Interleukin (IL)-7 is a secreted soluble globular protein 25 kDa in size encoded by the IL7 gene. It was first identified as a growth factor for murine B cell precursors found in the bone marrow. Other researchers found that mRNA encoding IL-7 was enriched in the thymus, the lymphoid organ in which T cells are made. Addition of recombinant IL-7 to in vitro cultures was found to promote survival and growth of thymocytes, the cells from the thymus that ultimately develop into mature T lymphocytes. These early experiments suggested that IL-7 was important for development of normal T lymphocytes in the thymus and B lymphocytes in bone marrow. The generation of genetically modified “knockout” mice, in which the IL-7 gene was specifically disrupted, ultimately confirmed these views (von Freeden-Jeffry et al. 1995). While these IL-7-deficient mice were grossly normal in appearance, the suggested importance of IL-7 for lymphocyte development was abundantly clear. These mice were virtually devoid of B lymphocytes and had greatly reduced numbers of T cells, but as will be discussed below, the highly distinctive phenotype of these mice revealed a number of other important functions of IL-7 in the immune system.
As well as activating the classical JAK-STAT signaling pathway, phosphorylation of the IL-7Rα cytoplasmic tail is also thought to allow recruitment and activation of phosphoinositide 3-kinase ( PI3K). This kinase phosphorylates phosphoinositides (PIs), phospholipid components of the cell membrane, which in turn provide recruitment sites for proteins with pleckstrin homology (PH) domains. Phosphoinositide-dependent protein kinase 1 (PDK1) and Akt (also known as PKB) are kinases with PH domains that are recruited following PI3K activation. Importantly, PDK1 activates Akt. Akt is a serine/threonine protein kinase that has potential to regulate multiple cellular processes, including cell growth, glucose metabolism, apoptosis, cell division, and cell migration. Thus, activation of PI3K by IL-7 is known to be essential for cell growth and glucose metabolism, but not survival, of T lymphocytes.
An additional layer of complexity to IL-7R signaling that must also be considered is that the IL-7Rα chain is not exclusive to the IL-7R heterodimer complex. Interestingly, IL-7Rα can also couple with another membrane protein, TSLPR, to form a receptor for another cytokine, thymic stromal lymhopoietin (TSLP). While TSLP has distinct functions to IL-7, there are some instances, such as B-cell development in humans, in which TSLP shares a common function with IL-7 and is in fact even more important than IL-7 in supporting cell survival and development.
Developmental Functions of IL-7
Insights into the cellular roles of IL-7 can be made by examining where, and by which cells, the cytokine is made and also by examining cells expressing the IL-7Rα chain that are in turn the targets of IL-7 activity. Studies of bone marrow chimeras, in which the complete bone marrow stem cell compartment of IL-7 knockout mice was replaced by normal bone marrow stem cells, revealed that such bone marrow transplantation could not cure the defects evident in IL-7 knockout mice. Conversely, similar chimeras in which normal mice had their bone marrow stem cells replaced with those from IL-7 knockout mice had none of the defects and problems inherent in the IL-7 knockout strains. These experiments clearly demonstrated that IL-7 is not produced by hematopoietic cells but must be produced by a stromal cell of mesenchymal origin. Recent studies have more clearly identified the cellular sources of IL-7 by generating mouse strains in which a specific reporter protein, in this case human CD25, is expressed from within the IL-7 gene locus (Repass et al. 2009). These studies show that IL-7 is indeed produced in the liver and small intestine and is most highly expressed in the lymphoid organs of bone marrow, thymus, and lymph nodes (LNs). The cells that express IL-7 in these lymphoid organs are stromal cell components that also contribute to the formation of the three-dimensional framework of these organs. Thus, the significant sites of IL-7 production are also the organs in which lymphocytes are generated and where they reside as mature cells. The thymus is the organ in which T lymphocytes develop, while LNs are home to mature T and B lymphocytes and are the organs in which immune responses are initiated. The bone marrow is the main site of hematopoiesis, in which pluripotent hematopoietic stem cells (HSCs) develop into all the different blood cell types.
Lymphoid progenitors destined to develop into T lineage cells leave the bone marrow and migrate to the thymus, another major site of IL-7 production. The thymus gives rise to numerous lymphoid lineages, including γδ T cells, and the various αβ T lymphocyte lineages: CD4 T cells, CD8 T cells, regulatory T cells, and NK T cells, amongst others. IL-7 signaling in developing thymocytes influences lineage fates and cellular development. Pre-T thymocyte progenitors entering the thymus lack expression of CD4 and CD8 molecules and are termed double negative (DN). DN cells can be further subdivided into four populations (DN1-4) on the basis of expression of two markers, CD44 and CD25. Development of γδ T cells from DN2 thymocytes is absolutely dependent on IL-7, and strong IL-7 signaling is thought to specifically favor γδ T-cell development over αβ T cells (Riera-Sans and Behrens 2007). However, even in αβ T-cell development, IL-7 is essential as it provides both survival and proliferative signals to developing DN3 and DN4 thymocytes while they rearrange their T-cell antigen receptor (TCR) genes to generate mature TCRs. Crucially, thymocytes must test their newly generated TCR to ensure it is functional. This is achieved by upregulating both CD4 and CD8 coreceptors to become double positive (DP), at which point DPs undergo the process of positive selection, in which only thymocytes with functional TCRs can survive and continue to develop into mature T lymphocytes. The criteria for a functional TCR are that it has the capacity to weakly recognize major histocompatibility complex (MHC) proteins on thymic epithelial cells. This proves to be the case for only a small minority of DP thymocytes, approximately 5%. It is therefore very significant that at the DP stage of thymocyte development, thymocytes completely lose IL-7R function. Doing so ensures that continued survival and development of thymocytes is entirely determined by their ability to successfully pass the stringent positive selection check point. This represents another important function of IL-7 during T-lineage development, albeit a negative one. IL-7 signaling is essential throughout lymphoid development. However, loss of IL-7R function ensures that continued development depends solely on the generation of a functional and useful TCR and, therefore, ensures that the peripheral repertoire of T cells is mostly composed of cells with useful TCRs.
Interestingly, mice and humans have evolved different mechanisms to inactivate IL-7R signaling. In mice, loss of IL-7R function is achieved by the near complete loss of IL-7Rα surface expression coupled with high-expression levels of suppressor of cytokine signaling ( SOCS) proteins that inhibit cytokine signal transduction. In contrast, human thymocytes express entirely normal levels of IL-7Rα but rather lose expression of STAT5 protein, the transcription factor target of IL-7 signaling. The fact that mice and humans have evolved different mechanisms of abating IL-7 signaling during thymic development may be viewed as an example of convergent evolution and may, therefore, represent a relatively recently evolved optimization of thymic selection. Cells that successfully pass the test of positive selection continue development to either CD4 or CD8 lineage T cells. DP thymocytes that successfully undergo positive selection by Class I MHC molecules lose expression of CD4 and become CD8 single positive (SP) thymocytes, while positive selection by Class II MHC induces commitment to the CD4 lineage and loss of CD8 expression so that cells become CD4 SP. In both cases, IL-7 function is restored in SP thymocytes, and mature T cells all express functional IL-7R. Thus, not only does IL-7 play a crucial role for development of both T and B lymphocytes, but it is also important in determining the direction that developing progenitor populations take during the development of these lymphocyte lineages.
The analysis of IL-7-deficient mice revealed another developmental role for IL-7 but one which has only recently begun to be understood. In addition to its characterized role in the lymphopoiesis, IL-7 is also essential for normal organogenesis of secondary lymphoid structures. IL-7-deficient mice have extremely small LNs, and other lymphoid structures, such as the Peyer’s patches that are located in the gut, are absent. While it was initially thought that the reduced LN size was merely secondary to the reduced numbers of lymphocytes in these mice, closer investigation revealed that the nonlymphocyte cellular structures that form the LNs were in fact defective. During embryonic development, LN structures actually form prior to development and generation of mature lymphocytes that only later populate the mature LNs. Formation of these early LN anlagen is initiated by the aggregation of so-called LN inducer (LNi) cells at the site of future LNs. These cells have now been identified as a member of the group 3 ILCs. These ILC3s, in turn, recruit a second population of cells, LN organizer (LNo) cells that are of nonhemopoetic mesenchymal origin. A final stage of LN formation occurs after population with mature lymphocytes. In IL-7-deficient mice, these structures initially form normally but fail to develop into normal LNs after birth, and it appears that IL-7 signaling either in the ILC3s or newly generated T cells that colonize the LNs is required for the final stage of LN formation (Repass et al. 2009).
Control of Homeostasis and Function of Mature Naïve T Cells by IL-7
Like other cells in the body, lymphocytes require specific signals to actively keep them alive. In the absence of such signals, cells undergo the process of controlled cell death, known as apoptosis. All naïve T cells express IL-7R, and IL-7 is one of several essential signals required for the long-term survival of these lymphocytes. Within the lymphoid system, naïve T cells are not static but rather actively migrate around LNs seeking antigen and also recirculate via efferent lymphatic vessels to the blood and, thence, to other LNs or the spleen. IL-7 is produced by stromal cells in LNs but is also detectable in high concentrations in the fluid in efferent lymphatics and in serum of blood. Naïve T cells are therefore constantly exposed to IL-7 during their recirculation through the blood and lymphatic system, providing them with the constant signal that they require to prevent them from undergoing apoptosis.
In providing survival signals to naïve T cells, IL-7 plays a key role as a master regulator of the size of the naïve T cell pool. Although the thymus is constantly generating new cells, this does not appear to be the limiting factor in setting the size of the naïve T-cell pool. This is particularly evident in experiments analyzing mice that can only make one of either CD4 or CD8 lineage T cells. In normal conditions, there are approximately two CD4 T cells for every one CD8 T cell. However, in mice that cannot make CD4 T cells, the total number of naïve T cells, all of which are CD8, is much the same as the total number of naïve T cells in normal mice, which comprise both CD4 and CD8 lineages. This is the case even though the rate of CD8 T-cell production by the thymus remains unchanged regardless of whether CD4 T cells are produced or not. Thus, the space created by the absence of CD4 T cells is filled by more CD8 lineage cells. For similar reasons, mice that cannot make CD8 T cells also have normal T-cell numbers but are all CD4 lineage. This ceiling on total naïve T-cell numbers is set by the amount of IL-7 available. Experimental mice that have artificially elevated IL-7 levels also have larger T-cell pools. Therefore, IL-7 is controlling T-cell homeostasis by its requirement as a survival resource. If there are too many T cells in an individual, they will not all get sufficient access to IL-7 signals and some will die, until the numbers of T cells reach a level that can be sustained by the amount of IL-7 available. As well as promoting T-cell survival, IL-7 signaling can also promote cell division, another property that lends itself well to the homeostatic role of this cytokine. In conditions of T-cell deficiency, as occurs in HIV-infected individuals or following radio- or chemo-ablative therapies, naïve T cells can sense the “space” in the T-cell compartment and start to undergo cell divisions in an effort to restore normal T-cell numbers. This response is highly dependent on IL-7 signaling. Such cell division is particularly important in humans, where recently generated naïve T cells undergo several cell divisions even in a normal individual (Jameson 2005).
IL-7 is thought to be produced at a constant rate by stromal cells in lymphoid organs. Whether these cells can dynamically regulate production in specific situations to modify T-cell homeostasis, for instance, during an immune response is not at present known. However, the dependence on IL-7 for naïve T-cell survival goes a long way toward explaining how the immune system solves a difficult homeostatic problem. The size of other organs in the body, such as the kidneys, the liver, and the brain are controlled through strict regulation of developmental processes. In contrast, T cells do not reside in any one place but are spread throughout the body and undergo dynamic changes in their rates of production and loss throughout life. The reliance on a fixed resource such as IL-7 explains how the T-cell pool can be so precisely regulated to prevent over- or underpopulation.
Memory Formation and Persistence
During an immune response, T cells that recognize specific antigen become activated, undergoing cell division and developing specific effector functions such as cytokine secretion for CD4 T cells and development of cytolytic activity in the case of CD8 T cells. Following T-cell activation, T cells rapidly lose expression of IL-7R. Maintenance of IL-7R expression in peripheral T cells is dependent on the activity of the Foxo1 transcription factor (Kerdiles et al. 2009). The activity of Foxo1 transcription factors is negatively regulated by their phosphorylation by Akt. TCR stimulation by antigen activates the PI3K pathway, resulting in activation of Akt and, therefore, causes loss of IL-7Rα expression by the repression of Foxo1 by Akt. An ongoing immune response represents a scenario that is analogous to thymic selection (Fig. 3). During such a response, the presence of antigen is a key determinant of T-cell behavior, inducing proliferation and differentiation. For an effective immune response, it is important that a large number of effector T cells are quickly generated. Therefore, it makes sense to uncouple effector T cells from the shackles of normal T-cell homeostatic control by IL-7 and switch to autocrine-regulated T-cell proliferation by factors, such as IL-2, synthesized by T cells following their activation by antigen. IL-2 is also a potent inhibitor of IL-7Rα expression, which may be mediated by similar mechanisms as TCR signaling since IL-2 strongly activates PI3K. However, similar to thymic positive selection, it is important that differentiation of T cells is strictly dependent on continued TCR signaling, and not IL-7, since persistence of the immune response should be closely allied to the ongoing presence of antigen to indicate the need for the response. This is very neatly achieved through the direct repression of IL-7Rα expression by antigen-dependent PI3K activation. Following the resolution of a successful immune response, most effector cells will undergo apoptosis. However, some survive to become long-term memory cells, a key feature of the adaptive immune system. IL-7 signaling plays a key role in the formation of this memory population. In the absence of antigen, TCR signaled repression of IL-7Rα expression is reversed, and effector T cells can start to reexpress IL-7Rα. In some experimental viral infections in mice, the effector cells that first reexpress IL-7Rα are the precursors of the long-term memory cells, suggesting that IL-7 could be instructing effectors to develop into memory cells. However, there is also evidence that effector T cells are far more predisposed to undergoing apoptosis than naïve or memory T cells. While reexpression of IL-7R can slow their death, the effector pool still undergoes a significant contraction because the effector cells are less able to compete and survive in response to IL-7 signaling than other T cells (Buentke et al. 2006). Nevertheless, IL-7 signaling represents a key gateway into the memory T-cell pool, ensuring only the most fit effectors persist. Like their naïve precursors, both CD4 (Seddon et al. 2003) and CD8 memory T cells (Schluns et al. 2000) are dependent on IL-7 for their long-term survival, although CD8 memory cells also have a strong reliance on the cytokine IL-15. Both CD4 and CD8 memory cells express higher levels of IL-7Rα than their naïve counterparts. However, in contrast to naive T cells, memory cells can also migrate to extralymphoid tissue sites in order to patrol them for the presence of their specific pathogen, returning to lymph nodes via the afferent lymphatic vessels. It is unclear whether these cells can obtain survival signals in such sites as IL-7 expression is largely limited to lymphoid organs. Therefore, higher cytokine receptor expression levels may be important for these cells to gain stronger survival signals sufficient to maintain their survival while patrolling peripheral T tissues and before their return to lymphoid tissue for “refueling.”
IL-7 and Disease
IL-7 has been implicated in a variety of different disease processes. There are several recognized severe combined immunodeficiencies (SCID) that arise from mutations in either the human IL7 or IL7R genes that affect production of IL-7 or signaling through IL-7R. Such patients have a phenotype similar to that described for IL-7 deficient mice, although one notable difference is that human B cells are less reliant on IL-7 than mouse B cells, and there appears to be some redundancy with TSLP. Therefore, IL-7RA mutations are more severe than IL-7 mutations. Given the potent survival and proliferative properties of IL-7, it is unsurprising that mutations in the IL7 gene are also implicated as susceptibility factors in certain T cell acute lymphoblastic leukemias (T-ALL), and experimental work has shown that IL-7 can accelerate disease progression of T-ALL transplanted into mice. More recently, IL7RA mutations have been identified in leukemic cells from T-ALL patients that result in spontaneous IL7R dimerization and signaling activity that does not require the receptor engagement by the cytokine (Zenatti et al. 2011).
There is also evidence to suggest that IL-7 may be involved in development of some autoimmune diseases. A genetic linkage study identified a point mutation in a noncoding region of the IL7R gene as strongly associated with development of multiple sclerosis (Gregory et al. 2007). Furthermore, recent experiments suggest that IL-7 signaling may exacerbate the T-cell mediated autoimmune process that is thought to contribute to the nerve damage in the disease.
IL-7 is also under consideration as a potential therapeutic. In HIV patients with active disease, reduction in T-cell counts is often accompanied with raised serum levels of IL-7, probably secondary to the reduction in T-cell numbers. However, IL-7 treatment has been tested in patients to see whether it could aid immune reconstitution in HIV patients with dwindling T-cell numbers. In some cases, a relatively short treatment with IL-7 was able to produce lasting increases in T-cell numbers. The potential for immune reconstitution by IL-7 is also considered in the context of aging. In humans, maximal thymic productivity is reached very early, at about 1 year of age, after which the thymus undergoes gradual atrophy, producing ever-decreasing numbers of T cells. Homeostatic mechanisms like those discussed earlier are successful in maintaining a diverse T-cell repertoire until old age, when there appears to be a sudden collapse in T-cell diversity. The lack of new thymically generated T cells is thought to be a contributing factor. One of the causes of thymic atrophy is a loss of IL-7 production by thymic stromal cells. Administering IL-7 to mice can successfully overcome some of the effects of reduced IL-7 production locally in the thymus. Therefore, immuno-reconstitution by IL-7 treatment is starting to be considered, at least for some specific conditions.
In conclusion, it is evident that IL-7 has a broad array of biological functions. It is vital for development of nearly all lymphoid lineages, but also has a role to play in controlling the lineage decisions at different stages of progenitor differentiation. The absolute number of mature T cells that an individual has is critically dependent on IL-7 availability, and all the mature subsets and lineages of T cells have at least some reliance on IL-7 for their survival. Even the absence of IL-7 signaling has a fundamental role to play in shaping the T-cell repertoire and memory populations. One of the greatest challenges for the future will be to understand how this one cytokine can influence such a diverse range of cellular properties. Our understanding of the molecular mechanisms of IL-7 signaling remains crude, and it is unclear whether all the potential signaling pathways are activated in different in vivo situations and, if not, which ones are important for different biological outcomes. Such understanding will be essential if the potent biological properties of IL-7 are to be fully exploited clinically.
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