The dynamics and longevity of circulating CD4+ memory T cells depend on cell age and not the chronological age of the host.

Quantifying the kinetics with which memory T cell populations are generated and maintained is essential for identifying the determinants of the duration of immunity. The quality and persistence of circulating CD4 effector memory (TEM) and central memory (TCM) T cells in mice appear to shift with age, but it is unclear whether these changes are driven by the aging host environment, by cell age effects, or both. Here, we address these issues by combining DNA labelling methods, established fate-mapping systems, a novel reporter mouse strain, and mathematical models. Together, these allow us to quantify the dynamics of both young and established circulating memory CD4 T cell subsets, within both young and old mice. We show that that these cells and their descendents become more persistent the longer they reside within the TCM and TEM pools. This behaviour may limit memory CD4 T cell diversity by skewing TCR repertoires towards clones generated early in life, but may also compensate for functional defects in new memory cells generated in old age.

The dynamics and longevity of circulating CD4+ memory T cells depend on cell age and not the chronological age of the host.

Quantifying the kinetics with which memory T cell populations are generated and maintained is essential for identifying the determinants of the duration of immunity. The quality and persistence of circulating CD4+ effector memory (TEM) and central memory (TCM) T cells in mice appear to shift with age, but it is unclear whether these changes are driven by the aging host environment, by cell age effects, or both. Here we address these issues by combining DNA labelling methods, established fate-mapping systems, a novel reporter mouse strain, and mathematical models. Together, these allow us to quantify the dynamics of both young and established circulating memory CD4+ T cell subsets, within both young and old mice. We show that that these cells and their descendents become more persistent the longer they reside within the TCM and TEM pools. This behaviour may limit memory CD4 T cell diversity by skewing TCR repertoires towards clones generated early in life, but may also compensate for functional defects in new memory cells generated in old age.

A linear ontogeny accounts for the development of naive, memory and tumour-infiltrating regulatory T cells in mice.

Foxp3 + Regulatory T cells (Treg) are a subset of CD4 + T cells that play critical functions in maintaining tolerance to self antigens and suppressing autoimmunity, regulating immune responses to pathogens and have a role in the pathophysiology of anti-tumoural immunity. Treg ontogeny is complex since they are generated following recognition of self antigens in the thymus during normal T cell development (thymic Treg), but are also induced from mature conventional T cells when activated by foreign antigen with appropriate additional cues (inducible Treg). How these distinct ontogenic pathways contribute to the maintenance and function of the mature Treg compartment in health and disease remains unclear. Here, we use a combination of fate mapping approaches in mice to map the ontogeny of Treg subsets throughout life and estimate rates of production, loss and self-renewal. We find that naive and effector/memory (EM) Treg subsets exhibit distinct dynamics but are both continuously replenished by de novo generation throughout life. Using an inducible Foxp3-dependent Cre fate reporter system, we show that naive Treg and not conventional T cells, are the predominant precursors of EM Treg in adults. Tonic development of new EM Treg is not influenced by foreign antigens from commensals, rather suggesting a role for self recognition. To investigate the ontogeny of Treg development in malignant disease, we used the same fate reporter systems to characterise the Treg infiltrate of three different model tumours. In all three cases, we found that Treg derived from pre-existing, EM Treg. Together, these results reveal a predominantly linear pathway of Treg development from thymic origin to EM Treg associated with pathophysiology of malignant disease, that is driven by self antigen recognition throughout.

A RIPK1-specific PROTAC degrader achieves potent antitumor activity by enhancing immunogenic cell death.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies.

Quantifying cellular dynamics in mice using a novel fluorescent division reporter system.

The dynamics of cell populations are frequently studied in vivo using pulse-chase DNA labeling techniques. When combined with mathematical models, the kinetic of label uptake and loss within a population of interest then allows one to estimate rates of cell production and turnover through death or onward differentiation. Here we explore an alternative method of quantifying cellular dynamics, using a cell fate-mapping mouse model in which dividing cells can be induced to constitutively express a fluorescent protein, using a Ki67 reporter construct. We use a pulse-chase approach with this reporter mouse system to measure the lifespans and division rates of naive CD4 and CD8 T cells using a variety of modeling approaches, and show that they are all consistent with estimates derived from other published methods. However we propose that to obtain unbiased parameter estimates and full measures of their uncertainty one should simultaneously model the timecourses of the frequencies of labeled cells within both the population of interest and its precursor. We conclude that Ki67 reporter mice provide a promising system for modeling cellular dynamics.

IKK promotes naïve T cell survival by repressing RIPK1-dependent apoptosis and activating NF-κB.

The inhibitor of κB kinase (IKK) complex regulates the activation of the nuclear factor κB (NF-κB) family of transcription factors. In addition, IKK represses extrinsic cell death pathways dependent on receptor-interacting serine/threonine-protein kinase 1 (RIPK1) by directly phosphorylating this kinase. Here, we showed that peripheral naïve T cells in mice required the continued expression of IKK1 and IKK2 for their survival; however, the loss of these cells was only partially prevented when extrinsic cell death pathways were blocked by either deleting Casp8 (which encodes the apoptosis-inducing caspase 8) or inhibiting the kinase activity of RIPK1. Inducible deletion of Rela (which encodes the NF-κB p65 subunit) in mature CD4+ T cells also resulted in loss of naïve CD4+ T cells and in reduced abundance of the interleukin-7 receptor (IL-7R) encoded by the NF-κB target Il7r, revealing an additional reliance upon NF-κB for the long-term survival of mature T cells. Together, these data indicate that the IKK-dependent survival of naïve CD4+ T cells depends on both repression of extrinsic cell death pathways and activation of an NF-κB-dependent survival program.

Phosphorylation of RIPK1 serine 25 mediates IKK dependent control of extrinsic cell death in T cells.

The Inhibitor of Kappa B Kinase (IKK) complex is a critical regulator of NF-κB activation. More recently, IKK has also been shown to repress RIPK1 dependent extrinsic cell death pathways by directly phosphorylating RIPK1 at serine 25. In T cells, IKK expression is essential for normal development in the thymus, by promoting survival of thymocytes independently of NF-κB activation. RIPK1 undergoes extensive phosphorylation following TNF stimulation in T cells, though which targets are required to repress RIPK1 has not been defined. Here, we show that TNF induced phosphorylation of RIPK1 at S25 is IKK dependent. We test the relevance of this phosphorylation event in T cells using mice with a RIPK1S25D phosphomimetic point mutation to endogenous RIPK1. We find that this mutation protects T cells from TNF induced cell death when IKK activity is inhibited in vitro, and can rescues development of IKK deficient thymocytes in vivo to a degree comparable with kinase dead RIPK1D138N. Together, these data show that phosphorylation of RIPK1S25 by IKK represents a key regulatory event promoting survival of T cells by IKK.

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.

Overexpression of wild-type IL-7Rα promotes T-cell acute lymphoblastic leukemia/lymphoma.

Tight regulation of IL-7Rα expression is essential for normal T-cell development. IL-7Rα gain-of-function mutations are known drivers of T-cell acute lymphoblastic leukemia (T-ALL). Although a subset of patients with T-ALL display high IL7R messenger RNA levels and cases with IL7R gains have been reported, the impact of IL-7Rα overexpression, rather than mutational activation, during leukemogenesis remains unclear. In this study, overexpressed IL-7Rα in tetracycline-inducible Il7r transgenic and Rosa26 IL7R knockin mice drove potential thymocyte self-renewal, and thymus hyperplasia related to increased proliferation of T-cell precursors, which subsequently infiltrated lymph nodes, spleen, and bone marrow, ultimately leading to fatal leukemia. The tumors mimicked key features of human T-ALL, including heterogeneity in immunophenotype and genetic subtype between cases, frequent hyperactivation of the PI3K/Akt pathway paralleled by downregulation of p27Kip1 and upregulation of Bcl-2, and gene expression signatures evidencing activation of JAK/STAT, PI3K/Akt/mTOR and Notch signaling. Notably, we also found that established tumors may no longer require high levels of IL-7R expression upon secondary transplantation and progressed in the absence of IL-7, but remain sensitive to inhibitors of IL-7R-mediated signaling ruxolitinib (Jak1), AZD1208 (Pim), dactolisib (PI3K/mTOR), palbociclib (Cdk4/6), and venetoclax (Bcl-2). The relevance of these findings for human disease are highlighted by the fact that samples from patients with T-ALL with high wild-type IL7R expression display a transcriptional signature resembling that of IL-7-stimulated pro-T cells and, critically, of IL7R-mutant cases of T-ALL. Overall, our study demonstrates that high expression of IL-7Rα can promote T-cell tumorigenesis, even in the absence of IL-7Rα mutational activation.

NF-κB and Extrinsic Cell Death Pathways - Entwined Do-or-Die Decisions for T cells.

NF-κB signaling is required at multiple stages of T cell development and function. The NF-κB pathway integrates signals from many receptors and involves diverse adapters and kinases. Recent advances demonstrate that kinases controlling NF-κB activation, such as the IKK complex, serve dual independent functions because they also control cell death checkpoints. Survival functions previously attributed to NF-κB are in fact mediated by these upstream kinases by novel mechanisms. This new understanding has led to a refined view of how NF-κB and cell death signaling are interlinked and how they regulate cell fate. We discuss how NF-κB activation and control of cell death signaling by common upstream triggers cooperate to regulate different aspects of T cell development and function.

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.

Regulatory T Cells Restrain Interleukin-2- and Blimp-1-Dependent Acquisition of Cytotoxic Function by CD4+ T Cells.

In addition to helper and regulatory potential, CD4+ T cells also acquire cytotoxic activity marked by granzyme B (GzmB) expression and the ability to promote rejection of established tumors. Here, we examined the molecular and cellular mechanisms underpinning the differentiation of cytotoxic CD4+ T cells following immunotherapy. CD4+ transfer into lymphodepleted animals or regulatory T (Treg) cell depletion promoted GzmB expression by tumor-infiltrating CD4+, and this was prevented by interleukin-2 (IL-2) neutralization. Transcriptional analysis revealed a polyfunctional helper and cytotoxic phenotype characterized by the expression of the transcription factors T-bet and Blimp-1. While T-bet ablation restricted interferon-γ (IFN-γ) production, loss of Blimp-1 prevented GzmB expression in response to IL-2, suggesting two independent programs required for polyfunctionality of tumor-reactive CD4+ T cells. Our findings underscore the role of Treg cells, IL-2, and Blimp-1 in controlling the differentiation of cytotoxic CD4+ T cells and offer a pathway to enhancement of anti-tumor activity through their manipulation.

Flip the coin: IL-7 and IL-7R in health and disease.

The cytokine IL-7 and its receptor, IL-7R, are critical for T cell and, in the mouse, B cell development, as well as differentiation and survival of naive T cells, and generation and maintenance of memory T cells. They are also required for innate lymphoid cell (ILC) development and maintenance, and consequently for generation of lymphoid structures and barrier defense. Here we discuss the central role of IL-7 and IL-7R in the lymphoid system and highlight the impact of their deregulation, placing a particular emphasis on their 'dark side' as promoters of cancer development. We also explore therapeutic implications and opportunities associated with either positive or negative modulation of the IL-7-IL-7R signaling axis.

Differential impact of self and environmental antigens on the ontogeny and maintenance of CD4+ T cell memory.

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.

Survival of Single Positive Thymocytes Depends upon Developmental Control of RIPK1 Kinase Signaling by the IKK Complex Independent of NF-κB.

NF-κB (nuclear factor κB) signaling is considered critical for single positive (SP) thymocyte development because loss of upstream activators of NF-κB, such as the IKK complex, arrests their development. We found that the compound ablation of RelA, cRel, and p50, required for canonical NF-κB transcription, had no impact upon thymocyte development. While IKK-deficient thymocytes were acutely sensitive to tumor necrosis factor (TNF)-induced cell death, Rel-deficient cells remained resistant, calling into question the importance of NF-κB as the IKK target required for thymocyte survival. Instead, we found that IKK controlled thymocyte survival by repressing cell-death-inducing activity of the serine/threonine kinase RIPK1. We observed that RIPK1 expression was induced during development of SP thymocytes and that IKK was required to prevent RIPK1-kinase-dependent death of SPs in vivo. Finally, we showed that IKK was required to protect Rel-deficient thymocytes from RIPK1-dependent cell death, underscoring the NF-κB-independent function of IKK during thymic development.

The natural history of naive T cells from birth to maturity.

Generating and maintaining a diverse repertoire of naive T cells is essential for protection against pathogens, and developing a mechanistic and quantitative description of the processes involved lies at the heart of our understanding of vertebrate immunity. Here, we review the biology of naive T cells from birth to maturity and outline how the integration of mathematical models and experiments has helped us to develop a full picture of their life histories.

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.

IL-7-dependent maintenance of ILC3s is required for normal entry of lymphocytes into lymph nodes.

IL-7 is essential for the development and homeostasis of T and B lymphocytes and is critical for neonatal lymph node organogenesis because Il7-/- mice lack normal lymph nodes. Whether IL-7 is a continued requirement for normal lymph node structure and function is unknown. To address this, we ablated IL-7 function in normal adult hosts. Either inducible Il7 gene deletion or IL-7R blockade in adults resulted in a rapid loss of lymph node cellularity and a corresponding defect in lymphocyte entry into lymph nodes. Although stromal and dendritic cell components of lymph nodes were present in normal numbers and representation, innate lymphoid cell (ILC) subpopulations were substantially decreased after IL-7 ablation. Testing lymphocyte homing in bone marrow chimeras reconstituted with Rorc-/- bone marrow confirmed that ILC3s in lymph nodes are required for normal lymphocyte homing. Collectively, our data suggest that maintenance of intact lymph nodes relies on IL-7-dependent maintenance of ILC3 cells.

Memory CD4 T cell subsets are kinetically heterogeneous and replenished from naive T cells at high levels.

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.

Generation of Busulfan Chimeric Mice for the Analysis of T Cell Population Dynamics.

This protocol was developed to generate chimeric mice in which T lymphocytes could be stratified by age on the basis of congenic marker expression. The conditioning drug busulfan is used to ablate host haematopoietic stem cells while leaving the peripheral immune system intact. Busulfan treatment is followed by bone marrow transplantation (BMT), with T-cell depleted donor bone marrow bearing a different congenic marker (CD45.2) to that of the host mouse (CD45.1). New cell production post-BMT can thus be tracked by measuring the fraction of CD45.2+ cells over time within a population of interest ( Hogan et al., 2015 ; Gossel et al., 2017 ).