news and views
Thymic IL-7 signaling goes beyond survival Benedict Seddon Interleukin 7 (IL-7) promotes the self-renewing ability of CD4– CD8– double-negative thymocytes by both supporting cell growth and repressing rearrangements of the locus encoding the T cell antigen receptor (TCR). he cytokine interleukin 7 (IL-7) has a critical role in the lymphoid compartment. Mice lacking either this cytokine or its receptor (IL-7R) have a striking array of lymphoid defects: mature follicular B cells and the γδ T cell lineage are absent, while the αβ T cells are profoundly reduced in number. Subsets of innate lymphoid cells, including lymphoid tissue–inducer cells, are also missing; without lymphoid tissue–inducer cells, normal organization and formation of lymph nodes and other lymphoid structures cannot occur and, consequently, the mice almost completely lack lymph nodes. Much of what is known about the molecular mechanisms of IL-7 can be distilled into a single word, ‘survival’, because one of the most obvious effects of IL-7 signaling in many cell types is induction of the pro-survival factor Bcl-2. However, this simplistic view is being challenged as understanding of the complex roles of IL-7 signaling increases. In this issue of Nature Immunology, Boudil et al. reveal that IL-7 signaling is crucial during the proliferative expansion of immature thymocytes through the provision of trophic signals to support their metabolic demands but also through the repression of their differentiation by inhibiting further rearrangements of loci encoding T cell antigen receptors (TCRs)1. The CD4–CD8– double-negative (DN) stage is a busy time for prospective T cells. Uncommitted progenitor cells entering the thymus become fixed to a T cell lineage fate and progress through various DN stages, during which they recombine the locus encoding their TCRβ chain, to the CD8+ immature singlepositive (ISP) and then to the CD4+CD8+ double positive (DP) stage, at which rearrangements of the locus encoding the TCRα chain and positive and negative selection occur. A fine balance among proliferation, maturation and gene rearrangement during this time is required for the generation of large numbers of DP cells all expressing different TCRs. Thus, expansion of the DN population is critical for the correct functioning of thymic selection. Benedict Seddon is at the Institute of Immunity and Transplantation, Division of Infection and Immunity, University College London, Royal Free Hospital, London, UK. e-mail: [email protected]
The Notch signaling pathway is a key participant in these processes. Notch signals commit progenitor cells to a T cell lineage fate, maintain the self-renewing ability of cycling thymocytes and, in concert with signaling via the pre-TCR, sustain continued development of cells in the DN3 and DN4 stages to the DP stage2. IL-7 signals also seem to play a non-redundant part in this developmental pathway, as indicated by the substantial loss of cells from the DN3 stage onward in the absence of IL-7. However, the mechanisms underlying the effects of IL-7 in the thymus have been less well understood than are those of Notch. Boudil et al. show that although Bcl-2 expression is indeed induced by IL-7 signaling in thymocytes at the DN3a stage, expression of a transgene encoding human Bcl-2 does not restore the number of DN3b or DN4 cells in mice lacking IL-7R1. This indicates that IL-7 is required for more than just the survival of DN thymocytes. A candidate pathway for investigation is signaling via phosphoinositol-3-OH kinase (PI(3)K), since signaling via IL-7 is known to enhance
DN3a
DN3b
PI(3)K activity3 and the PI(3)K pathway is also linked to the development of DN thymocytes: loss of expression of PTEN, the phosphatase that counterbalances the phosphoinositol-generating activity of PI(3)K, can circumvent the TCR β-selection checkpoint and ‘rescue’ the paucity of DN thymocytes that occurs both in the absence of Notch signaling2 and in IL-7-deficient mice4. Now, Boudil et al. show that in addition to activating PI(3)K activity downstream of IL-7R, IL-7 signaling also induces expression of the gene encoding the PI(3)K isoform required for β-selection and thereby mediates a feed-forward loop to facilitate cell growth. Moreover, the authors suggest that IL-7 signaling enhances cell proliferation by regulating expression of nutrient-transport proteins such as CD98 (an amino-acid transporter) and CD71 (a transferrin receptor). This activity was previously attributed to Notch signaling, although this was somewhat puzzling since neither CD98 nor CD71 is encoded by a known Notch target2. Given that the gene encoding IL-7R is a target of Notch5
DN4
TCRβ-chain rearrangement
ISP
DP TCRα-chain rearrangement
Il7r expression
PI(3)K
Nutrient-transport proteins (e.g., CD71, CD98)
Cell growth and proliferation
Katie Vicari/Nature Publishing Group
npg
© 2015 Nature America, Inc. All rights reserved.
T
Bcl-6
Cell differentiation
Pathway activity Transcriptional regulation
Figure 1 IL-7 signaling optimizes the proliferation of DN thymocytes. Expression of the gene encoding IL-7R (Il7r) is high in thymocytes as they progress from the DN3a stage to the DN4 stage of thymocyte development, during which rearrangement of the locus encoding the TCRβ chain occurs. Boudil et al. show that IL-7 signaling during these stages activates PI(3)K and expression of the gene encoding the PI(3)K isoform required for β-selection and promotes expression of nutrient-transport proteins such as CD98 and CD71. These combined activities promote the growth and proliferation of cells. Simultaneously, IL-7 signaling inhibits Bcl-6 expression and thereby inhibits the differentiation of DN3 and DN4 thymocytes and allows greater expansion of these populations. As expression of the gene encoding IL-7R then decreases from the ISP stage to the DP stage, during which the locus encoding the TCRα chain is rearranged, removal of the block to Bcl-6 expression allows differentiation of the DP cells into mature T cells.
nature immunology volume 16 number 4 april 2015
337
npg
© 2015 Nature America, Inc. All rights reserved.
news and views and it is known that amino-acid transporters such as CD98 are transcriptional targets of IL-7 (ref. 6), it is possible that IL-7 signaling may be the mediator of this Notch activity. Boudil et al. analyze gene expression in IL-7-stimulated DN thymocytes and show that IL-7 signaling influences expression of a battery of genes encoding regulators of cell growth1. Thus, it seems that IL-7 signaling ‘tunes’ the growth and metabolism of DN thymocytes at multiple levels and by both transcriptional mechanisms and non-transcriptional mechanisms (Fig. 1). Perhaps most intriguing, however, is the authors’ proposal that IL-7 signaling also plays a pivotal part in orchestrating the balance between cellular proliferation and cellular differentiation necessary for the delivery of large numbers of DP thymocytes with mature surface TCRs. They find that in the absence of IL-7 in vitro, Il7–/– DN3b thymocytes give rise to more DP thymocytes than do their wild-type counterparts. This seems to occur by the cells’ avoiding the DN4 stage, since the ISP cells retain CD25 expression, which is usually lost at the DN4 stage, and the few DN4 thymocytes present become DP cells very rapidly, skipping the ISP stage. Furthermore, they observe premature rearrangement of the Tcra locus in DN thymocytes from Il7–/– mice. Genetic analysis of IL-7-treated DN3a, DN3b and DN4 thymocytes reveals that the
gene encoding the transcriptional repressor Bcl-6 is the gene most significantly downregulated. In developing B cells, Bcl-6 is negatively regulated by the transcription factor STAT5 and is linked to inhibition of the survival of cells undergoing rearrangements of the locus encoding the immunoglobulin κ-chain7. In mature T cells, the repressive functions of Bcl-6 are thought steer activated cells toward a follicular helper T cell state by suppressing differentiation into other helper T cells subtypes. Boudil et al. therefore investigate whether restoration of Bcl-6 expression might underlie the developmental defects in DN thymocytes observed in the absence of IL-7 (ref. 1). In a culture system of OP9 stromal cells expressing the Delta-like ligand DL4, cultures of fetal liver progenitor cells from Bcl6–/– and wild-type donors both generate equal fractions of DN, ISP and DP thymocytes, but the Bcl6–/– cells give rise to more DN cells both with IL-7 and without IL-7, which suggests that the repression of Bcl-6 by IL-7 may be important for maintaining a degree of self-renewing ability in DN thymocytes (Fig. 1). Although IL-7 signaling is already known to be crucial to thymocyte development, Boudil et al. have now exposed the extent of mechanisms by which the IL-7 signaling pathway regulates the development of DN cells1. Future studies should assess whether IL-7 signaling serves these functions in other cell types or at other
stages of T cell differentiation. Aberrant development of DN thymocytes is associated with T cell leukemia, and the role of IL-7 in supporting both tumor survival and tumor growth is well recognized8. The authors’ proposed IL-7–Bcl-6 axis raises the intriguing possibility that this repressive IL-7 signaling circuit also contributes to tumor maintenance. If so, this might offer novel treatment opportunities. Evidence suggests that in mature T cells, IL-7 promotes, rather than represses, the development of follicular helper T cells9, which perhaps highlights the potential influence of the cellular context in which IL-7 signals are transmitted, since STAT5 seems to be repressive for Bcl-6 expression in developing T cells and B cells. Future studies of the targets of Bcl-6 activity will shed further light on the multiple roles of IL-7 signaling in lymphocytes. COMPETING FINANCIAL INTERESTS The author declares no competing financial interests. 1. Boudil, A. et al. Nat. Immunol. 16, 397–405 (2015). 2. Yashiro-Ohtani, Y., Ohtani, T. & Pear, W.S. Semin. Immunol. 22, 261–269 (2010). 3. Barata, J.T. et al. J. Exp. Med. 200, 659–669 (2004). 4. Hagenbeek, T.J. et al. J. Exp. Med. 200, 883–894 (2004). 5. González-Garciá, S. et al. J. Exp. Med. 206, 779–791 (2009). 6. Pearson, C., Silva, A. & Seddon, B. PLoS ONE 7, e33998 (2012). 7. Duy, C. et al. J. Exp. Med. 207, 1209–1221 (2010). 8 Ribeiro, D., Melão, A. & Barata, J.T. Adv. Biol. Regul. 53, 211–222 (2013). 9. Seo, Y.B. et al. J. Virol. 88, 8998–9009 (2014).
The lymph node filter revealed Miroslav Hons & Michael Sixt Stromal cells in the subcapsular sinus of the lymph node ‘decide’ which cells and molecules are allowed access to the deeper parenchyma. The glycoprotein PLVAP is a crucial component of this selector function.
L
ymph nodes are two-level filters. The first level is related mainly to innate immunological functions and operates at the subcapsular sinus, the site into which afferent lymph is discharged. The second level is at the interface between the sinus and the lymph node parenchyma. The parenchyma is where adaptive immunological functions are accommodated, and only ‘licensed’ cells and small solutes gain access to this region. In this issue of Nature Immunology, Rantakari et al. show that a
Miroslav Hons and Michael Sixt are with the Institute of Science and Technology Austria, Klosterneuburg, Austria. e-mail: [email protected]
338
diaphragm made up of the glycoprotein PLVAP (PV-1) spans the transendothelial channels that transect sinus-lining lymphatic endothelial cells (LECs) and show that this structure confers the selectivity of the sinus–parenchyma barrier1. Interstitial fluid, including cells and solutes, that arrives at the subcapsular sinus of lymph nodes is sampled by a resident network of cells of the innate immune system. Selection on the basis of these cells’ pattern-recognition receptors allows potentially dangerous particles to be retained and processed in the sinus, while other, potentially harmless, molecules pass through the chain of consecutive lymph nodes to ultimately reach the blood circulation2. This is the first filtering function of the lymph node.
The second filter level is much more selective: only cells carrying specific chemokine receptors are allowed to penetrate into the parenchyma, which houses the T cell and B cell zones2. For the entry of solutes into the parenchyma, size seems to be a critical parameter: all molecules above 70 kilodaltons are excluded from the parenchyma, and only smaller ones can enter it, traveling through a specialized interstitial compartment, the conduit system. Conduits form a delicate web of extracellular matrix comprising tubules ensheathed (and produced) by the fibroblastic reticular cell (FRC) network, the non–hematopoietic cell backbone of the lymph node. Conduits constitute an effective fluid shunt between the subcapsular sinus and the blood-vessel lumen. In 1975, Arthur Anderson,
volume 16 number 4 april 2015 nature immunology
Thymic IL-7 signaling goes beyond survival Benedict Seddon Interleukin 7 (IL-7) promotes the self-renewing ability of CD4– CD8– double-negative thymocytes by both supporting cell growth and repressing rearrangements of the locus encoding the T cell antigen receptor (TCR). he cytokine interleukin 7 (IL-7) has a critical role in the lymphoid compartment. Mice lacking either this cytokine or its receptor (IL-7R) have a striking array of lymphoid defects: mature follicular B cells and the γδ T cell lineage are absent, while the αβ T cells are profoundly reduced in number. Subsets of innate lymphoid cells, including lymphoid tissue–inducer cells, are also missing; without lymphoid tissue–inducer cells, normal organization and formation of lymph nodes and other lymphoid structures cannot occur and, consequently, the mice almost completely lack lymph nodes. Much of what is known about the molecular mechanisms of IL-7 can be distilled into a single word, ‘survival’, because one of the most obvious effects of IL-7 signaling in many cell types is induction of the pro-survival factor Bcl-2. However, this simplistic view is being challenged as understanding of the complex roles of IL-7 signaling increases. In this issue of Nature Immunology, Boudil et al. reveal that IL-7 signaling is crucial during the proliferative expansion of immature thymocytes through the provision of trophic signals to support their metabolic demands but also through the repression of their differentiation by inhibiting further rearrangements of loci encoding T cell antigen receptors (TCRs)1. The CD4–CD8– double-negative (DN) stage is a busy time for prospective T cells. Uncommitted progenitor cells entering the thymus become fixed to a T cell lineage fate and progress through various DN stages, during which they recombine the locus encoding their TCRβ chain, to the CD8+ immature singlepositive (ISP) and then to the CD4+CD8+ double positive (DP) stage, at which rearrangements of the locus encoding the TCRα chain and positive and negative selection occur. A fine balance among proliferation, maturation and gene rearrangement during this time is required for the generation of large numbers of DP cells all expressing different TCRs. Thus, expansion of the DN population is critical for the correct functioning of thymic selection. Benedict Seddon is at the Institute of Immunity and Transplantation, Division of Infection and Immunity, University College London, Royal Free Hospital, London, UK. e-mail: [email protected]
The Notch signaling pathway is a key participant in these processes. Notch signals commit progenitor cells to a T cell lineage fate, maintain the self-renewing ability of cycling thymocytes and, in concert with signaling via the pre-TCR, sustain continued development of cells in the DN3 and DN4 stages to the DP stage2. IL-7 signals also seem to play a non-redundant part in this developmental pathway, as indicated by the substantial loss of cells from the DN3 stage onward in the absence of IL-7. However, the mechanisms underlying the effects of IL-7 in the thymus have been less well understood than are those of Notch. Boudil et al. show that although Bcl-2 expression is indeed induced by IL-7 signaling in thymocytes at the DN3a stage, expression of a transgene encoding human Bcl-2 does not restore the number of DN3b or DN4 cells in mice lacking IL-7R1. This indicates that IL-7 is required for more than just the survival of DN thymocytes. A candidate pathway for investigation is signaling via phosphoinositol-3-OH kinase (PI(3)K), since signaling via IL-7 is known to enhance
DN3a
DN3b
PI(3)K activity3 and the PI(3)K pathway is also linked to the development of DN thymocytes: loss of expression of PTEN, the phosphatase that counterbalances the phosphoinositol-generating activity of PI(3)K, can circumvent the TCR β-selection checkpoint and ‘rescue’ the paucity of DN thymocytes that occurs both in the absence of Notch signaling2 and in IL-7-deficient mice4. Now, Boudil et al. show that in addition to activating PI(3)K activity downstream of IL-7R, IL-7 signaling also induces expression of the gene encoding the PI(3)K isoform required for β-selection and thereby mediates a feed-forward loop to facilitate cell growth. Moreover, the authors suggest that IL-7 signaling enhances cell proliferation by regulating expression of nutrient-transport proteins such as CD98 (an amino-acid transporter) and CD71 (a transferrin receptor). This activity was previously attributed to Notch signaling, although this was somewhat puzzling since neither CD98 nor CD71 is encoded by a known Notch target2. Given that the gene encoding IL-7R is a target of Notch5
DN4
TCRβ-chain rearrangement
ISP
DP TCRα-chain rearrangement
Il7r expression
PI(3)K
Nutrient-transport proteins (e.g., CD71, CD98)
Cell growth and proliferation
Katie Vicari/Nature Publishing Group
npg
© 2015 Nature America, Inc. All rights reserved.
T
Bcl-6
Cell differentiation
Pathway activity Transcriptional regulation
Figure 1 IL-7 signaling optimizes the proliferation of DN thymocytes. Expression of the gene encoding IL-7R (Il7r) is high in thymocytes as they progress from the DN3a stage to the DN4 stage of thymocyte development, during which rearrangement of the locus encoding the TCRβ chain occurs. Boudil et al. show that IL-7 signaling during these stages activates PI(3)K and expression of the gene encoding the PI(3)K isoform required for β-selection and promotes expression of nutrient-transport proteins such as CD98 and CD71. These combined activities promote the growth and proliferation of cells. Simultaneously, IL-7 signaling inhibits Bcl-6 expression and thereby inhibits the differentiation of DN3 and DN4 thymocytes and allows greater expansion of these populations. As expression of the gene encoding IL-7R then decreases from the ISP stage to the DP stage, during which the locus encoding the TCRα chain is rearranged, removal of the block to Bcl-6 expression allows differentiation of the DP cells into mature T cells.
nature immunology volume 16 number 4 april 2015
337
npg
© 2015 Nature America, Inc. All rights reserved.
news and views and it is known that amino-acid transporters such as CD98 are transcriptional targets of IL-7 (ref. 6), it is possible that IL-7 signaling may be the mediator of this Notch activity. Boudil et al. analyze gene expression in IL-7-stimulated DN thymocytes and show that IL-7 signaling influences expression of a battery of genes encoding regulators of cell growth1. Thus, it seems that IL-7 signaling ‘tunes’ the growth and metabolism of DN thymocytes at multiple levels and by both transcriptional mechanisms and non-transcriptional mechanisms (Fig. 1). Perhaps most intriguing, however, is the authors’ proposal that IL-7 signaling also plays a pivotal part in orchestrating the balance between cellular proliferation and cellular differentiation necessary for the delivery of large numbers of DP thymocytes with mature surface TCRs. They find that in the absence of IL-7 in vitro, Il7–/– DN3b thymocytes give rise to more DP thymocytes than do their wild-type counterparts. This seems to occur by the cells’ avoiding the DN4 stage, since the ISP cells retain CD25 expression, which is usually lost at the DN4 stage, and the few DN4 thymocytes present become DP cells very rapidly, skipping the ISP stage. Furthermore, they observe premature rearrangement of the Tcra locus in DN thymocytes from Il7–/– mice. Genetic analysis of IL-7-treated DN3a, DN3b and DN4 thymocytes reveals that the
gene encoding the transcriptional repressor Bcl-6 is the gene most significantly downregulated. In developing B cells, Bcl-6 is negatively regulated by the transcription factor STAT5 and is linked to inhibition of the survival of cells undergoing rearrangements of the locus encoding the immunoglobulin κ-chain7. In mature T cells, the repressive functions of Bcl-6 are thought steer activated cells toward a follicular helper T cell state by suppressing differentiation into other helper T cells subtypes. Boudil et al. therefore investigate whether restoration of Bcl-6 expression might underlie the developmental defects in DN thymocytes observed in the absence of IL-7 (ref. 1). In a culture system of OP9 stromal cells expressing the Delta-like ligand DL4, cultures of fetal liver progenitor cells from Bcl6–/– and wild-type donors both generate equal fractions of DN, ISP and DP thymocytes, but the Bcl6–/– cells give rise to more DN cells both with IL-7 and without IL-7, which suggests that the repression of Bcl-6 by IL-7 may be important for maintaining a degree of self-renewing ability in DN thymocytes (Fig. 1). Although IL-7 signaling is already known to be crucial to thymocyte development, Boudil et al. have now exposed the extent of mechanisms by which the IL-7 signaling pathway regulates the development of DN cells1. Future studies should assess whether IL-7 signaling serves these functions in other cell types or at other
stages of T cell differentiation. Aberrant development of DN thymocytes is associated with T cell leukemia, and the role of IL-7 in supporting both tumor survival and tumor growth is well recognized8. The authors’ proposed IL-7–Bcl-6 axis raises the intriguing possibility that this repressive IL-7 signaling circuit also contributes to tumor maintenance. If so, this might offer novel treatment opportunities. Evidence suggests that in mature T cells, IL-7 promotes, rather than represses, the development of follicular helper T cells9, which perhaps highlights the potential influence of the cellular context in which IL-7 signals are transmitted, since STAT5 seems to be repressive for Bcl-6 expression in developing T cells and B cells. Future studies of the targets of Bcl-6 activity will shed further light on the multiple roles of IL-7 signaling in lymphocytes. COMPETING FINANCIAL INTERESTS The author declares no competing financial interests. 1. Boudil, A. et al. Nat. Immunol. 16, 397–405 (2015). 2. Yashiro-Ohtani, Y., Ohtani, T. & Pear, W.S. Semin. Immunol. 22, 261–269 (2010). 3. Barata, J.T. et al. J. Exp. Med. 200, 659–669 (2004). 4. Hagenbeek, T.J. et al. J. Exp. Med. 200, 883–894 (2004). 5. González-Garciá, S. et al. J. Exp. Med. 206, 779–791 (2009). 6. Pearson, C., Silva, A. & Seddon, B. PLoS ONE 7, e33998 (2012). 7. Duy, C. et al. J. Exp. Med. 207, 1209–1221 (2010). 8 Ribeiro, D., Melão, A. & Barata, J.T. Adv. Biol. Regul. 53, 211–222 (2013). 9. Seo, Y.B. et al. J. Virol. 88, 8998–9009 (2014).
The lymph node filter revealed Miroslav Hons & Michael Sixt Stromal cells in the subcapsular sinus of the lymph node ‘decide’ which cells and molecules are allowed access to the deeper parenchyma. The glycoprotein PLVAP is a crucial component of this selector function.
L
ymph nodes are two-level filters. The first level is related mainly to innate immunological functions and operates at the subcapsular sinus, the site into which afferent lymph is discharged. The second level is at the interface between the sinus and the lymph node parenchyma. The parenchyma is where adaptive immunological functions are accommodated, and only ‘licensed’ cells and small solutes gain access to this region. In this issue of Nature Immunology, Rantakari et al. show that a
Miroslav Hons and Michael Sixt are with the Institute of Science and Technology Austria, Klosterneuburg, Austria. e-mail: [email protected]
338
diaphragm made up of the glycoprotein PLVAP (PV-1) spans the transendothelial channels that transect sinus-lining lymphatic endothelial cells (LECs) and show that this structure confers the selectivity of the sinus–parenchyma barrier1. Interstitial fluid, including cells and solutes, that arrives at the subcapsular sinus of lymph nodes is sampled by a resident network of cells of the innate immune system. Selection on the basis of these cells’ pattern-recognition receptors allows potentially dangerous particles to be retained and processed in the sinus, while other, potentially harmless, molecules pass through the chain of consecutive lymph nodes to ultimately reach the blood circulation2. This is the first filtering function of the lymph node.
The second filter level is much more selective: only cells carrying specific chemokine receptors are allowed to penetrate into the parenchyma, which houses the T cell and B cell zones2. For the entry of solutes into the parenchyma, size seems to be a critical parameter: all molecules above 70 kilodaltons are excluded from the parenchyma, and only smaller ones can enter it, traveling through a specialized interstitial compartment, the conduit system. Conduits form a delicate web of extracellular matrix comprising tubules ensheathed (and produced) by the fibroblastic reticular cell (FRC) network, the non–hematopoietic cell backbone of the lymph node. Conduits constitute an effective fluid shunt between the subcapsular sinus and the blood-vessel lumen. In 1975, Arthur Anderson,
volume 16 number 4 april 2015 nature immunology
