New insights emerge as antibody repertoire diversification meets chromosome conformation [version 1; peer review: 3 approved]

Vast repertoires of unique antigen receptors are created in developing lymphocytes. The antigen receptor loci contain many variable (V), diversity (D), and joining (J) gene segments that are arrayed across very large genomic expanses and are joined to form variable-region exons. This process creates the potential for an organism to respond to large numbers of different pathogens. Here, we consider the underlying molecular mechanisms that favor some V genes for recombination prior to selection of the final antigen receptor repertoire. We discuss chromatin structures that form in antigen receptor loci to permit spatial proximity among the V, D, and J gene segments and how these relate to the generation of antigen receptor diversity

Epigenetic Enhancer Marks and Transcription Factor Binding Influence Vκ Gene Rearrangement in Pre-B Cells and Pro-B Cells

To date there has not been a study directly comparing relative Igκ rearrangement frequencies obtained from genomic DNA (gDNA) and cDNA and since each approach has potential biases, this is an important issue to clarify. Here we used deep sequencing to compare the unbiased gDNA and RNA Igκ repertoire from the same pre-B cell pool. We find that ~20% of Vκ genes have rearrangement frequencies ≥2-fold up or down in RNA vs. DNA libraries, including many members of the Vκ3, Vκ4, and Vκ6 families. Regression analysis indicates Ikaros and E2A binding are associated with strong promoters. Within the pre-B cell repertoire, we observed that individual Vκ genes rearranged at very different frequencies, and also displayed very different Jκ usage. Regression analysis revealed that the greatly unequal Vκ gene rearrangement frequencies are best predicted by epigenetic marks of enhancers. In particular, the levels of newly arising H3K4me1 peaks associated with many Vκ genes in pre-B cells are most predictive of rearrangement levels. Since H3K4me1 is associated with long range chromatin interactions which are created during locus contraction, our data provides mechanistic insight into unequal rearrangement levels. Comparison of Igκ rearrangements occurring in pro-B cells and pre-B cells from the same mice reveal a pro-B cell bias toward usage of Jκ-distal Vκ genes, particularly Vκ10-96 and Vκ1-135. Regression analysis indicates that PU.1 binding is the highest predictor of Vκ gene rearrangement frequency in pro-B cells. Lastly, the repertoires of iEκ−/− pre-B cells reveal that iEκ actively influences Vκ gene usage, particularly Vκ3 family genes, overlapping with a zone of iEκ-regulated germline transcription. These represent new roles for iEκ in addition to its critical function in promoting overall Igκ rearrangement. Together, this study provides insight into many aspects of Igκ repertoire formation

Reduced receptor editing in lupus-prone MRL/lpr mice

The initial B cell repertoire contains a considerable proportion of autoreactive specificities. The first major B cell tolerance checkpoint is at the stage of the immature B cell, where receptor editing is the primary mode of eliminating self-reactivity. The cells that emigrate from the bone marrow have a second tolerance checkpoint in the transitional compartment in the spleen. Although it is known that the second checkpoint is defective in lupus, it is not clear whether there is any breakdown in central B cell tolerance in the bone marrow. We demonstrate that receptor editing is less efficient in the lupus-prone strain MRL/lpr. In an in vitro system, when receptor-editing signals are given to bone marrow immature B cells by antiidiotype antibody or after in vivo exposure to membrane-bound self-antigen, MRL/lpr 3-83 transgenic immature B cells undergo less endogenous rearrangement and up-regulate recombination activating gene messenger RNA to a lesser extent than B10 transgenic cells. CD19, along with immunoglobulin M, is down-regulated in the bone marrow upon receptor editing, but the extent of down-regulation is fivefold less in MRL/lpr mice. Less efficient receptor editing could allow some autoreactive cells to escape from the bone marrow in lupus-prone mice, thus predisposing to autoimmunity

Repertoire-based selection into the marginal zone compartment during B cell development

Marginal zone (MZ) B cells resemble fetally derived B1 B cells in their innate-like rapid responses to bacterial pathogens, but the basis for this is unknown. We report that the MZ is enriched in “fetal-type” B cell receptors lacking N regions (N−). Mixed bone marrow (BM) chimeras, made with adult terminal deoxynucleotidyl transferase (TdT)+/+ and TdT−/− donor cells, demonstrate preferential repertoire-based selection of N− B cells into the MZ. Reconstitution of irradiated mice with adult TdT+/+ BM reveals that the MZ can replenish N− B cells in adult life via repertoire-based selection and suggest the possibility of a TdT-deficient precursor population in the adult BM. The mixed chimera data also suggest repertoire-based bifurcations into distinct BM and splenic maturation pathways, with mature “recirculating” BM B cells showing a very strong preference for N+ complementarity-determining region (CDR) 3 compared with follicular B cells. Because the T1 and MZ compartments are both the most enriched for N− H-CDR3, we propose a novel direct T1→MZ pathway and identify a potential T1–MZ precursor intermediate. We demonstrate progressive but discontinuous repertoire-based selection throughout B cell development supporting multiple branchpoints and pathways in B cell development. Multiple differentiation routes leading to MZ development may contribute to the reported functional heterogeneity of the MZ compartment

Flexible ordering of antibody class switch and V(D)J joining during B-cell ontogeny

V(D)J joining is mediated by RAG recombinase during early B-lymphocyte development in the bone marrow (BM). Activation-induced deaminase initiates isotype switching in mature B cells of secondary lymphoid structures. Previous studies questioned the strict ontological partitioning of these processes. We show that pro-B cells undergo robust switching to a subset of immunoglobulin H (IgH) isotypes. Chromatin studies reveal that in pro-B cells, the spatial organization of the Igh locus may restrict switching to this subset of isotypes. We demonstrate that in the BM, V(D)J joining and switching are interchangeably inducible, providing an explanation for the hyper-IgE phenotype of Omenn syndrome

StrokeCog Markov Model Projected Prevalent and Incident Cases of Stroke and Poststroke Cognitive Impairment to 2035 in Ireland

Background and Purpose: Cognitive impairment no dementia (CIND) and dementia are common stroke outcomes, with significant health and societal implications for aging populations. These outcomes are not included in current epidemiological models. We aimed to develop an epidemiological model to project incidence and prevalence of stroke, poststroke CIND and dementia, and life expectancy, in Ireland to 2035, informing policy and service planning. Methods: We developed a probabilistic Markov model (the StrokeCog model) applied to the Irish population aged 40 to 89 years to 2035. Data sources included official population and hospital-episode statistics, longitudinal cohort studies, and published estimates. Key assumptions were varied in sensitivity analysis. Results were externally validated against independent sources. The model tracks poststroke progression into health states characterized by no cognitive impairment, CIND, dementia, disability, stroke recurrence, and death. Results: We projected 69 051 people with prevalent stroke in Ireland in 2035 (22.0 per 1000 population [95% CI, 20.8-23.1]), with 25 274 (8.0 per 1000 population [95% CI, 7.1-9.0]) of those projected to have poststroke CIND, and 12 442 having poststroke dementia (4.0 per 1000 population [95% CI, 3.2-4.8]). We projected 8725 annual incident strokes in 2035 (2.8 per 1000 population [95% CI, 2.7-2.9]), with 3832 of these having CIND (1.2 per 1000 population [95% CI, 1.1-1.3]), and 1715 with dementia (0.5 per 1000 population [95% CI, 0.5-0.6]). Life expectancy for stroke survivors at age 50 was 23.4 years (95% CI, 22.3-24.5) for women and 20.7 (95% CI, 19.5-21.9) for men. Conclusions: This novel epidemiological model of stroke, poststroke CIND, and dementia draws on the best available evidence. Sensitivity analysis indicated that findings were robust to assumptions, and where there was uncertainty a conservative approach was taken. The StrokeCog model is a useful tool for service planning and cost-effectiveness analysis and is available for adaptation to other national contexts.Peer reviewe

Identification of unique neoantigen qualities in long-term survivors of pancreatic cancer

Pancreatic ductal adenocarcinoma is a lethal cancer with fewer than 7% of patients surviving past 5 years. T-cell immunity has been linked to the exceptional outcome of the few long-term survivors1,2, yet the relevant antigens remain unknown. Here we use genetic, immunohistochemical and transcriptional immunoprofiling, computational biophysics, and functional assays to identify T-cell antigens in long-term survivors of pancreatic cancer. Using whole-exome sequencing and in silico neoantigen prediction, we found that tumours with both the highest neoantigen number and the most abundant CD8+ T-cell infiltrates, but neither alone, stratified patients with the longest survival. Investigating the specific neoantigen qualities promoting T-cell activation in long-term survivors, we discovered that these individuals were enriched in neoantigen qualities defined by a fitness model, and neoantigens in the tumour antigen MUC16 (also known as CA125). A neoantigen quality fitness model conferring greater immunogenicity to neoantigens with differential presentation and homology to infectious disease-derived peptides identified long-term survivors in two independent datasets, whereas a neoantigen quantity model ascribing greater immunogenicity to increasing neoantigen number alone did not. We detected intratumoural and lasting circulating T-cell reactivity to both high-quality and MUC16 neoantigens in long-term survivors of pancreatic cancer, including clones with specificity to both high-quality neoantigens and predicted cross-reactive microbial epitopes, consistent with neoantigen molecular mimicry. Notably, we observed selective loss of high-quality and MUC16 neoantigenic clones on metastatic progression, suggesting neoantigen immunoediting. Our results identify neoantigens with unique qualities as T-cell targets in pancreatic ductal adenocarcinoma. More broadly, we identify neoantigen quality as a biomarker for immunogenic tumours that may guide the application of immunotherapies

Mortality Among Adults With Cancer Undergoing Chemotherapy or Immunotherapy and Infected With COVID-19

Importance: Large cohorts of patients with active cancers and COVID-19 infection are needed to provide evidence of the association of recent cancer treatment and cancer type with COVID-19 mortality. // Objective: To evaluate whether systemic anticancer treatments (SACTs), tumor subtypes, patient demographic characteristics (age and sex), and comorbidities are associated with COVID-19 mortality. // Design, Setting, and Participants: The UK Coronavirus Cancer Monitoring Project (UKCCMP) is a prospective cohort study conducted at 69 UK cancer hospitals among adult patients (≥18 years) with an active cancer and a clinical diagnosis of COVID-19. Patients registered from March 18 to August 1, 2020, were included in this analysis. // Exposures: SACT, tumor subtype, patient demographic characteristics (eg, age, sex, body mass index, race and ethnicity, smoking history), and comorbidities were investigated. // Main Outcomes and Measures: The primary end point was all-cause mortality within the primary hospitalization. // Results: Overall, 2515 of 2786 patients registered during the study period were included; 1464 (58%) were men; and the median (IQR) age was 72 (62-80) years. The mortality rate was 38% (966 patients). The data suggest an association between higher mortality in patients with hematological malignant neoplasms irrespective of recent SACT, particularly in those with acute leukemias or myelodysplastic syndrome (OR, 2.16; 95% CI, 1.30-3.60) and myeloma or plasmacytoma (OR, 1.53; 95% CI, 1.04-2.26). Lung cancer was also significantly associated with higher COVID-19–related mortality (OR, 1.58; 95% CI, 1.11-2.25). No association between higher mortality and receiving chemotherapy in the 4 weeks before COVID-19 diagnosis was observed after correcting for the crucial confounders of age, sex, and comorbidities. An association between lower mortality and receiving immunotherapy in the 4 weeks before COVID-19 diagnosis was observed (immunotherapy vs no cancer therapy: OR, 0.52; 95% CI, 0.31-0.86). // Conclusions and Relevance: The findings of this study of patients with active cancer suggest that recent SACT is not associated with inferior outcomes from COVID-19 infection. This has relevance for the care of patients with cancer requiring treatment, particularly in countries experiencing an increase in COVID-19 case numbers. Important differences in outcomes among patients with hematological and lung cancers were observed