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IL-7 Engages Multiple Mechanisms to Overcome Chronic Viral Infection and Limit Organ Pathology - pdf attached
 
 
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Boosting Body's Immune Response May Hold Key To HIV Cure:
 
"Utilizing the clone 13 model, we have shown that IL-7 is able to overcome many of the factors that thwart an effective immune response during chronic overwhelming viremia.......These attributes of IL-7 have profound implications for its use as a therapeutic in the treatment of chronic viral diseases......Our data suggest that IL-7 therapy may be a useful adjuvant in chronic viral diseases like HIV. Antiretroviral therapy is successful in reducing viral loads to undetectable levels, and in this setting, T cells become more responsive to IL-7 through the restoration of IL-7R, thereby enabling their rescue. In this context of reduced viral load, IL-7 therapy can be used to produce and expand specific T cells and promote a broad and durable immune-mediated antiviral response. Furthermore, the secondary cytoprotective effects of IL-7 have therapeutic implications for the management of hepatitis C virus infections
......Our data provide insights into the inhibitory pathways that function to impede immune responses in chronic infection. We elucidate a molecular mechanism whereby IL-7 is able to repress Socs3 levels in immune cells to promote extensive expansion of naive and effector T cells. Furthermore, we highlight the attributes of IL-7 in augmenting recent thymic emigrants in the face of persistent infection and its ability to promote IL-22 production and hence limit bystander cytotoxicity"
 
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HIV-like infection banished from mice
 
newscientist.com
 
For the first time, an HIV-like infection has been cleared from an animal without the use of antiviral drugs. The infection was eliminated from mice using a human protein that peps up immune cells.
 
Marc Pellegrini from the Walter and Eliza Hall Institute of Medical Research in Melbourne, Australia, and colleagues infected mice with lymphocytic choriomeningitis virus (LCMV), which causes a chronic infection that spreads throughout the body. "The virus overwhelms mice, mimicking the massive viral loads associated with HIV infection in humans," says Pellegrini.
 
Eight days after infection, some of the mice were injected with human interleukin-7 (IL-7) - a chemical messenger that plays a role in the development of immune cells - once a day for three weeks. The others received a placebo instead.
 
"Usually mice never clear this virus," says Pellegrini. But 30 days later, those given treatment had cleared most of the infection and removed all of it by 60 days.
 
Overactive response
 
The explanation seems to lie in a protein called SOCS3, which blocks the function of T-cells - a type of immune cell and therefore part of the body's system for fighting infection. At the beginning of an acute infection, SOCS3 becomes highly activated, suppressing the body's immune response. That sounds just the opposite of what you'd want. But it's a good thing because it stops T-cells being overzealous, which can cause damage to body tissue, says Pellegrini.
 
It becomes a problem when the body is trying to fight an overwhelming infection like HIV, he says: then the immune system puts on the brakes too early and the infection persists.
 
Blood tests taken throughout the experiment showed that IL-7 seemed to switch off the production of SOCS3, thereby ramping up the T-cell response.
 
Start and stop
 
This explanation was confirmed when the team knocked out the SOCS3 gene in a separate group of mice and infected them with LCMV. Immune cell numbers in these mice skyrocketed - T-cells increased sixfold compared with normal mice.
 
But knocking out SOCS3 showed the dangers of taking the immune brakes off. "Initially the mice mounted a robust immune response, but then it got out of hand," says Pellegrini. The mice developed mass inflammation and increased incidences of autoimmune diseases.
 
However, because IL-7 only affects T-cells, and not other types of immune cell, the researchers suggest that drugs could be developed that turn off SOCS3 for very short periods to reinvigorate T-cells without causing damage to the body.
 
Sharon Lewin at the Burnet Institute in Melbourne says the finding that IL-7 can clear the virus without the help of antiviral drugs is very interesting. Viruses like HIV use the host's cells to replicate, which makes it difficult to design antivirals that stop the virus without harming healthy cells.
 
"[Stopping a virus] without antivirals is something we haven't seen before," she says.
 
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IL-7 Engages Multiple Mechanisms to Overcome Chronic Viral Infection and Limit Organ Pathology - pdf attached
 
Cell, 03 February 2011
 
Marc Pellegrini,1,2,3,10 Thomas Calzascia,3,10 Jesse G. Toe,1 Simon P. Preston,1 Amy E. Lin,3 Alisha R. Elford,3 Arda Shahinian,3 Philipp A. Lang,3 Karl S. Lang,3 Michel Morre,4 Brigitte Assouline,4 Katharina Lahl,5 Tim Sparwasser,6 Thomas F. Tedder,7 Ji-hye Paik,8 Ronald A. DePinho,8 Sameh Basta,9 Pamela S. Ohashi,3,11,* and Tak W. Mak3,11 1The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC 3052, Australia 2Department of Medical Biology, University of Melbourne, Melbourne 3050, Australia 3Departments of Medical Biophysics and Immunology, Campbell Family Cancer Research Institute, Ontario Cancer Institute, University Health Network, Toronto, Ontario M5G 2C1, Canada 4Cytheris Inc., 92130 Issy les Moulineaux, France 5Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA 6Institute for Infection Immunology, TWINCORE, Centre for Experimental and Clinical Infection Research, Feodor-Lynen-Str.7, 30625 Hannover, Germany 7Department of Immunology, Duke University Medical Center, Durham, NC 27708, USA 8Belfer Institute for Applied Cancer Science, Department of Medical Oncology, Department of Medicine and Department of Genetics, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA 9Department of Microbiology and Immunology, Queen's University, Kingston, ON K7L 396, Canada
 
Highlights
* Socs3 is upregulated in T cells during chronic active viral infection in mice
* Deletion of socs3 in T cells prevents immune failure and promotes viral clearance
* In vivo IL-7 therapy represses Socs3 in T cells and clears chronic infection
* IL-7 promotes IL-22 production to mitigate immunopathology in chronic infection
 
Summary
 
Understanding the factors that impede immune responses to persistent viruses is essential in designing therapies for HIV infection. Mice infected with LCMV clone-13 have persistent high-level viremia and a dysfunctional immune response. Interleukin-7, a cytokine that is critical for immune development and homeostasis, was used here to promote immunity toward clone-13, enabling elucidation of the inhibitory pathways underlying impaired antiviral immune response. Mechanistically, IL-7 downregulated a critical repressor of cytokine signaling, Socs3, resulting in amplified cytokine production, increased T cell effector function and numbers, and viral clearance. IL-7 enhanced thymic output to expand the naive T cell pool, including T cells that were not LCMV specific. Additionally, IL-7 promoted production of cytoprotective IL-22 that abrogated liver pathology. The IL-7-mediated effects were dependent on endogenous IL-6. These attributes of IL-7 have profound implications for its use as a therapeutic in the treatment of chronic viral diseases.
 
Introduction

 
Much attention has focused on modulating immune responses in an attempt to promote clearance of chronic viral infections. This is particularly relevant to human immunodeficiency virus (HIV) infections in which the immune system fails to clear virus and eventually succumbs to uncontrolled viral turnover ([Day et al., 2006] and [Klenerman and Hill, 2005]). Understanding the mechanisms that circumvent immune responses in cases of overwhelming viral replication and antigen expression underlies any rational attempt to augment immunity in HIV infection.
 
Lymphocytic chorimeningitis virus (LCMV) variant clone 13 establishes a chronic infection in mice with high viral turnover, hence mimicking the massive viral antigen levels associated with HIV infection in humans (Wherry et al., 2003). Clone 13 infection has served as a powerful tool in characterizing the dysfunctional immune response associated with chronic viremia, and numerous parallels with HIV, HCV, and HBV infection have been described (reviewed in Wilson and Brooks [2010]). In contrast to clone 13, LCMV Armstrong causes an acute infection in C57Bl/6 mice, and the immune response differs significantly from clone 13 infection both in magnitude and also in the specificity of the responding immunodominant CD8 T cell clones (Wherry et al., 2003 E.J. Wherry, J.N. Blattman, K. Murali-Krishna, R. van der Most and R. Ahmed, Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment, J. Virol. 77 (2003), pp. 4911-4927. View Record in Scopus | Cited By in Scopus (366)Wherry et al., 2003). In Armstrong infection, the most prominent and highest-affinity CD8 T cell response is directed against the dominant nucleoprotein NP396 epitope, followed by the glycopeptide GP33 epitope and then by GP276. Clone 13 infection is characterized by a deletion of the high-affinity NP396 clones and potentially a functional defect in the remaining LCMV-specific T cells clones (Wherry et al., 2003). Similar phenotypic and functional disturbances have been described in T cells of HIV-infected humans (Day et al., 2006).
 
Several host inhibitory pathways, which may circumvent effective immune responses, have been identified in mice that are chronically infected with LCMV clone 13 ([Barber et al., 2006], [Brooks et al., 2006], [Ejrnaes et al., 2006] and [Matter et al., 2006]). We hypothesized that nonredundant cytokines involved in homeostatic proliferation, and therefore apt at overcoming inhibitory networks that naturally limit expansion, would offer the most promise in promoting immune responses. IL-7 is unique for its critical and nonredundant role in immune development and homeostasis and therefore is a prime candidate for use as an immunotherapeutic to overcome immune inhibitory networks in chronic active infections. Indeed, IL-7 offers significant therapeutic promise ([Levy et al., 2009], [Sereti et al., 2009] and [Sportes et al., 2008]). It has been used in several nonhuman primate SIV infection models that have demonstrated its diverse immunological effects ([Beq et al., 2006], [Fry et al., 2003] and [Nugeyre et al., 2003]). However, the efficacy of IL-7 in promoting viral clearance has not been fully explored. Importantly, the ability of IL-7 to antagonize immune inhibitory networks and the mechanism underlying its immune modulatory actions remains an area of active investigation. Such knowledge holds the potential to illuminate points for rational therapeutic intervention.
 
In this study, administration of recombinant human IL-7 to mice that are chronically infected with clone 13 increased the magnitude of the immune response with rescue of the NP396-specific T cell clones. It increased the size of the naive T cell pool, including T cell clones directed against non-LCMV specific epitopes, in part by increasing thymic output. LCMV clone 13-specific T cells showed enhanced degranulation kinetics and cytokine production with IL-7 therapy, resulting in effective viral clearance and a downregulation of PD-1 on effector T cells. In addition, IL-7 promoted a cytokine milieu favoring immune activation and production of the cytoprotective cytokine IL-22, thus limiting hepatotoxicity. At the molecular level, IL-7 downregulated suppressor of cytokine signaling 3 (Socs3) expression in T cells. This was mediated by suppression of FoxO transcription factors. We show that conditional deficiency of Socs3 in T cells replicates aspects of the IL-7-induced phenotype in mice that are infected with LCMV clone 13. These findings have major implications for our understanding of chronic viremia and the therapeutic use of IL-7.
 
Discussion
 
Utilizing the clone 13 model, we have shown that IL-7 is able to overcome many of the factors that thwart an effective immune response during chronic overwhelming viremia. One host inhibitory pathway that may restrict immune responses involves the Socs family of proteins that critically regulate cytokine signaling. Socs3 has been identified as the major regulatory molecule that dampens IL-6 signaling (Yoshimura et al., 2007). Indeed, studies have shown that the pattern of gene expression induced by IL-6 in Socs3-deficient animals mimics that induced by IFN-γ ([Croker et al., 2003] and [Lang et al., 2003]). We have shown that IL-7 is able to downregulate Socs3 levels in T cells, and additionally, it promotes IL-6 production. Together, this would reprogram cells to promote an IFN-like response and facilitate viral clearance. Furthermore, Socs3 has recently been reported to function as an intrinsic negative regulator of T cell proliferation (Brender et al., 2007). Thus, by repressing Socs3, IL-7 may promote innate antiviral mechanisms and also promote expansion and function of the adaptive immune response.
 
We found that T cells in clone 13-infected mice have much higher levels of Socs3 protein compared to T cells from LCMV Armstrong-infected animals. The importance of both IL-6 and Socs3 repression in mediating the therapeutic potential of IL-7 was evidenced by the reduced efficacy of IL-7 therapy in IL-6-deficient animals. We were able to delete socs3 throughout the hemopoietic compartment of gene-targeted mice contemporaneously with clone 13 infection to avoid potential developmental confounders and found that many of the effects that we observed with IL-7 therapy were replicated in this system. The MxCre transgene was useful in our studies to coincide the deletion of socs3 with the time of infection, but the neutrophilia associated with broad deletion of socs3 across the hematopoietic lineage was associated with early lethality. This adverse event requires further investigation in the future. Notably, IL-7 does not induce a granulocytosis, as its effects are limited to cells that express IL-7R, and hence its ability to repress Socs3 would be restricted to T cells and perhaps other immune cell subsets, but not neutrophils. We observed a dramatic increase in LCMV clone 13-specific immune responses and viral clearance in mice lacking Socs3 just in T cells. Importantly, these mice showed no signs of increased collateral damage or pathology compared to controls. The ability of IL-7 to repress Socs3 in T cells and perhaps other IL-7 responsive cells has significant implications for its use in manipulating innate and adaptive responses to favor elimination of pathogens.
 
Despite the large increase in cytokine levels and the marked infiltration of lymphocytes in organs of clone 13-infected animals receiving IL-7 therapy, we found no significant indication of bystander hepatitis. We have shown that IL-7 favors in vivo Th17 differentiation with IL-17 production. This may be due to IL-7-induced repression of Socs3 and promotion of an IL-17-IL-6 feedback loop (Ogura et al., 2008). Th17 and other cells have been identified as a major source of IL-22 ([Colonna, 2009] and [Ouyang et al., 2008]), which is critically important in protecting hepatocytes and perhaps other cells from immune-mediated bystander damage ([Aujla et al., 2008], [Laurence et al., 2008], [Radaeva et al., 2004], [Sugimoto et al., 2008], [Zenewicz et al., 2007] and [Zheng et al., 2008]). Indeed, in our clone 13 model, we found that IL-7 promotes IL-22 production, and this mitigates collateral liver damage. These findings are particularly pertinent to the use of IL-7 in chronic viral hepatitis. In HIV infection, IL-7 also holds much promise. Systemic immune activation is a hallmark of HIV disease that has been attributed to circulating microbial products, which may be a consequence of bacterial translocation across damaged gut mucosa (Brenchley et al., 2006). Endogenous production of IL-22 has been shown to protect mucosal sites (Aujla et al., 2008 S.J. Aujla, Y.R. Chan, M. Zheng, M. Fei, D.J. Askew, D.A. Pociask, T.A. Reinhart, F. McAllister, J. Edeal and K. Gaus et al., IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia, Nat. Med. 14 (2008), pp. 275-281. View Record in Scopus | Cited By in Scopus (212)[Aujla et al., 2008], [Sugimoto et al., 2008] and [Zheng et al., 2008]).
 
Another limitation of the immune system in HIV infection is the inability to replete peripheral losses due to attrition of T lymphocytes caused by the virus itself or by secondary effects due to the high level of viremia (Chase et al., 2006). During clone 13 infection, this is most apparent with the deletion of NP396-specific T cell clones. IL-7 significantly increased the number of these clones, but there was also a more general increase in non-LCMV-specific CD4 and CD8 T cells. Additionally, IL-7 promoted thymic output and substantially expanded the naive T cell pool in infected mice.
 
Our data provide insights into the inhibitory pathways that function to impede immune responses in chronic infection. We elucidate a molecular mechanism whereby IL-7 is able to repress Socs3 levels in immune cells to promote extensive expansion of naive and effector T cells. Furthermore, we highlight the attributes of IL-7 in augmenting recent thymic emigrants in the face of persistent infection and its ability to promote IL-22 production and hence limit bystander cytotoxicity. Our data suggest that IL-7 therapy may be a useful adjuvant in chronic viral diseases like HIV. Antiretroviral therapy is successful in reducing viral loads to undetectable levels, and in this setting, T cells become more responsive to IL-7 through the restoration of IL-7R, thereby enabling their rescue. In this context of reduced viral load, IL-7 therapy can be used to produce and expand specific T cells and promote a broad and durable immune-mediated antiviral response. Furthermore, the secondary cytoprotective effects of IL-7 have therapeutic implications for the management of hepatitis C virus infections.
 
Results
 
IL-7 Increases Antiviral T Effector Responses and Organ Infiltration in Clone 13 Infection

 
Clone 13-infected mice were treated with IL-7 or PBS commencing 8 days after infection, when chronicity had been established. After 3 weeks of IL-7 therapy, the total number of splenic CD8 and CD4 T cells was increased 11- and 5-fold, respectively, compared to PBS control animals (Figure 1A). IL-7 treatment did not alter the number of granulocytes, macrophages, or dendritic cells (DCs) compared to controls. A dramatic 10- to 25-fold increase in virus-specific CD8 T cells recognizing GP276 and GP33, respectively, was observed in IL-7-treated mice compared to controls (Figure 1B). An equally robust 10-fold increase in NP396-specific T cells, which represented a very small proportion of virus-specific cells in control animals, was also observed (Figure 1B). The increase in splenic CD8 and CD4 T cell numbers and virus-specific T cells tracked with an equivalent increase in liver, kidney, brain, and lung infiltration by lymphocytes and virus-specific T cells (Figures 1C and 1D and data not shown). Therefore, IL-7 is able to expand and rescue T cell numbers, including CD8 T cell clones that are normally deleted in clone 13 infection, resulting in efficient lymphocyte infiltration into infected organs.
 
Enhanced Function of Virus-Specific T Cells, Viral Clearance, and Downregulation of PD-1 with IL-7 Treatment
 
To verify that the large increase in clone 13-specific T cells in IL-7-treated animals translated to a functional gain, we examined their ex vivo capacity to degranulate and produce cytokines. We observed a 2-fold increase in the proportion of clone 13-specific CD8 T cells in IL-7-treated animals at day 29 postinfection that were able to produce cytokines and degranulate in restimulation assays compared to controls (Figure 2A). This translates to a massive 30- to 50-fold increase in the number of functional GP33-specific splenic CD8 T cells in IL-7-treated animals compared to controls (Figure 2A). Similarly, the proportion of clone 13-specific CD4 T cells recognizing GP61 that produce both IFNγ and IL-2 was 4-fold higher in IL-7-treated animals compared to controls (Figure 2B). This translates to a 20-fold increase in the absolute number of functional CD4 T cells recognizing GP61 in the spleens of IL-7-treated animals (Figure 2B). These results highlight the potent effects of IL-7 treatment on the total number of functional CD8 and CD4 virus-specific T cells in a model of chronic infection.
 
Recently, high PD-1 levels on T cells from clone 13-infected animals have been shown to correlate with a dysfunctional phenotype (Barber et al., 2006 D.L. Barber, E.J. Wherry, D. Masopust, B. Zhu, J.P. Allison, A.H. Sharpe, G.J. Freeman and R. Ahmed, Restoring function in exhausted CD8 T cells during chronic viral infection, Nature 439 (2006), pp. 682-687. View Record in Scopus | Cited By in Scopus (660)Barber et al., 2006). We thus examined the level of PD-1 expression on both CD8 and CD4 T cells from IL-7- and PBS-treated animals and observed lower levels of PD-1 on both CD8 and CD4 T cells expressing this receptor, and hence activated by antigen, in IL-7-treated animals compared to controls (Figures 2C and 2D). Furthermore, an acute activation marker, CD69, was downregulated on PD-1-expressing T cells from IL-7-treated animals (Figures 2C and 2D). Collectively, these data indicate that clone 13-specific CD4 and CD8 T cells in IL-7-treated mice begin to lose their activation phenotype by day 29 after 3 weeks of therapy, most likely due to viral clearance. Indeed, IL-7 treatment facilitated viral clearance from spleen and liver by days 22-29 postinfection and resulted in elimination of virus from chronic reservoirs, including brain, lung, and kidney between days 36 and day 60 (Figure 2E). Control animals were not able to clear virus by the end of follow-up at day 60, consistent with previous reports that have demonstrated long-term chronicity in clone 13-infected mice (Wherry et al., 2003).
 
CD4 and CD8 T Cells, but Not B Cells, Are Required for IL-7's Capacity to Clear Virus
 
In addition to the expansion of activated T cells, IL-7 caused an increase in B cell numbers in infected mice. However, proportionally, T cells represented the largest increase in lymphocyte numbers (Figure S1 available online and Figure 1A). To determine which of the expanded lymphocyte populations may contribute to enhanced clearance of virus, we depleted the various subsets during the course of IL-7 therapy to maintain CD4 or CD8 T cell numbers at the same level as PBS clone 13-infected control mice (data not shown). Depletion of either CD4 or CD8 T cells abrogated IL-7's ability to clear virus (Figure S2).
 
Similarly, we depleted B cells with an anti-CD20 mAb (Uchida et al., 2004) during the course of IL-7 treatment (Figure S2A). Despite this depletion of B cells, the effects of IL-7 on viral clearance were not diminished (Figure S2B). This is consistent with previous studies showing that B cells play only minor roles in modulating T cell responses in cases in which there is abundant antigen (Bouaziz et al., 2007). Our data clearly demonstrate that IL-7 exerts its effect on viral clearance through the modulation of T cells rather than B cells. This has important implications in translating IL-7 therapies to humans, as this cytokine does not expand B cells in humans (Rosenberg et al., 2006).
 
IL-7 Expands Non-LCMV-Specific Naive T Cell Numbers in Clone 13-Infected Mice
 
We observed that the majority of splenic CD8 T cells in clone 13-infected mice treated with IL-7 or PBS expressed high levels of CD44 (Figure 3A and data not shown). We speculated that only a fraction of these cells were actually LCMV specific, and the remainder were non-LCMV-specific naive T cells that were upregulating CD44 in response to the inflammatory environment. This was supported, in the case of IL-7-treated animals, by the fact that only 15% of the expanded CD8 T cell population, most of which are CD44hi (data not shown) also expressed PD-1, which represents an antigen-driven activation marker (Figure 3B). To examine this further, we purified, phenotyped, and quantified CD8 T cells recognizing non-LCMV-specific epitopes directed against the SV40 large T antigen and the ovalbumin-derived SIINFEKL epitope (using pooled SV40/ova tetramers). These cells were expanded more than 14-fold in IL-7-treated animals compared to controls, and in both cases, although to a greater extent with IL-7 treatment, the levels of CD44 were upregulated on these cells following clone 13 infection (Figure 3C). Consistent with the fact that these cells were not activated by TCR triggering, as opposed to GP33 virus-specific T cells, there was minimal or no upregulation of PD-1 expression, hence indicating their naive origins (Figure 3C). Collectively, these data support the view that, in addition to expanding the number of activated virus-specific CD8 T cells, IL-7 also greatly expands the naive T cell pool, including T cells recognizing nonviral-related epitopes in chronically infected mice.
 
A similar finding has been reported with the use of IL-7 in uninfected humans (Sportes et al., 2008), but our data show that these effects are eminently translatable to chronic viral infections. These findings have important implications for the use of IL-7 in HIV-infected patients in which T cells of many different specificities unrelated to HIV also succumb to the disease ([Li et al., 2005] and [Mattapallil et al., 2005]). It is unlikely that the expansion of naive T cells contributes significantly to the clearance of virus in our model, as IL-7 is administered at the peak of immune activation, and its predominant effect is expansion of activated LCMV-specific T cells (Figure 3B).
 
IL-7 Enhances Thymic Output during Chronic Viral Infection to Expand the Naive T Cell Pool
 
The expanded naive T cell pool that we observed with IL-7 therapy may be a result of increased thymic output and/or peripheral expansion. IL-7 is thought to mobilize recent thymic emigrants from lymphoid tissues and promote thymic egress ([Chu et al., 2004] and Sportes et al., 2008 C. Sportes, F.T. Hakim, S.A. Memon, H. Zhang, K.S. Chua, M.R. Brown, T.A. Fleisher, M.C. Krumlauf, R.R. Babb and C.K. Chow et al., Administration of rhIL-7 in humans increases in vivo TCR repertoire diversity by preferential expansion of naive T cell subsets, J. Exp. Med. 205 (2008), pp. 1701-1714. View Record in Scopus | Cited By in Scopus (102)[Sportes et al., 2008]). However, there is no detailed analysis of thymic and peripheral effects on recent thymic emigrants in the setting of chronic viral infection. To address this issue, we utilized Rag-GFP transgenic mice, which express green fluorescent protein (GFP) under the control of the rag2 gene locus ([Yannoutsos et al., 2001] and [Yu et al., 1999]). Single positive cells in the thymus and those recently exiting the thymus silence the rag gene locus, so GFP levels slowly decline with time and with each division. We analyzed thymic subsets in clone 13-infected mice and found that, although the number of double-negative thymocytes did not increase with IL-7 treatment, the number of both CD8 and CD4 single positive thymocytes that were GFP bright increased about 3-fold (Figure 4A). These GFP bright cells are most likely in situ derived thymic cells rather than peripherally expanded recirculating cells, as the latter should be GFP negative or dull. Notably, these GFP+ cells have lower levels of GFP than cells in the thymus of naive uninfected controls and likely represent cells undergoing local thymic cycling and expansion promoted by IL-7. Therefore, the increase in GFP bright CD8 and CD4 single positive thymocytes may reflect a simple local thymic proliferation of newly generated CD8 and CD4 thymocytes. However, it may also be possible that enhanced thymopoiesis with de novo production of new single positive cells may contribute to the increased number of these cells in the thymus. Among the peripheral T cell population, in both uninfected and clone 13-infected animals, IL-7 increased the number of CD4 and CD8 single positive recent thymic emigrants by 3- to 6-fold, respectively (Figures 4B and 4C). These GFP+ cells were CD44loPD-1lo, indicating that they were truly naive and recent emigrants to the periphery (Figure 4C). These data show that IL-7 therapy in the setting of chronic infection can substantially expand the peripheral T cell pool both through mechanisms of peripheral homeostasis and enhanced thymic output.
 
IL-7 Augments IL-6, IL-12, IL-17, and IFNγ Serum Levels while Limiting TGF-ß Levels
 
We investigated the serum cytokine levels associated with the increased numbers of lymphocytes in IL-7-treated clone 13-infected mice. We found that the levels of several proinflammatory cytokines are greatly increased, particularly IL-6, IL-17, and IFNγ in IL-7-treated infected mice (Figure 5A). However, there was a modest decrease in the levels of TGFß (Figure 5A). The large increase in IL-17 serum levels likely reflects an expansion of Th17 cells, which may be a consequence of an IL-7-induced shift favoring IL-6 levels over TGFß and, in turn, favoring Th17 over T regulatory (Treg) cell differentiation ([Ogura et al., 2008] and [Zhou et al., 2008]). Interestingly, we observed a 2-fold increase in IL-10 serum levels at later time points during IL-7 treatment compared to controls (Figure 5A). Previous work suggests that IL-10 may abrogate immunity to chronic viruses, but in the present study, this inhibitory effect is likely overshadowed by the 17- to 18-fold increase in proinflammatory cytokines ([Brooks et al., 2006] and [Ejrnaes et al., 2006]). Despite the dramatic increases in proinflammatory cytokines and the heavy infiltration seen in organs of IL-7-treated animals (Figure 1C), we did not observe any significant immune-mediated pathology such as hepatitis (Figure 5B). Collectively, these data show that IL-7 promotes a proinflammatory milieu while limiting cytotoxicity and decreasing the levels of the inhibitory cytokine TGFß.
 
IL-7 Promotes IL-22-Mediated Cytoprotection during Clone 13 Infection
 
As a marker of immune-mediated damage and bystander pathology, we examined serum liver enzyme levels. Clone 13 causes a mild hepatitis, with an elevation of both serum aspartate transaminase (AST) and alanine transaminase (ALT) 8 days following infection. Compared to controls, IL-7 did not exacerbate liver disease, which resolved quickly in all animals (Figure 5B). IL-6 has been shown to protect hepatocytes from T cell-mediated injury, and this cytokine is significantly upregulated in IL-7-treated animals (Klein et al., 2005). IL-22 has also been identified as a cytoprotective cytokine that limits hepatotoxicity associated with ConA-induced cytokine storms ([Radaeva et al., 2004] and Zenewicz et al., 2007 L.A. Zenewicz, G.D. Yancopoulos, D.M. Valenzuela, A.J. Murphy, M. Karow and R.A. Flavell, Interleukin-22 but not interleukin-17 provides protection to hepatocytes during acute liver inflammation, Immunity 27 (2007), pp. 647-659. Article | PDF (1114 K) | View Record in Scopus | Cited By in Scopus (131)[Zenewicz et al., 2007]). We found a 2-fold increase in serum IL-22 levels in IL-7-treated mice compared to PBS treatment in clone 13-infected animals (Figure 5C). To determine whether the high levels of IL-22 in IL-7-treated clone 13-infected animals was responsible for the lack of significant liver pathology, we utilized anti-IL-22 mAb to neutralize the effect of this cytokine. Neutralizing IL-22 caused a significant hepatitis in IL-7-treated clone 13-infected mice (Figure 5D). This cytoprotective effect of IL-22 was not evident in PBS-treated control animals when IL-22 was neutralized (Figure 5D), most likely due to the lack of hepatic infiltration and the poor cytokine response associated with chronically infected PBS treated mice (Figure 1C and Figure 5A). These data indicate that IL-7 is able to limit its potential toxicity, which may be secondary to the augmented cytokine responses, by enhancing IL-22-mediated cytoprotection.
 
IL-22 is produced by Th17 cells (Zenewicz et al., 2007), lymphoid tissue inducer cells (LTi), and a subset of LTi-like cells in the gut that express the natural cytotoxic receptor NKp46 (reviewed in Colonna [2009]). To determine which cells were contributing to the increase in IL-22, we first determined whether this cytokine was locally produced in the liver. We found that IL-22 levels were 4-fold higher in the liver than in serum of IL-7-treated clone 13-infected mice (Figure S3A). This indicates that there is an endogenous source of IL-22 production in the liver. Major producers of IL-22 in the liver of IL-7-treated clone 13-infected animals were CD4+CD3+TCRαß+ T cells (Figure S3B and data not shown). Hence, conventional CD4 T cells, similar to those that secrete IL-17 (Figure S3B), are a significant source of IL-22 in the liver of IL-7-treated clone 13-infected mice.
 
IL-7 Limits T Regulatory Cell Numbers in Clone 13 Infection
 
Reports have suggested that IL-7 renders effector cells refractory to the effects of T regulatory cells (Treg) ([Pellegrini et al., 2009] and Ruprecht et al., 2005 C.R. Ruprecht, M. Gattorno, F. Ferlito, A. Gregorio, A. Martini, A. Lanzavecchia and F. Sallusto, Coexpression of CD25 and CD27 identifies FoxP3+ regulatory T cells in inflamed synovia, J. Exp. Med. 201 (2005), pp. 1793-1803. View Record in Scopus | Cited By in Scopus (184)[Ruprecht et al., 2005]). We investigated the effects of IL-7 on Treg physiology in the setting of chronic clone 13 infection. We found that the absolute number of Treg or Foxp3+CD4+ T cells was increased in the spleen of IL-7-treated clone 13-infected mice compared to controls. However, when compared to the overall increase in T cell numbers, Treg were dramatically underrepresented in IL-7-treated animals (Figure 6A-6D).
 
To determine the physiological relevance of Treg in the setting of chronic infection, we utilized BAC-transgenic DEREG mice that express a diphtheria toxin (DT) receptor-GFP fusion protein under the control of the foxp3 gene locus (Lahl et al., 2007). This allowed us to follow and selectively deplete Foxp3+ Treg cells by DT injection. We were able to efficiently delete GFP+ cells without overt adverse effect on the animals (data not shown). Clone 13-infected mice treated with PBS did not show any significant differences in absolute T cell numbers or GP- or NP-specific T cell numbers after Treg depletion (Figure 6E). Interestingly, IL-7-treated animals showed a 2- to 3-fold increase in the numbers of CD8-, CD4-, GP-, and NP-specific T cells, whereas B cell numbers were not affected after Treg depletion (Figure 6E). In all animals, the depletion of Treg did not cause short-term immune-mediated pathology, as liver function tests did not become elevated after depletion (Figure 6F). These data suggest that, at least in the short term, Treg do not impede an immune response in chronic clone 13 infection. However, Treg do partially abrogate IL-7's ability to expand T cells, but notably, viral titers in both IL-7- and PBS-treated animals were not altered in the absence of Treg (data not shown). Because previous experiments have shown that IL-7 renders T effector cells refractory to the inhibitory effects of Treg ([Pellegrini et al., 2009] and Ruprecht et al., 2005 C.R. Ruprecht, M. Gattorno, F. Ferlito, A. Gregorio, A. Martini, A. Lanzavecchia and F. Sallusto, Coexpression of CD25 and CD27 identifies FoxP3+ regulatory T cells in inflamed synovia, J. Exp. Med. 201 (2005), pp. 1793-1803. View Record in Scopus | Cited By in Scopus (184)[Ruprecht et al., 2005]), we postulated that the increase in T cell numbers observed only in IL-7-treated animals was perhaps due to the consumption of IL-2 by Treg (Pandiyan et al., 2007). In the absence of Treg, the abundance of this cytokine may further augment the expanded immune response seen in IL-7-treated animals. Indeed, when we examined the level of IL-2 compared to other cytokines, it was proportionally increased in IL-7-treated and Treg-depleted animals compared to nondepleted animals. This consumption of IL-2 in IL-7-treated animals is further supported by the high level of CD25 expression on both Treg and non-Treg CD4 T cell populations at day 29 postinfection (Figure 6C). Our data therefore suggest that depletion of Treg may compliment the effects of IL-7 by removing a potential cytokine sink.
 
IL-7 Reprograms T Cells to Repress Socs3 Inhibitory Pathways
 
In view of the dramatic increase in numerous cytokine levels and the ability of IL-7-treated mice to efficiently clear virus, we investigated whether inhibitors of cytokine signaling may be repressed. We focused on Socs3, given its prominent role in the negative regulation of IL-6 signaling ([Croker et al., 2003] and [Lang et al., 2003]) and prominent increase of this cytokine in the context of IL-7 treatment. Interestingly, the efficacy of IL-7 in promoting cytokine responses, including IL-22 and T cell expansion and viral clearance, is diminished in IL-6-deficient mice (Figure 7A and Figures S4A-S4C). Furthermore, it has been shown that, in the absence of Socs3, IL-6 signaling is reprogrammed to mimic IFN responses, and this may contribute to more efficient viral elimination ([Croker et al., 2003] and [Lang et al., 2003]). We first investigated whether Socs3 levels in T cells are aberrantly upregulated in clone 13 infection compared to acute LCMV Armstrong infection. Even at very early time points during clone 13 infection, the levels of Socs3 in T cells, purified and analyzed directly ex vivo, were substantially higher in clone 13-infected mice compared to Armstrong infected animals (Figure 7B). We then purified total T cells from PBS- or IL-7-treated clone 13-infected animals. Direct ex vivo western blot analysis was performed and established that IL-7 treatment resulted in a substantial reduction in Socs3 levels in T cells (Figure 7C). We did not observe any differences in Socs1 levels. These data elucidate a pathway downstream of IL-7 that interferes with Socs3 inhibition of cytokine-mediated signaling.
 
Socs3 Is Endogenously Regulated by FoxO Transcription Factors
 
It is generally accepted that Socs3 is induced with cytokine signaling via a Stat-dependent pathway. It is unusual then that IL-7, a potent inducer of Stat5 signaling, is able to repress Socs3. However, FoxO transcription factors can be negatively regulated by IL-7 and perhaps other cytokines that signal via the common γ chain receptor (reviewed in Kittipatarin and Khaled [2007]), and under certain circumstances, Stat5 transcriptional activity may negatively regulate FoxO1 (Stittrich et al., 2010). Therefore, we tested whether FoxO transcription factors are involved in Socs3 induction. Mice that had all six alleles of foxO conditionally targeted (foxO1/3/4) were crossed to MxCre transgenic mice to generate (foxO1/3/4)fl/flMxCre animals. These mice were injected with poly I:C to generate animals that were deficient in FoxO1/3/4 in the hemopoietic compartment (Paik et al., 2007). T cells were purified from these animals well before the development of any malignancies or abnormalities. T cells isolated from animals deficient in FoxO transcription factors had dramatically reduced levels of Socs3, as determined by western blot (Figure 7D). Collectively, these data indicate that IL-7 may repress Socs3 expression via inhibition of FoxO transcriptional activity.
 
Mice Conditionally Deficient in Socs3 Mimic the IL-7-Induced T Cell Phenotype in Clone 13-Infected Animals
 
To assess whether a deficiency in Socs3 in T cells could replicate some of the IL-7-induced effects that we observed in clone 13-infected animals, we used mice that had socs3 conditionally targeted (Croker et al., 2003). These socs3fl/fl mice were crossed with MxCre transgenic mice, and gene deletion throughout the hemopoietic compartment was induced by clone 13 infection, which induces a type I IFN response and activation of the IFN-responsive Mx1 promoter. Ex vivo analysis by PCR confirmed that socs3 was deleted in purified peripheral T cells by day 3 postinfection (data not shown). By day 6 postinfection, all mice had to be euthanized due to severe illness. At this time point, Socs3fl/flMxCre+ mice had a 2.5-fold increase in the number of splenic GP33-specific CD8 T cells and a 6-fold increase in the number of NP396-specific CD8 T cells compared to control animals (Figure 7E). Ex vivo analysis of Socs3fl/flMxCre+ GP33-specific CD8 T cells at day 6 postinfection demonstrated a 5.5-fold increase in the ability of these cells to secrete both IFN-γ and TNF-α compared to controls (Figure S5A). Clone 13-infected Socs3fl/flMxCre+ mice had a dramatic neutrophilia with a 3.5-fold increase in the number of splenic granulocytes (Figure S5B). This substantial neutrophilia, which is likely due to Socs3 deficiency in granulocytes induced by clone 13 infection, may explain the illness that the mice succumb to. Liver function tests indicated a prominent hepatitis in clone 13-infected Socs3fl/flMxCre+ mice compared to controls (Figure S5C). The exact cause of lethality in clone 13-infected Socs3fl/flMxCre+ is not clear but likely relates to the severe granulocytosis observed in these animals. To avoid the development of granulocytosis and early lethality, we next used Socs3fl/flLckCre+ mice to specifically delete socs3 in T cells.
 
T Cell-Specific Deletion of socs3 Augments Immunity to Efficiently Eliminate Virus
 
Analysis of Socs3fl/flLckCre+ mice on day 8 post-LCMV clone 13 infection showed a profound augmentation of immunity, with a 15-fold increase in the number of NP396 virus-specific CD8 T cells compared to controls (Figures 7F and 7G). These mice did not develop a granulocytosis postinfection (Figure S5D) and showed no signs of increased collateral liver pathology (Figure S5C), morbidity, or mortality compared to control animals. Ex vivo analysis of T cells from Socs3fl/flLckCre+-infected mice at day 8 postinfection showed a 4-fold increase in the ability of virus-specific CD4 T cells to secrete TNF-α and/or IL-2, a 7-fold increase in the ability of virus-specific CD8 T cells to secrete IFN-γ, and a 17-fold increase in the number of Th17 cells (Figures 7H and 7I). The severe granulocytosis and early mortality in LCMV clone 13-infected Socs3fl/flMxCre+ mice prevented the elimination of virus, but Socs3fl/flLckCre+ LCMV-infected mice progressively and efficiently cleared virus from organs with no mortality (Figure S5E, Figure 7J, and data not shown). Consistent with viral clearance, we observed a contraction of the immune response at day 14 postinfection in Socs3fl/flLckCre+ mice (Figures S5F and S5G). However, a 3-fold increase in the number of residual high-affinity NP396 virus-specific CD8 T cells persisted in Socs3fl/flLckCre+ mice compared to controls at day 14 postinfection. (Figure S5G). These data indicate that the T cell-intrinsic Socs3 induction is a major factor contributing to immunological failure in the setting of chronic active infection.
 
 
 
 
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