The marginal zone (MZ) of the spleen harbors B cells that play an indispensable role in immune defense, orchestrating rapid responses to bloodborne pathogens. Under steady-state conditions, MZ B cell numbers are maintained through the balance of de novo generation from precursors, proliferative self-renewal, and loss. The mechanisms governing this homeostatic control remain elusive. Further, the developmental pathways underlying the establishment and continued supplementation of the MZ B cell compartment are not fullyelucidated. To address these gaps, we combined multiple fate-mapping tools and mathematical models to study MZ B cell dynamics in mice across the life course. Our analyses find evidence of quorum sensing mechanisms that regulate both the accumulation of mature MZ B cells during early life and their maintenance throughout adulthood. Specifically, we demonstrate that they derive predominantly from transitional B cell precursors with an efficiency that increases with age, reaching stable levels only in adulthood. MZ B cells compensate for this early developmental inefficiency through cell density-dependent proliferation, ensuring the timely establishment of a stable pool. Collectively, these findings unveil critical roles of quorum sensing and immune system maturation in the maintenance of this vital B cell subset.

The inhibitor of kappa B kinase complex (IKK) is a critical regulator of cell death and inflammatory signaling in multiple cell types. Phosphorylation of IκB proteins by IKK results in their degradation and consequent activation of NF-κB transcription factors. RIPK1, a critical cell death regulator, is also a direct target of IKK kinase activity, thereby repressing its cell death activity. In αβ T cells, the RIPK1 kinase activity of IKK is critical for normal thymic development while mature αβ T cells require IKK for both activation of NF-κB dependent survival programmes, and repression of RIPK1. γδ T cells play a unique and versatile role in host immunity with specific effector functions that enables them to act as early responders in immune defence. The role of IKK regulated pathways in their development and survival is not known. Here, we use mouse genetics to dissect the function of IKK and downstream pathways in the normal homeostasis of γδ T cells. We find that IKK expression is critical to establish a replete γδ T cell compartment, but that requires vary between different subsets. Type 1 γδ T cells require IKK dependent NF-κB activation for their generation, while IKK is redundant for development of adaptive γδ T cells. Instead, IKK dependent NF-κB activation is required for their longterm survival. We also find evidence that IKK repression of RIPK1 is required for survival of peripheral but not thymic γδ T cells. Ablation of CASPASE8 did not rescue γδ T cells in the absence of IKK but rather revealed a potent sensitivity of all γδ subsets to necroptosis, that was rescued by kinase dead RIPK1. Overall, we reveal critical requirements for IKK regulated inflammatory pathways by γδ T cells that contrast with those of αβ T cells, and between different subsets, highlighting the complexity of the regulation of these pathways in the adaptive immune system.

Tissue-resident memory T cells (T RM ) protect from repeat infections within organs and barrier sites. The breadth and duration of such protection are defined at minimum by three quantities: the rate at which new T RM are generated from precursors, their rate of self-renewal, and their rate of loss through death, egress, or differentiation. Quantifying these processes individually is challenging. Here we combine genetic fate mapping tools and mathematical models to untangle these basic homeostatic properties of CD4 + T RM in the skin and gut lamina propria (LP) of healthy adult mice. We show that CD69 + CD4 + T RM in skin reside for ∼24 days and self-renew more slowly, such that clones halve in size approximately every 5 weeks, and approximately 2% of cells are replaced daily from precursors. CD69 + CD4 + T RM in LP have shorter residencies (∼14 days) and are maintained largely by immigration (4–6% per day). We also find evidence that the continuous replacement of CD69 + CD4 + T RM at both sites derives from circulating effector-memory CD4 + T cells, in skin possibly via a local CD9 − intermediate. Our approach maps the ontogeny of CD4 + T RM in skin and LP and exposes their dynamic and distinct behaviours, with continuous seeding and erosion potentially impacting the duration of immunity at these sites.

Regulatory T cells (T reg cells) are critical regulators of adaptive immunity and the pathophysiology of antitumoral immunity. T reg cells are both generated during thymic development and induced from peripheral conventional T cells. How these distinct pathways contribute to the homeostasis of circulating T reg cells in health and disease remains unclear. We addressed this question using multiple fate-mapping mouse systems and modeling. Naive and effector/memory (EM) T reg cells exhibit distinct dynamics but are both continuously replenished by de novo generation throughout life. The predominant precursors of circulating EM T reg cells are naive thymic T reg cells and not conventional T cells, a process driven by self rather than foreign antigen recognition. Using the same fate reporters and three tumor models, we demonstrate that infiltrating T reg cells specifically derive from preexisting EM T reg cells. In summary, we define a linear ontogeny of T reg cells from the thymus to EM, driven by self-antigen recognition, that then gives rise to tumor-infiltrating T reg cells.

Introduction:CASPASE8 promotes both cell death and survival by acting as a trigger of apoptosis and a repressor of necroptosis. In T cells, the function and mechanisms of CASPASE8 are incompletely understood. Methods:Here, we analysed mice in which Casp8 was conditionally deleted in T cells at different stages of development. Results:In mice with deletion early in T cell development, we observed a modest reduction in early thymic progenitors and a striking absence of NKT cells in the thymus. Amongst mature peripheral T cells, there was a substantial and specific reduction in the CD8 T cell compartment, which included naive, central memory and virtual memory subsets. Using a tamoxifen-inducible CD8CreERT to delete Casp8 revealed an acute requirement for continued CASPASE8 expression for survival of a fraction of mature CD8 T cells. Analysing Casp8-deficient mice that express a kinase dead RIPK1 suggested that in vivo, necroptosis contributed to death of thymic progenitors and CD8EM and CD8CM subsets. However, kinase dead RIPK1 failed to restore NKT cell development or rescue the loss of CD4EM and CD4CM in mixed bone marrow chimeras, and only partially rescued CD8 VM T cell. Conclusions:Together, these observations suggest that CASPASE8 promotes T cell survival independent of its established role in repressing RIPK1-dependent necroptosis.

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 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.

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.

IKK signalling is essential for survival of thymocytes by repressing RIPK1 induced cell death rather than its canonical function of activating NF-κB. The role of IKK signalling in activated T cells is unclear. To investigate this, we analysed activation of IKK2 deficient T cells. While TCR triggering was normal, proliferation and expansion was profoundly impaired. This was not due to defective cell cycle progression, rather dividing T cells became sensitised to TNF induced cell death, since inhibition of RIPK1 kinase activity rescued cell survival. Gene expression analysis of activated IKK2 deficient T cells revealed defective expression of Tnfaip3, that encodes A20, a negative regulator of NF-κB. To test whether A20 expression was required to protect IKK2 deficient T cells from cell death, we generated mice with T cells lacking both A20 and IKK2. Doing this resulted in near complete loss of peripheral T cells, in contrast to mice lacking one or other gene. Strikingly, this phenotype was completely reversed by inactivation of RIPK1 kinase activity in vivo. Together, our data show that IKK signalling in activated T cells protects against RIPK1 dependent death, both by direct phosphorylation of RIPK1 and through NF-κB mediated induction of A20, that we identify for the first time as a key modulator of RIPK1 activity in T cells.

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.

Supplementary Figures 1-4 from IL-7 Contributes to the Progression of Human T-cell Acute Lymphoblastic Leukemias

The Inhibitor of Kappa B Kinase (IKK) complex is a critical regulator of canonical NF-κB activation. More recently, RIPK1 has also been identified as a phosphorylation target of the IKK complex, resulting in repression of extrinsic cell death pathways. Our previous work shows that normal thymocyte development is exclusively reliant on repression of TNF triggered cell death pathways by IKK, and that NF-κB signalling is in fact redundant for development. The role of IKK signalling in activated T cells is unclear. To investigate this, we analysed activation of IKK2 deficient TCR transgenic T cells with cognate peptide. While early activation events were normal, proliferation of blasts was impaired. Surprisingly, cell cycle progression in IKK2 KO T cells was unperturbed. Instead, dividing cells were more sensitive to apoptosis triggered by extrinsic cell death pathways, since inhibition of RIPK1 kinase activity almost completely rescued cell survival. Transcriptomic analysis of activated IKK2 deficient T cells revealed defective expression of several NF-κB targets, including Tnfaip3, that encodes A20, a negative regulator of NF-κB in T cells. To test whether A20 expression was required to protect IKK2 deficient T cells from cell death, we generated mice with T cells lacking both A20 and IKK2. Conditional deletion of both Ikk2 and Tnfaip3 in T cells resulted in near complete ablation of peripheral naïve T cells, in contrast to mice lacking one or other gene. Strikingly, this phenotype was completely reversed by inhibition of RIPK1 kinase activity in vivo. Therefore, our data suggests that IKK signalling in T cells protects against RIPK1 dependent death, both by direct phosphorylation of RIPK1 and through NF-κB mediated induction of A20, that we identify for the first time as a modulator of RIPK1 function in T cells.

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, an established fate-mapping system, 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 find strong evidence that cell-age effects dominate host-age effects, and that clones become more quiescent and more persistent the longer they reside within the TCM and TEM pools. This behaviour will lead to an increasingly long-tailed distribution of clone sizes as an individual ages. Therefore, the age structure of CD4+ TCM and TEM clones can explain bulk changes in their dynamics and persistence across the lifespan. Author Summary Our long-term protection against infections depends in part on the maintenance of diverse populations of CD4 memory T cells, which are made in response to the initial exposure to the pathogen or a vaccine. These cells are not long-lived, but instead are maintained dynamically at a clonal level through loss and division. Understanding how immune memory persists therefore requires measuring these rates of these processes, and how they might change with age. Here we combine mouse experiments with mathematical models to show that CD4 memory T cell clones have a complex dynamical structure but become progressively more quiescent with time. Their expected cell lifespan increases as they age, leading to long-tailed survival curves. This dynamic implies that as individuals age, their CD4 memory populations become enriched for older clones that have a fitness advantage over newly generated ones. If these older clones are functionally impaired, this phenomenon may contribute to the waning of immunity in the elderly.

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.

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.

Naive CD4 and CD8 T cells are part of the foundation of adaptive immune responses, but multiple aspects of their behaviour remain elusive. Newly generated T cells continue to develop after they leave the thymus and their dynamics and ‘rules of entry’ into the mature naive population are challenging to define. The extents to which naive T cells’ capacities to survive or self-renew change as they age are also unclear. Further, much of what we know about their behaviour derives from studies in adults, both mouse and human. We know much less about naive T cell dynamics early in life, during which the thymus is highly active and peripheral T cell populations are rapidly established. For example, it has been suggested that neonatal mice are lymphopenic; if so, does this environment impact the behaviour of the earliest thymic emigrants, for example through altered rates of division and loss? In this study we integrate data from multiple experimental systems to construct models of naive CD4 and CD8 T cell population dynamics across the entire mouse lifespan. We infer that both subsets progressively increase their capacity to persist through survival mechanisms rather than through self-renewal, and find that this very simple model of adaptation describes the population dynamics of naive CD4 T cells from birth into old age. In addition, we find that newly generated naive CD8 T cells are lost at an elevated rate for the first 3-4 weeks of life, which may derive from transiently increased recruitment into conventional and virtual memory populations. We find no evidence for elevated rates of division of naive CD4 or CD8 T cells early in life and indeed estimate that these cells divide extremely rarely. Markers of proliferation within peripheral naive T cells are instead inherited from division during thymic development. We also find no evidence for feedback regulation of rates of division or loss of naive T cells at any age in healthy mice, challenging the dogma that their numbers are homeostatically regulated. Our analyses show how confronting an array of mechanistic mathematical models with diverse datasets can move us closer to a complete, and remarkably simple, picture of naive CD4 and CD8 T cell dynamics in mice.

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.

Tight regulation of IL-7R alpha expression is essential for normal T-cell development. IL-7R alpha gainof-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-7R alpha overexpression, rather than mutational activation, during leukemogenesis remains unclear. In this study, overexpressed IL-7R alpha 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 p27(Kip1) 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-7R alpha can promote T-cell tumorigenesis, even in the absence of IL-7R alpha mutational activation.