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.

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.

Targeting high-grade B cell lymphoma with CD19-specific T cells.

Adoptive T cell therapy is an important additional treatment option for malignant diseases resistant to chemotherapy. Using a murine high-grade B cell lymphoma model, we have addressed the question whether the B cell differentiation antigen CD19 can act as rejection antigen. CD19(-/-) mice inoculated with CD19(+) B cell lymphoma cells showed higher survival rates than WT mice and were protected against additional tumor challenge. T cell depletion prior to tumor transfer completely abolished the protective response. By heterotypic vaccination of CD19(-/-) mice against murine CD19, survival after tumor challenge was significantly increased. To define protective epitopes within the CD19 molecule, T cells collected from mice that had survived the tumor transfer were analyzed for IFNγ secretion in response to CD19-derived peptides. The majority of mice exhibited a CD4(+) T cell response to CD19 peptide 27, which was the most dominant epitope after CD19 vaccination. A peptide 27-specific CD4(+) T cell line protected CD19(-/-) mice against challenge with CD19(+) lymphoma and also cured a significant proportion of WT mice from recurrent disease in a model of minimal residual disease after chemotherapy. In conclusion, our data highlight CD19-specific CD4(+) T cells for adoptive T cell therapy of B cell lymphomas.

A safeguard eliminates T cell receptor gene-modified autoreactive T cells after adoptive transfer.

By transfer of T cell receptor (TCR) genes, antigen specificity of T cells can be redirected to target any antigen. Adoptive transfer of TCR-redirected T cells into patients has shown promising results. However, this immunotherapy bears the risk of autoreactive side effects if the TCR recognizes antigens on self-tissue. Here, we introduce a safeguard based on a TCR-intrinsic depletion mechanism to eliminate autoreactive TCR-redirected T cells in vivo. By the introduction of a 10-aa tag of the human c-myc protein into murine (OT-I, P14) and human (gp100) TCR sequences, we were able to deplete T cells that were transduced with these myc-tagged TCRs with a tag-specific antibody in vitro. T cells transduced with the modified TCR maintained equal properties compared with cells transduced with the wild-type receptor concerning antigen binding and effector function. More importantly, therapeutic in vivo depletion of adoptively transferred T cells rescued mice showing severe signs of autoimmune insulitis from lethal diabetes. This safeguard allows termination of adoptive therapy in case of severe side effects.

Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette.

The transfer of T cell receptor (TCR) genes allows to endow T cells with a new antigen specificity. For clinical applications of TCR-redirected T cells, efficient functional expression of the transgenic TCR is a key prerequisite. Here, we compared the influence of the transgene cassette on the expression and function of the murine TCR P14 (recognizing a LCMV gp33 epitope) and the human TCR WT-1 (recognizing an epitope of the tumor-associated antigen WT-1). We constructed different vectors, in which TCRalpha- and beta-chain genes were either (a) linked by an internal ribosomal entry site (IRES), (b) combined by a 2A peptide, or (c) introduced into two individual retroviral constructs. While in a TCR-deficient T cell line TCR P14 was expressed equally well by all constructs, we found that IRES- but not 2A-employing TCR expression is hampered in a TCR-bearing cell line and in primary murine T cells where the transgenic TCR has to compete with endogenous TCR chains. Similarly, 2A-linked TCR WT-1 genes yielded highest expression and function as measured by tetramer binding and peptide-specific IFN-gamma secretion. Differences in expression were independent of copy number integration as shown by real-time PCR. Thus, linking TCRalpha- and beta-chain genes by a 2A peptide is superior to an IRES for TCR expression and T cell function.

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.

Codon optimization of the human papillomavirus E7 oncogene induces a CD8+ T cell response to a cryptic epitope not harbored by wild-type E7.

Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine.

A method for the generation of TCR retrogenic mice.

Retrogenic mice provide a unique system for rapidly analyzing the function of genes in the hematopoietic system. Here, we provide a detailed protocol for the production of retrogenic mice expressing genes coding for T cell receptor (TCR) for antigen. This technology should be easy to establish in any laboratory and should allow for a rapid progress in our understanding of the functional roles of TCR repertoires in immunity.

Enhanced T cell receptor gene therapy for cancer.

IMPORTANCE OF THE FIELD: Adoptive therapy with T cell receptor- (TCR-) redirected T cells has shown efficacy in mouse tumor models and first responses in cancer patients. One prerequisite to elicit effective anti-tumor reactivity is the transfer of high-avidity T cells. Their generation, however, faces several technical difficulties. Target antigens are often expressed at low levels and their recognition requires the use of high-affine receptors. Yet, mainly low-affinity TCRs have been isolated from tumor-infiltrating lymphocytes. Furthermore, upon transfer into a T cell the introduced receptor has to compete with the endogenous TCR. AREAS COVERED IN THIS REVIEW: This review discusses how the functional avidity of TCR-modified T cells can be enhanced by i) increasing the amount of introduced TCR heterodimers on the cell surface; and ii) generating receptors with high affinity. Risks of TCR gene therapy and possible safety mechanisms are discussed. WHAT THE READER WILL GAIN: The reader will gain an overview of the technical developments in TCR and T cell engineering. TAKE HOME MESSAGE: Despite technical obstacles, many advances have been made in the generation of high-avidity T cells expressing enhanced TCRs. Mouse studies and clinical trials will evaluate the effect of these improvements.

Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy.

The transfer of T cell receptor (TCR) genes can be used to induce immune reactivity toward defined antigens to which endogenous T cells are insufficiently reactive. This approach, which is called TCR gene therapy, is being developed to target tumors and pathogens, and its clinical testing has commenced in patients with cancer. In this study we show that lethal cytokine-driven autoimmune pathology can occur in mouse models of TCR gene therapy under conditions that closely mimic the clinical setting. We show that the pairing of introduced and endogenous TCR chains in TCR gene-modified T cells leads to the formation of self-reactive TCRs that are responsible for the observed autoimmunity. Furthermore, we demonstrate that adjustments in the design of gene therapy vectors and target T cell populations can be used to reduce the risk of TCR gene therapy-induced autoimmune pathology.

Factors influencing subcellular localization of the human papillomavirus L2 minor structural protein.

Two structural proteins form the capsids of papillomaviruses. The major structural protein L1 is the structural determinant of the capsids and is present in 360 copies arranged in 72 pentamers. The minor structural protein L2 is estimated to be present in twelve copies per capsid. Possible roles for L2 in interaction with cell surface receptors and in virion uptake have been suggested. As previously reported, L2 localizes in subnuclear domains identified as nuclear domain 10 (ND10). As it was demonstrated that L2 is able to recruit viral and cellular proteins to ND10, a possible role for L2 as a mediator in viral assembly has been proposed. In this study, we determined factors influencing the localization of L2 at ND10. Under conditions of moderate L2 expression level and in the absence of heterologous viral components, we observed that, in contrast to previous reports, L2 is mainly distributed homogeneously throughout the nucleus. L2, however, is recruited to ND10 at a higher expression level or in the presence of viral components derived from vaccinia virus or from Semliki Forest virus. We observed that translocation of L2 to ND10 is not a concentration-dependent accumulation but rather seems to be triggered by yet unidentified cellular factors. In contrast to HPV 11 and 16 L2, the HPV 18 L2 protein seems to require L1 for efficient nuclear accumulation.