Megan A. Luckey

Intrathymic IL-7: The where, when, and why of IL-7 signaling during T cell development

The thymus is the birthplace of all T lineage cells. But the thymus is also a cradle as it provides the environment for further maturation and differentiation of immature thymocytes. While many factors contribute to make the thymus a unique place for T cell development, here we review the essential role of intrathymic interleukin-7 (IL-7). In the absence of IL-7 signaling, survival, proliferation and differentiation of immature thymocytes are all severely impaired. Consequently, IL-7 is critical to nurture and guide T precursor cells through the diverse steps of thymic maturation. Interestingly, even as IL-7 signaling is such a critical factor, IL-7 signaling must be also actively suppressed during specific stages of T cell differentiation. These contradictory observations are puzzling but can be satisfactorily explained when understanding the developmental context of IL-7 signaling. In this regard, here we will discuss the spatiotemporal expression of intrathymic IL-7 and address the stage-specific effects of IL-7 signaling in developing thymocytes. Specifically, we will review other facets of intrathymic IL-7 beyond its role as a pro-survival factor and so clarify and reaffirm the unique role of IL-7 as a prime factor in T cell development and differentiation.

An In Vivo IL-7 Requirement for Peripheral Foxp3+ Regulatory T Cell Homeostasis

All T cells are dependent on IL-7 for their development and for homeostasis. Foxp3+ regulatory T cells (Tregs) are unique among T cells in that they are dependent on IL-2. Whether such IL-2 dependency is distinct from or in addition to an IL-7 requirement has been a confounding issue, particularly because of the absence of an adequate experimental system to address this question. In this study, we present a novel in vivo mouse model where IL-2 expression is intact but IL-7 expression was geographically limited to the thymus. Consequently, IL-7 is not available in peripheral tissues. Such mice were generated by introducing a thymocyte-specific IL-7 transgene onto an IL-7 null background. In these mice, T cell development in the thymus, including Foxp3+ Treg numbers, was completely restored, which correlates with the thymus-specific expression of transgenic IL-7. In peripheral cells, however, IL-7 expression was terminated, which resulted in a general paucity of T cells and a dramatic reduction of Foxp3+ Treg numbers. Loss of Tregs was further accompanied by a significant reduction in Foxp3+ expression levels. These data suggest that peripheral IL-7 is not only necessary for Treg survival but also for upregulating Foxp3 expression. Collectively, we assessed the effect of a selective peripheral IL-7 deficiency in the presence of a fully functional thymus, and we document a critical requirement for in vivo IL-7 in T cell maintenance and specifically in Foxp3+ cell homeostasis.

The transcription factor ThPOK suppresses Runx3 and imposes CD4 + lineage fate by inducing the SOCS suppressors of cytokine signaling

Lineage fate in the thymus is determined by mutually exclusive expression of the transcription factors ThPOK and Runx3, with ThPOK imposing the CD4+ lineage fate and Runx3 promoting the CD8+ lineage fate. While it is known that cytokine signals induce thymocytes to express Runx3, it is not known how ThPOK prevents thymocytes from expressing Runx3 and adopting the CD8+ lineage fate, nor is it understood why ThPOK itself imposes the CD4+ lineage fate on thymocytes. We now report that genes encoding members of the SOCS (suppressor of cytokine signaling) family are critical targets of ThPOK and that their induction by ThPOK represses Runx3 expression and promotes the CD4+ lineage fate. Thus, induction of SOCS-encoding genes is the main mechanism by which ThPOK imposes the CD4+ lineage fate in the thymus.

Chronic Exposure to Type-I IFN under Lymphopenic Conditions Alters CD4 T Cell Homeostasis

HIV infection and the associated chronic immune activation alter T cell homeostasis leading to CD4 T cell depletion and CD8 T cell expansion. The mechanisms behind these outcomes are not totally defined and only partially explained by the direct cytopathic effect of the virus. In this manuscript, we addressed the impact of lymphopenia and chronic exposure to IFN-α on T cell homeostasis. In a lymphopenic murine model, this interaction led to decreased CD4 counts and CD8 T cell expansion in association with an increase in the Signal Transducer and Activator of Transcription 1 (STAT1) levels resulting in enhanced CD4 T cell responsiveness to IFN-α. Thus, in the setting of HIV infection, chronic stimulation of this pathway could be detrimental for CD4 T cell homeostasis.

Metabolic sensor AMPK directly phosphorylates RAG1 protein and regulates V(D)J recombination

The ability to sense metabolic stress is critical for successful cellular adaptation. In eukaryotes, the AMP-activated protein kinase (AMPK), a highly conserved serine/threonine kinase, functions as a critical metabolic sensor. AMPK is activated by the rising ADP/ATP and AMP/ATP ratios during conditions of energy depletion and also by increasing intracellular Ca2+. In response to metabolic stress, AMPK maintains energy homeostasis by phosphorylating and regulating proteins that are involved in many physiological processes including glucose and fatty acid metabolism, transcription, cell growth, mitochondrial biogenesis, and autophagy. Evidence is mounting that AMPK also plays a role in a number of pathways unrelated to energy metabolism. Here, we identify the recombination-activating gene 1 protein (RAG1) as a substrate of AMPK. The RAG1/RAG2 complex is a lymphoid-specific endonuclease that catalyzes specific DNA cleavage during V(D)J recombination, which is required for the assembly of the Ig and T-cell receptor genes of the immune system. AMPK directly phosphorylates RAG1 at serine 528, and the phosphorylation enhances the catalytic activity of the RAG complex, resulting in increased cleavage of oligonucleotide substrates in vitro, or increased recombination of an extrachromosomal substrate in a cellular assay. Our results suggest that V(D)J recombination can be regulated by AMPK activation, providing a potential new link between metabolic stress and development of B and T lymphocytes.

Pim1 permits generation and survival of CD4+ T cells in the absence of γc cytokine receptor signaling.

γ‐chain (γc) cytokine receptor signaling is required for the development of all lymphocytes. Why γc signaling plays such an essential role is not fully understood, but induction of the serine/threonine kinase Pim1 is considered a major downstream event of γc as Pim1 prevents apoptosis and increases metabolic activity. Consequently, we asked whether Pim1 overexpression would suffice to restore lymphocyte development in γc‐deficient mice. By analyzing Pim1‐transgenic γc‐deficient mice (Pim1TgγcKO), we show that Pim1 promoted T‐cell development and survival in the absence of γc. Interestingly, such effects were largely limited to CD4+ lineage αβ T cells as CD4+ T‐cell numbers improved to near normal levels but CD8+ T cells remained severely lymphopenic. Notably, Pim1 over‐expression failed to promote development and survival of any T‐lineage cells other than αβ T cells, as we observed complete lack of γδ, NKT, FoxP3+ T regulatory cells and TCR‐β+ CD8αα IELs in Pim1TgγcKO mice. Collectively, these results uncover distinct requirements for γc signaling between CD4+ αβ T cells and all other T‐lineage cells, and they identify Pim1 as a novel effector molecule sufficient to drive CD4+ αβ T‐cell development and survival in the absence of γc cytokine receptor signaling.

CD8 Lineage-specific Regulation of Interleukin-7 Receptor Expression by the Transcriptional Repressor Gfi1

Background: Expression of the IL-7Rα gene is up-/down-regulated during T/B-lymphocyte development. Results: IL-7Rα gene transcription is repressed by the transcription factor Gfi1, specifically in CD8+ T-lymphocytes. Conclusion: Treatment by dexamethasone down-regulates Gfi1, which contributes to glucocorticoid receptor mediated up-regulation of IL-7R expression. Significance: The mechanism by which the IL-7R gene gets turned on and off during development is a critical issue in biology. Interleukin-7 receptor α (IL-7Rα) is essential for T cell survival and differentiation. Glucocorticoids are potent enhancers of IL-7Rα expression with diverse roles in T cell biology. Here we identify the transcriptional repressor, growth factor independent-1 (Gfi1), as a novel intermediary in glucocorticoid-induced IL-7Rα up-regulation. We found Gfi1 to be a major inhibitory target of dexamethasone by microarray expression profiling of 3B4.15 T-hybridoma cells. Concordantly, retroviral transduction of Gfi1 significantly blunted IL-7Rα up-regulation by dexamethasone. To further assess the role of Gfi1 in vivo, we generated bacterial artificial chromosome (BAC) transgenic mice, in which a modified Il7r locus expresses GFP to report Il7r gene transcription. By introducing this BAC reporter transgene into either Gfi1-deficient or Gfi1-transgenic mice, we document in vivo that IL-7Rα transcription is up-regulated in the absence of Gfi1 and down-regulated when Gfi1 is overexpressed. Strikingly, the in vivo regulatory role of Gfi1 was specific for CD8+, and not CD4+ T cells or immature thymocytes. These results identify Gfi1 as a specific transcriptional repressor of the Il7r gene in CD8 T lymphocytes in vivo.

Soluble γc cytokine receptor suppresses IL-15 signaling and impairs iNKT cell development in the thymus

The soluble γc protein (sγc) is a naturally occurring splice isoform of the γc cytokine receptor that is produced by activated T cells and inhibits γc cytokine signaling. Here we show that sγc expression is also highly upregulated in immature CD4+CD8+ thymocytes but then downregulated in mature thymocytes. These results indicate a developmentally controlled mechanism for sγc expression and suggest a potential role for sγc in regulating T cell development in the thymus. Indeed, sγc overexpression resulted in significantly reduced thymocyte numbers and diminished expansion of immature thymocytes, concordant to its role in suppressing signaling by IL-7, a critical γc cytokine in early thymopoiesis. Notably, sγc overexpression also impaired generation of iNKT cells, resulting in reduced iNKT cell percentages and numbers in the thymus. iNKT cell development requires IL-15, and we found that sγc interfered with IL-15 signaling to suppress iNKT cell generation in the thymus. Thus, sγc represents a new mechanism to control cytokine availability during T cell development that constrains mature T cell production and specifically iNKT cell generation in the thymus.

Ikaros is required to survive positive selection and to maintain clonal diversity during T-cell development in the thymus

The zinc-finger protein Ikaros is a key player in T-cell development and a potent tumor suppressor in thymocytes. To understand the molecular basis of its function, we disabled Ikaros activity in vivo using a dominant negative Ikaros transgene (DN-IkTg). In DN-IkTg mice, T-cell development was severely suppressed, and positively selected thymocytes clonally expanded, resulting in a small thymus with a heavily skewed T-cell receptor (TCR) repertoire. Notably, DN-IkTg induced vigorous proliferation concomitant to downregulation of antiapoptotic factor expression such as Bcl2. Ikaros activity was required during positive selection, and specifically at the CD4(+)CD8(lo) intermediate stage of thymocyte differentiation, where it prevented persistent TCR signals from inducing aberrant proliferation and expansion. In particular, DN-IkTg induced the accumulation of CD4 single-positive (SP) thymocytes with a developmentally transitional phenotype, and it imposed a developmental arrest accompanied by massive apoptosis. Thus, we identified an in vivo requirement for Ikaros function, which is to suppress the proliferative potential of persistent TCR signals and to promote the survival and differentiation of positively selected thymocytes.

Interleukin-6 expands homeostatic space for peripheral T cells

T cell homeostasis and survival is dependent on interleukin-7 (IL-7). Immune activation, however, downregulates IL-7 receptor expression on T cells so that T cell survival during activation must be maintained independently of IL-7. The pro-inflammatory cytokine IL-6 shares common signaling pathways with IL-7 and can promote T cell survival in vitro. But whether IL-6 promotes T cell survival and homeostasis in vivo is not clear. Notably, IL-6 overexpression results in massive plasmacytosis and autoimmunity so that an IL-6 effect on in vivo T cell survival has remained untested. To overcome this limitation, here we generated IL-6 transgenic mice on an immunoglobulin heavy chain (IgH) deficient background which rendered them B cell deficient. Notably, such IgH(KO)IL6(Tg) mice were free of any signs of inflammation or autoimmunity and remained healthy throughout the course of analysis. In these mice, we found that IL-6 overexpression significantly increased peripheral T cell numbers, but importantly without increasing thymopoiesis. Moreover, IL-6 signaled T cells maintained their naïve phenotype and did not express activation/memory markers, suggesting that increased T cell numbers were due to increased T cell survival and not because of expansion of activated T cells. Mechanistically, we found that IL-6 signaling induced expression of pro-survival factors Mcl-1 and Pim-1/-2 but not Bcl-2. Thus, IL-6 is a T cell homeostatic cytokine that expands T cell space and can maintain the naïve T cell pool.