Notes

Sewall Wright's critique from the gene's-eye check out advancement.
© 2020 Elsevier Inc. All rights reserved.Dendritic cells have been widely investigated in cancer immunotherapy clinical trials for the last two decades mainly due to their robust ability to elicit an adaptive anticancer immune response of the cellular and humoral types. Immature DCs can be easily loaded with desired antigens. However, to become efficient antigen-presenting cells, DCs must first undergo a process of maturation. Protocols for the generation of DCs for use in cancer immunotherapy, including the generation of a large number of immature DCs for antigen pulsing and the selection of a well-defined immunostimulatory agent to achieve complete and reproducible maturation, which is a crucial step for further stimulation of T cell activation, must carefully consider the characteristics of DC physiology. In this report, we provided a detailed protocol for DC generation, pulsation and activation with the subsequent induction of T cell-specific immune responses. © 2020 Elsevier Inc. All rights reserved.Dendritic cells (DCs) are professional antigen-presenting cells, which are optimal for the priming of a T cell response against pathogens and tumors. Therefore, many efforts are made to develop therapeutic cancer vaccines which preferentially target the antigen to DC subsets. To this aim, we developed two types of recombinant fusion proteins, which favor antigen delivery to pro-inflammatory DCs as well as the crosstalk between specialized subpopulations of DCs. The first approach combines peptide/CpG vaccination with the recruitment of iNKT cells to the tumor site via CD1d-antitumor scFv fusion proteins. The second approach is targeting the tumor antigen to cross-presenting Xcr1+ DCs via a fusion protein made of Xcl1 fused to a synthetic long peptide followed by an IgG1 Fc fragment. Both strategies allow a potent tumor-specific CD8 T cell response associated with tumor regression or tumor growth delay depending on the model. In the case of iNKT cell activation, the strategy relies on a strong IL-12 release by splenic DCs, while in the second case, the T cell response is strictly dependent on the presence of Xcr1+ cross-presenting DCs. © 2020 Elsevier Inc. All rights reserved.The interaction strength between CD8+ T cells' TCR and cognate peptide-MHC (pMHC) impacts on the CD8+ T cell response against pathogens and tumors (Martinez-Usatorre, Donda, Zehn, & Romero, 2018; Zehn, Lee, & Bevan, 2009). CD8+ T cell responses against tumors are characterized by the presence of low affinity CD8+ T cells specific for nonmutated tumor associated self-antigens (TAA) and potentially high affinity tumor specific CD8+ T cells recognizing mutated self-antigens (Gros et al., 2016; Kvistborg et al., 2012; McMahan & Slansky, 2007). High affinity T cells display enhanced survival, expansion capacity and tumor control (Martinez-Usatorre et al., 2018; Schmid et al., 2010). In fact, recent clinical trials using neoantigen tumor vaccines showed prolonged progression free survival in melanoma patients (Ott et al., 2017; Sahin et al., 2017), while only modest clinical efficacy was obtained with TAA vaccines (Romero et al., 2016). However, the highly individual nature of neoantigens constitutes a major technical and economical hurdle for routine clinical application. Thus, the characterization of TAA-specific CD8+ T cell responses may reveal new strategies to enhance their anti-tumor properties. In parallel, the identification of high affinity antigens and CD8+ T cells may be essential to design effective tumor vaccines and adoptive cell transfer therapies. Therefore, in this chapter, we describe how to generate tumor cell lines with stable expression of affinity-ranged antigens and methods to assess T-cell affinity. © 2020 Elsevier Inc. All rights reserved.Understanding the interactions between immune and cancer cells occurring within the tumor microenvironment is a prerequisite for successful and personalized anti-cancer therapies. Microfluidic devices, coupled to advanced microscopy systems and automated analytical tools, can represent an innovative approach for high-throughput investigations on immune cell-cancer interactions. In order to study such interactions and to evaluate how therapeutic agents can affect this crosstalk, we employed two ad hoc fabricated microfluidic platforms reproducing advanced 2D or 3D tumor immune microenvironments. In the first type of chip, we confronted the capacity of tumor cells embedded in Matrigel containing one drug or Matrigel containing a combination of two drugs to attract differentially immune cells, by fluorescence microscopy analyses. In the second chip, we investigated the migratory/interaction response of naïve immune cells to danger signals emanated from tumor cells treated with an immunogenic drug, by time-lapse microscopy and automated tracking analysis. We demonstrate that microfluidic platforms and their associated high-throughput computed analyses can represent versatile and smart systems to (i) monitor and quantify the recruitment and interactions of the immune cells with cancer in a controlled environment, (ii) evaluate the immunogenic effects of anti-cancer therapeutic agents and (iii) evaluate the immunogenic efficacy of combinatorial regimens with respect to single agents. © 2020 Elsevier Inc. All rights reserved.Critical to the advancement of tumor immunotherapy is the reliable identification of responders and the quantification of the tumor-specific immune response elicited by treatments. In this regard, Enzyme-Linked Immunospot assay (ELISpot) is an ideal monitoring technique due to its high sensitivity, ease of execution and cost-effectiveness. Originally developed for the enumeration of B cells secreting antigen-specific antibodies, ELISpot assay has been adapted to detect and quantify cytokine-secreting immune cells present at low frequency in a variety of biological samples, including blood, in response to antigen-specific stimuli. The above-mentioned features emphasize the role of ELISpot as valuable assay for translational research and clinical applications. In the present chapter, we will focus on the use of ELISpot assay for monitoring the tumor-specific effector responses induced by different treatments in preclinical models and will provide some protocols and technical hints for its application. © 2020 Elsevier Inc. All rights reserved.Monoclonal antibody-based therapies are increasingly being used to treat cancer. Some mediate their therapeutic effects through modifying the function of immune cells globally, while others bind directly to tumor cells and can recruit immune effector cells through their Fc regions. As new direct-binding agents are developed, having the ability to test their Fc-mediated functions in a high-throughput manner is important for selecting antibodies with immune effector properties. Here, using monoclonal anti-CD20 antibody (rituximab) as an example and the CD20+ Raji cell line as tumor target, we describe flow cytometry-based assays for determining an antibody's capacity for mediating antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). These assays are sensitive, reliable, affordable and avoid the use of radioactivity. © 2020 Elsevier Inc. All rights reserved.APCs play a key role at initiating adaptive immune responses by presenting antigens to lymphocytes and DCs are professional APCs. It is critical to understand the differential antigen capture and presentation ability of different DC subsets, which is important for DC-targeted immunotherapy. Smoothened Agonist in vitro In this section, we give a brief introduction to different antigen presentation pathways and introduce the key concept of cross-presentation, the major antigen presentation pathway used for anti-viral and anti-tumoral immune responses. CD205, a DC restricted receptor, is highly expressed on certain DCs subsets. We find CD205-mediated antigen uptake to be a useful model for studying antigen uptake and defects. These methods provide an introduction to CD205-mediated pre-clinical delivery of antigens to cross-presenting DCs, which can be adapted to the study of targeting to multiple receptors and other C-type lectins. This is a promising strategy to detect the antigen capture capacity and to study the key players orchestrating tolerance and immunity ex vivo. © 2020 Elsevier Inc. All rights reserved.Dendritic cells (DCs) are specialized antigen presenting cells (APCs) able to intake and crosspresent antigens (Ags) on major histocompatibility complex (MHC) class I and II molecules to T cells thus initiating primary and memory immune responses. DC-mediated Ag uptake and crosspresentation represent crucial steps toward cancer recognition and eventually elimination. Cytofluorometry is a standardized procedure to study phagocytosis. By fast and reproducible single cell measurements, flow cytometry allows for simultaneous biochemical and functional analyses of Ag intake. In this chapter, we discuss a two-color flow cytometric analysis of DC-mediated uptake of apoptotic bodies. We also show data on the adjuvanticity of Type-I-interferons (Type-I-IFNs) during Ag retention as we offer a guideline and a range of advice on sample preparation and acquisition. © 2020 Elsevier Inc. All rights reserved.Supplementation of standard cancer therapies (radiotherapy, chemotherapy, surgery) with immunotherapies has revolutionized cancer treatment. In order to include recent improvements of multimodal therapies into clinical routine, knowledge about the immune status, the immune dynamics and the detailed composition and activation of patient's immune system is required. The here presented single-tube multicolor flow cytometry assay allows the discrimination of 20 clinically relevant immune cell subsets and their activation status in peripheral whole blood. It includes 15 different antibodies and can be established on a common 3 laser and 10 color flow-cytometer. Furthermore, this assay is easy to set-up and to perform as well as fast with only 40min of sample preparation time. Moreover, only 100μL of whole blood are sufficient for this precise determination of the individual immune status. It is already applied in translational programs of clinical studies and trials and can further be adapted for future ones. © 2020 Elsevier Inc. All rights reserved.Gliomas are the most common malignant brain tumors in the pediatric population. Even though great efforts have been made to understand their distinctive molecular characteristics, there has not been any improvements in the median survival in decades. In children, high-grade glial tumors have a median survival of 9-15 months. It has recently been demonstrated that pediatric high-grade gliomas (pHGG) are biologically and molecularly different from the adult counterparts, which could explain why conventional treatments universally fail. The development of an in vivo pHGG model harboring the specific genetic alterations encountered in pediatric gliomas is imperative in order to study the molecular basis that drives the progression and aggressiveness of these tumors. It would also enable harnessing these results for the development of novel therapeutic approaches. Our lab has implemented a method to induce brain tumors using transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mouse brain.
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