK/OTI + MHV-ova administration or CD4/NPM-ALK/OTI/RAG-/-). Conclusions: These data represent the first murine model resembling human ALCL and suggest that restriction of NPM-ALK to the CD8 T cell lineage results in a disease more closely resembling human ALCL. Furthermore, our data suggest that in the periphery, signaling though the TCR may be pejorative towards peripheral lymphoma development. Citation Format: Tim Malcolm, Camilla J. Fairbairn, Katherine Hughes, Lukas Kenner, Amos Burke, Suzanne Turner. Assessing the origins of anaplastic large cell lymphoma in murine models via forced lineage specificity of NPM-ALK expression in T cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 671.

Reduced TCR-dependent activation through citrullination of a T-cell epitope enhances Th17 development by disruption of the STAT3/5 balance.

Citrullination is a post‐translational modification of arginine that commonly occurs in inflammatory tissues. Because T‐cell receptor (TCR) signal quantity and quality can regulate T‐cell differentiation, citrullination within a T‐cell epitope has potential implications for T‐cell effector function. Here, we investigated how citrullination of an immunedominant T‐cell epitope affected Th17 development. Murine naïve CD4+ T cells with a transgenic TCR recognising p89‐103 of the G1 domain of aggrecan (agg) were co‐cultured with syngeneic bone marrow‐derived dendritic cells (BMDC) presenting the native or citrullinated peptides. In the presence of pro‐Th17 cytokines, the peptide citrullinated on residue 93 (R93Cit) significantly enhanced Th17 development whilst impairing the Th2 response, compared to the native peptide. T cells responding to R93Cit produced less IL‐2, expressed lower levels of the IL‐2 receptor subunit CD25, and showed reduced STAT5 phosphorylation, whilst STAT3 activation was unaltered. IL‐2 blockade in native p89‐103‐primed T cells enhanced the phosphorylated STAT3/STAT5 ratio, and concomitantly enhanced Th17 development. Our data illustrate how a post‐translational modification of a TCR contact point may promote Th17 development by altering the balance between STAT5 and STAT3 activation in responding T cells, and provide new insight into how protein citrullination may influence effector Th‐cell development in inflammatory disorders.

Adoptive Transfer of Tracer-Alloreactive CD4+ T Cell Receptor Transgenic T Cells Alters the Endogenous Immune Response to an Allograft.

T cell receptor transgenic (TCR-Tg) T cells are often used as tracer populations of antigen-specific responses to extrapolate findings to endogenous T cells. The extent to which TCR-Tg T cells behave purely as tracer cells or modify the endogenous immune response is not clear. To test the impact of TCR-Tg T cell transfer on endogenous alloimmunity, recipient mice were seeded with CD4+ or CD8+ TCR-Tg or polyclonal T cells at the time of cardiac allograft transplantation. Only CD4+ TCR-Tg T cells accelerated rejection and, unexpectedly, led to a dose-dependent decrease in both transferred and endogenous T cells infiltrating the graft. In contrast, recipients of CD4+ TCR-Tg T cells exhibited enhanced endogenous donor-specific CD8+ T cell activation in the spleen and accelerated alloantibody production. Introduction of CD4+ TCR-Tg T cells also perturbed the intragraft accumulation of innate cell populations. Transferred CD4+ TCR-Tg T cells alter many aspects of endogenous alloimmunity, suggesting that caution should be used when interpreting experiments using these adoptively transferred cells because the overall nature of allograft rejection may be altered. These results also may have implications for adoptive CD4+ T cell immunotherapy in tumor and infectious clinical settings because cell infusion may have additional effects on natural immune responses.

Tracking of TCR-Transgenic T Cells Reveals That Multiple Mechanisms Maintain Cardiac Transplant Tolerance in Mice.

Solid organ transplantation tolerance can be achieved following select transient immunosuppressive regimens that result in long-lasting restraint of alloimmunity without affecting responses to other antigens. Transplantation tolerance has been observed in animal models following costimulation or coreceptor blockade therapies, and in a subset of patients through induction protocols that include donor bone marrow transplantation, or following withdrawal of immunosuppression. Previous data from our lab and others have shown that proinflammatory interventions that successfully prevent the induction of transplantation tolerance in mice often fail to break tolerance once it has been stably established. This suggests that established tolerance acquires resilience to proinflammatory insults, and prompted us to investigate the mechanisms that maintain a stable state of robust tolerance. Our results demonstrate that only a triple intervention of depleting CD25+ regulatory T cells (Tregs), blocking programmed death ligand-1 (PD-L1) signals, and transferring low numbers of alloreactive T cells was sufficient to break established tolerance. We infer from these observations that Tregs and PD-1/PD-L1 signals cooperate to preserve a low alloreactive T cell frequency to maintain tolerance. Thus, therapeutic protocols designed to induce multiple parallel mechanisms of peripheral tolerance may be necessary to achieve robust transplantation tolerance capable of maintaining one allograft for life in the clinic.

Towards an Understanding of Protective Cellular Immunity - a TCR Transgenic Mouse Reactive with Chlamydia muridarum

Abstract A major limitation in understanding the development of protective T cell memory against Chlamydia is the difficulty in characterizing low frequencies of antigen-specific T cells. To better understand the kinetics and phenotype of specific CD4 T cells, we developed a C. muridarum-specific T-Cell Receptor (TCR) transgenic (Tg) mouse. Primed T cells from C57BL/6 mice immune to C. muridarum were stimulated with EB/RB for 5 days and fused with BW5147 cells. Specific clones producing IL-2 and IFN-γ were harvested and screened for TCR Vα and Vβ by qPCR. Cloned transgenes were extracted, purified, and co-microinjected into fertilized oocytes. PCR and FACS confirmed integration and expression. Tg T cells were analyzed ex vivo for activation markers and cytokine production after EB/RB stimulation. 90% of peripheral CD4+ T cells express TCR transgenes and exhibited enhanced proliferation and increased expression of IFN-γ, IL-2, and CD69, compared to controls. Naive T cells were adoptively transferred from CD45.2 Tg mice into CD45.1 mice one day before intravaginal inoculation with C. muridarum. Tg cells showed enhanced proliferation and TNF-α, IL-2, and IFN-γ production by day 5 compared to controls. Tg T cells collected from SLOs and oviducts on days 5, 8, 12, 22, 44, and 65 post-infection exhibited enhanced expression of memory and activation markers. Adoptive transfer of Tg or WT CD4 T cells to Rag1−/− mice rescued an otherwise lethal C. muridarum genital infection. WT and Tg CD4 T cells exhibited comparable chlamydial clearance from the genital tract. Adoptive transfer of Chlamydia-specific CD4+ T cells offers a powerful approach to characterize cellular activation, differentiation, and memory development.

Targeting human melanoma neoantigens by T cell receptor gene therapy.

In successful cancer immunotherapy, T cell responses appear to be directed toward neoantigens created by somatic mutations; however, direct evidence that neoantigen-specific T cells cause regression of established cancer is lacking. Here, we generated T cells expressing a mutation-specific transgenic T cell receptor (TCR) to target different immunogenic mutations in cyclin-dependent kinase 4 (CDK4) that naturally occur in human melanoma. Two mutant CDK4 isoforms (R24C, R24L) similarly stimulated T cell responses in vitro and were analyzed as therapeutic targets for TCR gene therapy. In a syngeneic HLA-A2-transgenic mouse model of large established tumors, we found that both mutations differed dramatically as targets for TCR-modified T cells in vivo. While T cells expanded efficiently and produced IFN-γ in response to R24L, R24C failed to induce an effective antitumor response. Such differences in neoantigen quality might explain why cancer immunotherapy induces tumor regression in some individuals, while others do not respond, despite similar mutational load. We confirmed the validity of the in vivo model by showing that the melan-A-specific (MART-1-specific) TCR DMF5 induces rejection of tumors expressing analog, but not native, MART-1 epitopes. The described model allows identification of those neoantigens in human cancer that serve as suitable T cell targets and may help to predict clinical efficacy.

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.

Mutation of the ER retention receptor KDELR1 leads to cell-intrinsic lymphopenia and a failure to control chronic viral infection

Endoplasmic reticulum (ER)-resident proteins are continually retrieved from the Golgi and returned to the ER by Lys-Asp-Glu-Leu (KDEL) receptors, which bind to an eponymous tetrapeptide motif at their substrate's C terminus. Mice and humans possess three paralogous KDEL receptors, but little is known about their functional redundancy, or if their mutation can be physiologically tolerated. Here, we present a recessive mouse missense allele of the prototypical mammalian KDEL receptor, KDEL ER protein retention receptor 1 (KDELR1). Kdelr1 homozygous mutants were mildly lymphopenic, as were mice with a CRISPR/Cas9-engineered frameshift allele. Lymphopenia was cell intrinsic and, in the case of T cells, was associated with reduced expression of the T-cell receptor (TCR) and increased expression of CD44, and could be partially corrected by an MHC class I-restricted TCR transgene. Antiviral immunity was also compromised, with Kdelr1 mutant mice unable to clear an otherwise self-limiting viral infection. These data reveal a nonredundant cellular function for KDELR1, upon which lymphocytes distinctly depend.

Spontaneous Ocular Autoimmunity in Mice Expressing a Transgenic T Cell Receptor Specific to Retina: A Tool to Dissect Mechanisms of Uveitis.

The "classical" EAU model induced by immunization of mice with the retinal protein IRBP or its peptides has been very useful to study basic mechanisms of ocular inflammation, but is inadequate for some types of studies due to the need for active immunization in the context of strong bacterial adjuvants. We generated transgenic (Tg) mice on the B10.RIII background that express a T cell receptor (TCR) specific for IRBP161-180. Three strains of TCR Tg mice were established. Spontaneous uveitis developed in two of the three strains by 2-3 months of age. Susceptibility correlated with a higher copy number of the transgenic TCR and a higher proportion of TCR Tg T cells in the peripheral repertoire. Even in mice with uveitis, peripheral IRBP-specific CD4(+) T cells displayed mostly a naïve phenotype. In contrast, T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells. These mice thus provide a new and distinct model of uveitis from the "classical" EAU, and may represent some types of uveitis more faithfully. Importantly, this new transgenic model of uveitis can serve as a template for therapeutic manipulations, and as a source of naïve retina-specific T cells for a variety of basic and pre-clinical studies. Several examples of such studies will be discussed.

Disregulated expression of the transcription factor ThPOK during T-cell development leads to high incidence of T-cell lymphomas

The transcription factor T-helper-inducing POZ/Krueppel-like factor (ThPOK, encoded by the Zbtb7b gene) plays widespread and critical roles in T-cell development, particularly as the master regulator of CD4 commitment. Here we show that mice expressing a constitutive T-cell-specific ThPOK transgene (ThPOK(const) mice) develop thymic lymphomas. These tumors resemble human T-cell acute lymphoblastic leukemia (T-ALL), in that they predominantly exhibit activating Notch1 mutations. Lymphomagenesis is prevented if thymocyte development is arrested at the DN3 stage by recombination-activating gene (RAG) deficiency, but restored by introduction of a T-cell receptor (TCR) transgene or by a single injection of anti-αβTCR antibody into ThPOK(const) RAG-deficient mice, which promotes development to the CD4(+)8(+) (DP) stage. Hence, TCR signals and/or traversal of the DN (double negative) > DP (double positive) checkpoint are required for ThPOK-mediated lymphomagenesis. These results demonstrate a novel link between ThPOK, TCR signaling, and lymphomagenesis. Finally, we present evidence that ectopic ThPOK expression gives rise to a preleukemic and self-perpetuating DN4 lymphoma precursor population. Our results collectively define a novel role for ThPOK as an oncogene and precisely map the stage in thymopoiesis susceptible to ThPOK-dependent tumor initiation.

T Cells Engineered to Express a T-Cell Receptor Specific for Glypican-3 to Recognize and Kill Hepatoma Cells In Vitro and in Mice

Cancer therapies are being developed based on our ability to direct T cells against tumor antigens. Glypican-3 (GPC3) is expressed by 75% of all hepatocellular carcinomas (HCC), but not in healthy liver tissue or other organs. We aimed to generate T cells with GPC3-specific receptors that recognize HCC and used them to eliminate GPC3-expressing xenograft tumors grown from human HCC cells in mice. We used mass spectrometry to obtain a comprehensive peptidome from GPC3-expressing hepatoma cells after immune-affinity purification of human leukocyte antigen (HLA)-A2 and bioinformatics to identify immunodominant peptides. To circumvent GPC3 tolerance resulting from fetal expression, dendritic cells from HLA-A2-negative donors were cotransfected with GPC3 and HLA-A2 RNA to stimulate and expand antigen-specific T cells. Peptide GPC3367 was identified as a predominant peptide on HLA-A2. We used A2-GPC3367 multimers to detect, select for, and clone GPC3-specific T cells. These clones bound the A2-GPC3367 multimer and secreted interferon-γ when cultured with GPC3367, but not with control peptide-loaded cells. By genomic sequencing of these T-cell clones, we identified a gene encoding a dominant T-cell receptor. The gene was cloned and the sequence was codon optimized and expressed from a retroviral vector. Primary CD8(+) T cells that expressed the transgenic T-cell receptor specifically bound GPC3367 on HLA-A2. These T cells killed GPC3-expressing hepatoma cells in culture and slowed growth of HCC xenograft tumors in mice. We identified a GPC3367-specific T-cell receptor. Expression of this receptor by T cells allows them to recognize and kill GPC3-positive hepatoma cells. This finding could be used to advance development of adoptive T-cell therapy for HCC.

562. Modification of TCR Specificity by TALEN and CRISPR

Adoptive T-cell transfer of T-cells with transgenic high avidity T-cell receptors (TCR) holds a lot of promise as therapeutic approach, however comprising certain challenges. Endogenous and transferred TCR-chains compete for surface expression and may pair inappropriately, potentially leading to autoimmunity. Designer nucleases like transcription activator-like effector nucleases (TALEN) and RNA-guided nucleases of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) system introduce specific DNA double strand breaks (DSB) at their target sites. This ability can be exploited for targeted genome editing. DSB repair through non-homologous end joining (NHEJ) can result in permanent gene knockout. Alternatively, if a homology-containing donor template is provided, transgenes can be inserted into the specific target locus by homology directed repair (HDR). In order to disrupt endogenous TCR expression, we assembled seven TALEN monomers and two guide RNAs targeting the constant regions of the TCR α-chain (TRAC) and the TCR β-chain (TRBC1 and TRBC2), respectively. Here we show the specific DSB induction by TALEN and CRISPR-Cas nucleases at their target sites, examined in K562 and T-cells using Cel1-assay and deep sequencing. Compared to nucleofection of TALEN-expressing plasmids, electroporation of mRNA considerably improved TCR knockout efficiency in primary T-cells. Using TRAC-TALEN and TRBC-TALEN mRNA, surface CD3 expression was successfully eliminated in about 75% and 60% of T-cells, respectively. The nuclease specificity was examined by identifying integrase-deficient lentiviral vector (IDLV)-marked DSB using LAM-PCR and deep sequencing. Although clustered IDLV integration sites were detected at the TALEN and CRISPR target sites, we did not detect any off-target activity, indicating high specificity. Additionally, K562 cells were nucleofected with TALEN-expressing plasmids and a donor template containing a GFP expression cassette flanked by 800bp homologous sequences to the TRAC locus. Thereby we were able to verify targeted gene addition through HDR of TALEN-mediated DSB in about 10% of treated cells. TALEN and CRISPR that show high efficiency and specificity to their target sequence will be used for generating T-cells with high avidity TCR.