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The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens 1 . In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1–CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens 2 – 22 . Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes 23 , 24 . We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.  Among naturally occurring antibodies that have adapted to antigen, those with similar heavy chains usually have similar light chains.

Clones are the fundamental building blocks of immune repertoires. The number of different clones relates to the diversity of the repertoire, whereas their size and sequence diversity are linked to selective pressures. Selective pressures act both between clones and within different sequence variants of a clone. Understanding how clonal selection shapes the immune repertoire is one of the most basic questions in all of immunology. But how are individual clones defined? Here we discuss different approaches for defining clones, starting with how antibodies are diversified during different stages of B cell development. Next, we discuss how clones are defined using different experimental methods. We focus on high-throughput sequencing datasets, and the computational challenges and opportunities that these data have for mining the antibody repertoire landscape. We discuss methods that visualize sequence variants within the same clone and allow us to consider collections of shared mutations to determine which sequences share a common ancestry. Finally, we comment on features of frequently encountered expanded B cell clones that may be of particular interest in the setting of autoimmunity and other chronic conditions.

Holländer and colleagues provide a map of genes that are direct targets of the transcription factor Foxn1 in thymic epithelial cells and show that Foxn1 controls genes encoding products involved in thymocyte development, antigen processing and thymocyte selection. Thymic epithelial cell differentiation, growth and function depend on the expression of the transcription factor Foxn1; however, its target genes have never been physically identified. Using static and inducible genetic model systems and chromatin studies, we developed a genome-wide map of direct Foxn1 target genes for postnatal thymic epithelia and defined the Foxn1 binding motif. We determined the function of Foxn1 in these cells and found that, in addition to the transcriptional control of genes involved in the attraction and lineage commitment of T cell precursors, Foxn1 regulates the expression of genes involved in antigen processing and thymocyte selection. Thus, critical events in thymic lympho-stromal cross-talk and T cell selection are indispensably choreographed by Foxn1.

Severe influenza A virus (IAV) infection is associated with immune dysfunction. Here, we show circulating CD8 + T-cell profiles from patients hospitalized with avian H7N9, seasonal IAV, and influenza vaccinees. Patient survival reflects an early, transient prevalence of highly activated CD38 + HLA-DR + PD-1 + CD8 + T cells, whereas the prolonged persistence of this set is found in ultimately fatal cases. Single-cell T cell receptor (TCR)-αβ analyses of activated CD38 + HLA-DR + CD8 + T cells show similar TCRαβ diversity but differential clonal expansion kinetics in surviving and fatal H7N9 patients. Delayed clonal expansion associated with an early dichotomy at a transcriptome level (as detected by single-cell RNAseq) is found in CD38 + HLA-DR + CD8 + T cells from patients who succumbed to the disease, suggesting a divergent differentiation pathway of CD38 + HLA-DR + CD8 + T cells from the outset during fatal disease. Our study proposes that effective expansion of cross-reactive influenza-specific TCRαβ clonotypes with appropriate transcriptome signatures is needed for early protection against severe influenza disease. Virus-specific CD8 + T cells are crucial during H7N9 influenza infection, but CD8 + T cell dysfunction is associated with poor prognosis. Here, the authors use molecular and phenotypic analysis to establish persistence of clonally diverse CD8 + T cell populations during fatal infection.

Germline coding mutations in different telomere-related genes have been linked to autosomal-dominant familial pulmonary fibrosis. Individuals with these inherited mutations demonstrate incomplete penetrance of clinical phenotypes affecting the lung, blood, liver, skin, and other organs. Here, we describe the somatic acquisition of promoter mutations in telomerase reverse transcriptase (TERT) in blood leukocytes of approximately 5% of individuals with inherited loss-of-function coding mutations in TERT or poly(A)-specific ribonuclease (PARN), another gene linked to telomerase function. While these promoter mutations were initially identified as oncogenic drivers of cancer, individuals expressing the mutations have no history of cancer. Neither promoter mutation was found in population-based cohorts of similar or advanced age. The TERT promoter mutations were found more frequently in cis with the WT allele than the TERT coding sequence mutation. EBV-transformed lymphoblastoid B cell lines (LCLs) derived from subjects with TERT promoter mutations showed increased telomerase expression and activity compared with cell lines from family members with identical coding mutations. TERT promoter mutations resulted in an increased proliferation of LCLs and demonstrated positive selection over time. The persistence and recurrence of noncoding gain-of-function mutations in these cases suggests that telomerase activation is not only safely tolerated but also advantageous for clonal expansion.

Unique molecular identifiers (MIDs) have been demonstrated to effectively improve immune repertoire sequencing (IR-seq) accuracy, especially to identify somatic hypermutations in antibody repertoire sequencing. However, evaluating the sensitivity to detect rare T cells and the degree of clonal expansion in IR-seq has been difficult due to the lack of knowledge of T cell receptor (TCR) RNA molecule copy number and a generalized approach to estimate T cell clone size from TCR RNA molecule quantification. This limited the application of TCR repertoire sequencing (TCR-seq) in clinical settings, such as detecting minimal residual disease in lymphoid malignancies after treatment, evaluating effectiveness of vaccination and assessing degree of infection. Here, we describe using an MID Clustering-based IR-Seq (MIDCIRS) method to quantitatively study TCR RNA molecule copy number and clonality in T cells. First, we demonstrated the necessity of performing MID sub-clustering to eliminate erroneous sequences. Further, we showed that MIDCIRS enables a sensitive detection of a single cell in as many as one million naïve T cells and an accurate estimation of the degree of T cell clonal expression. The demonstrated accuracy, sensitivity, and wide dynamic range of MIDCIRS TCR-seq provide foundations for future applications in both basic research and clinical settings.

Thymocytes bearing αβ T cell receptors (TCRαβ) with high affinity for self-peptide-MHC complexes undergo negative selection or are diverted to alternate T cell lineages, a process termed agonist selection. Among thymocytes bearing TCRs restricted to MHC class I, agonist selection can lead to the development of precursors that can home to the gut and give rise to CD8αα-expressing intraepithelial lymphocytes (CD8αα IELs). The factors that influence the choice between negative selection versus CD8αα IEL development remain largely unknown. Using a synchronized thymic tissue slice model that supports both negative selection and CD8αα IEL development, we show that the affinity threshold for CD8αα IEL development is higher than for negative selection. We also investigate the impact of peptide presenting cells and cytokines, and the migration patterns associated with these alternative cell fates. Our data highlight the roles of TCR affinity and the thymic microenvironments on T cell fate.

Positive selection of CD8 T cells in the thymus is thought to be a multistep process lasting 3–4 d; however, the discrete steps involved are poorly understood. Here, we examine phenotypic changes, calcium signaling, and intrathymic migration in a synchronized cohort of MHC class I-specific thymocytes undergoing positive selection in situ. Transient elevations in intracellular calcium concentration ([Ca ²⁺] ᵢ) and migratory pauses occurred throughout the first 24 h of positive selection, becoming progressively briefer and accompanied by a gradual shift in basal [Ca ²⁺] ᵢ over time. Changes in chemokine-receptor expression and relocalization from the cortex to medulla occurred between 12 and 24 h after the initial encounter with positive-selecting ligands, a time frame at which the majority of thymocytes retain CD4 and CD8 expression and still require T-cell receptor (TCR) signaling to efficiently complete positive selection. Our results identify distinct phases in the positive selection of MHC class I-specific thymocytes that are distinguished by their TCR-signaling pattern and intrathymic location and provide a framework for understanding the multistep process of positive selection in the thymus.

Costimulatory signals by CD28 are critical for thymic regulatory T‐cell (Treg) development. To determine the functional relevance of CD28 for peripheral Treg post thymic selection, we crossed the widely used Forkhead box protein 3 (Foxp3)‐CreYFP mice to mice bearing a conditional Cd28 allele. Treg‐specific CD28 deficiency provoked a severe autoimmune syndrome as a result of a strong disadvantage in competitive fitness and proliferation of CD28‐deficient Tregs. By contrast, Treg survival and lineage integrity were not affected by the lack of CD28. This data demonstrate that, even after the initial induction requirement, Treg maintain a higher dependency on CD28 signalling than conventional T cells for homeostasis. In addition, we found the Foxp3‐CreYFP allele to be a hypomorph, with reduced Foxp3 protein levels. Furthermore, we report here the stochastic activity of the Foxp3‐CreYFP allele in non‐Tregs, sufficient to recombine some conditional alleles (including Cd28) but not others (including R26‐RFP). This hypomorphism and ‘leaky’ expression of the Foxp3‐CreYFP allele should be considered when analysing the conditionally mutated Treg.

Selection of resistant clones following intensive chemotherapy is a common obstacle for cure in many cancers, particularly in acute myeloid leukemia (AML). In AML, clone-specific sensitivity to chemotherapy varies even within the same patient. Multiple mutations and genetic aberrations are associated with clones surviving chemotherapy. The current study explored the role of activated signaling pathways in chemoresistance as a function of cell maturation, reflected by CD34 expression. In-vitro , Kasumi-1 leukemic cell line, sorted by CD34 expression, showed increased apoptosis only in the CD34 − subpopulation after exposure to cytosine arabinoside (Ara-C) or daunorubicin. The resistant CD34 + subset demonstrated higher expression of ERK1/2 and BCL-2 proteins than CD34 − cells. MEK1/2 inhibition elevated Ara-C ability to induce apoptosis in CD34 + cells, suggesting that MEK1/2-ERK1/2 is surviving signaling, which correlates to cell maturation levels and plays a role in chemoresistance. Deep sequencing of sorted CD34 +/− populations, both derived from the same patient samples, demonstrated various subclonal distribution of NPM1, DNMT3A and FLT3-ITD mutations. Interestingly, in these samples, p-ERK levels and apoptosis rates following chemotherapy exposure significantly differed between CD34 +/− populations. Hence, clones may be selected due to their ability to escape apoptosis rather than a direct effect of chemotherapy on a specific mutated clone.