Notch2 controls developmental fate choices between germinal center and marginal zone B cells upon immunization.

Sustained Notch2 signals induce trans-differentiation of Follicular B (FoB) cells into Marginal Zone B (MZB) cells in mice, but the physiology underlying this differentiation pathway is still elusive. Here, we demonstrate that most B cells receive a basal Notch signal, which is intensified in pre-MZB and MZB cells. Ablation or constitutive activation of Notch2 upon T-cell-dependent immunization reveals an interplay between antigen-induced activation and Notch2 signaling, in which FoB cells that turn off Notch2 signaling enter germinal centers (GC), while high Notch2 signaling leads to generation of MZB cells or to initiation of plasmablast differentiation. Notch2 signaling is dispensable for GC dynamics but appears to be re-induced in some centrocytes to govern expansion of IgG1+ GCB cells. Mathematical modelling suggests that antigen-activated FoB cells make a Notch2 dependent binary fate-decision to differentiate into either GCB or MZB cells. This bifurcation might serve as a mechanism to archive antigen-specific clones into functionally and spatially diverse B cell states to generate robust antibody and memory responses.

Towards a unified model of naive T cell dynamics across the lifespan.

Naive CD4 and CD8 T cells are cornerstones of adaptive immunity, but the dynamics of their establishment early in life and how their kinetics change as they mature following release from the thymus are poorly understood. Further, due to the diverse signals implicated in naive T cell survival, it has been a long-held and conceptually attractive view that they are sustained by active homeostatic control as thymic activity wanes. Here we use multiple modelling and experimental approaches to identify a unified model of naive CD4 and CD8 T cell population dynamics in mice, across their lifespan. We infer that both subsets divide rarely, and progressively increase their survival capacity with cell age. Strikingly, this simple model is able to describe naive CD4 T cell dynamics throughout life. In contrast, we find that newly generated naive CD8 T cells are lost more rapidly during the first 3-4 weeks of life, likely due to increased recruitment into memory. We find no evidence for elevated division rates in neonates, or for feedback regulation of naive T cell numbers at any age. We show how confronting mathematical models with diverse datasets can reveal a quantitative and remarkably simple picture of naive T cell dynamics in mice from birth into old age.

Fate Mapping Quantifies the Dynamics of B Cell Development and Activation throughout Life.

Follicular mature (FM) and germinal center (GC) B cells underpin humoral immunity, but the dynamics of their generation and maintenance are not clearly defined. Here, we exploited a fate-mapping system in mice that tracks B cells as they develop into peripheral subsets, together with a cell division fate reporter mouse and mathematical models. We find that FM cells are kinetically homogeneous, recirculate freely, are continually replenished from transitional populations, and self-renew rarely. In contrast, GC B cell lineages persist for weeks with rapid turnover and site-specific dynamics. Those in the spleen derive from transitional cells and are kinetically homogeneous, while those in lymph nodes derive from FM B cells and comprise both transient and persistent clones. These differences likely derive from the nature of antigen exposure at the different sites. Our integrative approach also reveals how the host environment drives cell-extrinsic, age-related changes in B cell homeostasis.

Cell generation dynamics underlying naive T-cell homeostasis in adult humans.

Thymic involution and proliferation of naive T cells both contribute to shaping the naive T-cell repertoire as humans age, but a clear understanding of the roles of each throughout a human life span has been difficult to determine. By measuring nuclear bomb test-derived 14C in genomic DNA, we determined the turnover rates of CD4+ and CD8+ naive T-cell populations and defined their dynamics in healthy individuals ranging from 20 to 65 years of age. We demonstrate that naive T-cell generation decreases with age because of a combination of declining peripheral division and thymic production during adulthood. Concomitant decline in T-cell loss compensates for decreased generation rates. We investigated putative mechanisms underlying age-related changes in homeostatic regulation of CD4+ naive T-cell turnover, using mass cytometry to profile candidate signaling pathways involved in T-cell activation and proliferation relative to CD31 expression, a marker of thymic proximity for the CD4+ naive T-cell population. We show that basal nuclear factor κB (NF-κB) phosphorylation positively correlated with CD31 expression and thus is decreased in peripherally expanded naive T-cell clones. Functionally, we found that NF-κB signaling was essential for naive T-cell proliferation to the homeostatic growth factor interleukin (IL)-7, and reduced NF-κB phosphorylation in CD4+CD31- naive T cells is linked to reduced homeostatic proliferation potential. Our results reveal an age-related decline in naive T-cell turnover as a putative regulator of naive T-cell diversity and identify a molecular pathway that restricts proliferation of peripherally expanded naive T-cell clones that accumulate with age.

Spatiotemporal Modeling of the Key Migratory Events During the Initiation of Adaptive Immunity.

Initiation of adaptive immunity involves distinct migratory cell populations coming together in a highly dynamic and spatially organized process. However, we lack a detailed spatiotemporal map of these events due to our inability to track the fate of cells between anatomically distinct locations or functionally identify cell populations as migratory. We used photo-convertible transgenic mice (Kaede) to spatiotemporally track the fate and composition of the cell populations that leave the site of priming and enter the draining lymph node to initiate immunity. We show that following skin priming, the lymph node migratory population is principally composed of cells recruited to the site of priming, with a minor contribution from tissue resident cells. In combination with the YAe/Eα system, we also show that the majority of cells presenting antigen are CD103+CD11b+ dendritic cells that were recruited to the site of priming during the inflammatory response. This population has previously only been described in relation to mucosal tissues. Comprehensive phenotypic profiling of the cells migrating from the skin to the draining lymph node by mass cytometry revealed that in addition to dendritic cells, the migratory population also included CD4+ and CD8+ T cells, B cells, and neutrophils. Taking our complex spatiotemporal data set, we then generated a model of cell migration that quantifies and describes the dynamics of arrival, departure, and residence times of cells at the site of priming and in the draining lymph node throughout the time-course of the initiation of adaptive immunity. In addition, we have identified the mean migration time of migratory dendritic cells as they travel from the site of priming to the draining lymph node. These findings represent an unprecedented, detailed and quantitative map of cell dynamics and phenotypes during immunization, identifying where, when and which cells to target for immunomodulation in autoimmunity and vaccination strategies.

Correlation of cell-surface CD8 levels with function, phenotype and transcriptome of naive CD8 T cells.

We have previously demonstrated co-receptor level-associated functional heterogeneity in apparently homogeneous naive peripheral CD4 T cells, dependent on MHC-mediated tonic signals. Maturation pathways can differ between naive CD4 and naive CD8 cells, so we tested whether the latter showed similar co-receptor level-associated functional heterogeneity. We report that, when either polyclonal and T-cell receptor (TCR)-transgenic monoclonal peripheral naive CD8 T cells from young mice were separated into CD8hi and CD8lo subsets, CD8lo cells responded poorly, but CD8hi and CD8lo subsets of CD8 single-positive (SP) thymocytes responded similarly. CD8lo naive CD8 T cells were smaller and showed lower levels of some cell-surface molecules, but higher levels of the negative regulator CD5. In addition to the expected peripheral decline in CD8 levels on transferred naive CD8 T cells in wild-type (WT) but not in MHC class I-deficient recipient mice, short-duration naive T-cell-dendritic cell (DC) co-cultures in vitro also caused co-receptor down-modulation in CD8 T cells but not in CD4 T cells. Constitutive pZAP70/pSyk and pERK levels ex vivo were lower in CD8lo naive CD8 T cells and dual-specific phosphatase inhibition partially rescued their hypo-responsiveness. Bulk mRNA sequencing showed major differences in the transcriptional landscapes of CD8hi and CD8lo naive CD8 T cells. CD8hi naive CD8 T cells showed enrichment of genes involved in positive regulation of cell cycle and survival. Our data show that naive CD8 T cells show major differences in their signaling, transcriptional and functional landscapes associated with subtly altered CD8 levels, consistent with the possibility of peripheral cellular aging.

Age is not just a number: Naive T cells increase their ability to persist in the circulation over time.

The processes regulating peripheral naive T-cell numbers and clonal diversity remain poorly understood. Conceptually, homeostatic mechanisms must fall into the broad categories of neutral (simple random birth-death models), competition (regulation of cell numbers through quorum-sensing, perhaps via limiting shared resources), adaptation (involving cell-intrinsic changes in homeostatic fitness, defined as net growth rate over time), or selection (involving the loss or outgrowth of cell populations deriving from intercellular variation in fitness). There may also be stably maintained heterogeneity within the naive T-cell pool. To distinguish between these mechanisms, we confront very general models of these processes with an array of experimental data, both new and published. While reduced competition for homeostatic stimuli may impact cell survival or proliferation in neonates or under moderate to severe lymphopenia, we show that the only mechanism capable of explaining multiple, independent experimental studies of naive CD4+ and CD8+ T-cell homeostasis in mice from young adulthood into old age is one of adaptation, in which cells act independently and accrue a survival or proliferative advantage continuously with their post-thymic age. However, aged naive T cells may also be functionally impaired, and so the accumulation of older cells via 'conditioning through experience' may contribute to reduced immune responsiveness in the elderly.

Peripheral residence of naïve CD4 T cells induces MHC class II-dependent alterations in phenotype and function.

BACKGROUND: As individual naïve CD4 T lymphocytes circulate in the body after emerging from the thymus, they are likely to have individually varying microenvironmental interactions even in the absence of stimulation via specific target recognition. It is not clear if these interactions result in alterations in their activation, survival and effector programming. Naïve CD4 T cells show unimodal distribution for many phenotypic properties, suggesting that the variation is caused by intrinsic stochasticity, although underlying variation due to subsets created by different histories of microenvironmental interactions remains possible. To explore this possibility, we began examining the phenotype and functionality of naïve CD4 T cells differing in a basic unimodally distributed property, the CD4 levels, as well as the causal origin of these differences. RESULTS: We examined separated CD4hi and CD4lo subsets of mouse naïve CD4 cells. CD4lo cells were smaller with higher CD5 levels and lower levels of the dual-specific phosphatase (DUSP)6-suppressing micro-RNA miR181a, and responded poorly with more Th2-skewed outcomes. Human naïve CD4lo and CD4hi cells showed similar differences. Naïve CD4lo and CD4hi subsets of thymic single-positive CD4 T cells did not show differences whereas peripheral naïve CD4lo and CD4hi subsets of T cell receptor (TCR)-transgenic T cells did. Adoptive transfer-mediated parking of naïve CD4 cells in vivo lowered CD4 levels, increased CD5 and reactive oxygen species (ROS) levels and induced hyporesponsiveness in them, dependent, at least in part, on availability of major histocompatibility complex class II (MHCII) molecules. ROS scavenging or DUSP inhibition ameliorated hyporesponsiveness. Naïve CD4 cells from aged mice showed lower CD4 levels and cell sizes, higher CD5 levels, and hyporesponsiveness and Th2-skewing reversed by DUSP inhibition. CONCLUSIONS: Our data show that, underlying a unimodally distributed property, the CD4 level, there are subsets of naïve CD4 cells that vary in the time spent in the periphery receiving MHCII-mediated signals and show resultant alteration of phenotype and functionality via ROS and DUSP activity. Our findings also suggest the feasibility of potential pharmacological interventions for improved CD4 T cell responses during vaccination of older people via either anti-oxidant or DUSP inhibitor small molecules.

T cell ageing: effects of age on development, survival & function.

Age associated decline of the immune system continues to be a major health concern. All components of innate and adaptive immunity are adversely affected to lesser or greater extent by ageing resulting in an overall decline of immunocompetence. As a result in the aged population, there is increased susceptibility to infection, poor responses to vaccination, and increased incidence of autoreactivity. There is an increasing focus on the role of T cells during ageing because of their impact on the overall immune responses. A steady decline in the production of fresh naïve T cells, more restricted T cell receptor (TCR) repertoire and weak activation of T cells are some of the effects of ageing. In this review we summarize our present understanding of the effects of ageing on naïve CD4 T cells and potential approaches for therapeutic interventions to restore protective immunity in the aged population.