Susan M. Schlenner

Fundamental properties of unperturbed haematopoiesis from stem cells in vivo

Haematopoietic stem cells (HSCs) are widely studied by HSC transplantation into immune- and blood-cell-depleted recipients. Single HSCs can rebuild the system after transplantation. Chromosomal marking, viral integration and barcoding of transplanted HSCs suggest that very low numbers of HSCs perpetuate a continuous stream of differentiating cells. However, the numbers of productive HSCs during normal haematopoiesis, and the flux of differentiating progeny remain unknown. Here we devise a mouse model allowing inducible genetic labelling of the most primitive Tie2+ HSCs in bone marrow, and quantify label progression along haematopoietic development by limiting dilution analysis and data-driven modelling. During maintenance of the haematopoietic system, at least 30% or ∼5,000 HSCs are productive in the adult mouse after label induction. However, the time to approach equilibrium between labelled HSCs and their progeny is surprisingly long, a time scale that would exceed the mouse’s life. Indeed, we find that adult haematopoiesis is largely sustained by previously designated ‘short-term’ stem cells downstream of HSCs that nearly fully self-renew, and receive rare but polyclonal HSC input. By contrast, in fetal and early postnatal life, HSCs are rapidly used to establish the immune and blood system. In the adult mouse, 5-fluoruracil-induced leukopenia enhances the output of HSCs and of downstream compartments, thus accelerating haematopoietic flux. Label tracing also identifies a strong lineage bias in adult mice, with several-hundred-fold larger myeloid than lymphoid output, which is only marginally accentuated with age. Finally, we show that transplantation imposes severe constraints on HSC engraftment, consistent with the previously observed oligoclonal HSC activity under these conditions. Thus, we uncover fundamental differences between the normal maintenance of the haematopoietic system, its regulation by challenge, and its re-establishment after transplantation. HSC fate mapping and its linked modelling provide a quantitative framework for studying in situ the regulation of haematopoiesis in health and disease.

Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3 + regulatory T cells

Foxp3+ regulatory T (Treg) cells are a crucial immunosuppressive population of CD4+ T cells, yet the homeostatic processes and survival programs that maintain the Treg cell pool are poorly understood. Here we report that peripheral Treg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2–dependent and costimulation-dependent process. By contrast, excess Treg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-xL and Bcl-2 were dispensable for survival of Treg cells, whereas Mcl-1 was critical for survival of Treg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which Treg cells maintain homeostasis via critical survival pathways.

Genetic predisposition for beta cell fragility underlies type 1 and type 2 diabetes

Type 1 (T1D) and type 2 (T2D) diabetes share pathophysiological characteristics, yet mechanistic links have remained elusive. T1D results from autoimmune destruction of pancreatic beta cells, whereas beta cell failure in T2D is delayed and progressive. Here we find a new genetic component of diabetes susceptibility in T1D non-obese diabetic (NOD) mice, identifying immune-independent beta cell fragility. Genetic variation in Xrcc4 and Glis3 alters the response of NOD beta cells to unfolded protein stress, enhancing the apoptotic and senescent fates. The same transcriptional relationships were observed in human islets, demonstrating the role of beta cell fragility in genetic predisposition to diabetes.

The microRNA-29 Family Dictates the Balance Between Homeostatic and Pathological Glucose Handling in Diabetes and Obesity

The microRNA-29 (miR-29) family is among the most abundantly expressed microRNA in the pancreas and liver. Here, we investigated the function of miR-29 in glucose regulation using miR-29a/b-1 (miR-29a)-deficient mice and newly generated miR-29b-2/c (miR-29c)-deficient mice. We observed multiple independent functions of the miR-29 family, which can be segregated into a hierarchical physiologic regulation of glucose handling. miR-29a, and not miR-29c, was observed to be a positive regulator of insulin secretion in vivo, with dysregulation of the exocytotic machinery sensitizing β-cells to overt diabetes after unfolded protein stress. By contrast, in the liver both miR-29a and miR-29c were important negative regulators of insulin signaling via phosphatidylinositol 3-kinase regulation. Global or hepatic insufficiency of miR-29 potently inhibited obesity and prevented the onset of diet-induced insulin resistance. These results demonstrate strong regulatory functions for the miR-29 family in obesity and diabetes, culminating in a hierarchical and dose-dependent effect on premature lethality.

Promiscuous Foxp3-cre activity reveals a differential requirement for CD28 in Foxp3 + and Foxp3 − T cells

Costimulatory signals by CD28 are critical for thymic regulatory T‐cell (Treg) development. To determine the functional relevance of CD28 for peripheral Treg post thymic selection, we crossed the widely used Forkhead box protein 3 (Foxp3)‐CreYFP mice to mice bearing a conditional Cd28 allele. Treg‐specific CD28 deficiency provoked a severe autoimmune syndrome as a result of a strong disadvantage in competitive fitness and proliferation of CD28‐deficient Tregs. By contrast, Treg survival and lineage integrity were not affected by the lack of CD28. This data demonstrate that, even after the initial induction requirement, Treg maintain a higher dependency on CD28 signalling than conventional T cells for homeostasis. In addition, we found the Foxp3‐CreYFP allele to be a hypomorph, with reduced Foxp3 protein levels. Furthermore, we report here the stochastic activity of the Foxp3‐CreYFP allele in non‐Tregs, sufficient to recombine some conditional alleles (including Cd28) but not others (including R26‐RFP). This hypomorphism and ‘leaky’ expression of the Foxp3‐CreYFP allele should be considered when analysing the conditionally mutated Treg.

Premature thymic involution is independent of structural plasticity of the thymic stroma

The thymus is the organ devoted to T‐cell production. The thymus undergoes multiple rounds of atrophy and redevelopment before degenerating with age in a process known as involution. This process is poorly understood, despite the influence the phenomenon has on peripheral T‐cell numbers. Here we have investigated the FVB/N mouse strain, which displays premature thymic involution. We find multiple architectural and cellular features that precede thymic involution, including disruption of the epithelial–endothelial relationship and a progressive loss of pro‐T cells. The architectural features, reminiscent of the human thymus, are intrinsic to the nonhematopoietic compartment and are neither necessary nor sufficient for thymic involution. By contrast, the loss of pro‐T cells is intrinsic to the hematopoietic compartment, and is sufficient to drive premature involution. These results identify pro‐T‐cell loss as the main driver of premature thymic involution, and highlight the plasticity of the thymic stroma, capable of maintaining function across diverse interstrain architectures.

A novel Zap70 mutation with reduced protein stability demonstrates the rate-limiting threshold for Zap70 in T-cell receptor signalling.

Loss of ζ‐associated protein 70 (Zap70) results in severe immunodeficiency in humans and mice because of the critical role of Zap70 in T‐cell receptor (TCR) signalling. Here we describe a novel mouse strain generated by N‐ethyl‐N‐nitrosourea mutagenesis, with the reduced protein stability (rps) mutation in Zap70. The A243V rps mutation resulted in decreased Zap70 protein and a reduced duration of TCR‐induced calcium responses, equivalent to that induced by a 50% decrease in catalytically active Zap70. The reduction of signalling through Zap70 was insufficient to substantially perturb thymic differentiation of conventional CD4 and CD8 T cells, although Foxp3+ regulatory T cells demonstrated altered thymic production and peripheral homeostasis. Despite the mild phenotype, the Zap70A243V variant lies just above the functional threshold for TCR signalling competence, as T cells relying on only a single copy of the Zap70rps allele for TCR signalling demonstrated no intracellular calcium response to TCR stimulation. This addition to the Zap70 allelic series indicates that a rate‐limiting threshold for Zap70 protein levels exists at which signalling capacity switches from nearly intact to effectively null.

The thymoprotective function of leptin is indirectly mediated via suppression of obesity

Leptin is an adipokine that regulates metabolism and plays an important role as a neuroendocrine hormone. Leptin mediates these functions via the leptin receptor, and deficiency in either leptin or its receptor leads to obesity in humans and mice. Leptin has far reaching effects on the immune system, as observed in obese mice, which display decreased thymic function and increased inflammatory responses. With expression of the leptin receptor on T cells and supporting thymic epithelium, aberrant signalling through the leptin receptor has been thought to be the direct cause of thymic involution in obese mice. Here, we demonstrate that the absence of leptin receptor on either thymic epithelial cells or T cells does not lead to the loss of thymic function, demonstrating that the thymoprotective effect of leptin is mediated by obesity suppression rather than direct signalling to the cellular components of the thymus.

Defective germinal center B-cell response and reduced arthritic pathology in microRNA-29a-deficient mice

MicroRNA (miR) are short non-coding RNA sequences of 19–24 nucleotides that regulate gene expression by binding to mRNA target sequences. The miR-29 family of miR (miR-29a, b-1, b-2 and c) is a key player in T-cell differentiation and effector function, with deficiency causing thymic involution and a more inflammatory T-cell profile. However, the relative roles of different miR-29 family members in these processes have not been dissected. We studied the immunological role of the individual members of the miR-29 family using mice deficient for miR-29a/b-1 or miR-29b-2/c in homeostasis and during collagen-induced arthritis. We found a definitive hierarchy of immunological function, with the strong phenotype of miR-29a-deficiency in thymic involution and T-cell activation being reduced or absent in miR-29c-deficient mice. Strikingly, despite elevating the Th1 and Th17 responses, loss of miR-29a conferred near-complete protection from collagen-induced arthritis (CIA), with profound defects in B-cell proliferation and antibody production. Our results identify the hierarchical structure of the miR-29 family in T-cell biology, and identify miR-29a in B cells as a potential therapeutic target in arthritis.

The Thymic Microenvironment Differentially Regulates Development and Trafficking of Invariant NKT Cell Sublineages

The regulatory role of the thymic microenvironment during trafficking and differentiation of the invariant NKT (iNKT) cell lineage remains poorly understood. In this study, we show that fractalkine receptor expression marks emigrating subpopulations of the NKT1, NKT2, and NKT17 sublineages in the thymus and peripheral organs of naive mice. Moreover, NKT1 sublineage cells can be subdivided into two subsets, namely NKT1a and NKT1b, which exhibit distinct developmental and tissue-specific distribution profiles. More specifically, development and trafficking of the NKT1a subset are selectively dependent upon lymphotoxin (LT)α1β2-LTβ receptor–dependent differentiation of thymic stroma, whereas the NKT1b, NKT2, and NKT17 sublineages are not. Furthermore, we identify a potential cellular source for LTα1β2 during thymic organogenesis, marked by expression of IL-7Rα, which promotes differentiation of the NKT1a subset in a noncell-autonomous manner. Collectively, we propose a mechanism by which thymic differentiation and retention of the NKT1 sublineage are developmentally coupled to LTα1β2-LTβ receptor–dependent thymic organogenesis.