Analysis of Wilms' tumor protein 1 specific TCR repertoire in AML patients uncovers higher diversity in patients in remission than in relapsed.

The Wilms' tumor protein 1 (WT1) is a well-known and prioritized tumor-associated antigen expressed in numerous solid and blood tumors. Its abundance and immunogenicity have led to the development of different WT1-specific immune therapies. The driving player in these therapies, the WT1-specific T-cell receptor (TCR) repertoire, has received much less attention. Importantly, T cells with high affinity against the WT1 self-antigen are normally eliminated after negative selection in the thymus and are thus rare in peripheral blood. Here, we developed computational models for the robust and fast identification of WT1-specific TCRs from TCR repertoire data. To this end, WT137-45 (WT1-37) and WT1126-134 (WT1-126)-specific T cells were isolated from WT1 peptide-stimulated blood of healthy individuals. The TCR repertoire from these WT1-specific T cells was sequenced and used to train a pattern recognition model for the identification of WT1-specific TCR patterns for the WT1-37 or WT1-126 epitopes. The resulting computational models were applied on an independent published dataset from acute myeloid leukemia (AML) patients, treated with hematopoietic stem cell transplantation, to track WT1-specific TCRs in silico. Several WT1-specific TCRs were found in AML patients. Subsequent clustering analysis of all repertoires indicated the presence of more diverse TCR patterns within the WT1-specific TCR repertoires of AML patients in complete remission in contrast to relapsing patients. We demonstrate the possibility of tracking WT1-37 and WT1-126-specific TCRs directly from TCR repertoire data using computational methods, eliminating the need for additional blood samples and experiments for the two studied WT1 epitopes.

Training of epitope-TCR prediction models with healthy donor-derived cancer-specific T cells.

Discovery of epitope-specific T-cell receptors (TCRs) for cancer therapies is a time consuming and expensive procedure that usually requires a large amount of patient cells. To maximize information from and minimize the need of precious samples in cancer research, prediction models have been developed to identify in silico epitope-specific TCRs. In this chapter, we provide a step-by-step protocol to train a prediction model using the user-friendly TCRex webtool for the nearly universal tumor-associated antigen Wilms' tumor 1 (WT1)-specific TCR repertoire. WT1 is a self-antigen overexpressed in numerous solid and hematological malignancies with a high clinical relevance. Training of computational models starts from a list of known epitope-specific TCRs which is often not available for new cancer epitopes. Therefore, we describe a workflow to assemble a training data set consisting of TCR sequences obtained from WT137-45-reactive CD8 T cell clones expanded and sorted from healthy donor peripheral blood mononuclear cells.

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.

A systematic review on poly(I:C) and poly-ICLC in glioblastoma: adjuvants coordinating the unlocking of immunotherapy.

Immunotherapy is currently under intensive investigation as a potential breakthrough treatment option for glioblastoma. Given the anatomical and immunological complexities surrounding glioblastoma, lymphocytes that infiltrate the brain to develop durable immunity with memory will be key. Polyinosinic:polycytidylic acid, or poly(I:C), and its derivative poly-ICLC could serve as a priming or boosting therapy to unleash lymphocytes and other factors in the (immuno)therapeutic armory against glioblastoma. Here, we present a systematic review on the effects and efficacy of poly(I:C)/poly-ICLC for glioblastoma treatment, ranging from preclinical work on cellular and murine glioblastoma models to reported and ongoing clinical studies. MEDLINE was searched until 15 May 2021 to identify preclinical (glioblastoma cells, murine models) and clinical studies that investigated poly(I:C) or poly-ICLC in glioblastoma. A systematic review approach was conducted according to PRISMA guidelines. ClinicalTrials.gov was queried for ongoing clinical studies. Direct pro-tumorigenic effects of poly(I:C) on glioblastoma cells have not been described. On the contrary, poly(I:C) changes the immunological profile of glioblastoma cells and can also kill them directly. In murine glioblastoma models, poly(I:C) has shown therapeutic relevance as an adjuvant therapy to several treatment modalities, including vaccination and immune checkpoint blockade. Clinically, mostly as an adjuvant to dendritic cell or peptide vaccines, poly-ICLC has been demonstrated to be safe and capable of eliciting immunological activity to boost therapeutic responses. Poly-ICLC could be a valuable tool to enhance immunotherapeutic approaches for glioblastoma. We conclude by proposing several promising combination strategies that might advance glioblastoma immunotherapy and discuss key pre-clinical aspects to improve clinical translation.

PD-L1 siRNA-mediated silencing in acute myeloid leukemia enhances anti-leukemic T cell reactivity.

Acute myeloid leukemia (AML) is an immune-susceptible malignancy, as demonstrated by its responsiveness to allogeneic stem cell transplantation (alloSCT). However, by employing inhibitory signaling pathways, including PD-1/PD-L1, leukemia cells suppress T cell-mediated immune attack. Notably, impressive clinical efficacy has been obtained with PD-1/PD-L1 blocking antibodies in cancer patients. Yet, these systemic treatments are often accompanied by severe toxicity, especially after alloSCT. Here, we investigated RNA interference technology as an alternative strategy to locally interfere with PD-1/PD-L1 signaling in AML. We demonstrated efficient siRNA-mediated PD-L1 silencing in HL-60 and patients' AML cells. Importantly, WT1-antigen T cell receptor+ PD-1+ 2D3 cells showed increased activation toward PD-L1 silenced WT1+ AML. Moreover, PD-L1 silenced AML cells significantly enhanced the activation, degranulation, and IFN-γ production of minor histocompatibility antigen-specific CD8+ T cells. Notably, PD-L1 silencing was equally effective as PD-1 antibody blockade. Together, our study demonstrates that PD-L1 silencing may be an effective strategy to augment AML immune-susceptibility. This provides rationale for further development of targeted approaches to locally interfere with immune escape mechanisms in AML, thereby minimizing severe toxicity. In combination with alloSCT and/or adoptive T cell transfer, this strategy could be very appealing to boost graft-versus-leukemia immunity and improve outcome in AML patients.

Epidemiology of Functional Abdominal Pain Disorders and Functional Defecation Disorders in Adolescents in Curacao.

OBJECTIVES: Functional abdominal pain disorders (FAPDs) and functional defecation disorders (FDDs) are common in children and adolescents, but prevalence rates from the Caribbean are lacking. Therefore, our aim was to determine the prevalence of FAPDs and FDDs in adolescents in Curacao and to assess the influence of psychosocial factors on the prevalence of FAPDs and FDDs. METHODS: The prevalence of FAPDs and FDDs in children and adolescents living in Curacao, ages 11 to 18 years, was assessed using the Rome IV Questionnaire on Pediatric Gastrointestinal Disorders (RIV-QPGD). FAPDs and FDDs were diagnosed according to the Rome IV criteria. Sociodemographic characteristics, somatic symptoms, early adverse life events, stressful life events, and physical and emotional abuse were evaluated as associated factors. RESULTS: Out of 946 questionnaires distributed, 783 were included for further analysis. The mean age of adolescents was 14.7 years (±1.6) with 61.7% being girls. A total of 266 adolescents (34%, 95% confidence interval [CI] 30.7-37.5) met Rome IV criteria for at least 1 FAPD or FDD. Twenty-nine adolescents (3.7%) qualified for 2 functional gastrointestinal disorders. Functional constipation (18.6%) and irritable bowel syndrome (12.3%) were the most prevalent disorders. After multivariate logistic regression analyses, dizziness (odds ratio [OR] 1.84, 95% CI 1.28-2.64) was significantly associated with having a FAPD or FDD. CONCLUSIONS: FAPDs and FDDs are common in adolescents in Curacao. Dizziness is associated with the presence of a FAPD or FDD.

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.