Multiplex immunofluorescence IHC is a powerful technique to address the increasing research needs in immuno-oncology to better understand the tumor microenvironment. In this webinar, we will address how a rapid microfluidic IHC stainer enables fast multi-staining delivering high-quality results and great reproducibility.

Learning Objectives:

1. Introduction to precision microfluidics for rapid tissue staining

2. How to perform a 6-plex TSA-based multiplex staining in 4h12

3. How to transfer a protocol transfer to a different tissue

The RNAscope in situ hybridization technology allows high sensitivity, specificity and spatial resolution providing pivotal single-cell gene expression information to gain better insights into the complex and heterogeneous tumor microenvironment. Using the RNAscope technology, it is possible to simultaneously visualize and assess the gene expression of cytokines, chemokines, immune cell markers, tumor cell markers and checkpoint markers within the tumor in order to:

Learning Objectives:

1. Understand complex spatial relationship among different cell types within the TME

2. Profile immune checkpoint markers and other therapeutic targets

3. Characterize immune cell types and activation states

Neoantigen based personalized immuno-oncology drugs are a relatively new therapeutic avenue that have shown strong anti-tumor efficacy in the clinic.  Neoantigen peptides have been widely reported to be difficult to synthesize due to their hydrophobicity, length, and charge.  Leveraging extensive peptide synthesis experience GenScript has developed NeoPreTM, a predictive algorithm which is able to determine peptide synthesis difficulty based on sequence alone.  NeoPreTM can then recommend the most efficient approach to successfully synthesizing peptides using one of GenScript’s many synthesis platforms.  This presentation will highlight how NeoPreTM identifies synthesis difficulty and review successful cases of difficult neoantigen peptide synthesis from several researchers.  The presentation will also review how these neoantigen peptides are used during various stages of personalized therapeutic development to increase treatment efficacy.

Learning Objectives:

1. What neoantigen personalized therapeutics are and why they are gaining so much interest amongst the immuno-oncology community 

2. How neoantigen peptides are utilized during personalized therapeutic development

3. How NeoPreTM identifies synthesis difficulty and recommends the most efficient approach for successful neoantigen peptide synthesis

While providing indispensable insight into immune processes, the live cell imaging of immune cells poses unique challenges due to their non-adherent nature. Suspension cells such as immune cells can easily move away from the FOV (field of view) during imaging by perturbation such as reagent introduction, often necessitating the use of surface coating to artificially immobilize cells. In addition, unlike adherent cells, interacting suspension cells form aggregates, making it very difficult to track and analyze individual cells during imaging. Here we describe the novel, microfluidic-based dynamic live cell imaging platform CellASIC® ONIX2, where microenvironmental parameters such as the perfusion of media/reagent, temperature, and gas compositions can be precisely controlled on-demand via software during the entire imaging experiment. Unlike most live imaging platforms merely aiming to maintain temperature and gas at certain levels, CellASIC® ONIX2 platform’s ability to control fluidics and perfusion offers true on-demand control of cellular microenvironment during live cell imaging, and its standard plate form factor can instantly upgrade any existing inverted microscope into a fully capable live cell imaging platform. Overview of applications covering hypoxia, apoptosis, migration, and suspension immune cell imaging will be presented. Specific emphasis will be given to microfluidic designs targeted for use with different cell types as well as fluorescent probes for live cell imaging. Any scientists planning to start live cell imaging experiments, as well as experienced imaging scientists wanting to broaden their applications will benefit from this session. 

Learning Objectives:

1. Understand the advantages and limitations of current live cell imaging platforms

2. Learn about the emerging field of microfluidic based dynamic imaging

3. Review current state of the art regarding microfluidics based dynamic imaging

Gene manipulation studies in primary human immune cells can answer important biological questions within a clinically relevant cellular context. In this webinar we explore the use of several gene editing or modulation tools, including CRISPRko, RNAi and base editing, to modify various primary human immune cell types. In addition, we will discuss numerous applications, such as cell therapy, arrayed and pooled screening, for various gene edited primary immune cell types.

Learning Objectives:

1. Understand the usefulness of gene editing and modulation experiments in various primary cell types

2. Understand the different tools available for gene silencing, knockout and editing

3. Understand the potential research and therapeutic applications for gene edited cell lines

A key step in the clinical production of CAR T cells is the expansion of engineered T cells. To generate enough cells for viable adoptive cell therapy, cells must be robustly stimulated, which raises the risk of inducing T-cell exhaustion and reducing therapeutic efficacy. We sought to answer fundamental questions about the impact of in vitro manipulation on T-cell identity to by performing single-cell multiomic analysis using BD® AbSeq and BD Rhapsody™ Single-Cell Analysis system to simultaneously measure expression of 38 proteins and 399 genes in human T cells expanded in vitro. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states and the identification of a donor-specific subset of terminally differentiated T cells that would have been otherwise overlooked using canonical cell classification schema. As expected, T-cell activation induced downregulation of naïve-associated markers and upregulation of effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation defined by markers temporarily expressed upon 3 days of stimulation, markers constitutively expressed throughout chronic activation, and markers uniquely up-regulated upon 14 days of stimulation. These data indicate heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. We demonstrate the power of a single-cell multiomic approach to comprehensively characterize T cells and to precisely monitor changes in differentiation, activation and exhaustion signatures in response to different activation protocols. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2020 BD. All rights reserved.

Learning Objectives:

1. The complexity of T-cell exhaustion and utility of multiomic approaches in studying complex biological processes such as T-cell exhaustion

2. Use of unsupervised data analysis approaches in analyzing heterogeneous cell population

3. Comparing and contrasting data from traditional flow cytometry and BD® AbSeq

Immune checkpoint blockade has revolutionized the treatment for patients with cancer.  Great efforts remain to better understand resistant mechanisms to treatment as well as means to improve outcomes via combinatorial approaches.  Work included will highlight the success of immune checkpoint blockade in melanoma as an example as well as propose further combinations by targeting innate immunity and angiogenesis for potential synergistic effects.  Proposed novel targets will be discussed with correlative data.

Learning Objectives:

1. To identify patients and biomarkers predictive of responses to checkpoint blockade

2. To better understand combinatorial approached with immune checkpoint blockade by further targeting the tumor microenvironment

Cancer cells can acquire immune resistance resulting in a loss of immunosurveillance. Tumor cells can upregulate PDL-1 which binds to PD-1. This complex inhibits CD8 Tumor Infiltrating cells ( TILs) response to tumor antigens inducing immune blockade. Anti-blockade immunotherapy blocks the blockade and restores immunosurveillance (self-healing).This presentation emphasizes clinical examples of immunotherapy with both an individual patient with malignant melanoma and population-based examples including triple negative breast cancer.. The cases demonstrate the importance of intact tissue chemistry, in particular multi-analyte immunohistochemistry to inform treatment.. It emphasizes the future need to add more markers and cancer types to the testing repertoire and the need to go beyond diagnosis to prediction and monitoring of immunotherapy responses.

Learning Objectives:

1. Understand the concepts of tumor immunosurveillance, immune blockade, and immune restoration via immunotherapy

2. Understand the pivotal role of the diagnostic laboratory in informing immunotherapy by using intact multi-analyte immunohistochemistry.

Cancer initiation and virulence depends on mechanisms to evade host immunosurveillance and suppression of early invasion and growth.  Distinct phenotypes of the immune infiltrates in and around cancers derive from differing mechanisms.  Some very good prognosis cancers are immune "deserts" with very little immune cell infiltration.  These tumors register as normal or self to the immune system, accompanied by a more normal (less aggressive) behavior.  Meanwhile, the most aggressive appearing cancers elicit the most inflammation.  These tumors employ a variety of mechanisms to evade immune suppression.  The now well known immune checkpoint PDL1 aberrantly expressed by tumor cells (or induced in surrounding immune cells) has provided a critical target for therapy, effecting cures in a subset of previously incurable patients.  Overall, however, this remains a small subset. Advanced generation mouse models provide an opportunity to explore new treatment protocols.  For example, we have shown that immune "priming" augments PDL1 checkpoint inhibitor therapy.  In another model, we find that tumors with an "immune excluded" phenotype, where a brisk infiltrate is seen surrounding but not infiltrating a tumor, can be attributed to the simultaneous expression of macrophage chemoattracting and repelling paracrine factors.  HER2 amplification, seen in 15% of invasive breast cancers but twice as many intraductal cancers (DCIS) provides a prototype tumor antigen and another opportunity to explore rational combinations of immune and cell targeted therapies.  There is growing list of additional immune synapse targets with drugs in development and clinical trials now.  Meanwhile, predictive biomarkers for specific targets or combinations of targets are also in active development.  As we and others have shown, advanced generation mouse models of human disease play a critical role in the development of mechanistic evidence for new therapies and new diagnostic and predictive biomarkers.

Learning Objectives:

1. Review patterns and mechanisms of the tumor immune microenvironment

2. Understand examples of experimental pathology in immune intact models of disease

3. Promote active interest in the evolving field of cancer immunotherpy as a personalized medicine approach

Immune checkpoint therapies targeting the CTLA-4 and PD-(L)1 pathways have been largely clinically disappointing for patients with prostate cancer. This can be attributed to the highly immunosuppressive prostate tumor microenvironment, reflected in the paucity of T cells and enrichment of myeloid cells. However, a subset of patients with metastatic prostate cancer respond to ipilimumab (anti-CTLA-4). We recently identified markers associated with Th1 effector T cells responses, such as a high intratumoral CD8 density and/or expression of a high IFN-gamma-response gene signature, which correlated with prolonged clinical responses to ipilimumab.

To elucidate mechanisms of resistance to ipilimumab, we evaluated pre- and post-treatment prostate tumor tissues. Ipilimumab increased T cell infiltration within the prostate tumor microenvironment, converting it to an immunologically “hot” tumor. However, this was countered by upregulation of the inhibitory immune checkpoints, PD-L1 and VISTA. Simultaneously targeting the PD-(L)1 and CTLA-4 improved survival in murine models of prostate cancer. These data led to the design and accrual of an international study evaluating the combination of nivolumab (anti-PD-1) plus ipilimumab in patients with metastatic prostate cancer. The combination induced durable radiographic responses in a subset of patients, suggesting that adaptive resistance to ipilimumab monotherapy could be overcome by targeting more than one immune checkpoint.

Despite the striking responses in certain patients, that most did not benefit from the combination of nivolumab plus Ipilimumab implies other mechanisms of resistance to ipilimumab monotherapy. We found high levels of the immunosuppressive cytokine TGF-beta within the metastatic prostate bone tumor microenvironment. In a preclinical model of prostate cancer, concurrently targeting TGF-beta and CTLA-4 promoted Th1 responses associated with improved overall survival. This suggested that high levels of TGF-beta may be a mechanism of primary resistance to ipilimumab, which will need to be confirmed in patients. Taken together, our data supports the rational development of combination therapies to overcome the immunosuppressive prostate tumor microenvironment.

Learning Objectives:

1. Explain the mechanisms of action for immune checkpoint therapies

2. Identify primary and adaptive resistance mechanisms to immune checkpoint therapies

3. Explain current approaches to combination therapy in the clinic

Programmed death-1 (PD-1) inhibits T and B cell function upon ligand binding. PD-1 blockade revolutionized cancer treatment, and while numerous patients respond, some develop autoimmune-like symptoms or overt autoimmunity characterized by autoantibody production.  PD-1 inhibition accelerates autoimmunity in mice, but its role in regulating germinal centers is controversial. To address the role of PD-1 in the germinal center reaction in autoimmune type 1 diabetes, we used tetramers to phenotype insulin-specific CD4+ T and B cells in diabetes-prone non-obese diabetic (NOD) mice and diabetes-resistant B6.IAg7 mice. While insulin-reactive lymphocytes were detected in both strains at baseline, they were only antigen-experienced in NOD mice. PD-1 or PD-L1 deficiency, or PD-1  blockade, further unleashed insulin-specific T follicular helper CD4+ T cells and enhanced their survival in NOD mice. This was concomitant with an increase in germinal center B cells and augmented insulin autoantibody production. The effect of PD-1 blockade on germinal centers was reduced when mice were treated with a monoclonal antibody targeting the insulin peptide:MHCII complex. In B6.IAg7 mice, PD-1 blockade did not have any effect, and mice remained autoantibody- and diabetes-free. This work provides an explanation for autoimmune side effects following PD-1 pathway inhibition, and suggests that the state of CD4+ T cell differentiation predetermines its response to PD-1 blockade. Additionally, our work suggests that targeting the self-peptide:MHCII complex may limit autoimmunity arising from checkpoint blockade.

Learning Objectives:

1. Explain the clinical benefits and challenges associated with checkpoint blockade

2. Describe the role of programmed death-1 in mouse models of diabetes

3. Identify ongoing efforts to develop reliable markers for safe and effective use of checkpoint blockade

Platinum-based chemotherapy has been the cornerstone for treating metastatic urothelial cancer, which compromises the majority of bladder cancer. While urothelial cancer is chemosensitive, patients often do not have a durable response and ultimately progress. Until recently patients were left with less effective second line chemotherapy options, but this has changed with the advent of immunotherapy. The United States Food and Drug Administration approved two programmed death-1 (PD-1) inhibitors and three programmed death ligand-1 (PD-L1) in 2016-2017 for the second line setting. Adding check point inhibitors to our armamentarium has been revolutionary, however there are opportunities to improve with strategies to increase response rates and apply immunotherapy to earlier disease stages.

Learning Objectives:

1. Understand the current role of checkpoint inhibitors in metastatic urothelial cancer

2. Identify areas where immunotherapy could impact urothelial cancer treatment

B cells have recently come into vogue as important contributors to the anti-tumor immune response in cancer patients. Specifically, B cells and the immune structures in which they reside (tertiary lymphoid structures) correlate with increased survival and immunotherapeutic response for several solid tumor types. Our research is focused on linking anti-tumor function of B cells with their location and phenotype utilizing high level immunological techniques.

Learning objectives:

1. Understand the importance of B cells and tertiary lymphoid structures (TLS) in human cancer

2. Define heterogeneity of B cells and TLS in human cancer

3. Identify viable immunotherapeutics to target B cells and TLS in cancer patients

Cancer immunotherapy had revolutionized the field of oncology, providing noveltherapeutic options for cancer patients with dramatically improved outcomes. However, the development of the new field of immuno-oncology requires a deep understanding of the inflammatory tumor microenvironment with the goal to identify predictive biomarkers for immunotherapy.   Multiplex immunofluorescencetechniques had become a key tool for the immune profiling of cancer tissue samplesfrom patients; however, these techniques are still in development, offering newchallenges   to   the   laboratory   scientist.   The   first   aim   of   this   talk   is   to   defineapplications of approved and experimental tissue-based biomarker assessment inImmuno-Oncology. We will explore the methodology of multiplex immune profilingfor   formalin   fixed   biopsy   specimens   for   immuno-oncology   studies,   including   adescription of the basic concepts, general workflow, optimization, and validationprocess. The second aim is to define bottlenecks and areas of improvement atdifferent levels of this biomarker strategy.

Learning Objectives:

1. Define applications of approved and experimental (multiplexing IF) biomarker assessment in Immuno-Oncology

2. Define bottlenecks and areas of improvement to current tissue-based biomarker strategies.

NCI estimates that cancer will be the leading cause of death in 2030, worldwide.  Checkpoint inhibitors and adoptive cell therapies (ACTs) cost up to ~$2 million/patient and have shown durable responses in a few patients while many patients experience life-threatening side-effects. So, there is a critical need to identify cost-effective therapies to treat cancer with minimal to no side-effects.  Deep mapping of cancer antigens and their cognate immune cells is one approach that has the potential for more universal/targeted treatment. However, no methods exist that can identify cancer antigens and their cognate immune cells functionally and link phenotypic readouts to antigen and immune cell DNA sequences, 100k-1M cell combinations at a time. We will show proof-of-concept for the MOD platform that integrates three technologies after fluorescently-labeled single-cells are encapsulated in droplets – droplet-based cell-sorting and merging, single-cell-based interferon-gamma assays and selective sequencing of T-cell receptor variable regions based on interferon-gamma signals.

Learning Objectives:

1. Importance of linked neoantigen/TCR sequence information

2. Microenvironment on demand technology’s role in screening for adoptive cancer cell therapies (T-cell and CAR-Ts)

Adoptive cell immunotherapy (ACT) with genetically-modified T cells has shown impressive efficacy against some cancers, particularly CD19+ leukemias. However, ACT efficacy in solid tumors can be limited by restrictive tumor microenvironments (TMEs), with increased inhibitory signals, reduced T cell infiltration/accumulation, and inadequate metabolic substrates. FasL is a death receptor ligand that is overexpressed in the majority of human TMEs and can protect tumor cells from immunity. To overcome this particular barrier, engineered immunomodulatory fusion proteins (IFPs), combining the Fas receptor ectodomain with the 4-1BB costimulatory signaling domain, thereby converting a negative signal into a positive one. T cells engineered with the Fas-4-1BB IFP exhibited enhanced proliferation and anti-tumor function, and reduced exhaustion in vitro. Fas-4-1BB T cells displayed characteristics consistent with 4-1BB signaling, including altered metabolism, increased mitochondrial density, and expression of pro-survival signaling molecules. In vivo, Fas-4-1BB T cells exhibited improved persistence and eradicated lethal murine leukemia. In the autochthonous KPC pancreatic tumor model, IFP ACT resulted in increased T cell accumulation in the tumor and significantly improved survival. The Fas-4-1BB IFP also enhanced primary human T cell expansion and anti-tumor function in vitro, supporting clinical translation.

Learning Objectives:

1. Identify 3 barriers to immune cell response to cancer

2. List 3 approaches to overcome immunosuppressive barriers to bolster the immune response to cancer

3. Explain how fusion proteins can convert a negative signal to a positive boost for T cells

One of the main challenges in cancer immunotherapy is the resistance to I-O therapies. Primary resistance can occur before treatment and acquired resistance can occur in patients who responded to treatment but relapse after a period of effectiveness. Most cancer patients will develop primary resistance to checkpoint inhibitors, while 40% of those who don’t have primary resistance have an objective response. Although immunotherapy can induce tumor cell death and provoke significant clinical results, there are still unknown mechanisms that limit cancer immunotherapy.

Learning Objectives:

1. Understanding Primary and Adaptive Resistance to Immunotherapy

2. Monitoring Resistance Mechanisms

3. Overcoming Resistance to Immunotherapy