Characterisation of high and low avidity peptide specific CD8+ T cells using immunologic, transcriptomic and proteomic tools

One of the hallmarks of successful immunotherapy is the generation of high avidity cytotoxic T cells which can recognise and respond to very low concentration of antigens. This sensitivity of T cells is usually determined by peptide titration ELISpot assays. Even though these assays are generally useful, they are laborious and sample demanding. The assays become even more difficult if the peptide(s) accountable for the generation of vaccine specific responses are unknown such as whole protein or cell vaccines. Therefore, there is a need to identify markers which can quickly and reliably identify a high avidity T cell response in cancer vaccination settings. To achieve this goal, this study utilised a C57Bl/6J mouse model which could efficiently generate high and low avidity T cell responses, when immunisation was undertaken with two form of vaccines to deliver the target antigens. The antigenic epitopes used for this study were derived from TRP-2 ‘self’ and ovalbumin (OVA) ‘foreign’ antigens. Immunisation of animals with these antigens in a DNA vaccine format induces a high avidity T cell response, in contrast to the response when these are administered in the peptide vaccine format. However, both the immunisations produced same number of peptide specific CD8+ T cells, which was assessed my multimer staining. When these cells were subjected to in vitro stimulations with the target peptides, the functionality of the low avidity T cells was restored whereas the high avidity T cells failed to respond to lower peptide concentrations. This showed the plasticity of antigen specific T cells and their ability to modulate their functionality according to the stimulation they have received

T-Cell manipulation strategies to prevent graft-versus-host disease in haploidentical stem cell transplantation

Allogeneic haematopoietic stem cell transplantation (HSCT) from an human leukocyte antigen (HLA)-identical donor can be curative for eligible patients with non-malignant and malignant haematological disorders. HSCT from alternative donor sources, such as HLA-mismatched haploidentical donors, is increasingly considered as a viable therapeutic option for patients lacking HLA-matched donors. Initial attempts at haploidentical HSCT were associated with vigorous bidirectional alloreactivity, leading to unacceptably high rates of graft rejection and graft-versus-host disease (GVHD). More recently, new approaches for mitigating harmful T-cell alloreactivity that mediates GVHD, while preserving the function of tumour-reactive natural killer (NK) cells and γδ T cells, have led to markedly improved clinical outcomes, and are successfully being implemented in the clinic. This article will provide an update on in vitro strategies and in vivo approaches aimed at preventing GVHD by selectively manipulating key components of the adaptive immune response, such as T-cell receptor (TCR)- αβ T cells and CD45RA-expressing naive T cells

Discovery and application of immune biomarkers for hematological malignancies

Introduction: Haematological malignancies originate and progress in primary and secondary lymphoid organs, where they establish a uniquely immune-suppressive tumour microenvironment. Although high-throughput transcriptomic and proteomic approaches are being employed to interrogate immune surveillance and escape mechanisms in patients with solid tumours, and to identify actionable targets for immunotherapy, our knowledge of the immunological landscape of haematological malignancies, as well as our understanding of the molecular circuits that underpin the establishment of immune tolerance, is not comprehensive. Areas covered: This article will discuss how multiplexed immunohistochemistry, flow cytometry/mass cytometry, proteomic and genomic techniques can be used to dynamically capture the complexity of tumour-immune interactions. Moreover, the analysis of multi-dimensional, clinically annotated data sets obtained from public repositories such as Array Express, TCGA and GEO is crucial to identify immune biomarkers, to inform the rational design of immune therapies and to predict clinical benefit in individual patients. We will also highlight how artificial neural network models and alternative methodologies integrating other algorithms can support the identification of key molecular drivers of immune dysfunction. Expert comment: High-dimensional technologies have the potential to enhance our understanding of immune-cancer interactions and will support clinical decision making and the prediction of therapeutic benefit from immune-based interventions

Flow cytometry and targeted immune transcriptomics identify distinct profiles in patients with chronic myeloid leukemia receiving tyrosine kinase inhibitors with or without interferon-α

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Immune-phenotyping and transcriptomic profiling of peripheral blood mononuclear cells from patients with breast cancer: identification of a 3 gene signature which predicts relapse of triple negative breast cancer

Background: Interactions between the immune system and tumors are highly reciprocal in nature, leading to speculation that tumor recurrence or therapeutic resistance could be influenced or predicted by immune events that manifest locally, but can be detected systemically. Methods: Multi-parameter flow cytometry was used to examine the percentage and phenotype of natural killer (NK) cells, myeloid-derived suppressor cells (MDSCs), monocyte subsets and regulatory T (Treg) cells in the peripheral blood of of 85 patients with breast cancer (50 of whom were assessed before and after one cycle of anthracycline-based chemotherapy), and 23 controls. Transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) in 23 patients were generated using a NanoString gene profiling platform. Results: An increased percentage of immunosuppressive cells such as granulocytic MDSCs, intermediate CD14++CD16+ monocytes and CD127negCD25highFoxP3+ Treg cells was observed in patients with breast cancer, especially patients with stage 3 and 4 disease, regardless of ER status. Following neoadjuvant chemotherapy, B cell numbers decreased significantly, whereas monocyte numbers increased. Although chemotherapy had no effect on the percentage of Treg, MDSC and NK cells, the expression of inhibitory receptors CD85j, LIAR and NKG2A and activating receptors NKp30 and NKp44 on NK cells increased, concomitant with a decreased expression of NKp46 and DNAM-1 activating receptors. Transcriptomic profiling revealed a distinct group of 3 patients in the triple negative breast cancer (TNBC) cohort who expressed high levels of mRNA encoding genes predominantly involved in inflammation. The analysis of a large transcriptomic dataset derived from the tumors of patients with TNBC revealed that the expression of CD163, CXCR4, THBS1 predicted relapse-free survival. Conclusions: The peripheral blood immunome of patients with breast cancer is influenced by the presence and stage of cancer, but not by molecular subtypes. Furthermore, immune profiling coupled with transcriptomic analyses of peripheral blood cells may identify patients with TNBC that are at risk of relapse after chemotherapy

Multi-omic analysis of two common p53 mutations: Proteins regulated by mutated p53 as potential targets for immunotherapy

The p53 protein is mutated in more than 50% of human cancers. Mutated p53 proteins not only lose their normal function but often acquire novel oncogenic functions, a phenomenon termed mutant p53 gain-of-function. Mutant p53 has been shown to affect the transcription of a range of genes, as well as protein–protein interactions with transcription factors and other effectors; however, no one has intensively investigated and identified these proteins, or their MHC presented epitopes, from the viewpoint of their ability to act as targets for immunotherapeutic interventions. We investigated the molecular changes that occurred after the TP53 null osteosarcoma cells, SaOS-2, were transfected with one of two conformational p53-mutants, either R175H or R273H. We then examined the phenotypic and functional changes using macroscopic observations, proliferation, gene expression and proteomics alongside immunopeptidome profiling of peptide antigen presentation in the context of major histocompatibility complex (MHC) class I molecules. We identified several candidate proteins in both TP53 mutant cell lines with differential expression when compared to the TP53 null vector control, SaOS-V. Quantitative SWATH proteomics combined with immune-peptidome analysis of the class-I eluted peptides identified several epitopes presented on pMHC and in silico analysis shortlisted which antigens were expressed in a range of cancerous but not adjacent healthy tissues. Out of all the candidates, KLC1 and TOP2A showed high levels of expression in every tumor type examined. From these proteins, three A2 and four pan HLA-A epitopes were identified in both R175H and R273H from TOP2A. We have now provided a short list of future immunotherapy targets for the treatment of cancers harboring mutated TP53

A parsimonious 3-gene signature predicts clinical outcomes in an acute myeloid leukemia multicohort study

Acute myeloid leukemia (AML) is a genetically heterogeneous hematological malignancy with variable responses to chemotherapy. Although recurring cytogenetic abnormalities and gene mutations are important predictors of outcome, 50% to 70% of AMLs harbor normal or risk-indeterminate karyotypes. Therefore, identifying more effective biomarkers predictive of treatment success and failure is essential for informing tailored therapeutic decisions. We applied an artificial neural network (ANN)–based machine learning approach to a publicly available data set for a discovery cohort of 593 adults with nonpromyelocytic AML. ANN analysis identified a parsimonious 3-gene expression signature comprising CALCRL, CD109, and LSP1, which was predictive of event-free survival (EFS) and overall survival (OS). We computed a prognostic index (PI) using normalized gene-expression levels and β-values from subsequently created Cox proportional hazards models, coupled with clinically established prognosticators. Our 3-gene PI separated the adult patients in each European LeukemiaNet cytogenetic risk category into subgroups with different survival probabilities and identified patients with very high–risk features, such as those with a high PI and either FLT3 internal tandem duplication or nonmutated nucleophosmin 1. The PI remained significantly associated with poor EFS and OS after adjusting for established prognosticators, and its ability to stratify survival was validated in 3 independent adult cohorts (n = 905 subjects) and 1 cohort of childhood AML (n = 145 subjects). Further in silico analyses established that AML was the only tumor type among 39 distinct malignancies for which the concomitant upregulation of CALCRL, CD109, and LSP1 predicted survival. Therefore, our ANN-derived 3-gene signature refines the accuracy of patient stratification and the potential to significantly improve outcome prediction

A mutated prostatic acid phosphatase (PAP) peptide-based vaccine induces PAP-specific CD8+ T cells with ex vivo cytotoxic capacities in HHDII/DR1 transgenic mice

Background: Current treatments for castrate (hormone)-resistant prostate cancer (CRPC) remain limited and are not curative, with a median survival from diagnosis of 23 months. The PAP-specific Sipuleucel-T vaccine, which was approved by the FDA in 2010, increases the Overall Survival (OS) by 4 months, but is extremely expensive. We have previously shown that a 15 amino acid (AA) PAP sequence-derived peptide could induce strong immune responses and delay the growth of murine TRAMP-C1 prostate tumors. We have now substituted one amino acid and elongated the sequence to include epitopes predicted to bind to several additional HLA haplotypes. Herein, we present the immunological properties of this 42mer-mutated PAP-derived sequence (MutPAP42mer). Methods: The presence of PAP-135-143 epitope-specific CD8+ T cells in the blood of patients with prostate cancer (PCa) was assessed by flow cytometry using Dextramer ™ technology. HHDII/DR1 transgenic mice were immunized with mutated and non-mutated PAP-derived 42mer peptides in the presence of CAF®09 or CpG ODN1826 (TLR-9 agonist) adjuvants. Vaccine-induced immune responses were measured by assessing the proportion and functionality of splenic PAP-specific T cells in vitro. Results: PAP-135-143 epitope-specific CD8+ T cells were detected in the blood of patients with PCa and stimulation of PBMCs from patients with PCa with mutPAP42mer enhanced their capacity to kill human LNCaP PCa target cells expressing PAP. The MutPAP42mer peptide was significantly more immunogenic in HHDII/DR1 mice than the wild type sequence, and immunogenicity was further enhanced when combined with the CAF®09 adjuvant. The vaccine induced secretory (IFNγ and TNFα) and cytotoxic CD8+ T cells and effector memory splenic T cells. Conclusions: The periphery of patients with PCa exhibits immune responsiveness to the MutPAP42mer peptide and immunization of mice induces/expands T cell-driven, wild-type PAP immunity, and therefore, has the potential to drive protective anti-tumor immunity in patients with PCa

Immune landscapes predict chemotherapy resistance and immunotherapy response in acute myeloid leukemia

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous hematological malignancy. Although immunotherapy may be an attractive modality to exploit in patients with AML, the ability to predict the groups of patients and the types of cancer that will respond to immune targeting remains limited. This study dissected the complexity of the immune architecture of AML at high resolution and assessed its influence on therapeutic response. Using 442 primary bone marrow samples from three independent cohorts of children and adults with AML, we defined immune-infiltrated and immune-depleted disease classes and revealed critical differences in immune gene expression across age groups and molecular disease subtypes. Interferon (IFN)–γ–related mRNA profiles were predictive for both chemotherapy resistance and response of primary refractory/relapsed AML to flotetuzumab immunotherapy. Our compendium of microenvironmental gene and protein profiles provides insights into the immuno-biology of AML and could inform the delivery of personalized immunotherapies to IFN-γ–dominant AML subtypes

TP53 abnormalities correlate with immune infiltration and associate with response to flotetuzumab immunotherapy in AML

Somatic TP53 mutations and 17p deletions with genomic loss of TP53 occur in 37% to 46% of acute myeloid leukemia (AML) with adverse-risk cytogenetics and correlate with primary induction failure, high risk of relapse, and dismal prognosis. Herein, we aimed to characterize the immune landscape of TP53-mutated AML and determine whether TP53 abnormalities identify a patient subgroup that may benefit from immunotherapy with flotetuzumab, an investigational CD123 × CD3 bispecific dual-affinity retargeting antibody (DART) molecule. The NanoString PanCancer IO360 assay was used to profile 64 diagnostic bone marrow (BM) samples from patients with TP53-mutated (n = 42) and TP53-wild-type (TP53-WT) AML (n = 22) and 45 BM samples from patients who received flotetuzumab for relapsed/refractory (R/R) AML (15 cases with TP53 mutations and/or 17p deletion). The comparison between TP53-mutated and TP53-WT primary BM samples showed higher expression of IFNG, FOXP3, immune checkpoints, markers of immune senescence, and phosphatidylinositol 3-kinase-Akt and NF-κB signaling intermediates in the former cohort and allowed the discovery of a 34-gene immune classifier prognostic for survival in independent validation series. Finally, 7 out of 15 patients (47%) with R/R AML and TP53 abnormalities showed complete responses to flotetuzumab (less than 5% BM blasts) on the CP-MGD006-01 clinical trial (NCT #02152956) and had significantly higher tumor inflammation signature, FOXP3, CD8, inflammatory chemokine, and PD1 gene expression scores at baseline compared with nonresponders. Patients with TP53 abnormalities who achieved a complete response experienced prolonged survival (median, 10.3 months; range, 3.3-21.3 months). These results encourage further study of flotetuzumab immunotherapy in patients with TP53-mutated AML