T-cell Stimulation Assays Research Articles

A novel mass cytometry protocol optimized for immunophenotyping of low-frequency antigen-specific T cells.

Understanding antigen-specific T-cell responses, for example, following virus infections or allergen exposure, is of high relevance for the development of vaccines and therapeutics. We aimed on optimizing immunophenotyping of T cells after antigen stimulation by improving staining procedures for flow and mass cytometry. Our method can be used for primary cells of both mouse and human origin for the detection of low-frequency T-cell response using a dual-barcoding system for individual samples and conditions. First, live-cell barcoding was performed using anti-CD45 antibodies prior to an in vitro T-cell stimulation assay. Second, to discriminate between stimulation conditions and prevent cell loss, sample barcoding was combined with a commercial barcoding solution. This dual-barcoding approach is cell sparing and, therefore, particularly relevant for samples with low cell numbers. To further reduce cell loss and to increase debarcoding efficiency of multiplexed samples, we combined our dual-barcoding approach with a new centrifugation-free washing system by laminar flow (Curiox™). Finally, to demonstrate the benefits of our established protocol, we assayed virus-specific T-cell response in SARS-CoV-2-vaccinated and SARS-CoV-2-infected patients and compared with healthy non-exposed individuals by a high-parameter CyTOF analysis. We could reveal a heterogeneity of phenotypes among responding CD4, CD8, and gd-T cells following antigen-specific stimulations. Our protocol allows to assay antigen-specific responses of minute populations of T cells to virus-derived peptides, allergens, or other antigens from the same donor sample, in order to investigate qualitative and quantitative differences.

An Overview of Peptides and Peptide Pools for Antigen-Specific Stimulation in T-Cell Assays.

The analysis of antigen-specific T-cell responses has become routine in many laboratories. Functional T-cell assays like enzyme-linked-immuno-spot (ELISPOT), which depend on antigen-specific stimulation, increasingly use peptides to represent the antigen of interest. Besides single peptides, mixtures of peptides (peptide pools) are very frequently applied. Such peptide pools may, for example, represent entire proteins (with overlapping peptides covering a protein sequence) or include noncontiguous peptides such as a collection of T-cell-stimulating peptides. The optimum specification of single peptides or peptide pools for T-cell stimulation assays will depend on the purpose of the test, the target T-cell population, the availability of sample, requirements regarding reproducibility, and, last but not least, the available budget, to mention only the most important factors. Because of the way peptides are produced, they will always contain certain amounts of impurities such as peptides with deletions or truncated peptides, and there may be additional by-products of peptide synthesis. Optimized synthesis protocols as well as purification help reduce impurities that might otherwise cause false-positive assay results. However, specific requirements with respect to purity will vary depending on the purpose of an assay. Finally, storage conditions significantly affect the shelf life of peptides, which is relevant especially for longitudinal studies. The present book chapter addresses all of these aspects in detail. It should provide the researcher with all necessary background knowledge for making the right decisions when it comes to choosing, using, and storing peptides for ELISPOT and other T-cell stimulation assays.

Influence of the isolation method on characteristics and functional activity of mesenchymal stromal cell-derived extracellular vesicles

Background aimsExtracellular vesicle (EV) isolation methods are based on different physicochemical properties and may result in the purification of distinct EV populations. We compared two different isolation methods suitable for producing clinical-grade mesenchymal stromal cell-derived EVs (MSC-EVs)—ion exchange chromatography (IEX) and ultrafiltration (UF)—and evaluated their impact on the composition and functional properties of EVs. MethodsEVs were purified from conditioned culture medium using an anion exchange resin (IEX) or Amicon filters with a 100-kDa cutoff (UF) (MilliporeSigma, Burlington, MA, USA). We assessed nanoparticle size and distribution by nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS) and morphology by transmission electron microscopy. We also measured protein, lipid and total RNA concentration and immunophenotyped both EV populations by flow cytometry (MACSPlex assay; Miltenyi Biotec, Bergisch Gladbach, Germany). Moreover, immunomodulatory activity was tested using a standardized macrophage polarization assay and T-cell stimulation assay. Finally, proteomic analysis and cytokine quantification were carried out to better characterize both EV populations. ResultsWe found by both TRPS and NTA that IEX and UF yielded a comparable amount of total particles with similar size and distribution. In addition, a similar quantity of lipids was obtained with the two procedures. However, IEX yielded 10-fold higher RNA quantity and a larger amount of proteins than UF. MSC-EVs isolated from IEX and UF were positive for the exosome markers CD9, CD63 and CD81 and showed a comparable surface marker expression pattern. Both populations demonstrated immunomodulatory activity in vitro, as they prevented acquisition of the M1 phenotype in lipopolysaccharide-stimulated macrophages and inhibited acquisition of the activation markers CD69 and CD25 on T cells, but the IEX-EVs exerted a significantly greater immunomodulatory effect on both macrophages and T cells compared with UF-EVs. Proteomic analysis and gene ontology enrichment analysis revealed no major differences between the preparations. Finally, cytokine quantification revealed that IEX-EVs were more enriched in some crucial anti-inflammatory and immunomodulatory cytokines (e.g., IL-2, IL-10, transforming growth factor beta and vascular endothelial growth factor) compared with UF-EVs. ConclusionsMSC-EVs isolated by IEX and UF displayed similar physicochemical, phenotypic and functional characteristics. In our conditions, both EV populations demonstrated important anti-inflammatory activity in macrophages and T cells. However, IEX-EVs were more potent than UF-EVs, which may indicate the superiority of this method for the production of clinical-grade EVs.

Obesity in adolescents does not influence early immune responses to influenza vaccination

Background Obesity has been linked to reduced vaccine responses against tetanus, hepatitis B and influenza. Data on the influence of paediatric obesity on influenza vaccine response is still lacking and this study aims to fill the gap. Methods A total of 30 children with obesity and 30 children with normal weight, aged 12–18 years, were recruited. Participants were vaccinated with a tetravalent influenza vaccine. Blood was collected prior to the vaccination and again four weeks later. The humoral response was assessed with haemagglutinin inhibition assay. The cellular response was assessed with T-cell stimulation assays measuring TNF-α, IFN-γ, IL-2 and IL-13. Results Of the 29/30 from the study group and 30/30 from the control group finished both visits. Seroconversion occurred for > 90% of participants in both groups for the A/H1N1, A/H3N2 and B/Victoria strains, but the B/Yamagata strain had lower seroconversion rates (93% in the study group and 80% in the control group). 97-100% of participants from both groups had adequate serological responses following vaccination. Cellular responses were similar between the two groups post-vaccination. Conclusions Early humoral and cellular immune responses to influenza vaccinations are similar among adolescents with obesity and normal weight.

Clinical, Virologic, and Immunologic Evaluation of Symptomatic Coronavirus Disease 2019 Rebound Following Nirmatrelvir/Ritonavir Treatment.

Nirmatrelvir/ritonavir, the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protease inhibitor, reduces the risk of hospitalization and death by coronavirus disease 2019 (COVID-19) but has been associated with symptomatic rebound after therapy completion. Six individuals with relapse of COVID-19 symptoms after treatment with nirmatrelvir/ritonavir, 2 individuals with rebound symptoms without prior antiviral therapy and 7 patients with acute Omicron infection (controls) were studied. Soluble biomarkers and serum SARS-CoV-2 nucleocapsid protein were measured. Nasal swabs positive for SARS-CoV-2 underwent viral isolation and targeted viral sequencing. SARS-CoV-2 anti-spike, anti-receptor-binding domain, and anti-nucleocapsid antibodies were measured. Surrogate viral neutralization tests against wild-type and Omicron spike protein, as well as T-cell stimulation assays, were performed. High levels of SARS-CoV-2 anti-spike immunoglobulin G (IgG) antibodies were found in all participants. Anti-nucleocapsid IgG and Omicron-specific neutralizing antibodies increased in patients with rebound. Robust SARS-CoV-2-specific T-cell responses were observed, higher in rebound compared with early acute COVID-19 patients. Inflammatory markers mostly decreased during rebound. Two patients sampled longitudinally demonstrated an increase in activated cytokine-producing CD4+ T cells against viral proteins. No characteristic resistance mutations were identified. SARS-CoV-2 was isolated by culture from 1 of 8 rebound patients; Polybrene addition increased this to 5 of 8. Nirmatrelvir/ritonavir treatment does not impede adaptive immune responses to SARS-CoV-2. Clinical rebound corresponds to development of a robust antibody and T-cell immune response, arguing against a high risk of disease progression. The presence of infectious virus supports the need for isolation and assessment of longer treatment courses. NCT04401436.

TIGIT and PD1 Co-blockade Restores ex vivo Functions of Human Tumor-Infiltrating CD8+ T Cells in Hepatocellular Carcinoma

Background & AimsTIGIT is a co-inhibitory receptor, and its suitability as a target for cancer immunotherapy in HCC is unknown. PD1 blockade is clinically effective in about 20% of advanced HCC patients. Here we aim to determine whether co-blockade of TIGIT/PD1 has added value to restore functionality of HCC tumor-infiltrating T cells (TILs).MethodsMononuclear leukocytes were isolated from tumors, paired tumor-free liver tissues (TFL) and peripheral blood of HCC patients, and used for flow cytometric phenotyping and functional assays. CD3/CD28 T-cell stimulation and antigen-specific assays were used to study the ex vivo effects of TIGIT/PD1 single or dual blockade on T-cell functions.ResultsTIGIT was enriched, whereas its co-stimulatory counterpart CD226 was down-regulated on PD1high CD8+ TILs. PD1high TIGIT+ CD8+ TILs co-expressed exhaustion markers TIM3 and LAG3 and demonstrated higher TOX expression. Furthermore, this subset showed decreased capacity to produce IFN-γ and TNF-α. Expression of TIGIT-ligand CD155 was up-regulated on tumor cells compared with hepatocytes in TFL. Whereas single PD1 blockade preferentially enhanced ex vivo functions of CD8+ TILs from tumors with PD1high CD8+ TILs (high PD1 expressers), co-blockade of TIGIT and PD1 improved proliferation and cytokine production of CD8+ TILs from tumors enriched for PD1int CD8+ TILs (low PD1 expressers). Importantly, ex vivo co-blockade of TIGIT/PD1 improved proliferation, cytokine production, and cytotoxicity of CD8+ TILs compared with single PD1 blockade.ConclusionsEx vivo, co-blockade of TIGIT/PD1 improves functionality of CD8+ TILs that do not respond to single PD1 blockade. Therefore co-blockade of TIGIT/PD1 could be a promising immune therapeutic strategy for HCC patients.

Prolongation of rat-to-mouse islets xenograft survival by co-transplantation of autologous IL-10 differentiated murine tolerogenic dendritic cells.

Tolerogenic dendritic cells (DCs) represent a promising approach to promote transplantation tolerance. In this study, the potential of autologous bone marrow (BM)-derived murine DC to protect rat-to-mouse islets xenografts was analyzed. Tolerogenic DCs were generated by differentiating BM cells in the presence of granulocyte-macrophage colony-stimulating factor and interleukin 10 (IL-10, IL-10 DC). The phenotype of IL-10 DC was characterized in vitro by expression of costimulatory/inhibitory molecules (flow cytometry) and cytokines (Luminex and ELISA), their function by phagocytosis and T-cell stimulation assays. To study transplant tolerance in vivo, rat islets were transplanted alone or in combination with autologous murine IL-10 DC under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. Xenograft survival was evaluated by monitoring glycemia, cellular infiltration of xenografts by microscopy and flow cytometry 10days post-transplantation. Compared with control DC, IL-10 DC exhibited lower levels of major histocompatibility complex class II, costimulatory molecules (CD40, CD86, CD205), lower production of pro-inflammatory cytokines (IL-12p70, TNF, IL-6), and higher production of IL-10. Phagocytosis of xenogeneic rat splenocytes was not impaired in IL-10 DC, whereas stimulation of T-cell proliferation was reduced in the presence of IL-10 DC. Xenograft survival of rat islets in diabetic mice co-transplanted with autologous murine IL-10 DC was significantly prolonged from 12 to 21days, without additional immunosuppressive treatment. Overall, infiltration of xenografts by T cells and myeloid cells was not different in IL-10 DC recipient mice, but enriched for CD8+ T cells and myeloid cells with suppressor-associated phenotype. Autologous IL-10-differentiated DC with tolerogenic properties prolong rat-to-mouse islets xenograft survival, potentially by locally inducing immune regulatory cells, indicating their potential for regulatory immune cell therapy in xenotransplantation.

Rapid Assessment of Functional Avidity of Tumor-Specific T Cell Receptors Using an Antigen-Presenting Tumor Cell Line Electroporated with Full-Length Tumor Antigen mRNA.

The functional avidity of T-cell receptor (TCR)-engineered T cells towards their cognate epitope plays a crucial role in successfully targeting and killing tumor cells expressing the tumor-associated antigen (TAA). When evaluating in vitro functional T-cell avidity, an important aspect that is often neglected is the antigen-presenting cell (APC) used in the assay. Cell-based models for antigen-presentation, such as tumor cell lines, represent a valid alternative to autologous APCs due to their availability, off-the-shelf capabilities, and the broad range of possibilities for modification via DNA or messenger RNA (mRNA) transfection. To find a valuable model APC for in vitro validation of TAA Wilms’ tumor 1 (WT1)-specific TCRs, we tested four different WT1 peptide-pulsed HLA-A2+ tumor cell lines commonly used in T-cell stimulation assays. We found the multiple myeloma cell line U266 to be a suitable model APC to evaluate differences in mean functional avidity (EC50) values of transgenic TCRs following transfection in 2D3 Jurkat T cells. Next, to assess the dose-dependent antigen-specific responsiveness of WT1 TCR-engineered 2D3 T cells to endogenously processed epitopes, we electroporated U266 cells with different amounts of full-length antigen WT1 mRNA. Finally, we analyzed the functional avidity of WT1 TCR-transfected primary CD8 T cells towards WT1 mRNA-electroporated U266 cells. In this study, we demonstrate that both the APC and the antigen loading method (peptide pulsing versus full-length mRNA transfection) to analyze T-cell functional avidity have a significant impact on the EC50 values of a given TCR. For rapid assessment of the functional avidity of a cloned TCR towards its endogenously processed MHC I-restricted epitope, we showcase that the TAA mRNA-transfected U266 cell line is a suitable and versatile model APC.

Rapid Production of Physiologic Dendritic Cells (phDC) for Immunotherapy.

Generation of large numbers of dendritic cells (DC) for research or immunotherapeutic purposes typically involves in vitro conversion of murine bone marrow precursors or humanblood monocytes to DC via cultivation with supraphysiologic concentrations of cytokines such as GM-CSF and IL-4 for up to 7days. Alternatively, our group has recently established a new approach, based on the underlying mechanism of action of a widely used cancer immunotherapy termed Extracorporeal Photochemotherapy (ECP). Our method of rapid and cytokine-free production of therapeutically relevant DC populations,leveragingthe innate physiologic programs likely responsible for DC differentiation from blood monocytes in vivo, potentially offers a novel, inexpensive, and easily accessiblesource of DC for clinical and research uses. This approach involves ex vivo physiologic reprogramming of blood monocytes to immunologically tunable dendritic antigen-presenting cells, which we term "phDC," for physiological DC. To facilitate access and utilization of these new DC populations by the research community, in this chapter, we describethe use of a scaled-down version of the clinical ECP leukocyte-treatment devicetermed the Transimmunization (TI) chamberor plate, suitable for processing both mouse and human samples. We highlight the methodological sequences necessary to isolate mouse or human peripheral blood mononuclear cell (PBMC) from whole blood, and to expose those PBMC to the TI chamber for facilitating monocyte activation and conversion to physiological DC (phDC) through interaction with blood proteins and activated platelets under controlled flow conditions. We thenprovide sample protocols for potential applications of the generated DC, including their use as vaccinating antigen-presenting cells (APC) in murine in vivo antitumor models, and in human ex vivo T-cell stimulation and antigen cross-presentation assays which mimic clinical vaccination. We additionally highlight the technical aspects of loading mouse or human phDC with tumor-associated antigens (TAA) in the form of peptides or apoptotic tumor cells. We provide a simple and clinically relevant means to reprogram blood monocytes into functional APC, potentially replacing the comparatively expensive and clinically disappointing cytokine-derived DC which have previously dominated the dendritic cell landscape.

Immune checkpoint inhibitors reverse T-cell functional suppression in the bone marrow of a subset of AML patients

Abstract Acute Myeloid Leukemia is a blood cancer with poor prognosis despite decades of therapeutic development. Immune checkpoint inhibitors (ICIs) have been shown to be effective in many cancers, but their utility in AML has not been clearly established. To better understand the immune landscape of the AML microenvironment, we performed immunophenotyping on fresh bone marrow cells from AML patients using mass-spec based flow cytometry. In addition, we performed functional assays to evaluate T-cell proliferative capacity and determine whether AML bone marrow T cells can be modified by ICI treatment. Sixty two bone marrow samples from patients with AML were analyzed. We performed T-cell stimulation assays to measure proliferation and cytokine production in the presence and absence of ICIs. Samples were sub-categorized as being proliferators or non-proliferators based on the percent of T-cell division in response to TCR stimulation. Phenotypic analysis showed proliferation status correlated with a specific T-cell profile. Additionally, within the non-proliferator group, 70% overcame immune suppression and responded to at least one of the ICIs tested. In conclusion, we show that T-cell dysfunction is present in a subset of patients with AML. Nonetheless, this suppression can be reversed by ICIs in many cases, supporting the use of ICIs in AML therapy.

Abstract 5669: Establishment of a human CD3ε transgenic mouse model to assess anti-tumor efficacy of human T-cell-redirecting bispecific antibodies

Abstract T-cell redirecting therapy has taken a prominent area in immuno-oncology. T cells driven by a tumor-specific antigen, traffic into the tumor and initiate tumor killing. However, this is often hampered by inhibitory factors in the tumor microenvironment. In recent years, researchers have been actively developing T-cell redirecting bispecific antibodies, which binds to a specific tumor associated antigen (TAA) on tumor cells and CD3 (usually epsilon chain, CD3E) on T cells. This physically links the tumor cell to the T-cell which leads to MHC-independent recognition and killing of cells carrying the TAA. Several CD3 recruiting bispecific antibodies that have been approved are now in clinical trials and demonstrate promising efficacy. However, research on new therapeutic T-cell redirecting antibodies is often hampered by a lack of proper in vivo models, due to the absence of cross-reactivity with mouse CD3ε. We have now built a human CD3E BAC transgenic model in BALB/c background to address this issue. These transgenic mice express both human- and mouse CD3ε in more than 80% of the T cells. Unlike previously developed transgenic lines, where early T lymphocyte and natural killer cell development were blocked in mice with high copy numbers of the human CD3ε gene, our model is phenotypically normal with levels of T , B , and NK cells comparable to those in wild-type BALB/c mice. In an ex vivo T-cell stimulation assay, spleen-derived T cells could be activated by either anti-human CD3 antibody (OKT3) or anti-mouse CD3 antibody (145-2C11), indicated by significantly elevated CD25+/CD69+ population, as well as IL-2 and IFNγ release. In an efficacy assay, we inoculated syngeneic mouse CD20-expressing A20 lymphoma cells into the transgenic mice and treated with mCD20xmCD3 or mCD20xhCD3 bispecific antibodies, containing a human- or mouse CD3ε-specific CD3 arm, respectively. We found complete depletion of peripheral B cells 48 hours after dosing of either bispecific antibody. Treatment also induced 33% and 39% tumor growth inhibition at day 10, following 3 doses of 1 mg/kg mCD20xhCD3 and mCD20xmCD3 bispecific antibody, respectively. Taken together, our human CD3ε transgenic mice offer a novel model to assess the preclinical in vivo efficacy of human CD3-T-cell redirecting therapeutics. Citation Format: Mengmeng Yang, Mingkun Zhang, Sandra Verploegen, Patrick Engelberts, Yuxi Zhang, Lei Zheng, Keyi Zhu, Annie Xiaoyu An, Cunxiang Ju, Jing Zhao, Xiang Gao, Qian Shi, Davy Xuesong Ouyang. Establishment of a human CD3ε transgenic mouse model to assess anti-tumor efficacy of human T-cell-redirecting bispecific 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 5669.

Anti-Drug Antibodies: Emerging Approaches to Predict, Reduce or Reverse Biotherapeutic Immunogenicity.

The development of anti-drug antibodies (ADAs) following administration of biotherapeutics to patients is a vexing problem that is attracting increasing attention from pharmaceutical and biotechnology companies. This serious clinical problem is also spawning creative research into novel approaches to predict, avoid, and in some cases even reverse such deleterious immune responses. CD4+ T cells are essential players in the development of most ADAs, while memory B-cell and long-lived plasma cells amplify and maintain these responses. This review summarizes methods to predict and experimentally identify T-cell and B-cell epitopes in therapeutic proteins, with a particular focus on blood coagulation factor VIII (FVIII), whose immunogenicity is clinically significant and is the subject of intensive current research. Methods to phenotype ADA responses in humans are described, including T-cell stimulation assays, and both established and novel approaches to determine the titers, epitopes and isotypes of the ADAs themselves. Although rational protein engineering can reduce the immunogenicity of many biotherapeutics, complementary, novel approaches to induce specific tolerance, especially during initial exposures, are expected to play significant roles in future efforts to reduce or reverse these unwanted immune responses.

ITOC2 – 021. The melanoma immune-peptidome for T-cell-based anti-tumour immunotherapies

Metastatic malignant melanoma (MMM) is a highly aggressive disease. Immune checkpoint inhibitors have proved immunotherapeutic efficacy; however, not all patients profit and severe side effects may occur. Currently, little is known about the tumour-specific targets and the tumour-reactive effector cells that are involved. Aiming to characterise the melanoma immune-peptidome, we purified HLA-I and HLA-II complexes from MMM patient tumours and accurately identified thousands of unique HLA binding peptides per tissue sample, using state-of-the-art mass spectrometry. We applied algorithms for database search in combination with de-novo sequencing and identified many novel cancer-associated peptides from known melanoma-specific antigens (PRAME, Tyrosinase, MAGEA3, PMEL, CTAG1A, PAX3 etc.). We further extended our search to post-translationally modified peptides that potentially serve as neo-antigens. After in vitro T-cell stimulation assays, we detected elevated levels of peptide-specific T-cells that displayed functional responses against peptide-pulsed target cells as well as tumour reactivity. We established this large-scale resource of the melanoma in vivo presented immune-peptidome that contains many novel targets validated by immune reactivity. These data provide the fundament for further optimisation of treatment modalities for this life-threatening disease.

Distinct phenotypic features of neonatal murine macrophages.

Neonates rely on their innate immune system. Resident tissue macrophages are considered to be initiators and regulators of the innate immune response and thus, appear to be especially important to neonates. We hypothesized that the phenotype and function of neonatal tissue macrophages differ from their adult counterparts. Peritoneal macrophages from neonatal (<24 h) and adult (6 weeks old) C57BL/6J mice were isolated and analyzed by high-content chipcytometry. After stimulation for 6 h with LPS (0, 1, 10, 100 ng/mL), macrophage transcriptome was analyzed by microarray and cytokine release was measured using multiparametric bead assays. Antigen presenting capacity was compared by T-cell stimulation assays. We observed that neonatal murine peritoneal macrophages are characterized by selective lack of expression of F4/80, MHC class II, and costimulatory molecules (CD80, CD86). Furthermore, we found differences in the transcriptome between neonatal and adult macrophages, unstimulated and after LPS stimulation. Although neonatal macrophages showed a significantly increased secretion of proinflammatory cytokines upon LPS stimulation, their potential to induce T-cell proliferation was significantly reduced. In conclusion, we observed a distinct phenotype of the neonatal macrophage population. The specific functions of this macrophage population could help to understand the excessive inflammatory reactions observed in the very young.

EmTIP, a T-Cell Immunomodulatory Protein Secreted by the Tapeworm Echinococcus multilocularis Is Important for Early Metacestode Development

BackgroundAlveolar echinococcosis (AE), caused by the metacestode of the tapeworm Echinococcus multilocularis, is a lethal zoonosis associated with host immunomodulation. T helper cells are instrumental to control the disease in the host. Whereas Th1 cells can restrict parasite proliferation, Th2 immune responses are associated with parasite proliferation. Although the early phase of host colonization by E. multilocularis is dominated by a potentially parasitocidal Th1 immune response, the molecular basis of this response is unknown.Principal FindingsWe describe EmTIP, an E. multilocularis homologue of the human T-cell immunomodulatory protein, TIP. By immunohistochemistry we show EmTIP localization to the intercellular space within parasite larvae. Immunoprecipitation and Western blot experiments revealed the presence of EmTIP in the excretory/secretory (E/S) products of parasite primary cell cultures, representing the early developing metacestode, but not in those of mature metacestode vesicles. Using an in vitro T-cell stimulation assay, we found that primary cell E/S products promoted interferon (IFN)-γ release by murine CD4+ T-cells, whereas metacestode E/S products did not. IFN-γ release by T-cells exposed to parasite products was abrogated by an anti-EmTIP antibody. When recombinantly expressed, EmTIP promoted IFN-γ release by CD4+ T-cells in vitro. After incubation with anti-EmTIP antibody, primary cells showed an impaired ability to proliferate and to form metacestode vesicles in vitro.ConclusionsWe provide for the first time a possible explanation for the early Th1 response observed during E. multilocularis infections. Our data indicate that parasite primary cells release a T-cell immunomodulatory protein, EmTIP, capable of promoting IFN-γ release by CD4+ T-cells, which is probably driving or supporting the onset of the early Th1 response during AE. The impairment of primary cell proliferation and the inhibition of metacestode vesicle formation by anti-EmTIP antibodies suggest that this factor fulfills an important role in early E. multilocularis development within the intermediate host.

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended. Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3. In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity (1-8). Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines (9-12). Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication (13). Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended. Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSC(high)) cell population and are separated by fluorescence activated cell sorting (3). In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads (14, 15), flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses (3, 4).

F8 and HLA-II Haplotypes in the Hispanic Population: Implications for Inhibitor Risk Development in Hispanic Hemophilia A Patients

AbstractAbstract 3365 Background:A recent study suggested an increased rate of inhibitor development among Hispanic compared to White hemophilia A (HA) patients; however, a possible mechanism was not proposed or explored. In light of findings implicating a role for race-specific distributions of factor VIII (F8) and class-II human-leukocyte antigen (HLA-II) haplotypes on the disparate risk of inhibitor development in the Black HA population, we sought to determine the distributions of F8 and HLA-II haplotypes among Hispanic Americans and compare them to that of Black and White Americans. Methods:Using archived genomic DNA samples from the 194 unrelated founders of the San Antonio Family Heart Study (SAFHS), a collection of 1431 people from 41 large Mexican American families, we re-sequenced all exons of F8 to genotype the known nonsynonymous-single nucleotide polymorphisms (ns-SNPs) and identify any novel ns-SNPs. We then performed high-resolution HLA-II genotyping to identify each founders' pair of HLA-DRB1 alleles to compare them against those found in other ethnic groups. Results:Among the 291 potentially distinct Mexican American X-chromosomes evaluated, we identified the H2 F8 haplotype, defined by D1241E, in 25.0% of the subjects, which is in between that observed in the White (7.4%) and Black (37.4%) populations. We also found H3 F8, defined by D1241E and M2238V, in two subjects, who represent the first non-Black individuals reported to carry this haplotype. Furthermore, we discovered a previously unreported ns-SNP (H1919N), whose minor allele was found in only one male and defines a ninth wild-type F8 haplotype (H9). Regarding HLA-II alleles, the distribution among Mexican Americans in the present study was quite different from those found in Black and White individuals in the National Marrow Donor Program registry (see Figure). Three of the 4 most common HLA-II alleles in the Mexican Americans (DRB1*0802, DRB1*0407 and DRB1*1406) were seen in < 1% of both Blacks and Whites. Discussion:This is the first study to report the haplotypic prevalence of F8 and HLA-II alleles in Mexican Americans, the largest Hispanic ethnic group in the United States. By informing specific wild-type factor VIII (FVIII) peptides for use in HLA-II binding and T-cell stimulation assays, these results may help to identify high risk combinations of FVIII therapeutics and individual HLA-II repertoires that contribute to the higher rate of inhibitor development observed in Mexican versus Caucasian American HA patients. [Display omitted] Disclosures:Viel:Histonis, Incorporated: Employment. Howard:Haplomics, Inc.: Equity Ownership.

Silencing the expression of platelet endothelial cell adhesion molecule‐1 prevents allogeneic T‐cell cytotoxicity

After solid organ transplantation, the endothelium is the first allorecognition checkpoint of the recipient's immune system. A major player at this interface is the platelet endothelial cell adhesion molecule-1 (PECAM-1), which is an immunoglobulin-like glycoprotein, involved in white blood cell migration, cellular adhesion, and signal transduction. The potential of preventing allorecognition at this interface was explored by knocking down PECAM-1 expression using RNA interference. Lentiviral-based vectors encoding short-hairpin RNA sequences specific for PECAM-1 transcripts were used for the transduction of monocytic and endothelial cell lines. Expression of PECAM-1 decreased by up to 80% at mRNA and protein levels on monocytic and endothelial cell lines. Antigen-binding capacity assays likewise showed up to 80% reduction of PECAM-1 expression on cell surfaces. In allogeneic T-cell stimulation assays, T cells stimulated with PECAM-1-silenced monocytes had granzyme B mRNA levels up to 85% lower than those in T cells stimulated with monocytes expressing nonspecific shRNA. Also, T-cell cytotoxicity showed to decrease significantly against PECAM-1-silenced monocytes versus those nonsilenced for PECAM-1. Moreover, the former T cells did not secrete interferon-γ and exhibited reduced proliferation. These findings suggest that permanent silencing of PECAM-1 expression is feasible and contributes to efficiently inhibiting the cytotoxic T-cell response to allogeneic target cells. Thus, targeting both the immunologic synapse at the regulatory interface and the lymphocyte migration might provide a new strategy to overcome allogeneic transplant rejection.

Nonsteroidal anti‐inflammatory drugs do not interfere with imiquimod treatment for usual type vulvar intraepithelial neoplasia

Imiquimod has been shown to be an effective treatment for usual type vulvar intraepithelial neoplasia (uVIN). Since local inflammation and burning are common side effects, patients often use nonsteroidal anti-inflammatory drugs (NSAIDs). Our study investigated whether NSAID-use, which has been documented to inhibit the cell-mediated immune response, interferes with the outcome of imiquimod treatment. Monocyte-derived dendritic cells (moDCs) and Langerhans cells (moLCs) were cultured in the presence of NSAIDs. The expression of relevant surface markers (CD80, CD86, CD40, HLA-DR, CCR6 and CCR7), stimulatory function, and cytokine production were evaluated. Furthermore, we analyzed in uVIN patients whether frequent NSAID-use had an effect on the clinical response and on immunocompetent cell counts before and after imiquimod treatment. Although an effect was observed on the expression of moDC and moLC maturation markers, NSAIDs did not affect the ability of moDCs and moLCs to stimulate allogeneic T-cell proliferation, or the production of cytokines in an allogeneic T-cell stimulation assay. In agreement with this, in uVIN patients treated with imiquimod, no interference of frequent NSAID-use with clinical outcome was observed. However, we did notice that high CD1a(+) and CD207(+) cell counts in frequent NSAID-users before treatment seemed to predict a favourable response to imiquimod treatment. Our data indicate that NSAID-use does not seem to interfere with moDC and moLC function and does not interfere with immunomodulatory properties of imiquimod in uVIN patients. Therefore, NSAIDs can safely be used to reduce imiquimod side effects in uVIN patients during treatment.