Lack of functional TCR-epitope interaction is associated with herpes zoster through reduced downstream T cell activation.

The role of T cell receptor (TCR) diversity in infectious disease susceptibility is not well understood. We use a systems immunology approach on three cohorts of herpes zoster (HZ) patients and controls to investigate whether TCR diversity against varicella-zoster virus (VZV) influences the risk of HZ. We show that CD4+ T cell TCR diversity against VZV glycoprotein E (gE) and immediate early 63 protein (IE63) after 1-week culture is more restricted in HZ patients. Single-cell RNA and TCR sequencing of VZV-specific T cells shows that T cell activation pathways are significantly decreased after stimulation with VZV peptides in convalescent HZ patients. TCR clustering indicates that TCRs from HZ patients co-cluster more often together than TCRs from controls. Collectively, our results suggest that not only lower VZV-specific TCR diversity but also reduced functional TCR affinity for VZV-specific proteins in HZ patients leads to lower T cell activation and consequently affects the susceptibility for viral reactivation.

Unravelling the immune signature of herpes zoster: Insights into pathophysiology and the HLA risk profile.

The varicella-zoster virus (VZV) infects over 95% of the population. VZV reactivation causes herpes zoster (HZ), known as shingles, primarily affecting the elderly and immunocompromised individuals. However, HZ can also occur in otherwise healthy individuals. We analyzed the immune signature and risk profile in HZ patients using a genome-wide association study across different UK Biobank HZ cohorts. Additionally, we conducted one of the largest HZ HLA association studies to date, coupled with transcriptomic analysis of pathways underlying HZ susceptibility. Our findings highlight the significance of the MHC locus for HZ development, identifying five protective and four risk HLA alleles. This demonstrates that HZ susceptibility is largely governed by variations in the MHC. Furthermore, functional analyses revealed the upregulation of type I interferon and adaptive immune responses. These findings provide fresh molecular insights into the pathophysiology and the activation of innate and adaptive immune responses triggered by symptomatic VZV reactivation.

Diagnosing Viral Infections Through T-Cell Receptor Sequencing of Activated CD8+ T Cells.

T-cell-based diagnostic tools identify pathogen exposure but lack differentiation between recent and historical exposures in acute infectious diseases. Here, T-cell receptor (TCR) RNA sequencing was performed on HLA-DR+/CD38+CD8+ T-cell subsets of hospitalized coronavirus disease 2019 (COVID-19) patients (n = 30) and healthy controls (n = 30; 10 of whom had previously been exposed to severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]). CDR3α and CDR3ß TCR regions were clustered separately before epitope specificity annotation using a database of SARS-CoV-2-associated CDR3α and CDR3ß sequences corresponding to >1000 SARS-CoV-2 epitopes. The depth of the SARS-CoV-2-associated CDR3α/ß sequences differentiated COVID-19 patients from the healthy controls with a receiver operating characteristic area under the curve of 0.84 ± 0.10. Hence, annotating TCR sequences of activated CD8+ T cells can be used to diagnose an acute viral infection and discriminate it from historical exposure. In essence, this work presents a new paradigm for applying the T-cell repertoire to accomplish TCR-based diagnostics.

In vitro expansion of Wilms' tumor protein 1 epitope-specific primary T cells from healthy human peripheral blood mononuclear cells.

Wilms' tumor protein 1 (WT1) is a tumor-associated antigen overexpressed in various cancers. As a self-antigen, negative selection reduces the number of WT1-specific T cell receptors (TCRs). Here, we provide a protocol to generate WT137-45-specific TCRs using healthy human peripheral blood mononuclear cells. We describe the expansion of WT1-specific T cell clones by two consecutive in vitro stimulations with autologous WT137-45-pulsed dendritic cells and peripheral blood lymphocytes. We then detail the detection with human leukocyte antigen/WT137-45 tetramers.

Activation of Interferon-Stimulated Genes following Varicella-Zoster Virus Infection in a Human iPSC-Derived Neuronal In Vitro Model Depends on Exogenous Interferon-α.

Varicella-zoster virus (VZV) infection of neuronal cells and the activation of cell-intrinsic antiviral responses upon infection are still poorly understood mainly due to the scarcity of suitable human in vitro models that are available to study VZV. We developed a compartmentalized human-induced pluripotent stem cell (hiPSC)-derived neuronal culture model that allows axonal VZV infection of the neurons, thereby mimicking the natural route of infection. Using this model, we showed that hiPSC-neurons do not mount an effective interferon-mediated antiviral response following VZV infection. Indeed, in contrast to infection with Sendai virus, VZV infection of the hiPSC-neurons does not result in the upregulation of interferon-stimulated genes (ISGs) that have direct antiviral functions. Furthermore, the hiPSC-neurons do not produce interferon-α (IFNα), a major cytokine that is involved in the innate antiviral response, even upon its stimulation with strong synthetic inducers. In contrast, we showed that exogenous IFNα effectively limits VZV spread in the neuronal cell body compartment and demonstrated that ISGs are efficiently upregulated in these VZV-infected neuronal cultures that are treated with IFNα. Thus, whereas the cultured hiPSC neurons seem to be poor IFNα producers, they are good IFNα responders. This could suggest an important role for other cells such as satellite glial cells or macrophages to produce IFNα for VZV infection control.

Anti-Tumor Potency of Short-Term Interleukin-15 Dendritic Cells Is Potentiated by In Situ Silencing of Programmed-Death Ligands.

Dendritic cell (DC) vaccines have proven to be a valuable tool in cancer immune therapy. With several DC vaccines being currently tested in clinical trials, knowledge about their therapeutic value has been significantly increased in the past decade. Despite their established safety, it has become clear that objective clinical responses are not yet robust enough, requiring further optimization. Improvements of this advanced therapy medicinal product encompass, among others, regulating their immune stimulating capacity by in situ gene engineering, in addition to their implementation in combination therapy regimens. Previously, we have reported on a superior monocyte-derived DC preparation, including interleukin-15, pro-inflammatory cytokines and immunological danger signals in the culture process. These so-called IL-15 DCs have already proven to exhibit several favorable properties as cancer vaccine. Evolving research into mechanisms that could further modulate the immune response towards cancer, points to programmed death-1 as an important player that dampens anti-tumor immunity. Aiming at leveraging the immunogenicity of DC vaccines, we hypothesized that additional implementation of the inhibitory immune checkpoint molecules programmed death-ligand (PD-L)1 and PD-L2 in IL-15 DC vaccines would exhibit superior stimulatory potential. In this paper, we successfully implemented PD-L silencing at the monocyte stage in the 3-day IL-15 DC culture protocol resulting in substantial downregulation of both PD-L1 and PD-L2 to levels below 30%. Additionally, we validated that these DCs retain their specific characteristics, both at the level of phenotype and interferon gamma secretion. Evaluating their functional characteristics, we demonstrate that PD-L silencing does not affect the capacity to induce allogeneic proliferation. Ultimately designed to induce a durable tumor antigen-specific immune response, PD-L silenced IL-15 DCs were capable of surpassing PD-1-mediated inhibition by antigen-specific T cells. Further corroborating the superior potency of short-term IL-15 DCs, the combination of immune stimulatory components during DC differentiation and maturation with in situ checkpoint inhibition supports further clinical translation.

Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination.

Antigen recognition through the T cell receptor (TCR) αß heterodimer is one of the primary determinants of the adaptive immune response. Vaccines activate naïve T cells with high specificity to expand and differentiate into memory T cells. However, antigen-specific memory CD4 T cells exist in unexposed antigen-naïve hosts. In this study, we use high-throughput sequencing of memory CD4 TCRß repertoire and machine learning to show that individuals with preexisting vaccine-reactive memory CD4 T cell clonotypes elicited earlier and higher antibody titers and mounted a more robust CD4 T cell response to hepatitis B vaccine. In addition, integration of TCRß sequence patterns into a hepatitis B epitope-specific annotation model can predict which individuals will have an early and more vigorous vaccine-elicited immunity. Thus, the presence of preexisting memory T cell clonotypes has a significant impact on immunity and can be used to predict immune responses to vaccination.

Immune cells called CD4 T cells help the body build immunity to infections caused by bacteria and viruses, or after vaccination. Receptor proteins on the outside of the cells recognize pathogens, foreign molecules called antigens, or vaccine antigens. Vaccine antigens are usually inactivated bacteria or viruses, or fragments of these pathogens. After recognizing an antigen, CD4 T cells develop into memory CD4 T cells ready to defend against future infections with the pathogen. People who have never been exposed to a pathogen, or have never been vaccinated against it, may nevertheless have preexisting memory cells ready to defend against it. This happens because CD4 T cells can recognize multiple targets, which enables the immune system to be ready to defend against both new and familiar pathogens. Elias, Meysman, Bartholomeus et al. wanted to find out whether having preexisting memory CD4 T cells confers an advantage for vaccine-induced immunity. Thirty-four people who were never exposed to hepatitis B or vaccinated against it participated in the study. These individuals provided blood samples before vaccination, with 2 doses of the hepatitis B vaccine, and at 3 time points afterward. Using next generation immune sequencing and machine learning techniques, Elias et al. analyzed the individuals' memory CD4 T cells before and after vaccination. The experiments showed that preexisting memory CD4 T cells may determine vaccination outcomes, and people with more preexisting memory cells develop quicker and stronger immunity after vaccination against hepatitis B. This information may help scientists to better understand how people develop immunity to pathogens. It may guide them develop better vaccines or predict who will develop immunity after vaccination.

Novel Insights on MRGPRX2-Mediated Hypersensitivity to Neuromuscular Blocking Agents And Fluoroquinolones.

Neuromuscular blocking agents (NMBAs) like atracurium and rocuronium as well as fluoroquinolones (FQs) cause mast cell-mediated anaphylaxis by activating Mas-related G protein-coupled receptor X2 (MRGPRX2), but many questions remain unanswered. Here, we address three of them, namely whether primary human mast cells show similar activation by these drugs as murine mast cells and mast cell lines, how sugammadex protects from atracurium-induced MRGPRX2-mediated mast cell activation, and why some but not all patients treated with rocuronium develop anaphylaxis. We used peripheral blood-derived cultured mast cells from healthy donors and patients, assessed mast cell activation and degranulation by quantifying intracellular calcium and CD63 expression, respectively, and made use of MRGPRX2-silencing, via electroporation with Dicer-substrate small interfering RNAs, and single cell flow cytometric analyses. Atracurium, ciprofloxacin, and levofloxacin activated and degranulated primary human mast cells, but only MRGPRX2-positive and not MRGPRX2-negative or -silenced mast cells. Sugammadex attenuated the atracurium-induced and MRGPRX2-mediated activation and degranulation of human mast cells by reducing free atracurium levels. The mast cells of patients with IgE-independent anaphylaxis to rocuronium were similar, in their MRGPRX2 expression and function, to those of patients with IgE-mediated anaphylaxis. These findings further improve our understanding of the role and relevance of MRGPRX2-driven mast cell activation in anaphylactic reactions to NMBAs and FQs and may help to improve their prediction, prevention, and treatment.

The Ins and Outs of Messenger RNA Electroporation for Physical Gene Delivery in Immune Cell-Based Therapy.

Messenger RNA (mRNA) electroporation is a powerful tool for transient genetic modification of cells. This non-viral method of genetic engineering has been widely used in immunotherapy. Electroporation allows fine-tuning of transfection protocols for each cell type as well as introduction of multiple protein-coding mRNAs at once. As a pioneering group in mRNA electroporation, in this review, we provide an expert overview of the ins and outs of mRNA electroporation, discussing the different parameters involved in mRNA electroporation as well as the production of research-grade and production and application of clinical-grade mRNA for gene transfer in the context of cell-based immunotherapies.

Transcriptomic profiling of different responder types in adults after a Priorix® vaccination.

Thanks to the recommendation of a combined Measles/Mumps/Rubella (MMR) vaccine, like Priorix®, these childhood diseases are less common now. This is beneficial to limit the spread of these diseases and work towards their elimination. However, the measles, mumps and rubella antibody titers show a large variability in short- and long-term immunity. The recent outbreaks worldwide of measles and mumps and previous studies, which mostly focused on only one of the three virus responses, illustrate that there is a clear need for better understanding the immune responses after vaccination. Our healthy cohort was already primed with the MMR antigens in their childhood. In this study, the adult volunteers received one Priorix® vaccine dose at day 0. First, we defined 4 different groups of responders, based on their antibody titers' evolution over 4 time points (Day 0, 21, 150 and 365). This showed a high variability within and between individuals. Second, we determined transcriptome profiles using 3'mRNA sequencing at day 0, 3 and 7. Using two analytical approaches, "one response group per time point" and "a time comparison per response group", we correlated the short-term gene expression profiles to the different response groups. In general, the list of differentially expressed genes is limited, however, most of them are clearly immune-related and upregulated at day 3 and 7, compared to the baseline day 0. Depending on the specific response group there are overlapping signatures for two of the three viruses. Antibody titers and transcriptomics data showed that an additional Priorix vaccination does not facilitate an equal immune response against the 3 viruses or among different vaccine recipients.

Serum profiling identifies ibrutinib as a treatment option for young adults with B-cell acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) is a haematological malignancy that is characterized by the uncontrolled proliferation of immature lymphocytes. 80% of cases occur in children where ALL is well understood and treated. However it has a devastating affects on adults, where multi-agent chemotherapy is the standard of care with allogeneic stem cell transplantation for those who are eligible. New treatments are required to extend remission and prevent relapse to improve patient survival rates. We used serum profiling to compare samples from presentation adult B-ALL patients with age- and sex-matched healthy volunteer (HV) sera and identified 69 differentially recognised antigens (P ≤ 0·02). BMX, DCTPP1 and VGLL4 showed no differences in transcription between patients and healthy donors but were each found to be present at higher levels in B-ALL patient samples than HVs by ICC. BMX plays a crucial role in the Bruton's Tyrosine Kinase (BTK) pathway which is bound by the BTK inhibitor, ibrutinib, suggesting adult B-ALL would also be a worthy target patient group for future clinical trials. We have shown the utility of proto-array analysis of B-ALL patient sera, predominantly from young adults, to help characterise the B-ALL immunome and identified a new target patient population for existing small molecule therapy.

New targets for therapy: antigen identification in adults with B-cell acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) in adults is a rare and difficult-to-treat cancer that is characterised by excess lymphoblasts in the bone marrow. Although many patients achieve remission with chemotherapy, relapse rates are high and the associated impact on survival devastating. Most patients receive chemotherapy and for those whose overall fitness supports it, the most effective treatment to date is allogeneic stem cell transplant that can improve overall survival rates in part due to a 'graft-versus-leukaemia' effect. However, due to the rarity of this disease, and the availability of mature B-cell antigens on the cell surface, few new cancer antigens have been identified in adult B-ALL that could act as targets to remove residual disease in first remission or provide alternative targets for escape variants if and when current immunotherapy strategies fail. We have used RT-PCR analysis, literature searches, antibody-specific profiling and gene expression microarray analysis to identify and prioritise antigens as novel targets for the treatment of adult B-ALL.

Activation-induced surface proteins in the identification of antigen-responsive CD4 T cells.

Identification of antigen specificity of CD4 T cells is instrumental in understanding adaptive immune responses in health and disease. The high diversity of CD4 T cell repertoire combined with the functional heterogeneity of the compartment poses a challenge to the assessment of CD4 T cell responses. In spite of that, multiple technologies allow direct or indirect interrogation of antigen specificity of CD4 T cells. In the last decade, multiple surface proteins have been established as cytokine-independent surrogates of in vitro CD4 T cell activation, and have found applications in the live identification and isolation of antigen-responsive CD4 T cells. Here we review the current knowledge of the surface proteins that permit identification of viable antigen-responsive CD4 T cells with high specificity, including those capable of identifying specialized CD4 T subsets such as germinal center follicular helper T cells and CD4 regulatory T cells.

Dendritic Cell-Based and Other Vaccination Strategies for Pediatric Cancer.

Dendritic cell-based and other vaccination strategies that use the patient's own immune system for the treatment of cancer are gaining momentum. Most studies of therapeutic cancer vaccination have been performed in adults. However, since cancer is one of the leading causes of death among children past infancy in the Western world, the hope is that this form of active specific immunotherapy can play an important role in the pediatric population as well. Since children have more vigorous and adaptable immune systems than adults, therapeutic cancer vaccines are expected to have a better chance of creating protective immunity and preventing cancer recurrence in pediatric patients. Moreover, in contrast to conventional cancer treatments such as chemotherapy, therapeutic cancer vaccines are designed to specifically target tumor cells and not healthy cells or tissues. This reduces the likelihood of side effects, which is an important asset in this vulnerable patient population. In this review, we present an overview of the different therapeutic cancer vaccines that have been studied in the pediatric population, with a main focus on dendritic cell-based strategies. In addition, new approaches that are currently being investigated in clinical trials are discussed to provide guidance for further improvement and optimization of pediatric cancer vaccines.

Chimeric Antigen Receptor-Modified T Cell Therapy in Multiple Myeloma: Beyond B Cell Maturation Antigen.

Chimeric antigen receptor (CAR)-modified T cell therapy is a rapidly emerging immunotherapeutic approach that is revolutionizing cancer treatment. The impressive clinical results obtained with CAR-T cell therapy in patients with acute lymphoblastic leukemia and lymphoma have fueled the development of CAR-T cells targeting other malignancies, including multiple myeloma (MM). The field of CAR-T cell therapy for MM is still in its infancy, but remains promising. To date, most studies have been performed with B cell maturation antigen (BCMA)-targeted CARs, for which high response rates have been obtained in early-phase clinical trials. However, responses are usually temporary, and relapses have frequently been observed. One of the major reasons for relapse is the loss or downregulation of BCMA expression following CAR-T therapy. This has fostered a search for alternative target antigens that are expressed on the MM cell surface. In this review, we provide an overview of myeloma target antigens other than BCMA that are currently being evaluated in pre-clinical and clinical studies.

CD56 Homodimerization and Participation in Anti-Tumor Immune Effector Cell Functioning: A Role for Interleukin-15.

A particularly interesting marker to identify anti-tumor immune cells is the neural cell adhesion molecule (NCAM), also known as cluster of differentiation (CD)56. Namely, hematopoietic expression of CD56 seems to be confined to powerful effector immune cells. Here, we sought to elucidate its role on various killer immune cells. First, we identified the high motility NCAM-120 molecule to be the main isoform expressed by immune cells. Next, through neutralization of surface CD56, we were able to (1) demonstrate the direct involvement of CD56 in tumor cell lysis exerted by CD56-expressing killer cells, such as natural killer cells, gamma delta (γδ) T cells, and interleukin (IL)-15-cultured dendritic cells (DCs), and (2) reveal a putative crosstalk mechanism between IL-15 DCs and CD8 T cells, suggesting CD56 as a co-stimulatory molecule in their cell-to-cell contact. Moreover, by means of a proximity ligation assay, we visualized the CD56 homophilic interaction among cancer cells and between immune cells and cancer cells. Finally, by blocking the mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase (PI3K)-Akt pathway, we showed that IL-15 stimulation directly led to CD56 upregulation. In conclusion, these results underscore the previously neglected importance of CD56 expression on immune cells, benefiting current and future immune therapeutic options.

Tracking Dye-Independent Approach to Identify and Isolate In Vitro Expanded T Cells.

T cell proliferation is routinely identified in vitro using tracking dyes or through detecting intracellular upregulation of the nuclear protein, Ki-67. However, labeling with tracking dyes is cumbersome, associated with cellular toxicity, while Ki-67 cannot be used to identify and isolate viable T cells, and both techniques are incompatible with MACS technology. Here, we introduce a simple tool to identify and isolate in vitro T cell expansion that is tracking dye-independent and allows for sorting of viable T cells. We show that CD71, a transferrin receptor, and CD98, a heterodimer glycoprotein involved in both integrin signaling and amino-acid transport, are both highly upregulated on proliferating T cells upon in vitro stimulation, and that CD71 expression is maximal on the more recent progeny T cells, while CD98 upregulation remains stable across different generations of progeny T cells. Moreover, we demonstrate that the upregulation of CD71 and CD98 identifies CFSElow T cells and provides further proof of the antigen-specificity of T cells identified by CD71 and CD98 dual upregulation based on tetramer staining. We further show that CD71 can be used to enrich for in vitro expanding T cells using MACS technology. In conclusion, we show that CD71 and CD98 can be used to identify and isolate expanded T cells following in vitro stimulation and that CD71 is an MACS-compatible alternative to tracking dyes or Ki-67 detection. © 2019 International Society for Advancement of Cytometry.

Dendritic Cell-Based Immunotherapy of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a type of blood cancer characterized by the uncontrolled clonal proliferation of myeloid hematopoietic progenitor cells in the bone marrow. The outcome of AML is poor, with five-year overall survival rates of less than 10% for the predominant group of patients older than 65 years. One of the main reasons for this poor outcome is that the majority of AML patients will relapse, even after they have attained complete remission by chemotherapy. Chemotherapy, supplemented with allogeneic hematopoietic stem cell transplantation in patients at high risk of relapse, is still the cornerstone of current AML treatment. Both therapies are, however, associated with significant morbidity and mortality. These observations illustrate the need for more effective and less toxic treatment options, especially in elderly AML and have fostered the development of novel immune-based strategies to treat AML. One of these strategies involves the use of a special type of immune cells, the dendritic cells (DCs). As central orchestrators of the immune system, DCs are key to the induction of anti-leukemia immunity. In this review, we provide an update of the clinical experience that has been obtained so far with this form of immunotherapy in patients with AML.

Memory CD4+ T cell receptor repertoire data mining as a tool for identifying cytomegalovirus serostatus.

Pathogens of past and current infections have been identified directly by means of PCR or indirectly by measuring a specific immune response (e.g., antibody titration). Using a novel approach, Emerson and colleagues showed that the cytomegalovirus serostatus can also be accurately determined by using a T cell receptor repertoire data mining approach. In this study, we have sequenced the CD4+ memory T cell receptor repertoire of a Belgian cohort with known cytomegalovirus serostatus. A random forest classifier was trained on the CMV specific T cell receptor repertoire signature and used to classify individuals in the Belgian cohort. This study shows that the novel approach can be reliably replicated with an equivalent performance as that reported by Emerson and colleagues. Additionally, it provides evidence that the T cell receptor repertoire signature is to a large extent present in the CD4+ memory repertoire.

Efficient and Non-genotoxic RNA-Based Engineering of Human T Cells Using Tumor-Specific T Cell Receptors With Minimal TCR Mispairing.

Genetic engineering of T cells with tumor specific T-cell receptors (TCR) is a promising strategy to redirect their specificity against cancer cells in adoptive T cell therapy protocols. Most studies are exploiting integrating retro- or lentiviral vectors to permanently introduce the therapeutic TCR, which can pose serious safety issues when treatment-related toxicities would occur. Therefore, we developed a versatile, non-genotoxic transfection method for human unstimulated CD8+ T cells. We describe an optimized double sequential electroporation platform whereby Dicer-substrate small interfering RNAs (DsiRNA) are first introduced to suppress endogenous TCR α and ß expression, followed by electroporation with DsiRNA-resistant tumor-specific TCR mRNA. We demonstrate that double sequential electroporation of human primary unstimulated T cells with DsiRNA and TCR mRNA leads to unprecedented levels of transgene TCR expression due to a strongly reduced degree of TCR mispairing. Importantly, superior transgenic TCR expression boosts epitope-specific CD8+ T cell activation and killing activity. Altogether, DsiRNA and TCR mRNA double sequential electroporation is a rapid, non-integrating and highly efficient approach with an enhanced biosafety profile to engineer T cells with antigen-specific TCRs for use in early phase clinical trials.