Antigen receptor-engineered Tregs inhibit CNS autoimmunity in cell therapy using nonredundant immune mechanisms in mice.

CD4+ FOXP3+ Tregs are currently explored to develop cell therapies against immune-mediated disorders, with an increasing focus on antigen receptor-engineered Tregs. Deciphering their mode of action is necessary to identify the strengths and limits of this approach. Here, we addressed this issue in an autoimmune disease of the CNS, EAE. Following disease induction, autoreactive Tregs upregulated LAG-3 and CTLA-4 in LNs, while IL-10 and amphiregulin (AREG) were increased in CNS Tregs. Using genetic approaches, we demonstrated that IL-10, CTLA-4, and LAG-3 were nonredundantly required for the protective function of antigen receptor-engineered Tregs against EAE in cell therapy whereas AREG was dispensable. Treg-derived IL-10 and CTLA-4 were both required to suppress acute autoreactive CD4+ T-cell activation, which correlated with disease control. These molecules also affected the accumulation in the recipients of engineered Tregs themselves, underlying complex roles for these molecules. Noteworthy, despite the persistence of the transferred Tregs and their protective effect, autoreactive T cells eventually accumulated in the spleen of treated mice. In conclusion, this study highlights the remarkable power of antigen receptor-engineered Tregs to appropriately provide multiple suppressive factors nonredundantly necessary to prevent autoimmune attacks.

Gene Therapy "Made in Germany": A Historical Perspective, Analysis of the Status Quo, and Recommendations for Action by the German Society for Gene Therapy.

Gene therapies have been successfully applied to treat severe inherited and acquired disorders. Although research and development are sufficiently well funded in Germany and while the output of scientific publications and patents is comparable with the leading nations in gene therapy, the country lags noticeably behind with regard to the number of both clinical studies and commercialized gene therapy products. In this article, we give a historical perspective on the development of gene therapy in Germany, analyze the current situation from the standpoint of the German Society for Gene Therapy (DG-GT), and define recommendations for action that would enable our country to generate biomedical and economic advantages from innovations in this sector, instead of merely importing advanced therapy medicinal products. Inter alia, we propose (1) to harmonize and simplify regulatory licensing processes to enable faster access to advanced therapies, and (2) to establish novel coordination, support and funding structures that facilitate networking of the key players. Such a center would provide the necessary infrastructure and know-how to translate cell and gene therapies to patients on the one hand, and pave the way for commercialization of these promising and innovative technologies on the other. Hence, these courses of action would not only benefit the German biotech and pharma landscape but also the society and the patients in need of new treatment options.

Characterization and modulation of anti-αßTCR antibodies and their respective binding sites at the ßTCR chain to enrich engineered T cells.

T cell engineering strategies offer cures to patients and have entered clinical practice with chimeric antibody-based receptors; αßT cell receptor (αßTCR)-based strategies are, however, lagging behind. To allow a more rapid and successful translation to successful concepts also using αßTCRs for engineering, incorporating a method for the purification of genetically modified T cells, as well as engineered T cell deletion after transfer into patients, could be beneficial. This would allow increased efficacy, reduced potential side effects, and improved safety of newly to-be-tested lead structures. By characterizing the antigen-binding interface of a good manufacturing process (GMP)-grade anti-αßTCR antibody, usually used for depletion of αßT cells from stem cell transplantation products, we developed a strategy that allows for the purification of untouched αßTCR-engineered immune cells by changing 2 amino acids only in the TCRß chain constant domain of introduced TCR chains. Alternatively, we engineered an antibody that targets an extended mutated interface of 9 amino acids in the TCRß chain constant domain and provides the opportunity to further develop depletion strategies of engineered immune cells.

Generation of Epstein-Barr Virus Antigen-Specific T Cell Receptors Recognizing Immunodominant Epitopes of LMP1, LMP2A, and EBNA3C for Immunotherapy.

Epstein-Barr virus (EBV) infections in healthy individuals are usually cleared by immune cells, wherein CD8+ T lymphocytes play the most important role. However, in some immunocompromised individuals, EBV infections can lead to the development of cancer in B, T, natural killer (NK) cells and epithelial cells. Most EBV-associated cancers express a limited number of virus-specific antigens such as latent membrane proteins (LMP1 and LMP2) and nuclear proteins (EBNA1, -2, EBNA3A, -B, -C, and EBNA-LP). These antigens represent true tumor-specific antigens and can be considered useful targets for T cell receptor (TCR) gene therapy to treat EBV-associated diseases. We used a TCR isolation platform based on a single major histocompatibility complex class I (MHC I) K562 cell library for the detection, isolation, and re-expression of TCRs targeting immunodominant peptide MHC (pMHC). Mature dendritic cells (mDCs) were pulsed with in vitro-transcribed (ivt) RNA encoding for the selected antigen to stimulate autologous T cells. The procedure allowed the mDCs to select an immunogenic epitope of the antigen for processing and presentation on the cell surface in combination with the most suitable MHC I molecule. We isolated eight EBV-specific TCRs. They recognize various pMHCs of EBV antigens LMP1, LMP2A, and EBNA3C, some of them described previously and some newly identified in this study. The TCR genes were molecularly cloned into retroviral vectors and the resultant TCR-engineered T cells secreted interferon-γ after antigen contact and were able to lyse tumor cells. The EBV-specific TCRs can be used as a basis for the generation of a TCR library, which provides a valuable source of TCRs for the production of EBV-specific T cells to treat EBV-associated diseases in patients with different MHC I types.

CXCR5 CAR-T cells simultaneously target B cell non-Hodgkin's lymphoma and tumor-supportive follicular T helper cells.

CAR-T cell therapy targeting CD19 demonstrated strong activity against advanced B cell leukemia, however shows less efficacy against lymphoma with nodal dissemination. To target both B cell Non-Hodgkin's lymphoma (B-NHLs) and follicular T helper (Tfh) cells in the tumor microenvironment (TME), we apply here a chimeric antigen receptor (CAR) that recognizes human CXCR5 with high avidity. CXCR5, physiologically expressed on mature B and Tfh cells, is also highly expressed on nodal B-NHLs. Anti-CXCR5 CAR-T cells eradicate B-NHL cells and lymphoma-supportive Tfh cells more potently than CD19 CAR-T cells in vitro, and they efficiently inhibit lymphoma growth in a murine xenograft model. Administration of anti-murine CXCR5 CAR-T cells in syngeneic mice specifically depletes endogenous and malignant B and Tfh cells without unexpected on-target/off-tumor effects. Collectively, anti-CXCR5 CAR-T cells provide a promising treatment strategy for nodal B-NHLs through the simultaneous elimination of lymphoma B cells and Tfh cells of the tumor-supporting TME.

CD8-Specific Designed Ankyrin Repeat Proteins Improve Selective Gene Delivery into Human and Primate T Lymphocytes.

Adoptive T cell immunotherapy in combination with gene therapy is a promising treatment concept for chronic infections and cancer. Recently, receptor-targeted lentiviral vectors (LVs) were shown to enable selective gene transfer into particular types of lymphocytes both in vitro and in vivo. This approach might facilitate the genetic engineering of a patient's own T lymphocytes, possibly even shifting this concept from personalized medicine to an off-the shelf therapy in future. Here, we describe novel high-affinity binders for CD8 consisting of designed ankyrin repeat proteins (DARPins), which were selected to bind to the CD8 receptor of human and nonhuman primate (NHP) cells. These binders were identified by ribosome display screening of DARPin libraries using recombinant human CD8 followed by receptor binding analysis on primary lymphocytes. CD8-targeted LVs (CD8-LVs) were then generated that delivered genes exclusively and specifically to human and NHP T lymphocytes by using the same targeting domain. These CD8-LVs were as specific for human T lymphocytes as their single-chain variable fragment-based counterpart, but they could be produced to higher titers. Moreover, they were superior in transducing cytotoxic T cells both in vitro and in vivo when equal particle numbers were applied. Since the here described CD8-LVs transduced primary T lymphocytes from NHP and human donors equally well, they offer the opportunity for preclinical studies in different animal models including large animals such as NHPs without the need for modifications in vector design.

ER-aminopeptidase 1 determines the processing and presentation of an immunotherapy-relevant melanoma epitope.

Dissecting the different steps of the processing and presentation of tumor-associated antigens is a key aspect of immunotherapies enabling to tackle the immune response evasion attempts of cancer cells. The immunodominant glycoprotein gp100209-217 epitope, which is liberated from the melanoma differentiation antigen gp100PMEL17 , is part of immunotherapy trials. By analyzing different human melanoma cell lines, we here demonstrate that a pool of N-terminal extended peptides sharing the common minimal epitope is generated by melanoma proteasome subtypes. In vitro and in cellulo experiments indicate that ER-resident aminopeptidase 1 (ERAP1)-but not ERAP2-defines the processing of this peptide pool thereby modulating the T-cell recognition of melanoma cells. By combining the outcomes of our studies and others, we can sketch the complex processing and endogenous presentation pathway of the gp100209-217 -containing epitope/peptides, which are produced by proteasomes and are translocated to the vesicular compartment through different pathways, where the precursor peptides that reach the endoplasmic reticulum are further processed by ERAP1. The latter step enhances the activation of epitope-specific T lymphocytes, which might be a target to improve the efficiency of anti-melanoma immunotherapy.

CAR T Cells with Enhanced Sensitivity to B Cell Maturation Antigen for the Targeting of B Cell Non-Hodgkin's Lymphoma and Multiple Myeloma.

Autologous T cells genetically modified with a chimeric antigen receptor (CAR) redirected at CD19 have potent activity in the treatment of B cell leukemia and B cell non-Hodgkin's lymphoma (B-NHL). Immunotherapies to treat multiple myeloma (MM) targeted the B cell maturation antigen (BCMA), which is expressed in most cases of MM. We developed a humanized CAR with specificity for BCMA based on our previously generated anti-BCMA monoclonal antibody. The targeting single-chain variable fragment (scFv) domain exhibited a binding affinity in the low nanomolar range, conferring T cells with high functional avidity. Redirecting T cells by this CAR allowed us to explore BCMA as an alternative target for mature B-NHLs. We validated BCMA expression in diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia. BCMA CAR T cells triggered target cell lysis with an activation threshold in the range of 100 BCMA molecules, which allowed for an efficient eradication of B-NHL cells in vitro and in vivo. Our data corroborate BCMA is a suitable target in B cell tumors beyond MM, providing a novel therapeutic option for patients where BCMA is expressed at low abundance or where anti-CD19 immunotherapies have failed due to antigen loss.

Efficient Non-Viral T-Cell Engineering by Sleeping Beauty Minicircles Diminishing DNA Toxicity and miRNAs Silencing the Endogenous T-Cell Receptors.

Transposon-based vectors have entered clinical trials as an alternative to viral vectors for genetic engineering of T cells. However, transposon vectors require DNA transfection into T cells, which were found to cause adverse effects. T-cell viability was decreased in a dose-dependent manner, and DNA-transfected T cells showed a delayed response upon T-cell receptor (TCR) stimulation with regard to blast formation, proliferation, and surface expression of CD25 and CD28. Gene expression analysis demonstrated a DNA-dependent induction of a type I interferon response and interferon-ß upregulation. By combining Sleeping Beauty transposon minicircle vectors with SB100X transposase-encoding RNA, it was possible to reduce the amount of total DNA required, and stable expression of therapeutic TCRs was achieved in >50% of human T cells without enrichment. The TCR-engineered T cells mediated effective tumor cell killing and cytokine secretion upon antigen-specific stimulation. Additionally, the Sleeping Beauty transposon system was further improved by miRNAs silencing the endogenous TCR chains. These miRNAs increased the surface expression of the transgenic TCR, diminished mispairing with endogenous TCR chains, and enhanced antigen-specific T-cell functionality. This approach facilitates the rapid non-viral generation of highly functional, engineered T cells for immunotherapy.

Distinguishing features of microglia- and monocyte-derived macrophages after stroke.

After stroke, macrophages in the ischemic brain may be derived from either resident microglia or infiltrating monocytes. Using bone marrow (BM)-chimerism and dual-reporter transgenic fate mapping, we here set out to delimit the responses of either cell type to mild brain ischemia in a mouse model of 30 min transient middle cerebral artery occlusion (MCAo). A discriminatory analysis of gene expression at 7 days post-event yielded 472 transcripts predominantly or exclusively expressed in blood-derived macrophages as well as 970 transcripts for microglia. The differentially regulated genes were further collated with oligodendrocyte, astrocyte, and neuron transcriptomes, resulting in a dataset of microglia- and monocyte-specific genes in the ischemic brain. Functional categories significantly enriched in monocytes included migration, proliferation, and calcium signaling, indicative of strong activation. Whole-cell patch-clamp analysis further confirmed this highly activated state by demonstrating delayed outward K+ currents selectively in invading cells. Although both cell types displayed a mixture of known phenotypes pointing to the significance of 'intermediate states' in vivo, blood-derived macrophages were generally more skewed toward an M2 neuroprotective phenotype. Finally, we found that decreased engraftment of blood-borne cells in the ischemic brain of chimeras reconstituted with BM from Selplg-/- mice resulted in increased lesions at 7 days and worse post-stroke sensorimotor performance. In aggregate, our study establishes crucial differences in activation state between resident microglia and invading macrophages after stroke and identifies unique genomic signatures for either cell type.

Unbiased Identification of T-Cell Receptors Targeting Immunodominant Peptide-MHC Complexes for T-Cell Receptor Immunotherapy.

T-cell receptor (TCR) immunotherapy uses T cells engineered with new TCRs to enable detection and killing of cancer cells. Efficacy of TCR immunotherapy depends on targeting antigenic peptides that are efficiently presented by the best-suited major histocompatibility complex (MHC) molecules of cancer cells. However, efficient strategies are lacking to easily identify TCRs recognizing immunodominant peptide-MHC (pMHC) combinations utilizing any of the six possible MHC class I alleles of a cancer cell. We generated an MHC cell library and developed a platform approach to detect, isolate, and re-express TCRs specific for immunodominant pMHCs. The platform approach was applied to identify a human papillomavirus (HPV16) oncogene E5-specific TCR, recognizing a novel, naturally processed pMHC (HLA-B*15:01) and a cytomegalovirus-specific TCR targeting an immunodominant pMHC (HLA-B*07:02). The platform provides a useful tool to isolate in an unbiased manner TCRs specific for novel and immunodominant pMHC targets for use in TCR immunotherapy.

Isolation and functional characterization of hepatitis B virus-specific T-cell receptors as new tools for experimental and clinical use.

T-cell therapy of chronic hepatitis B is a novel approach to restore antiviral T-cell immunity and cure the infection. We aimed at identifying T-cell receptors (TCR) with high functional avidity that have the potential to be used for adoptive T-cell therapy. To this end, we cloned HLA-A*02-restricted, hepatitis B virus (HBV)-specific T cells from patients with acute or resolved HBV infection. We isolated 11 envelope- or core-specific TCRs and evaluated them in comprehensive functional analyses. T cells were genetically modified by retroviral transduction to express HBV-specific TCRs. CD8+ as well as CD4+ T cells became effector T cells recognizing even picomolar concentrations of cognate peptide. TCR-transduced T cells were polyfunctional, secreting the cytokines interferon gamma, tumor necrosis factor alpha and interleukin-2, and effectively killed hepatoma cells replicating HBV. Notably, our collection of HBV-specific TCRs recognized peptides derived from HBV genotypes A, B, C and D presented on different HLA-A*02 subtypes common in areas with high HBV prevalence. When co-cultured with HBV-infected cells, TCR-transduced T cells rapidly reduced viral markers within two days. Our unique set of HBV-specific TCRs with different affinities represents an interesting tool for elucidating mechanisms of TCR-MHC interaction and dissecting specific anti-HBV mechanisms exerted by T cells. TCRs with high functional avidity might be suited to redirect T cells for adoptive T-cell therapy of chronic hepatitis B and HBV-induced hepatocellular carcinoma.

Chimeric PD-1:28 Receptor Upgrades Low-Avidity T cells and Restores Effector Function of Tumor-Infiltrating Lymphocytes for Adoptive Cell Therapy.

Inherent intermediate- to low-affinity T-cell receptors (TCR) that develop during the natural course of immune responses may not allow sufficient activation for tumor elimination, making the majority of T cells suboptimal for adoptive T-cell therapy (ATT). TCR affinity enhancement has been implemented to provide stronger T-cell activity but carries the risk of creating undesired cross-reactivity leading to potential serious adverse effects in clinical application. We demonstrate here that engineering of low-avidity T cells recognizing a naturally processed and presented tumor-associated antigen with a chimeric PD-1:28 receptor increases effector function to levels seen with high-avidity T cells of identical specificity. Upgrading the function of low-avidity T cells without changing the TCR affinity will allow a large arsenal of low-avidity T cells previously thought to be therapeutically inefficient to be considered for ATT. PD-1:28 engineering reinstated Th1 function in tumor-infiltrating lymphocytes that had been functionally disabled in the human renal cell carcinoma environment without unleashing undesired Th2 cytokines or IL10. Involved mechanisms may be correlated to restoration of ERK and AKT signaling pathways. In mouse tumor models of ATT, PD-1:28 engineering enabled low-avidity T cells to proliferate stronger and prevented PD-L1 upregulation and Th2 polarization in the tumor milieu. Engineered T cells combined with checkpoint blockade secreted significantly more IFNγ compared with T cells without PD-1:28, suggesting a beneficial combination with checkpoint blockade therapy or other therapeutic strategies. Altogether, the supportive effects of PD-1:28 engineering on T-cell function make it an attractive tool for ATT. Cancer Res; 77(13); 3577-90. ©2017 AACR.

Tumour ischaemia by interferon-γ resembles physiological blood vessel regression.

The relative contribution of the effector molecules produced by T cells to tumour rejection is unclear, but interferon-γ (IFNγ) is critical in most of the analysed models. Although IFNγ can impede tumour growth by acting directly on cancer cells, it must also act on the tumour stroma for effective rejection of large, established tumours. However, which stroma cells respond to IFNγ and by which mechanism IFNγ contributes to tumour rejection through stromal targeting have remained unknown. Here we use a model of IFNγ induction and an IFNγ-GFP fusion protein in large, vascularized tumours growing in mice that express the IFNγ receptor exclusively in defined cell types. Responsiveness to IFNγ by myeloid cells and other haematopoietic cells, including T cells or fibroblasts, was not sufficient for IFNγ-induced tumour regression, whereas responsiveness of endothelial cells to IFNγ was necessary and sufficient. Intravital microscopy revealed IFNγ-induced regression of the tumour vasculature, resulting in arrest of blood flow and subsequent collapse of tumours, similar to non-haemorrhagic necrosis in ischaemia and unlike haemorrhagic necrosis induced by tumour necrosis factor. The early events of IFNγ-induced tumour ischaemia resemble non-apoptotic blood vessel regression during development, wound healing or IFNγ-mediated, pregnancy-induced remodelling of uterine arteries. A better mechanistic understanding of how solid tumours are rejected may aid the design of more effective protocols for adoptive T-cell therapy.

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

T cells engineered to express a tumor-specific αß T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αß chains with endogenous αß 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 ß 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 αß 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.

In vivo transduction of primitive mobilized hematopoietic stem cells after intravenous injection of integrating adenovirus vectors.

Current protocols for hematopoietic stem/progenitor cell (HSPC) gene therapy, involving the transplantation of ex vivo genetically modified HSPCs are complex and not without risk for the patient. We developed a new approach for in vivo HSPC transduction that does not require myeloablation and transplantation. It involves subcutaneous injections of granulocyte-colony-stimulating factor/AMD3100 to mobilize HSPCs from the bone marrow (BM) into the peripheral blood stream and the IV injection of an integrating, helper-dependent adenovirus (HD-Ad5/35++) vector system. These vectors target CD46, a receptor that is uniformly expressed on HSPCs. We demonstrated in human CD46 transgenic mice and immunodeficient mice with engrafted human CD34+ cells that HSPCs transduced in the periphery home back to the BM where they stably express the transgene. In hCD46 transgenic mice, we showed that our in vivo HSPC transduction approach allows for the stable transduction of primitive HSPCs. Twenty weeks after in vivo transduction, green fluorescent protein (GFP) marking in BM HSPCs (Lin-Sca1+Kit- cells) in most of the mice was in the range of 5% to 10%. The percentage of GFP-expressing primitive HSPCs capable of forming multilineage progenitor colonies (colony-forming units [CFUs]) increased from 4% of all CFUs at week 4 to 16% at week 12, indicating transduction and expansion of long-term surviving HSPCs. Our approach was well tolerated, did not result in significant transduction of nonhematopoietic tissues, and was not associated with genotoxicty. The ability to stably genetically modify HSPCs without the need of myeloablative conditioning is relevant for a broader clinical application of gene therapy.

Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity.

Pediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients' immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology, targetable tumor-associated antigens (TAA) are identified and exploited for engineered T-cell therapy. Here, the specific recognition and lytic potential of transgenic allo-restricted CD8(+) T cells, directed against the ES-associated antigen 6-transmembrane epithelial antigen of the prostate 1 (STEAP1), was examined. Following repetitive STEAP1(130) peptide-driven stimulations with HLA-A*02:01(+) dendritic cells (DC), allo-restricted HLA-A*02:01(-) CD8(+) T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, T cell receptors' (TCR) α- and ß-chains were identified, cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01(-) primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2(-/-)γc(-/-) mouse model. Initially generated transgenic T cells specifically recognized STEAP1(130)-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific interferon-γ (IFNγ) release, lysed cells and inhibited growth of HLA-A*02:01(+) ES lines more effectively than HLA-A*02:01(-) ES lines. In vivo tumor growth was inhibited more effectively with transgenic STEAP1(130)-specific T cells than with unspecific T cells. Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1-expressing HLA-A*02:01(+) ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1-expressing tumors.

Targeting B-cell neoplasia with T-cell receptors recognizing a CD20-derived peptide on patient-specific HLA.

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

ERK1 as a Therapeutic Target for Dendritic Cell Vaccination against High-Grade Gliomas.

Glioma regression requires the recruitment of potent antitumor immune cells into the tumor microenvironment. Dendritic cells (DC) play a role in immune responses to these tumors. The fact that DC vaccines do not effectively combat high-grade gliomas, however, suggests that DCs need to be genetically modified specifically to promote their migration to tumor relevant sites. Previously, we identified extracellular signal-regulated kinase (ERK1) as a regulator of DC immunogenicity and brain autoimmunity. In the current study, we made use of modern magnetic resonance methods to study the role of ERK1 in regulating DC migration and tumor progression in a model of high-grade glioma. We found that ERK1-deficient mice are more resistant to the development of gliomas, and tumor growth in these mice is accompanied by a higher infiltration of leukocytes. ERK1-deficient DCs exhibit an increase in migration that is associated with sustained Cdc42 activation and increased expression of actin-associated cytoskeleton-organizing proteins. We also demonstrated that ERK1 deletion potentiates DC vaccination and provides a survival advantage in high-grade gliomas. Considering the therapeutic significance of these results, we propose ERK1-deleted DC vaccines as an additional means of eradicating resilient tumor cells and preventing tumor recurrence. Mol Cancer Ther; 15(8); 1975-87. ©2016 AACR.

Thymus-Derived Regulatory T Cells Are Positively Selected on Natural Self-Antigen through Cognate Interactions of High Functional Avidity.

Regulatory T (Treg) cells expressing Foxp3 transcripton factor are essential for immune homeostasis. They arise in the thymus as a separate lineage from conventional CD4(+)Foxp3(-) T (Tconv) cells. Here, we show that the thymic development of Treg cells depends on the expression of their endogenous cognate self-antigen. The formation of these cells was impaired in mice lacking this self-antigen, while Tconv cell development was not negatively affected. Thymus-derived Treg cells were selected by self-antigens in a specific manner, while autoreactive Tconv cells were produced through degenerate recognition of distinct antigens. These distinct modes of development were associated with the expression of T cell receptor of higher functional avidity for self-antigen by Treg cells than Tconv cells, a difference subsequently essential for the control of autoimmunity. Our study documents how self-antigens define the repertoire of thymus-derived Treg cells to subsequently endow this cell type with the capacity to undermine autoimmune attack.