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T cell activation upon TCR signaling can lead to development of effector and memory cells. Roychoudhuri and colleagues show that the transcription factor BACH2 promotes memory CD8 + T cell generation by blocking access to genomic regulatory sites recognized by AP-1. T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8 + T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.

BACKGROUNDThere is increasing evidence, in transgenic mice and in vitro, that inhibitory killer cell immunoglobulin-like receptors (iKIRs) can modulate T cell responses. Furthermore, we have previously shown that iKIRs are an important determinant of T cell-mediated control of chronic viral infection and that these results are consistent with an increase in the CD8+ T cell lifespan due to iKIR-ligand interactions. Here, we tested this prediction and investigated whether iKIRs affect T cell lifespan in humans in vivo.METHODSWe used stable isotope labeling with deuterated water to quantify memory CD8+ T cell survival in healthy individuals and patients with chronic viral infections.RESULTSWe showed that an individual's iKIR-ligand genotype was a significant determinant of CD8+ T cell lifespan: in individuals with 2 iKIR-ligand gene pairs, memory CD8+ T cells survived, on average, for 125 days; in individuals with 4 iKIR-ligand gene pairs, the memory CD8+ T cell lifespan doubled to 250 days. Additionally, we showed that this survival advantage was independent of iKIR expression by the T cell of interest and, further, that the iKIR-ligand genotype altered the CD8+ and CD4+ T cell immune aging phenotype.CONCLUSIONSTogether, these data reveal an unexpectedly large effect of iKIR genotype on T cell survival.FUNDINGWellcome Trust; Medical Research Council; EU Horizon 2020; EU FP7; Leukemia and Lymphoma Research; National Institute of Health Research (NIHR) Imperial Biomedical Research Centre; Imperial College Research Fellowship; National Institutes of Health; Jefferiss Trust.

The use of deuterated water (also known as ‘heavy water’) as a tracer to measure human in vivo cell proliferation rates for specific cell subsets has expanded significantly in recent years. Although there have been several published methods papers, investigators developing new applications may be confused by differences in study design and deuterated water dose/duration. Furthermore, this approach may be met with regulatory difficulties and participant concerns about toxicity. This scoping review explores lessons that can be learnt from the current literature on the use of deuterated water in human in vivo studies measuring cell proliferation. We identified 29 such studies involving 535 study participants, both healthy volunteers and those with specific clinical conditions. Wide variations in protocols were noted with doses ranging from 40-100 ml/day of pure deuterated water (or equivalent) and durations from 4-12 weeks. Study design usually reflected the kinetics of the cell of interest. No clinical toxicity signals were noted in any studies although four studies did report transient dizziness, a recognized symptom of changing water density. These published studies provide a strong safety signal for potential participants and regulatory authorities and can act as templates for the development of new research applications.

Pathogenesis in chronic lymphocytic leukemia (CLL) is strongly linked to the potential for leukemic cells to migrate to and proliferate within lymph-nodes. Previous in vivo studies suggest that all leukemic cells participate in cycles of migration and proliferation. In vitro studies, however, have shown heterogeneous migration patterns.To investigate tumor subpopulation kinetics, we performed in vivo isotope-labeling studies in ten patients with IgVH-mutated CLL (M-CLL). Using deuterium-labeled glucose, we investigated proliferation in sub-populations defined by CXCR4/CD5 and surface (sIgM) expression. Mathematical modeling was performed to test the likelihood that leukemic cells exist as distinct sub-populations or as a single population with the same proliferative capacity. Further labeling studies in two patients with M-CLL commencing idelalisib investigated the effect of B-cell receptor (BCR) antagonists on sub-population kinetics.Modeling revealed that data were more consistent with a model comprising distinct sub-populations (p = 0.008) with contrasting, characteristic kinetics. Following idelalisib therapy, similar labeling suppression across all sub-populations suggested that the most proliferative subset is the most sensitive to treatment. As the quiescent sub-population precedes treatment, selection likely explains the persistence of such residual non-proliferating populations during BCR-antagonist therapy. These findings have clinical implications for discontinuation of long-term BCR-antagonist treatment in selected patients.

Stable isotope labelling is one of the best methods currently available for quantifying cell dynamics in vivo , particularly in humans where the absence of toxicity makes it preferable over other techniques such as CFSE or BrdU. Interpretation of stable isotope labelling data (as for BrdU and CFSE) necessitates simplifying assumptions. Here we investigate the impact of three of the most commonly used simplifying assumptions: (i) that the cell population of interest is closed, (ii) that the population of interest is kinetically homogeneous, and (iii) that the population is spatially homogeneous and suggest pragmatic ways in which the resulting errors can be reduced.

Long-term T cell-mediated protection depends upon the formation of a pool of memory cells to protect against future pathogen challenge. In this review we argue that looking at T cell memory from a dynamic viewpoint can help in understanding how memory populations are maintained following pathogen exposure or vaccination. For example, a dynamic view resolves the apparent paradox between the relatively short lifespans of individual memory cells and very long-lived immunological memory by focussing on the persistence of clonal populations, rather than individual cells. Clonal survival is achieved by balancing proliferation, death and differentiation rates within and between identifiable phenotypic pools; such pools correspond broadly to sequential stages in the linear differentiation pathway. Each pool has its own characteristic kinetics, but only when considered as a population; single cells exhibit considerable heterogeneity. In humans, we tend to concentrate on circulating cells, but memory T cells in non-lymphoid tissues and bone marrow are increasingly recognised as critical for immune defence; their kinetics, however, remain largely unexplored. Considering vaccination from this viewpoint shifts the focus from the size of the primary response to the survival of the clone and enables identification of critical system pinch-points and opportunities to improve vaccine efficacy.

We investigated the dynamics of seroconversion in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. During March 29-May 22, 2020, we collected serum samples and associated clinical data from 177 persons in London, UK, who had SARS-CoV-2 infection. We measured IgG against SARS-CoV-2 and compared antibody levels with patient outcomes, demographic information, and laboratory characteristics. We found that 2.0%-8.5% of persons did not seroconvert 3-6 weeks after infection. Persons who seroconverted were older, were more likely to have concurrent conditions, and had higher levels of inflammatory markers. Non-White persons had higher antibody concentrations than those who identified as White; these concentrations did not decline during follow-up. Serologic assay results correlated with disease outcome, race, and other risk factors for severe SARS-CoV-2 infection. Serologic assays can be used in surveillance to clarify the duration and protective nature of humoral responses to SARS-CoV-2 infection.

The lymphatic system has a pivotal role in immune homeostasis. To better understand this, we investigated the impact of Primary Lymphatic Anomalies (PLA) on lymphocyte numbers and phenotype. The study comprised (i) a retrospective cohort: 177 PLA subjects from the National Primary Lymphatic Anomaly Register with clinical and laboratory data, and (ii) a prospective cohort: 28 patients with PLA and 20 healthy controls. Patients were subdivided using established phenotypic diagnostic categories and grouped into simplex (localised tissue involvement only) and systemic (involvement of central lymphatics). Further grouping variables included genital involvement and the likelihood of co-existent intestinal lymphangiectasia. Haematology laboratory parameters were analysed in both cohorts. In the prospective cohort, prospective blood samples were analysed by flow cytometry for markers of proliferation, differentiation, activation, skin-homing, and for regulatory (CD4 Foxp3 ) T cells (Treg). In patients with PLA, lymphopaenia was frequent (22% of subjects), affected primarily the CD4 T cell subset, and was more severe in subjects with systemic versus simplex patterns of disease (36% vs 9% for lymphopaenia; 70% vs 33% for CD4 cells). B cells, NK cells and monocytes were better conserved (except in deficiency characterised by monocytopaenia). Genital oedema and likelihood of concomitant intestinal lymphangiectasia independently predicted CD4 T cell depletion. Analysing CD4 and CD8 T cells by differentiation markers revealed disproportionate depletion of naïve cells, with a skewing towards a more differentiated effector profile. Systemic PLA conditions were associated with: increased expression of Ki67, indicative of recent cell division, in naïve CD4 , but not CD8 T cells; increased levels of activation in CD4 , but not CD8 T cells; and an increased proportion of Treg. Skin-homing marker (CCR10, CLA and CCR4) expression was reduced in some patients with simplex phenotypes. Patients with PLA who have dysfunctional lymphatics have a selective reduction in circulating lymphocytes which preferentially depletes naïve CD4 T cells. The presence of systemic disease, genital oedema, and intestinal lymphangiectasia independently predict CD4 lymphopaenia. The association of this depletion with immune activation and increased circulating Tregs suggests lymphatic-lymphocyte interactions and local inflammatory changes are pivotal in driving immunopathology.

T stem cell-like memory cells (T SCM cells) are considered to be essential for the maintenance of immune memory. The T SCM population has been shown to have the key properties of a stem cell population: multipotency, self-renewal and clonal longevity. Here we show that no single population has all these stem cell properties, instead the properties are distributed. We show that the human T SCM population consists of two distinct cell subpopulations which can be distinguished by the level of their CD95 expression (CD95int and CD95hi). Crucially, using long-term in vivo labelling of human volunteers, we establish that these are distinct populations rather than transient states of the same population. These two subpopulations have different functional profiles ex vivo , different transcriptional patterns, and different tissue distributions. They also have significantly different TREC content indicating different division histories and we find that the frequency of CD95hi T SCM increases with age. Most importantly, CD95hi and CD95int T SCM cells also have very different dynamics in vivo with CD95hi cells showing considerably higher proliferation but significantly reduced clonal longevity compared with CD95int T SCM . While both T SCM subpopulations exhibit considerable multipotency, no single population of T SCM cells has both the properties of self-renewal and clonal longevity. Instead, the “stemness” of the T SCM population is generated by the complementary dynamic properties of the two subpopulations: CD95int T SCM which have the property of clonal longevity and CD95hi T SCM which have the properties of expansion and self-renewal. We suggest that together, these two populations function as a stem cell population.

FUNDING. Wellcome Trust; Medical Research Council; EU Horizon 2020; EU FP7; Leukemia and Lymphoma Research; National Institute of Health Research (NIHR) Imperial Biomedical Research Centre; Imperial College Research Fellowship; National Institutes of Health; Jefferiss Trust.