Thea Hogan

Laboratory mice develop populations of circulating memory CD4⁺ T cells in the absence of overt infection. We have previously shown that these populations are replenished from naive precursors at high levels throughout life (Gossel et al., 2017). However, the nature, relative importance and timing of the forces generating these cells remain unclear. Here, we tracked the generation of memory CD4⁺ T cell subsets in mice housed in facilities differing in their ‘dirtiness’. We found evidence for sequential naive to central memory to effector memory development, and confirmed that both memory subsets are heterogeneous in their rates of turnover. We also inferred that early exposure to self and environmental antigens establishes persistent memory populations at levels determined largely, although not exclusively, by the dirtiness of the environment. After the first few weeks of life, however, these populations are continuously supplemented by new memory cells at rates that are independent of environment.

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. The body maintains large populations of naive T cells, a type of white blood cell that is able to respond specifically to pathogens. This arsenal is essential for our capacity to fight novel infections throughout our lifespan, and their numbers remain quite stable despite a gradual decline in the production of new naive T cells as we age. However, the mechanisms that underlie this stability are not well understood. In this study, we address this problem by testing a variety of potential mechanisms, each framed as a mathematical model, against multiple datasets obtained from experiments performed in mice. Our analysis supports a mechanism by which naïve T cells gradually increase their ability to survive the longer they reside in the circulation. Paradoxically, however, naïve T cells may also lose their ability to respond effectively to infections as they age. Together, these processes may drive the accumulation of older, functionally impaired T cells, potentially at the expense of younger and more immunologically potent cells, as we age.

Characterising the longevity of immunological memory requires establishing the rules underlying the renewal and death of peripheral T cells. However, we lack knowledge of the population structure and how self-renewal and de novo influx contribute to the maintenance of memory compartments. Here, we characterise the kinetics and structure of murine CD4 T cell memory subsets by measuring the rates of influx of new cells and using detailed timecourses of DNA labelling that also distinguish the behaviour of recently divided and quiescent cells. We find that both effector and central memory CD4 T cells comprise subpopulations with highly divergent rates of turnover, and show that inflows of new cells sourced from the naive pool strongly impact estimates of memory cell lifetimes and division rates. We also demonstrate that the maintenance of CD4 T cell memory subsets in healthy mice is unexpectedly and strikingly reliant on this replenishment.

Significance: T cells are essential components of vertebrate immune systems, but the mechanisms by which they are maintained are still poorly defined. Existing methods infer cell lifetimes and division rates using DNA labeling of dividing cells, but do not resolve heterogeneity in population dynamics well. We present a novel experimental system that, when combined with mathematical models, yields kinetic parameters and allows us to measure the effect of a cell’s age on its ability to survive and divide. Our approach quantifies lymphocyte dynamics over a year of a mouse’s life and reveals a first-in, last-out structure in which subpopulations of naive T cells generated early in life persist with slower kinetics and resist displacement by newer specificities.

The rate at which a cytotoxic T lymphocyte (CTL) can survey for infected cells is a key ingredient of models of vertebrate immune responses to intracellular pathogens. Estimates have been obtained using in vivo cytotoxicity assays in which peptide-pulsed splenocytes are killed by CTL in the spleens of immunised mice. However the spleen is a heterogeneous environment and splenocytes comprise multiple cell types. Are some cell types intrinsically more susceptible to lysis than others? Quantitatively, what impacts are made by the spatial distribution of targets and effectors, and the level of peptide-MHC on the target cell surface? To address these questions we revisited the splenocyte killing assay, using CTL specific for an epitope of influenza virus. We found that at the cell population level T cell targets were killed more rapidly than B cells. Using modeling, quantitative imaging and in vitro killing assays we conclude that this difference in vivo likely reflects different migratory patterns of targets within the spleen and a heterogeneous distribution of CTL, with no detectable difference in the intrinsic susceptibilities of the two populations to lysis. Modeling of the stages involved in the detection and killing of peptide-pulsed targets in vitro revealed that peptide dose influenced the ability of CTL to form conjugates with targets but had no detectable effect on the probability that conjugation resulted in lysis, and that T cell targets took longer to lyse than B cells. We also infer that incomplete killing in vivo of cells pulsed with low doses of peptide may be due to a combination of heterogeneity in peptide uptake and the dissociation, but not internalisation, of peptide-MHC complexes. Our analyses demonstrate how population-averaged parameters in models of immune responses can be dissected to account for both spatial and cellular heterogeneity. Measurements of the rates at which a single cytotoxic T lymphocyte (CTL) can survey for infected cells, and kill them upon encounter, are important for constructing predictive models of vertebrate immune responses to intracellular pathogens. The surveillance rate has been estimated previously using combinations of modeling and experiment, making the assumption that CTL and target cells are well-mixed and that all cell types are killed with equal efficiency. In this study we take an iterative approach with theory and experiment to go beyond such models and detail the effects of cellular heterogeneity, the spatial organisation of the tissue within which killing is taking place, and the influence of the level of expression of peptides on the target cell surface. We demonstrate that determining the degree of co-localisation of effector and target cells, and the level of peptide expression on targets, are most important for improving estimates of CTL killing rates. Further, while the probabilities of killing upon conjugation of CTL with T and B cell targets are similar, T cells take substantially longer to kill than B cells, an effect that may be important when CTL numbers are limiting.

The expression of the Ikaros transcription factor family member, Helios, has been shown to be associated with T‐cell tolerance in both the thymus and the periphery. To better understand the importance of Helios in tolerance pathways, we have examined the expression of Helios in TCR‐transgenic T cells specific for the gastric H⁺/K⁺ ATPase, the autoantigen target in autoimmune gastritis. Analysis of H⁺/K⁺ ATPase‐specific T cells in mice with different patterns of H⁺/K⁺ ATPase expression revealed that, in addition to the expression of Helios in CD4⁺Foxp3⁺ regulatory T (Treg) cells, Helios is expressed by a large proportion of CD4⁺Foxp3⁻ T cells in both the thymus and the paragastric lymph node (PgLN), which drains the stomach. In the thymus, Helios was expressed by H⁺/K⁺ ATPase‐specific thymocytes that were undergoing negative selection. In the periphery, Helios was expressed in H⁺/K⁺ ATPase‐specific CD4⁺ T cells following H⁺/K⁺ ATPase presentation and was more highly expressed when T‐cell activation occurred in the absence of inflammation. Analysis of purified H⁺/K⁺ ATPase‐specific CD4⁺Foxp3⁻Helios⁺ T cells demonstrated that they were functionally anergic. These results demonstrate that Helios is expressed by thymic and peripheral T cells that are being driven to tolerance in response to a genuine autoantigen.

The production of protective antibody requires effective signalling of naive B cells following encounter with antigen, and the divergence of responding B lymphocytes into distinct lineages. Polarity proteins have recently been proposed as important mediators of both the initial B cell response, and potentially of asymmetric cell division. Here we show that, although polarity proteins of the Scribble complex, Scribble, Dlg1 and Lgl1, are expressed and polarized during early B cell activation, their deficiency has no effect on the in vivo outcome of immunization or challenge with influenza infection. Furthermore, we find a striking correlation in the differentiation outcome of daughters of single founder B cells in vitro. Taken together, our results indicate that B cell differentiation does not require polarity proteins of the Scribble complex, and the findings do not support a role for asymmetric cell division in B cell activation and differentiation.

Lymphopenia induces T cells to undergo cell divisions as part of a homeostatic response mechanism. The clonal response to lymphopenia is extremely diverse, and it is unknown whether this heterogeneity represents distinct mechanisms of cell-cycle control or whether a common mechanism can account for the diversity. We addressed this question by combining in vivo and mathematical modeling of lymphopenia-induced proliferation (LIP) of two distinct T cell clonotypes. OT-I T cells undergo rapid LIP accompanied by differentiation that superficially resembles Ag-induced proliferation, whereas F5 T cells divide slowly and remain naive. Both F5 and OT-I LIP responses were most accurately described by a single stochastic division model where the rate of cell division was exponentially decreased with increasing cell numbers. The model successfully identified key biological parameters of the response and accurately predicted the homeostatic set point of each clone. Significantly, the model was successful in predicting interclonal competition between OT-I and F5 T cells, consistent with competition for the same resource(s) required for homeostatic proliferation. Our results show that diverse and heterogenous clonal T cell responses can be accounted for by a single common model of homeostasis.

IL‐17, produced by a distinct lineage of CD4⁺ helper T (Th) cells termed Th17 cells, induces the production of pro‐inflammatory cytokines from resident cells and it has been demonstrated that over‐expression of IL‐17 plays a crucial role in the onset of several auto‐immune diseases. Here we examined the role of IL‐17 in the pathogenesis of autoimmune gastritis, a disease that was previously believed to be mediated by IFN‐γ. Significantly higher levels of IL‐17 and IFN‐γ were found in the stomachs and stomach‐draining lymph nodes of mice with severe autoimmune gastritis. Unlike IL‐17, which was produced solely by CD4⁺ T cells in gastritic mice, the majority of IFN‐γ‐producing cells were CD8⁺ T cells. However, CD8⁺ T cells alone were not able to induce autoimmune gastritis. T cells that were deficient in IL‐17 or IFN‐γ production were able to induce autoimmune gastritis but to a much lower extent compared with the disease induced by wild‐type T cells. These data demonstrate that production of neither IL‐17 nor IFN‐γ by effector T cells is essential for the initiation of autoimmune gastritis, but suggest that both are required for the disease to progress to the late pathogenic stage that includes significant tissue disruption.