106 research outputs found
Improving MHC-I ligand identification by incorporating targeted searches of mass spectrometry data
No full textEffective immunotherapies rely on specific activation of immune cells. Class I major histocompatibility complex (MHC-I) bound peptide ligands play a major role in dictating the specificity and activation of CD8+ T cells and hence are important in developing T cell-based immunotherapies. Mass spectrometry-based approaches are most commonly used for identifying these MHC-bound peptides, wherein the MS/MS spectra are compared against a reference proteome database. Unfortunately, the effectiveness of matching the immunopeptide MS/MS spectra to a reference proteome database is hindered by inflated search spaces attributed to a lack of enzyme restriction in searches. These large search spaces limit the efficiency with which MHC-I peptides are identified. Here, we describe the implementation of a targeted database search approach and accompanying tool, SpectMHC, that is based on a priori-predicted MHC-I peptides. We have previously shown that this targeted search strategy improved peptide identifications for both mouse and human MHC ligands by greater than two-fold and is superior to traditional "no enzyme" search of reference proteomes (Murphy et al. J Res Proteome 16:1806-1816, 2017)
Enhancing mass spectrometry-based MHC-I peptide identification through a targeted database search approach
No full textMHC-bound peptide ligands dictate the activation and specificity of CD8+ T- cells-based and thus are important for devising T-cell immunotherapies. In recent times, advances in mass spectrometry (MS) have enabled the precise identification of these peptides, wherein MS/MS spectra are compared against a reference proteome. Unfortunately, matching immunopeptide MS/MS to reference proteome databases is hindered by inflated search spaces attributed to the number of matches that need to be considered due to a lack of enzyme restriction. These large search spaces limit the efficiency with which MHC-I peptides are identified. Here we offer a solution to this problem whereby we describe a targeted database search approach and accompanying tool SpectMHC that is based on a priori predicted MHC-I peptide
Improving MHC-I ligand identifications from LC-MS/MS data by incorporating allelic peptide motifs
No full textMHC class I (MHC-I)-bound ligands play a pivotal role in CD8 T cell immunity and are hence of major interest in understanding and designing immunotherapies. One of the most commonly utilized approaches for detecting MHC ligands is LC-MS/MS. Unfortunately, the effectiveness of current algorithms to identify MHC ligands from LC-MS/MS data is limited because the search algorithms used were originally developed for proteomics approaches detecting tryptic peptides. Consequently, the analysis often results in inflated false discovery rate (FDR) statistics and an overall decrease in the number of peptides that pass FDR filters. Andreatta et al. describe a new scoring tool (MS-rescue) for peptides from MHC-I immunopeptidome datasets. MS-rescue incorporates the existence of MHC-I peptide motifs to rescore peptides from ligandome data. The tool is demonstrated here using peptides assigned from LC-MS/MS data with PEAKs software but can be deployed on data from any search algorithm. This new approach increased the number of peptides identified by up to 20-30% and promises to aid the discovery of novel MHC-I ligands with immunotherapeutic potential
MHC-I ligand discovery using targeted database searches of mass spectrometry data: Implications for T-cell immunotherapies
No full textClass I major histocompatibility complex (MHC-I)-bound peptide ligands dictate the activation and specificity of CD8+ T cells and thus are important for devising T-cell immunotherapies. In recent times, advances in mass spectrometry (MS) have enabled the precise identification of these MHC-I peptides, wherein MS spectra are compared against a reference proteome. Unfortunately, matching these spectra to reference proteome databases is hindered by inflated search spaces attributed to a lack of enzyme restriction in the searches, limiting the efficiency with which MHC ligands are discovered. Here we offer a solution to this problem whereby we developed a targeted database search approach and accompanying tool SpectMHC, that is based on a priori-predicted MHC-I peptides. We first validated the approach using MS data from two different allotype-specific immunoprecipitates for the C57BL/6 mouse background. We then developed allotype-specific HLA databases to search previously published MS data sets of human peripheral blood mononuclear cells (PBMCs). This targeted search strategy improved peptide identifications for both mouse and human ligandomes by greater than 2-fold and is superior to traditional “no enzyme” searches of reference proteomes. Our targeted database search promises to uncover otherwise missed novel T-cell epitopes of therapeutic potential
Therapy-induced MHC I ligands shape neo-antitumor CD8 T cell responses during oncolytic virus-based cancer immunotherapy
No full textOncolytic viruses (OVs), known for their cancer-killing characteristics, also overturn tumor-associated defects in antigen presentation through the MHC class I pathway and induce protective neo-antitumor CD8 T cell responses. Nonetheless, whether OVs shape the tumor MHC-I ligandome remains unknown. Here, we investigated if an OV induces the presentation of novel MHC I-bound tumor antigens (termed tumor MHC-I ligands). Using comparative mass spectrometry (MS)-based MHC-I ligandomics, we determined differential tumor MHC-I ligand expression following treatment with oncolytic reovirus in a murine ovarian cancer model. In vitro, we found that reovirus changes the tumor ligandome of cancer cells. Concurrent multiplexed quantitative proteomics revealed that the reovirus-induced changes in tumor MHC-I ligand presentation were mostly independent of their source proteins. In an in vivo model, tumor MHC-I ligands induced by reovirus were detectable not only in tumor tissues but also the spleens (a source of antigen-presenting cells) of tumor-bearing mice. Most importantly, therapy-induced MHC-I ligands stimulated antigen-specific IFNγ responses in antitumor CD8 T cells from mice treated with reovirus. These data show that therapy-induced MHC-I ligands may shape underlying neo-antitumor CD8 T cell responses. As such, they should be considered in strategies promoting the efficacy of OV-based cancer immunotherapies
UNDERSTANDING THE ROLE OF T CELL MEDIATED IMMUNE RESPONSES IN CANCER IMMUNOTHERAPIES
No full textImproving MHC peptide identification and immunogenic cell death of melanoma.The field of cancer immunotherapy has been transformed over the last decade, with a significant emphasis on T cell-based therapies due to their ability to attack cancer cells specifically. However, despite substantial progress in the development of T cell-based cancer immunotherapies, a large proportion of patients do not respond favorably, particularly in ‘cold’ tumors, which are typically categorized by a lack of tumor antigens, and defective antigen-presenting cell (APC) and T cell priming, activation, or infiltration. Methods for characterizing and modulating tumor microenvironments (TME) could help develop future immunotherapies. The current thesis investigates two avenues of research: developing new methods for detecting tumor antigens and developing novel therapeutics to make tumors ‘hot’ and boost anticancer immunity.
The first project focuses on discovering class I major histocompatibility complex (MHC-I)-bound tumor antigens that govern the specificity and activation of CD8+ T cells. Traditional methods using mass spectrometry (LC-MS/MS) based MHC-peptide identification suffer from inflated search spaces, leading to limited efficiency and poor statistical power in peptide mapping and identification. The current thesis addresses these shortcomings by employing a targeted database search strategy and developing an accompanying tool, SpectMHC, which is based on previously predicted MHC-I peptides. This unique technique improved the identification rates and statistical power of MHC-I peptides in human and mouse models in an MS-based peptide discovery platform.
The later projects focus on utilizing immunogenic cell death (ICD) of cancer, a regulatory form of cell death characterized by enhanced antigenicity and adjuvanticity, to modulate the TME and initiate specific anticancer immune responses mediated by APCs and T cells. We created novel photodynamic therapies that target cancer cells directly via cytotoxic and indirectly via inflammatory responses, induction of the hallmarks of ICD, and activation of dendritic cells resulting in protective anticancer immunity. This research resulted in the development of promising immunogenic photodynamic therapies for the treatment of melanoma, which have the potential to be translated from bench to bedside. Overall, the current thesis presents novel strategies for understanding and inducing T cell-mediated anticancer immune responses
Multiplexed relative quantitation with isobaric tagging mass spectrometry reveals class I major histocompatibility complex ligand dynamics in response to doxorubicin
No full textMHC-I peptides are intracellular-cleaved peptides, usually 8–11 amino acids in length, which are presented on the cell surface and facilitate CD8+ T cell responses. Despite the appreciation of CD8+ T-cell antitumor immune responses toward improvement in patient outcomes, the MHC-I peptide ligands that facilitate the response are poorly described. Along these same lines, although many therapies have been recognized for their ability to reinvigorate antitumor CD8+ T-cell responses, whether these therapies alter the MHC-I peptide repertoire has not been fully assessed due to the lack of quantitative strategies. We develop a multiplexing platform for screening therapy-induced MHC-I ligands by employing tandem mass tags (TMTs). We applied this approach to measuring responses to doxorubicin, which is known to promote antitumor CD8+ T-cell responses during its therapeutic administration in cancer patients. Using both in vitro and in vivo systems, we show successful relative quantitation of MHC-I ligands using TMT-based multiplexing and demonstrate that doxorubicin induces MHC-I peptide ligands that are largely derived from mitotic progression and cell-cycle proteins. This high-throughput MHC-I ligand discovery approach may enable further explorations to understand how small molecules and other therapies alter MHC-I ligand presentation that may be harnessed for CD8+ T-cell-based immunotherapies
Positron emission tomography-computed tomography in subcutaneous panniculitis-like T-cell lymphoma
No full textSubcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare and poorly differentiated type of cutaneous T-cell lymphoma. In this variant, the lymphoma cells infiltrate preferentially into the subcutaneous adipose tissue. It is an indolent type of non-Hodgkin's lymphoma and can be mistaken for panniculitis. Here, we describe the case of a 59-year-old female patient who presented with altered skin pigmentation with diffuse plaque-like patches in the skin around the thighs and legs. A skin biopsy revealed subcutaneous lobular panniculitis composed of lymphocytes, epithelioid histiocytes, and occasional giant cells admixed with atypical lymphoid cells, which were suggestive of cutaneous lymphoma. Immunohistochemistry showed CD3 positive, CD20 negative, CD8 positive, CD4 occasional cells positive, CD56 negative, and CD5 few cells positive, confirming the diagnosis of SPTCL. Therefore, cases with atypical and nonresolving dermatological lesions should raise a suspicion of SPTCL as diagnosis against other benign conditions
Refugee children : supporting families in transitions
No full textThe chapter will discuss the difficulties refugee families face in transitioning across countries, and how community support can enhance their experience. It begins with a brief introduction, followed by an explanation of transition framed within a 'transitional and life course' perspective. The challenges families with children experience are then described as this relates to resettlement, parenting, and children's transition to school. The chapter concludes with recommendations for policies and practices to benefit families and children. Throughout the chapter, the author draws on published literature and examples from ongoing research being undertaken with Professor Carol Reid, involving interviews with people working with refugee families and children in community hubs, early childhood, school, and community organisations in three Australian states. While these sources provide keen insights into the refugee experiences in Australia, the author acknowledges the absence of the active refugee voice and her positionality of 'outsider researcher', taking account of the insights generated by scholars exploring the sensitivities of the insider/outsider dive in migration research (Carling et al., 2014)
The development of imaging techniques for analyzing cervical and lumbar vertebrae
No full textImage analysis techniques are developed for detection and analysis of osteophytes in cervical and lumbar vertebrae. Detection of osteophytes helps in identifying conditions of arthritis such as osteoarthritis and osteoporosis. A size-invariant feature based on disc space narrowing for the detection of claw in cervical vertebrae is introduced. Using K-means clustering, the proposed feature, along with the previously developed size invariant features, is used for the detection of claw in cervical vertebrae.
A three-dimensional (3D) model for normal lumbar vertebrae, L1-L5, is developed from computerized tomography (CT) scans. Using the models as a reference, techniques are explored to estimate the orientation angles of individual vertebrae from two-dimensional lumbar X-rays. Experimental results are presented, and the scope of future work is discussed --Abstract, page iii
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