Introduction: Malignant pleural mesothelioma is a disease primarily associated with exposure to the carcinogen asbestos. Whereas other carcinogen-related tumors are associated with a high tumor mutation burden, mesothelioma is not. We sought to resolve this discrepancy. Methods: We used mate-pair (n ¼ 22), RNA (n ¼ 28), and T cell receptor sequencing along with in silico predictions and immunologic assays to understand how structural variants of chromosomes affect the transcriptome. Results: We observed that inter-or intrachromosomal rearrangements were present in every specimen and were frequently in a pattern of chromoanagenesis such as chromoplexy or chromothripsis. Transcription of rearrangementrelated junctions was predicted to result in many potential neoantigens, some of which were proven to bind patient-specific major histocompatibility complex molecules and to expand intratumoral T cell clones. T cells responsive to these predicted neoantigens were also present in a patient's circulating T cell repertoire. Analysis of genomic array data from the mesothelioma cohort in The Cancer Genome Atlas suggested that multiple chromothriptic-like events negatively impact survival. Conclusions: Our findings represent the discovery of potential neoantigen expression driven by structural chromosomal rearrangements. These results may have implications for the development of novel immunotherapeutic strategies and the selection of patients to receive immunotherapies.
Very little is known about how the adaptive immune system responds to clonal evolution and tumor heterogeneity in non-small cell lung cancer. We profiled the T-cell receptor β complementarity determining region 3 in 20 patients with fully resected non-small cell lung cancer primary lesions and paired brain metastases. We characterized the richness, abundance and overlap of T cell clones between pairs, in addition to the tumor mutation burden and predicted neoantigens. We found a significant contraction in the number of unique T cell clones in brain metastases compared to paired primary cancers. The vast majority of T cell clones were specific to a single lesion, and there was minimal overlap in T cell clones between paired lesions. Despite the contraction in the number of T cell clones, brain metastases had higher non-synonymous mutation burdens than primary lesions. Our results suggest that there is greater richness of T cell clones in primary lung cancers than their paired metastases despite the higher mutation burden observed in metastatic lesions. These results may have implications for immunotherapy.
Light chain (AL) amyloidosis is an incurable human disease characterized by the misfolding, aggregation, and systemic deposition of amyloid composed of immunoglobulin light chains (LC). This work describes our studies on potential mechanisms of AL cytotoxicity. We have studied the internalization of AL soluble proteins and amyloid fibrils into human AC16 cardiomyocytes by using real time live cell image analysis. Our results show how external amyloid aggregates rapidly surround the cells and act as a recruitment point for soluble protein, triggering the amyloid fibril elongation. Soluble protein and external aggregates are internalized into AC16 cells via macropinocytosis. AL amyloid fibrils are shown to be highly cytotoxic at low concentrations. Additionally, caspase assays revealed soluble protein induces apoptosis, demonstrating different cytotoxic mechanisms between soluble protein and amyloid aggregates. This study emphasizes the complex immunoglobulin light chain-cell interactions that result in fibril internalization, protein recruitment, and cytotoxicity that may occur in AL amyloidosis. Light chain (AL)2 amyloidosis is a protein misfolding disease characterized by extracellular deposition of immunoglobulin light chains (LC) as amyloid fibrils. LC proteins are comprised of two distinct domains: the variable (V L ) and constant (C L ) domains (also called LC full-length (FL) protein to differentiate with the V L domain). In patients with AL amyloidosis, the LC aggregate and deposit in vital organs, causing organ failure and death (1). The factors governing deposition in individual tissues are unknown. Patients with cardiac AL amyloidosis have the worst prognosis, with a median survival of less than a year (2, 3).The finding that the V L was the primary component of amyloid fibrils influenced previous biophysical studies (4,5). Recent proteomic studies have demonstrated that amyloid deposits are likely heterogeneous in nature and can be formed by FL, V L , C L , or mixtures of all types of LC fragments (6 -8). Thermodynamic studies proposed a stabilizing role for the 3C L domain in the stability and a modulating effect on fibril formation (9). Recently, our laboratory has demonstrated that the C L domain modulates the amyloid formation reaction but has no effect on the stability of the protein (10).Soluble monoclonal LC, isolated from patients with amyloidosis, can impair rat cardiomyocyte function (11) and induce apoptotic events in mouse cardiomyocytes (12, 13). Also, urinederived LC can be internalized into primary rat cardiac fibroblasts (14) and primary human renal mesangial cells (15) through a pinocytic pathway (16) or via receptor, clathrin-mediated mechanisms, respectively (15).Within the amyloid field, it is widely accepted that oligomeric species are potentially more toxic than mature fibrils (17-20). However, toxicity associated with amyloid fibrils may also be pathologically relevant. Engel et al. (21) described a mechanism in which growth of islet amyloid associated polypeptides fibrils is re...
Using personalized peptide vaccines (PPVs) to target tumor-specific nonself-antigens (neoantigens) is a promising approach to cancer treatment. However, the development of PPVs is hindered by the challenge of identifying tumor-specific neoantigens, in part because current in silico methods for identifying such neoantigens have limited effectiveness. In this article, we report the results of molecular dynamics simulations of 12 oligopeptides bound with an HLA, revealing a previously unrecognized association between the inability of an oligopeptide to elicit a T cell response and the contraction of the peptide-binding groove upon binding of the oligopeptide to the HLA. Our conformational analysis showed that this association was due to incompatibility at the interface between the contracted groove and its αβ–T cell Ag receptor. This structural demonstration that having the capability to bind HLA does not guarantee immunogenicity prompted us to develop an atom-based method (SEFF12MC) to predict immunogenicity through using the structure and energy of a peptide·HLA complex to assess the propensity of the complex for further complexation with its TCR. In predicting the immunogenicities of the 12 oligopeptides, SEFF12MC achieved a 100% success rate, compared with success rates of 25–50% for 11 publicly available residue-based methods including NetMHC-4.0. Although further validation and refinements of SEFF12MC are required, our results suggest a need to develop in silico methods that assess peptide characteristics beyond their capability to form stable binary complexes with HLAs to help remove hurdles in using the patient tumor DNA information to develop PPVs for personalized cancer immunotherapy.
Using personalized peptide vaccines (PPVs) to target tumorspecific non-self antigens (neoantigens) is a promising approach to cancer treatment. However, the development of PPVs is hindered by the challenge of identifying tumor-specific neoantigens, in part because current in silico methods for identifying such neoantigens have limited effectiveness. Here we report the results of molecular dynamics simulations of 12 oligopeptides bound with a human leukocyte antigen (HLA), revealing a previously unrecognized association between the inability of an oligopeptide to elicit a T-cell response and the contraction of the peptide-binding groove upon binding of the oligopeptide to the HLA. Our conformational analysis showed that this association was due to incompatibility at the interface between the contracted groove and its α αβ β-T-cell antigen receptor (TCR). This structural demonstration that having the capability to bind HLA does not guarantee immunogenicity prompted us to develop an atom-based method (SE FF12MC ) to predict immunogenicity through using the structure and energy of a peptide• •HLA complex to assess the propensity of the complex for forming a ternary complex with its TCR. In predicting the immunogenicities of the 12 oligopeptides, SE FF12MC achieved a 100% success rate compared with success rates of 25-50% for 11 publicly available residue-based methods including NetMHC-4.0. While further validation and refinements of SE FF12MC are required, our results suggest a need to develop in silico methods that assess peptide characteristics beyond their capability to form stable binary complexes with HLAs to help remove hurdles in using the patient tumor DNA information to develop PPVs for personalized cancer immunotherapy.
Malignant melanoma is the most aggressive form of skin cancer, accounting for 10,000 deaths annually. Largely resistant to conventional cytotoxic chemotherapy and radiation, immune checkpoint blockade (ICB) therapies have revolutionized patient care, accounting for partial or complete responses in up to 70% of patients. An important aspect of cancer elimination is activation of the cytotoxic T lymphocyte (CTL) response. Recent data suggest that ICB mediated broadening of the peripheral blood CTL repertoire correlates with good clinical outcomes. Thus, recent studies in cancer immunotherapy have focused on targeting tumor neoantigens (tNeoAg) with cancer vaccines for use in combination with ICB. Computational algorithm-driven predictions of immunogenic tNeoAgs yield vast numbers of potential peptide targets, only a fraction of which may be immunogenic in patients. This likely contributed to the modest success of cancer vaccines targeting predicted, high-avidity tNeoAgs, emphasizing the importance of identifying true tumor rejection antigens, including sub-dominant antigens, outside the scope of predictive models. In addition to promoting T cell-mediated tumor rejection, ICB often comes at the expense of treatment-induced immune-related adverse effects (irAE) that frequently require discontinuation of treatment. Modulation of ICB towards antitumor immunity and away from autoimmunity may dramatically improve the therapeutic index of modern cancer therapy. This appears feasible considering the clinical observation that antitumor efficacy does not correlate with type/severity of irAE, suggesting that the mechanisms of both processes, though likely related (CTL mediated), may not be identical. We aim to identify and separate non-cross-reacting antigenic targets mediating tumor rejection from those mediating irAEs to enable therapeutic interventions that maximize the efficacy of ICB, expanding tumor-specific CTLs with vaccines while minimizing irAEs through desensitization. We have access to samples from responding/nonresponding melanoma patients subjected to ICB with varying degrees of irAEs. We have designed an experimental approach that combines established mass spectrometry and sequencing techniques to identify peptides and matching TCR clones with a novel strategy that targets the TCR-associated CD3 complex to allow inclusion of subdominant antigens in our studies. T-cell “co-potentiation” is achieved when anti-CD3 monovalent Fabs induce a conformational change in the CD3 complex that sustains the T-cell response to weak antigenic stimulation. We have successfully used anti-human CD3 Fabs to co-potentiate in vitro the activation and subsequent response to weak peptide-HLA/TCR interactions of human T cells found in PBMCs isolated from healthy donors. Our preliminary data suggest T-cell co-potentiation may allow identification of ICB-induced CTL clones specific for subdominant tNeoAgs and irAE targets in patients with melanoma undergoing active immunotherapy. Citation Format: Laura Elsbernd, Alfreda Nelson, Hien Huynh, Michele Hoffmann, Christopher Parks, Wendy Nevala, Shari Sutor, Larry Pease, Adam Schrum, Diana Gil, Svetomir N. Markovic. Co-potentiation of human T cells to identify subdominant tumor neoantigens from melanoma patients responding to immune checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B78.
We recently described the concept of T cell co-potentiation as means to target antigen recognition by T cells to achieve antitumor immunotherapy in mouse models. Normally, T cell antigen receptor (TCR) engagement by a strong immunogenic antigen induces a CD3 conformational change that exposes a proline-rich sequence (PRS) in the cytosolic domain of CD3 epsilon (CD3e) to dock cytosolic proteins and contribute to the assembly of the signaling cascade that activates T cell immune function. While induction of CD3 conformational change is required by T cells to achieve productive TCR/CD3 signaling, this molecular event fails to occur when the TCR interacts with poorly immunogenic antigens, like most tumor-associated antigens (TAAs). We found that CD3 conformational change can be exogenously induced on T cells using monovalent Fab fragments (Mono-Fabs) specific for the CD3 complex. Anti-CD3 Mono-Fabs expose the CD3e PRS, but fail to crosslink the TCR/CD3, turning them incompetent to trigger T cell activation in the absence of additional stimulation. However, if TCR has bound a weak antigen, one that fails to induce CD3 conformational change intrinsically, then anti-CD3 Mono-Fabs enable TCR/CD3 signaling, and as a result co-potentiate T cell responses to weak antigenic stimulation. Using a mouse model for melanoma (B6 mice and B16-F10 melanoma cell line), we found that treatment of mice with anti-CD3e Mono-Fabs improved antitumor T cell responses against melanoma in an antigen-dependent manner, and dramatically improved the outcome of complementary T cell immunotherapies. Based on these observations, we hypothesize that T cell co-potentiation using anti-human CD3 Mono-Fabs can be exploited as a novel immunotherapy for cancer patients. We have recently identified one anti-human CD3 Mono-Fab that is capable to induce CD3 conformational change in human T cells and allows the co-potentiation of T cell response to weak antigenic stimulation. Here we present data from human T cells and humanized mouse models using this Mono-Fab that support the potential therapeutic value of exploiting T cell co-potentiation to treat cancer patients. Citation Format: Alfreda D. Nelson, Laura Elsbernd, Adam G. Schrum, Diana Gil Pages. Testing T cell co-potentiation as an antitumor therapeutic strategy in humanized mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 962.
Tip-enhanced Raman Scattering (TERS) is an advanced technique allowing to measure Raman signals with high spatial resolution. By utilizing SERS effect in a probe coupled with Scanning Probe Microscopy the resolution reaches only a few nanometers breaking diffraction limit of optical microscopy. We have used TERS to study conformational features of amyloid aggregates formed by a peptide (CGNNQQNY) from the yeast prion protein Sup35. TERS results allowed us to identify a set of conformations that composes surfaces of morphologically different amyloid fibrils. Analysis of characteristic Raman bands in the amide III region showed that fibrils formed at pH 5.6 are composed of a mixture of peptide conformations (b-sheets, unordered and a-helix) while fibrils formed at pH 2 have primarily b-sheets. Additionally, by correlating the Raman peak positions corresponding to b-sheet structure in the amide III region with J dihedral angle obtained from Molecular Dynamics simulations of 16-mer fibrils, we proposed that peptides have parallel arrangement of b-sheets for pH 2 fibrils and antiparallel arrangement for fibrils formed at pH 5.6. We developed a methodology for detailed analysis of the peptide secondary structure that enabled us to correlate intensity changes in TERS spectra. Extensive cross correlation analysis of Raman peak intensities corresponding to a-helix, b-sheet and unordered secondary structures in amide III region and amide I region indicated that both regions provide adequate representation of structural characteristics of amyloid surfaces. Furthermore, 2D correlation with the band corresponding to Ca-H in plane bending suggested high localization of structural properties and large contribution of unordered non-helical secondary structures. Here we also discuss various ways to prepare TERS active probes, their longevity and enhancement capabilities suitable for protein aggregation studies.
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