Target Immune Cells Research Articles

Abstract 4648: Using the IntelliCyt iQue Screener to simultaneously profile antibody dependent cell cytotoxicity and cytokine release of therapeutic antibodies

Abstract Therapeutic antibodies can mediate cancer cell killing through a variety of mechanisms including antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and complement dependent killing (CDC). ADCC is induced through the binding of the antibody Fc region to Fc receptors (FcRs) expressed on effector cells of the immune system. Granule exocytosis of perforin, granzyme B, the induction of Fas ligand (FasL) expression on immune cells and the release of cytokines are major mechanisms involved in targeted cancer cell killing. Traditional approaches to screen for antibody Fc effector functions focus on ADCC using a homogenous live/dead readout, which greatly limits the contextual and correlative value of the screening data. To overcome this limitation, a multiplex screening assay profiling ADCC using multiple cell death endpoints, and quantitating secreted proteins /cytokines was developed and analyzed using Intellicyt's iQue Screener PLUS and integrated ForeCyt software. The iQue Screener Plus is a high throughput flow cytometry platform featuring 3 lasers with 13 fluorescent channels and can sample a 384-well plate in 20 minutes. As a proof of concept, a small set of therapeutic antibodies directed against the same tumor antigen were used to induce immune cell-mediated killing of tumor cells using different effector to target cell ratios and a range of antibody concentrations. To demonstrate antibody specificity, antigen positive target cells, negative control target cells and effector immune cells were barcoded with different encoding dyes and included in the same well. At specific times, a small aliquot from each well was transferred to a new assay plate to run a multiplexed cell/bead mixture assay by the addition of a cocktail of fluorescent immunophenotyping antibodies, fluorescent dyes measuring unique apoptosis parameters, and a panel of QBeads for secreted protein detection. The samples were assayed on the IntelliCyt iQue Screener PLUS with simultaneous data analysis including multi-plate analysis for cell killing kinetics. Positive/negative target cells and immune cells were digitally segregated by differently encoded fluorescence. Time-dependent cell killing of antigen positive target cells were observed using the apoptosis markers mitochondria depolarization staining and cell membrane integrity, whereas little killing was measured in antigen negative cells. The antibody set showed a range of ADCC mediated killing and granule exocytosis/ cytokine release suggesting differences in Fc effector functions. These results highlight the streamlined workflow on IntelliCyt iQue Screener PLUS platform to profile therapeutic antibodies in a functional ADCC/cytokine release assay predicting their clinical efficacy. Citation Format: John O'Rourke. Using the IntelliCyt iQue Screener to simultaneously profile antibody dependent cell cytotoxicity and cytokine release of therapeutic antibodies [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 4648.

Abstract 2089: Highly multiplexed analysis of immune cell subsets in non-small cell lung cancer: validation of protein and RNA analysis by the Nanostring Digital Spatial Profiling (DSP) platform

Abstract Background: Highly multiplexed spatial analysis of tumor tissue is necessary to improve understanding of anti-tumor immunity. Moreover, spatial interrogation of protein and nucleic acid targets simultaneously can better characterize immune cell subsets and targets such as cytokines that are difficult to detect by immunohistochemistry (IHC). NanoString's Digital Spatial Profıling (DSP) technology can detect and quantify proteins and RNA at highly multiplexed levels. We compared DSP to flow cytometry (FCM), IHC and in-situ hybridization (ISH) to assess its sensitivity and specificity. Ongoing studies will assess the prevalence and distribution of immune cell subsets, cytokines and tumor cells in 10 non-small cell lung cancer (NSCLC) cases. Methods: 20 FFPE NSCLC cases and 1 peripheral blood mononuclear cell (PBMC) pellet were assayed on the DSP platform. Regions of interest (300 um x 400 um) or single cell masks were selected from H&E or fluorescence-stained sections. Serial sections were then probed with a cocktail of primary antibodies conjugated to DNA oligos via a UV-cleavable linker for 29 protein and probes for 39 RNA targets. Oligos from the ROIs were then released via UV-mediated linker cleavage and quantitated on the nCounter® Analysis System. Repeat analysis was performed on 10 cases to assess reproducibility between serial sections. A subset of cases was stained for CD3, CD4, CD8, CD45, PD-1, and PD-L1 by IHC and counted by image analysis. FCM was performed on the PBMC sample. Counts from both methods were compared to nCounter counts. Results: Comparison of DSP to FCM in PBMC showed good correlation (R2=0.65). Correlation between DSP counts and IHC was robust for CD3 (R2=0.72), CD4 (R2=0.80), and CD45 (R2=0.90). 21 of 29 protein markers showed good/excellent reproducibility (R2 >0.70) on separate DSP runs. 7 of 29 showed fair/good reproducibility across tissue sections (R2=0.50-0.70). RNA analysis of NSCLC samples by both DSP and ISH is ongoing. Analysis of both PBMC and NSCLC samples suggests that target abundance and section-to-section variability may explain differences in performance and reproducibility between sections. For example, low abundance in NSCLC tissue and section-to-section variability was observed for CD19 and CD56 with corresponding lower correlation between replicates (R2=0.68 and R2=0.52, respectively). Conclusion: DSP shows high section to section reproducibility and fair/good correlation with FCM and IHC. We find performance can be affected by differences in antibody clones or lots and/or section to section variability, particularly for low abundance cell types. Multiplexed RNA analysis of NSCLC samples is ongoing and will document the sensitivity and specificity of DSP compared to traditional ISH and the potential utility of DSP in assessing cytokines in the tumor microenvironment. Citation Format: James Ziai, Patrick Caplazi, Jérémie Decalf, Yan Liang, Patricia de Almeida, Daniel Zollinger, Alison Van Schoiack, Joseph Beechem, Jane Grogan, Matthew Albert. Highly multiplexed analysis of immune cell subsets in non-small cell lung cancer: validation of protein and RNA analysis by the Nanostring Digital Spatial Profiling (DSP) platform [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 2089.

Abstract 5562: A 3D image-based quantification of T cell-mediated killing of CRC organoids in the presence of immuno-modulators

Abstract BACKGROUND Cancer immunotherapy has already yielded promising clinical results but most patients still do not respond. The mechanisms of action of these treatment modalities are also not fully understood and the progress in this direction is hampered by a lack of appropriate pre-clinical testing models. To investigate the role of novel therapeutics targeting the immune cell compartment to kill tumor cells, we developed an in vitro assay based on 3D cultures and image based analysis in a 384-wells plate format. Immune cells are added to recapitulate the tumor micro-environment and its complex interactions between different cellular players. Specifically, infiltration of immune cells into the tumoroids and their killing are visualized and measured, enabling a better understanding of the immune-modulatory profile of different treatments. METHODS Autologous patient derived colon organoids from normal and tumor tissue from several patients were cultured in a 3D environment. HLA-matched PBMCs with and without activation were added and immune-cell infiltration and killing of the CRC organoids was visualized in 3D using automated microscopy. Quantification of immune cell effects was achieved with morphometric analysis with OMinerTM software. RESULTS 3D image data analysis enabled the discrimination of immune-tumor cell interactions and revealed a higher immune cell infiltration and tumoroid killing upon activation. In addition, we were able to compare both normal and tumor colon tissue from the same patient and compare between patients, demonstrating patient related differences and elucidating the effect of immune cell targeting on normal colon tissue. CONCLUSIONS The 3D assay presented here allows the analysis of different cell types that engage in a more realistic setting than when culturing them in traditional 2D cultures. Using image-based analysis, immune-tumor interactions can be visualized and quantified. The 3D environment, both for the cell culture and image analysis, allows for measurement of spatially resolved information, not accessible by monolayer cultures or biochemical assays. This new and innovative platform can empower immunotherapy drug developers to select the most promising candidates and better understand their mechanism of action. Citation Format: Gera Goverse, Lidia Daszkiewicz, Kuan Yan, Mohamed Tleis, Mariusz Madej, Lucia Salinaro, Leo Price. A 3D image-based quantification of T cell-mediated killing of CRC organoids in the presence of immuno-modulators [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 5562.

PO-374 A 3D image-based quantification of organoid-immune cell interactions in the presence of immuno-modulators

IntroductionIncreasing the number of cancer patients that benefit from immunotherapies and maintain durable responses requires understanding of cellular mechanisms that govern anti-tumour immune responses. However, this is hampered by a lack of appropriate pre-clinical testing models that are both clinically relevant and practical. Therefore, we have set up a robust in vitro assay that allows image based high-throughput analysis of 3D cultures. Here, immune cells are co-cultured with cancer cells in a 3D environment which recapitulates the tumour micro-environment and its complex cellular interactions. Specifically, infiltration of immune cells into the organoids and their killing are visualised and measured, which enables a better understanding of the immune-modulatory profile of different immunotherapies.Material and methodsAutologous colon organoids from normal and tumour tissue (Hubrecht Organoid Technology) from several patients were cultured in a 3D environment. Immune cells with and without activation were added and their infiltration into organoids and subsequent killing was visualised using high-content microscopy. Quantification of immune cell effects was achieved with morphometric analysis with OMinerTM software.Results and discussions3D image-based analysis of organoid-immune cell interactions demonstrated differential responses to immune-modulation between normal and tumour tissues and highlighted patient related differences. These results elucidate the effect of immune cell targeting normal colon tissue and discriminate immune-tumour cell interactions depending on activation status of T cells. The 3D environment, both for the cell culture and image analysis, allows for measurement of spatially resolved information, not accessible by monolayer cultures or biochemical assays.ConclusionThe 3D assay presented here allows the analysis of immuno-oncology drug effects on different cell types that engage in a more physiologically relevant spatial setting than when culturing them in traditional 2D cultures. Using image-based analysis, immune-tumour interactions can be visualised and quantified. Analysis of both healthy and tumour tissue from the same patient allows for better prediction of clinical outcome versus non-specific cytotoxic reactions. This new and innovative platform can empower immunotherapy drug developers with a tool to select the most promising candidates and understand their mechanism of action, which ultimately translate to better clinical performance.

Advance in Targeted Immunotherapy for Graft-Versus-Host Disease.

Graft-versus-host disease (GVHD) is a serious and deadly complication of patients, who undergo hematopoietic stem cell transplantation (HSCT). Despite prophylactic treatment with immunosuppressive agents, 20–80% of recipients develop acute GVHD after HSCT. And the incidence rates of chronic GVHD range from 6 to 80%. Standard therapeutic strategies are still lacking, although considerable advances have been gained in knowing of the predisposing factors, pathology, and diagnosis of GVHD. Targeting immune cells, such as regulatory T cells, as well as tolerogenic dendritic cells or mesenchymal stromal cells (MSCs) display considerable benefit in the relief of GVHD through the deletion of alloactivated T cells. Monoclonal antibodies targeting cytokines or signaling molecules have been demonstrated to be beneficial for the prevention of GVHD. However, these remain to be verified in clinical therapy. It is also important and necessary to consider adopting individualized treatment based on GVHD subtypes, pathological mechanisms involved and stages. In the future, it is hoped that the identification of novel therapeutic targets and systematic research strategies may yield novel safe and effective approaches in clinic to improve outcomes of GVHD further. In this article, we reviewed the current advances in targeted immunotherapy for the prevention of GVHD.

Targeting and Modulation of Liver Myeloid Immune Cells by Hard-Shell Microbubbles.

Poly n-butylcyanoacrylate (PBCA)-based hard-shell microbubbles (MB) have manifold biomedical applications, including targeted drug delivery or contrast agents for ultrasound (US)-based liver imaging. MB and their fragments accumulate in phagocytes, especially in the liver, but it is unclear if MB affect the function of these immune cells. We herein show that human primary monocytes internalize different PBCA-MB by phagocytosis, which transiently inhibits monocyte migration in vertical chemotaxis assays and renders monocytes susceptible to cytotoxic effects of MB during US-guided destruction. Conversely, human macrophage viability and function, including cytokine release and polarization, remain unaffected after MB uptake. After i.v. injection in mice, MB predominantly accumulate in liver, especially in hepatic phagocytes (monocytes and Kupffer cells). Despite efficiently targeting myeloid immune cells in liver, MB or MB after US-elicited burst do not cause overt hepatotoxicity or inflammation. Furthermore, MB application with or without US-guided burst does not aggravate the course of experimental liver injury in mice or the inflammatory response to liver injury in vivo. In conclusion, PBCA-MB have immunomodulatory effects on primary human myeloid cells in vitro, but do not provoke hepatotoxicity, inflammation or altered response to liver injury in vivo, suggesting the safety of these MB for diagnostic and therapeutic purposes.

Biomimetic Targeting of Nanoparticles to Immune Cell Subsets via Cognate Antigen Interactions.

Within the body, cellular recognition is mediated in large part by receptor-ligand interactions that result from the surface marker expression of the participant cells. In the case of immune cells, these interactions can be highly specific, enabling them to carry out their protective functions in fighting off infection and malignancy. In this work, we demonstrate the biomimetic targeting of antigen-specific immune cell populations by using nanoparticles functionalized with natural membrane derived from cells expressing the cognate antigen. Using red blood cell (RBC)-specific B cells as a model target, it is shown that RBC membrane-coated nanoparticles exhibit enhanced affinity compared with control nanoparticles. The concept is further demonstrated using murine models of alloimmunity and autoimmunity, where B cells elicited against RBCs can be positively labeled using the biomimetic nanoparticles. This strategy for antigen-specific immune cell targeting may have utility for the detection and treatment of various autoimmune conditions, and it may additionally have implications for the prevention of immune cell malignancies.

Role of the Immune System in Diabetic Kidney Disease.

The purpose of this review is to examine the proposed role of immune modulation in the development and progression of diabetic kidney disease (DKD). Diabetic kidney disease has not historically been considered an immune-mediated disease; however, increasing evidence is emerging in support of an immune role in its pathophysiology. Both systemic and local renal inflammation have been associated with DKD. Infiltration of immune cells, predominantly macrophages, into the kidney has been reported in a number of both experimental and clinical studies. In addition, increased levels of circulating pro-inflammatory cytokines have been linked to disease progression. Consequently, a variety of therapeutic strategies involving modulation of the immune response are currently being investigated in diabetic kidney disease. Although no current therapies for DKD are directly based on immune modulation many of the therapies in clinical use have anti-inflammatory effects along with their primary actions. Macrophages emerge as the most likely beneficial immune cell target and compounds which reduce macrophage infiltration to the kidney have shown potential in both animal models and clinical trials.

Combining Immunotherapy and Radiotherapy for Cancer Treatment: Current Challenges and Future Directions.

Since the approval of anti-CTLA4 therapy (ipilimumab) for late-stage melanoma in 2011, the development of anticancer immunotherapy agents has thrived. The success of many immune-checkpoint inhibitors has drastically changed the landscape of cancer treatment. For some types of cancer, monotherapy for targeting immune checkpoint pathways has proven more effective than traditional therapies, and combining immunotherapy with current treatment strategies may yield even better outcomes. Numerous preclinical studies have suggested that combining immunotherapy with radiotherapy could be a promising strategy for synergistic enhancement of treatment efficacy. Radiation delivered to the tumor site affects both tumor cells and surrounding stromal cells. Radiation-induced cancer cell damage exposes tumor-specific antigens that make them visible to immune surveillance and promotes the priming and activation of cytotoxic T cells. Radiation-induced modulation of the tumor microenvironment may also facilitate the recruitment and infiltration of immune cells. This unique relationship is the rationale for combining radiation with immune checkpoint blockade. Enhanced tumor recognition and immune cell targeting with checkpoint blockade may unleash the immune system to eliminate the cancer cells. However, challenges remain to be addressed to maximize the efficacy of this promising combination. Here we summarize the mechanisms of radiation and immune system interaction, and we discuss current challenges in radiation and immune checkpoint blockade therapy and possible future approaches to boost this combination.

Visualizing the Immune System: Providing Key Insights into HIV/SIV Infections.

Immunological inductive tissues, such as secondary lymphoid organs, are composed of distinct anatomical microenvironments for the generation of immune responses to pathogens and immunogens. These microenvironments are characterized by the compartmentalization of highly specialized immune and stromal cell populations, as well as the presence of a complex network of soluble factors and chemokines that direct the intra-tissue trafficking of naïve and effector cell populations. Imaging platforms have provided critical contextual information regarding the molecular and cellular interactions that orchestrate the spatial microanatomy of relevant cells and the development of immune responses against pathogens. Particularly in HIV/SIV disease, imaging technologies are of great importance in the investigation of the local interplay between the virus and host cells, with respect to understanding viral dynamics and persistence, immune responses (i.e., adaptive and innate inflammatory responses), tissue structure and pathologies, and changes to the surrounding milieu and function of immune cells. Merging imaging platforms with other cutting-edge technologies could lead to novel findings regarding the phenotype, function, and molecular signatures of particular immune cell targets, further promoting the development of new antiviral treatments and vaccination strategies.

Selective liposome targeting of folate receptor positive immune cells in inflammatory diseases

Activated macrophages play a key role in the development and maintenance of inflammatory diseases such as atherosclerosis, lupus, psoriasis, rheumatoid arthritis, ulcerative colitis, and many others. These activated macrophages, but not resting or quiescent macrophages highly up-regulate folate receptor beta (FR-β). This differential expression of FR-β provides a mechanism to selectively deliver imaging and therapeutic agents utilizing folate as a targeting molecule. In an effort to determine whether inflammatory diseases can be targeted utilizing a folate-linked nanosize carrier, a PEG-coated liposome was prepared that incorporated a folate conjugated PEG that also could transport imaging or therapeutic cargo. We demonstrate that these folate-liposomes specifically bind to folate receptor positive cells and accumulate at sites of inflammation in mouse models of colitis and atherosclerosis. These two animal models show that folate-targeted liposomes could be successfully utilized to deliver fluorescent molecules and an anti-inflammatory drug (betamethasone) for diagnostic and therapeutic applications.

Immunology: Is Actin at the Lytic Synapse a Friend or a Foe?

Cytotoxic Tcells and natural killer cells defend us against disease by secreting lytic granules. Whether actin facilitates or thwarts lytic granule secretion has been an open question. Recent results now indicate that the answer depends on the maturation stage of the immune cell-target cell contact.

P076 MICRORNA SAFEGUARDING THE INFLAMMATORY COLON STEM CELL NICHE

Inflammation triggers stem cell proliferation to repair the tissue damage, while abnormal gene expression can transform temporary hyperplasia into a fertile ground for tumorigenesis. We investigated the role and mechanism of microRNA miR-34a deficiency in inflammation-induced colon tumorigenesis. We treated wildtype and miR-34a-/- mice with DSS or infected them with Citrobacter rodentium and examined how miR-34a deficiency affects inflammation-induced colon tumorigenesis. We analyzed type and level of T helper cells infiltrated in infected colon. We investigated how Th17 cells and IL-17 regulate colon organoid growth. We systemically analyzed and validated miR-34a targets involved in Th17 cell infiltration, colon organoid growth and tumorigenesis. miR-34a deficiency leads to colon tumorigenesis after DSS treatment or Citrobacter rodentium infection. miR-34a targets both immune and epithelial stem cells to restrain inflammatory cytokine-induced stem cell proliferation (Fig. 1). miR-34a targets Interleukin 6 receptor (IL-6R) to suppress T helper 17 (Th17) cell differentiation, targets Interleukin 23 receptor (IL-23R) to block Th17 cell expansion and IL-17 production, targets chemokine CCL22 production to hinder Th17 cell recruitment to the colon epithelium, and targets an orphan receptor Interleukin 17 receptor D (IL-17RD) in colon stem cells to inhibit IL-17 induced stem cell proliferation. IP injection of IL-17 neutralizing antibody abrogates stem cell proliferation. Deletion of miR-34a in either colon epithelial cells alone (by generating a Lgr5-EGFP-CreERT2/miR-34aflox/flox strain) or in immune cells alone (bone marrow transplantation from miR-34a-/- mice) promoted tumorigenesis at much lower incidence rates. miR-34a acts as a central safeguard to protect the inflammatory colon stem cell niche by targeting both Th17 cells and colon epithelial stem cells during IBD reparative regeneration (Fig. 2).

Targeted Strategies for Mucosal Vaccination.

Mucosal immune responses are in the first line of defense against most infections and protective mucosal immunity can be achieved by mucosal vaccination. However, mucosal tolerance and physicochemical features of the mucosal environment pose challenging obstacles to the development of mucosal vaccines. Vaccine formulations must be designed to enhance stability at the mucosae and incorporate features that induce innate immunity at mucosal inductive sites. To face these challenges, a number of novel delivery systems for targeting of mucosal vaccines to specific mucosal locations have been developed. In addition, specific mucosal immune cell targeting can potentially be achieved with ligand-antigen bioconjugates, in particular, those directed to specific receptors expressed on Microfold (M) cells, mucosal epithelial cells, or mucosal antigen presenting cells (APCs). In this topical review, targeted strategies to enhance the effectiveness of mucosal vaccines are addressed, and obstacles to the design and progression of effective ligand-mediated mucosal vaccines are highlighted.

P076 MICRORNA SAFEGUARDING THE INFLAMMATORY COLON STEM CELL NICHE

Inflammation triggers stem cell proliferation to repair the tissue damage, while abnormal gene expression can transform temporary hyperplasia into a fertile ground for tumorigenesis. We investigated the role and mechanism of microRNA miR-34a deficiency in inflammation-induced colon tumorigenesis. We treated wildtype and miR-34a-/- mice with DSS or infected them with Citrobacter rodentium and examined how miR-34a deficiency affects inflammation-induced colon tumorigenesis. We analyzed type and level of T helper cells infiltrated in infected colon. We investigated how Th17 cells and IL-17 regulate colon organoid growth. We systemically analyzed and validated miR-34a targets involved in Th17 cell infiltration, colon organoid growth and tumorigenesis. miR-34a deficiency leads to colon tumorigenesis after DSS treatment or Citrobacter rodentium infection. miR-34a targets both immune and epithelial stem cells to restrain inflammatory cytokine-induced stem cell proliferation (Fig. 1). miR-34a targets Interleukin 6 receptor (IL-6R) to suppress T helper 17 (Th17) cell differentiation, targets Interleukin 23 receptor (IL-23R) to block Th17 cell expansion and IL-17 production, targets chemokine CCL22 production to hinder Th17 cell recruitment to the colon epithelium, and targets an orphan receptor Interleukin 17 receptor D (IL-17RD) in colon stem cells to inhibit IL-17 induced stem cell proliferation. IP injection of IL-17 neutralizing antibody abrogates stem cell proliferation. Deletion of miR-34a in either colon epithelial cells alone (by generating a Lgr5-EGFP-CreERT2/miR-34aflox/flox strain) or in immune cells alone (bone marrow transplantation from miR-34a-/- mice) promoted tumorigenesis at much lower incidence rates. miR-34a acts as a central safeguard to protect the inflammatory colon stem cell niche by targeting both Th17 cells and colon epithelial stem cells during IBD reparative regeneration (Fig. 2).View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Unsupervised capture and profiling of rare immune cells using multi-directional magnetic ratcheting.

Immunotherapies (IT) require induction, expansion, and maintenance of specific changes to a patient's immune cell repertoire which yield a therapeutic benefit. Recently, mechanistic understanding of these changes at the cellular level has revealed that IT results in complex phenotypic transitions in target cells, and that therapeutic effectiveness may be predicted by monitoring these transitions during therapy. However, monitoring will require unique tools that enable capture, manipulation, and profiling of rare immune cell populations. In this study, we introduce a method of automated and unsupervised separation and processing of rare immune cells, using high-force and multidimensional magnetic ratcheting (MR). We demonstrate capture of target immune cells using samples with up to 1 : 10 000 target cell to background cell ratios from input volumes as small as 25 microliters (i.e. a low volume and low cell frequency sample sparing assay interface). Cell capture is shown to achieve up to 90% capture efficiency and purity, and captured cell analysis is shown using both on-chip culture/activity assays and off-chip ejection and nucleic acid analysis. These results demonstrate that multi-directional magnetic ratcheting offers a unique separation system for dealing with blood cell samples that contain either rare cells or significantly small volumes, and the "sample sparing" capability leads to an expanded spectrum of parameters that can be measured. These tools will be paramount to advancing techniques for immune monitoring under conditions in which both the sample volume and number of antigen-specific target cells are often exceedingly small, including during IT and treatment of allergy, asthma, autoimmunity, immunodeficiency, cell based therapy, transplantation, and infection.

Generation of Innate Immune Reporter Cells Using Retroviral Transduction.

Innate immune cells are notoriously difficult to transfect; however, retroviruses can be used to stably integrate genes of interest into the host genome of primary or immortalized immune cells resulting in the generation of reporter cells. Here, we provide a detailed protocol covering the production of retroviruses, retroviral infection of innate immune target cells (including isolation and differentiation of murine bone marrow cells to macrophages), and several methods for enrichment of positively transduced cells.

Dual pH-responsive multifunctional nanoparticles for targeted treatment of breast cancer by combining immunotherapy and chemotherapy.

Combination of immunotherapy and chemotherapy is becoming a promising new treatment for cancer. The major challenge is to target cancer and immune cells simultaneously and specifically. In this study, a dual pH-responsive multifunctional nanoparticle system based on poly(L-histidine) and hyaluronic acid was designed for co-loading R848 (immune-regulator) and doxorubicin (chemotherapeutic drug) through different encapsulation modes. By responding to the acidic pHs of tumor microenvironment and intracellular organelles, this multifunctional nanoparticle system could release R848 extracellularly and deliver DOX targetedly to breast cancer cells, thus achieving synergistic effects of immunotherapy and chemotherapy against breast cancer.

HIV target cells in the female reproductive tract: effects of hormonal or intrauterine contraception and pregnancy

Objectives: Observational data suggest that depot medroxyprogesterone acetate (DMPA) and pregnancy increase HIV susceptibility. We compared proportions of immune and HIV target cells in the female reproductive tract from women who were and were not using hormonal or intrauterine contraceptives, and from pregnant women.

Pro-inflammatory chitosan/poly(γ-glutamic acid) nanoparticles modulate human antigen-presenting cells phenotype and revert their pro-invasive capacity.

The immune system is responsible to detect and destroy abnormal cells preventing the development of cancer. However, the immunosuppressive tumor microenvironment can compromise the immune response favoring tumor progression. Thus, immune system modulation towards an immunostimulatory profile can improve anticancer therapies. This research focus on the development of chitosan/poly(γ-glutamic acid) nanoparticles (NPs) to modulate human antigen-presenting cells (APCs) phenotype and to counteract their pro-invasive capacity. Interestingly, Ch/γ-PGA NPs had a prominent effect in inducing macrophages and dendritic cells immunostimulatory phenotype, thus favoring T cell proliferation and inhibiting colorectal cancer cell invasion. We propose that their combination with other immunomodulatory drugs or conventional anticancer therapies can improve patients' outcome.