Abstract Background For many people public transport is the only mode of travel, and it can be challenging to keep the necessary distances in such a restricted space. The exact role of public transportation and risk of SARS-CoV-2 transmission is not known. Methods Participants (n = 121,374) were untested adult Norwegian residents recruited through social media who in the spring of 2020 completed a baseline questionnaire on demographics and the use of public transport. Incident cases (n = 1069) had a positive SARS-CoV-2 polymerase chain reaction test registered at the Norwegian Messaging System for Infectious Diseases by January 27, 2021. We investigated the association between the use of public transport and SARS-CoV-2 using logistic regression. Odds ratios (ORs) with 95% confidence intervals (CIs) adjusted for age, calendar time, gender, municipality, smoking, income level, fitness and underlying medical conditions were estimated. Frequency of the use of public transport was reported for 2 week-periods. Results Before lockdown, those who tested positive on SARS-CoV-2 were more likely to have used public transport 1–3 times (OR = 1.28, CI 1.09–1.51), 4–10 times (OR = 1.49, CI 1.26–1.77) and ≥ 11 times (OR = 1.50, CI 1.27–1.78, p for trend < 0.0001) than those who had not tested positive. Conclusion The use of public transport was positively associated with contracting SARS-CoV-2 both before and after lockdown.

Chronic inflammation of the small intestine in celiac disease is driven by activation of CD4+ T cells that recognize gluten peptides presented by disease-associated HLA-DQ molecules. We have performed direct cell cloning of duodenal biopsies from five untreated and one refractory celiac disease patients, and three non-celiac disease control subjects in order to assess, in an unbiased fashion, the frequency of gluten-reactive T cells in the disease-affected tissue as well as the antigen fine specificity of the responding T cells. From the biopsies of active disease lesions of five patients, 19 T-cell clones were found to be gluten-reactive out of total 1,379 clones tested. This gave an average of 1.4% (range 0.7% - 1.9%) of gluten-reactive T cells in lamina propria of active celiac lesions. Interestingly, also the patient with refractory celiac disease had gluten-reactive T cell clones in the lamina propria (5/273; 1.8%). In comparison, we found no gluten-reactive T cells in any of the total 984 T-cell clones screened from biopsies from three disease control donors. Around two thirds of the gluten-reactive clones were reactive to a panel of peptides representing known gluten T-cell epitopes, of which two thirds were reactive to the immunodominant DQ2.5-glia-α1/DQ2.5-glia-α2 and DQ2.5-glia-ω1/DQ2.5-glia-ω2 epitopes. This study shows that gluten-reactive T cells in the inflamed duodenal tissue are prevalent in the active disease lesion, and that many of these T cells are reactive to T-cell epitopes that are not yet characterized. Knowledge of the prevalence and epitope specificity of gluten-specific T cells is a prerequisite for therapeutic efforts that target disease-specific T cells in celiac disease.

Gut intraepithelial γδ and CD8+ αβ T lymphocytes have been connected to celiac disease (CeD) pathogenesis. Based on the previous observation that activated (CD38+), gut-homing (CD103+) γδ and CD8+ αβ T cells increase in blood upon oral gluten challenge, we wanted to shed light on the pathogenic involvement of these T cells by examining the clonal relationship between cells of blood and gut during gluten exposure. Of 20 gluten-challenged CeD patients, 8 and 10 had increase in (CD38+CD103+) γδ and CD8+ αβ T cells, respectively, while 16 had increase in gluten-specific CD4+ T cells. We obtained γδ and αβ TCR sequences of >2500 single cells from blood and gut of 5 patients, before and during challenge. We observed extensive sharing between blood and gut γδ and CD8+ αβ T-cell clonotypes even prior to gluten challenge. In subjects with challenge-induced surge of γδ and/or CD8+ αβ T cells, as larger populations of cells analyzed, we observed more expanded clonotypes and clonal sharing, yet no discernible TCR similarities between expanded and/or shared clonotypes. Thus, CD4+ T cells appear to drive expansion of clonally diverse γδ or CD8+ αβ T-cell clonotypes that may not be specific for the gluten antigen.

We compared the αβ T-cell receptor repertoires of CD8+ αβ intraepithelial lymphocytes from celiac disease patients and healthy subjects by single-cell sequencing. We demonstrate that the repertoires of untreated celiac disease patients were more polyclonal and more diverse than what was observed in both treated patients and healthy subjects.

Disease-specific plasma cells (PCs) reactive with transglutaminase 2 (TG2) or deamidated gluten peptides (DGPs) are abundant in celiac disease (CeD) gut lesions. Their contribution toward CeD pathogenesis is unclear. We assessed expression of markers associated with PC longevity in 15 untreated and 26 treated CeD patients in addition to 13 non-CeD controls and performed RNA sequencing with clonal inference and transcriptomic analysis of 3,251 single PCs. We observed antigen dependent V-gene selection and stereotypic antibodies. Generation of recombinant DGP-specific antibodies revealed a key role of a heavy chain residue that displays polymorphism, suggesting that immunoglobulin gene polymorphisms may influence CeD-specific antibody responses. We identified transcriptional differences between CeD-specific and non-disease-specific PCs and between short-lived and long-lived PCs. The short-lived CD19'CD45' phenotype dominated in untreated and shortterm-treated CeD, in particular among disease-specific PCs but also in the general PC population. Thus, the disease lesion of untreated CeD is characterized by massive accumulation of short-lived PCs that are not only directed against disease-specific antigens.

A hallmark of celiac disease (CeD), a chronic condition driven by cereal gluten exposure, is increase of gut intraepithelial γδ T cells. This may indicate pathogenic involvement of γδ T cells and existence of disease-specific γδ T-cell receptors (TCRs) recognizing defined antigen(s). We performed high-throughput and paired γδ TCR sequencing of single intraepithelial γδ T cells of untreated CeD patients (n = 8; 1821 cells), CeD patients treated with a gluten-free diet (n = 5; 436 cells) and controls (n = 7; 1068 cells). We found that CeD patients, both untreated and treated, had larger and more diverse γδ TCR repertoires, more frequent usage of TRDV1 and TRDV3 and different patterns of TCRγ/TCRδ-pairing compared with controls. Although we observed no public CDR3δ sequences, there were several public CDR3γ sequences—many of which were shared by not only the CeD patients, but also by the controls. These public CDR3s were characterized by few N/P nucleotide insertions with germline and near-germline configuration, hence being easy to generate. Previous findings of CeD-specific CDR3 motifs were not replicated. Thus, being unable to raise evidence for CeD-specific γδ TCRs in this first large, paired γδ TCR single-cell sequencing study, we project challenges for identification of CeD-relevant γδ TCR ligands.

Inactivation of the tumor suppressor gene encoding the transcriptional regulator Ikaros (IKZF1) is a hallmark of BCR-ABL1+ precursor B cell acute lymphoblastic leukemia (pre-B ALL). However, the mechanisms by which Ikaros functions as a tumor suppressor in pre-B ALL remain poorly understood. Here, we analyzed a mouse model of BCR-ABL1+ pre-B ALL together with a new model of inducible expression of wild-type Ikaros in IKZF1 mutant human BCR-ABL1+ pre-B ALL. We performed integrated genome-wide chromatin and expression analyses and identified Ikaros target genes in mouse and human BCR-ABL1+ pre-B ALL, revealing novel conserved gene pathways associated with Ikaros tumor suppressor function. Notably, genetic depletion of different Ikaros targets, including CTNND1 and the early hematopoietic cell surface marker CD34, resulted in reduced leukemic growth. Our results suggest that Ikaros mediates tumor suppressor function by enforcing proper developmental stage-specific expression of multiple genes through chromatin compaction at its target genes.

Abstract Inactivation of the transcriptional factor Ikaros (IKZF1) correlates with poor prognosis in progenitor B-cell acute lymphoblastic leukemia (pre-B ALL), and is a hallmark of the BCR-ABL1+ subgroup of pre-B ALL. Ikaros is a critical regulator of hematopoietic development and required for B-cell development, however the mechanisms by which Ikaros functions as a tumor suppressor in pre-B ALL remain poorly understood. We analyzed recently developed mouse models of BCR-ABL1+ pre-B ALL containing targeted deletions of Ikaros DNA-binding zinc finger domains together with a new model of inducible expression of WT Ikaros in IKZF1-mutant human BCR-ABL1+ pre-B ALL. We found that both the mouse and human Ikaros-mutated leukemic cells displayed a less mature cell surface phenotype and failed to downregulate the developmentally restricted cell surface receptors c-kit and CD34, respectively. In addition, Ctnnd1, a gene that is also expressed in earlier hematopoietic progenitor cells and normally downregulated as cells differentiate down the B-cell lineage, was found to be a conserved Ikaros target gene, with increased expression in Ikaros-mutated leukemic cells. RNA sequencing defined the Ikaros target genes in both mouse and human Ikaros-mutated pre-B ALL cells and revealed additional conserved target genes and biological functions. Loss of Ikaros tumor suppression was associated with deregulated adhesion pathways and stem-cell signatures. Furthermore, our results presented herein suggest that Ikaros mediates tumor suppressor function, at least in part, by enforcing proper developmental-stage specific expression of multiple genes involved in a network of cadherin-dependent, Rho-regulated Wnt/b-catenin pathways.

Abstract Ikaros is a zinc finger transcription factor required for B-cell development and proper hematopoiesis, and an important tumor suppressor in developing lymphocytes. To understand the mechanism of Ikaros tumor suppressor function and potentially develop new and improved targeted therapies, it is important to elucidate the downstream target genes involved. With this aim, we combine mouse models of pre-B ALL and in vitro culture of human patient-derived pre-B ALL cells. A mouse model with targeted deletion of the fourth DNA-binding zinc finger of Ikaros resulted in loss of tumor suppressor function, with a limited set of deregulated genes useful to narrow down the list of putative relevant Ikaros target genes (Schjerven et al., 2013). To specifically address the role of Ikaros as a tumor suppressor in human pre-B ALL, we developed TET-regulated Ikaros expression in human pre-B ALL cells. This enables us to test specific Ikaros target genes from the mouse model, and allows for genome-wide expression analysis by RNA-seq to elucidate all genes downstream of Ikaros in human pre-B ALL cells. To help distinguish direct from indirect target genes, we have mapped the genome-wide binding sites of Ikaros in these human pre-B ALL cells by ChIP-Seq. This approach has identified the Ikaros target gene Ctnnd1. Ongoing experiments explore the potential role of Ctnnd1 and test the current hypothesis that Ikaros-mediated repression of Ctnnd1 limits CyclinD levels and leukemic growth in pre-B ALL.