Carsten Linnemann

High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4 + T cells in human melanoma

Tumor-specific neo-antigens that arise as a consequence of mutations are thought to be important for the therapeutic efficacy of cancer immunotherapies. Accumulating evidence suggests that neo-antigens may be commonly recognized by intratumoral CD8+ T cells, but it is unclear whether neo-antigen–specific CD4+ T cells also frequently reside within human tumors. In view of the accepted role of tumor-specific CD4+ T-cell responses in tumor control, we addressed whether neo-antigen–specific CD4+ T-cell reactivity is a common property in human melanoma.

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.

Anti–CTLA-4 therapy broadens the melanoma-reactive CD8+ T cell response

Anti–CTLA-4 treatment increases the diversity of the melanoma-specific CD8 T cell response. Anti–CTLA-4 Therapy Expands T Cell Range An antibody to the immune inhibitory molecule CTLA-4, ipilimumab, can improve survival in patients with advanced melanoma. However, how anti–CTLA-4 works to improve the tumor immune response in humans remains unclear. Now, Kvistborg et al. show that although the magnitude of T cell responses was largely unaltered after therapy, the number of different T cell responses was significantly increased. Indeed, this increased breadth suggests that anti–CTLA-4 may work by increasing priming of T cells to tumor-related antigens rather than boosting preexisting immune responses. If so, other strategies that improve the range of T cells may have similar success battling cancer. Anti–CTLA-4 treatment improves the survival of patients with advanced-stage melanoma. However, although the anti–CTLA-4 antibody ipilimumab is now an approved treatment for patients with metastatic disease, it remains unknown by which mechanism it boosts tumor-specific T cell activity. In particular, it is unclear whether treatment amplifies previously induced T cell responses or whether it induces new tumor-specific T cell reactivities. Using a combination ultraviolet (UV)–induced peptide exchange and peptide–major histocompatibility complex (pMHC) combinatorial coding, we monitored immune reactivity against a panel of 145 melanoma-associated epitopes in a cohort of patients receiving anti–CTLA-4 treatment. Comparison of pre- and posttreatment T cell reactivities in peripheral blood mononuclear cell samples of 40 melanoma patients demonstrated that anti–CTLA-4 treatment induces a significant increase in the number of detectable melanoma-specific CD8 T cell responses (P = 0.0009). In striking contrast, the magnitude of both virus-specific and melanoma-specific T cell responses that were already detected before start of therapy remained unaltered by treatment (P = 0.74). The observation that anti–CTLA-4 treatment induces a significant number of newly detected T cell responses—but only infrequently boosts preexisting immune responses—provides strong evidence for anti–CTLA-4 therapy–enhanced T cell priming as a component of the clinical mode of action.

Towards error-free profiling of immune repertoires

Deep profiling of antibody and T cell–receptor repertoires by means of high-throughput sequencing has become an attractive approach for adaptive immunity studies, but its power is substantially compromised by the accumulation of PCR and sequencing errors. Here we report MIGEC (molecular identifier groups–based error correction), a strategy for high-throughput sequencing data analysis. MIGEC allows for nearly absolute error correction while fully preserving the natural diversity of complex immune repertoires.

TIL therapy broadens the tumor-reactive CD8+ T cell compartment in melanoma patients

There is strong evidence that both adoptive T cell transfer and T cell checkpoint blockade can lead to regression of human melanoma. However, little data are available on the effect of these cancer therapies on the tumor-reactive T cell compartment. To address this issue we have profiled therapy-induced T cell reactivity against a panel of 145 melanoma-associated CD8+ T cell epitopes. Using this approach, we demonstrate that individual tumor-infiltrating lymphocyte cell products from melanoma patients contain unique patterns of reactivity against shared melanoma-associated antigens, and that the combined magnitude of these responses is surprisingly low. Importantly, TIL therapy increases the breadth of the tumor-reactive T cell compartment in vivo, and T cell reactivity observed post-therapy can almost in full be explained by the reactivity observed within the matched cell product. These results establish the value of high-throughput monitoring for the analysis of immuno-active therapeutics and suggest that the clinical efficacy of TIL therapy can be enhanced by the preparation of more defined tumor-reactive T cell products.

Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7

Major histocompatibility complex (MHC) class I multimer technology has become an indispensable immunological assay system to dissect antigen-specific cytotoxic CD8+ T cell responses by flow cytometry. However, the development of high-throughput assay systems, in which T cell responses against a multitude of epitopes are analyzed, has been precluded by the fact that for each T cell epitope, a separate in vitro MHC refolding reaction is required. We have recently demonstrated that conditional ligands that disintegrate upon exposure to long-wavelength UV light can be designed for the human MHC molecule HLA-A2. To determine whether this peptide-exchange technology can be developed into a generally applicable approach for high throughput MHC based applications we set out to design conditional ligands for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Here, we describe the development and characterization of conditional ligands for this set of human MHC molecules and apply the peptide-exchange technology to identify melanoma-associated peptides that bind to HLA-A3 with high affinity. The conditional ligand technology developed here will allow high-throughput MHC-based analysis of cytotoxic T cell immunity in the vast majority of Western European individuals.

High-throughput identification of antigen-specific TCRs by TCR gene capture

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen–reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

Pairing of T-cell receptor chains via emulsion PCR

Our ability to analyze adaptive immunity and engineer its activity has long been constrained by our limited ability to identify native pairs of heavy–light antibody chains and alpha–beta T‐cell receptor (TCR) chains — both of which comprise coupled “halves of a key”, collectively capable of recognizing specific antigens. Here, we report a cell‐based emulsion RT‐PCR approach that allows the selective fusion of the native pairs of amplified TCR alpha and beta chain genes for complex samples. A new type of PCR suppression technique was developed that makes it possible to amplify the fused library with minimal noise for subsequent analysis by high‐throughput paired‐end Illumina sequencing. With this technique, single analysis of a complex blood sample allows identification of multiple native TCR chain pairs. This approach may be extended to identify native antibody chain pairs and, more generally, pairs of mRNA molecules that are coexpressed in the same living cells.

TCR repertoires of intratumoral T-cell subsets.

The infiltration of human tumors by T cells is a common phenomenon, and over the past decades, it has become increasingly clear that the nature of such intratumoral T‐cell populations can predict disease course. Furthermore, intratumoral T cells have been utilized therapeutically in clinical studies of adoptive T‐cell therapy. In this review, we describe how novel methods that are either based on T‐cell receptor (TCR) sequencing or on cancer exome analysis allow the analysis of the tumor reactivity and antigen‐specificity of the intratumoral TCR repertoire with unprecedented detail. Furthermore, we discuss studies that have started to utilize these techniques to probe the link between cancer exomes and the intratumoral TCR pool. Based on the observation that both the cancer epitope repertoire and intratumoral TCR repertoire appear highly individual, we outline strategies, such as ‘autologous TCR gene therapy’, that exploit the tumor‐resident TCR repertoire for the development of personalized immunotherapy.

T-Cell Receptor Gene Therapy: Critical Parameters for Clinical Success

T-cell receptor (TCR) gene therapy aims to induce immune reactivity against tumors by introducing genes encoding a tumor-reactive TCR into patient T cells. This approach has been extensively tested in preclinical mouse models, and initial clinical trials have demonstrated the feasibility and potential of TCR gene therapy as a cancer treatment. However, data obtained from preclinical and clinical studies suggest that both the therapeutic efficacy and the safety of TCR gene therapy can be and needs to be further enhanced. This review highlights those strategies that can be followed to develop TCR gene therapy into a clinically relevant treatment option for cancer patients.