Immune Responses to Retroviral Infection

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1 This information is current as of March 5, Negative Impact of IFN-γ on Early Host Immune Responses to Retroviral Infection Amanda K. Duley, Mickaël J.-Y. Ploquin, Urszula Eksmond, Christoph G. Ammann, Ronald J. Messer, Lara Myers, Kim J. Hasenkrug and George Kassiotis July J Immunol 2012; 189: ; Prepublished online ; doi: /jimmunol References Subscription Permissions Alerts This article cites 49 articles, 29 of which you can access for free at: Why The JI? Submit online. Rapid Reviews! 30 days* from submission to initial decision No Triage! Every submission reviewed by practicing scientists Fast Publication! 4 weeks from acceptance to publication *average Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: Downloaded from by guest on March 5, 2019 The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD All rights reserved. Print ISSN: Online ISSN:

2 The Journal of Immunology Negative Impact of IFN-g on Early Host Immune Responses to Retroviral Infection Amanda K. Duley,*,1 Mickaël J.-Y. Ploquin,,1,2 Urszula Eksmond, Christoph G. Ammann,* Ronald J. Messer,* Lara Myers,* Kim J. Hasenkrug,*,1 and George Kassiotis,1 The immune system is tasked with defending against a myriad of microbial infections, and its response to a given infectious microbe may be strongly influenced by coinfection with another microbe. It was shown that infection of mice with lactate dehydrogenase-elevating virus (LDV) impairs early adaptive immune responses to Friend virus (FV) coinfection. To investigate the mechanism of this impairment, we examined LDV-induced innate immune responses and found LDV-specific induction of IFNa and IFN-g. LDV-induced IFN-a had little effect on FV infection or immune responses, but unexpectedly, LDV-induced IFN-g production dampened Th1 adaptive immune responses and enhanced FV infection. Two distinct effects were identified. First, LDV-induced IFN-g signaling indirectly modulated FV-specific CD8 + T cell responses. Second, intrinsic IFN-g signaling in B cells promoted polyclonal B cell activation and enhanced early FV infection, despite promotion of germinal center formation and neutralizing Ab production. Results from this model reveal that IFN-g production can have detrimental effects on early adaptive immune responses and virus control. The Journal of Immunology, 2012, 189: *Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840; and Division of Immunoregulation, Medical Research Council National Institute for Medical Research, London NW7 1AA, United Kingdom 1 A.K.D., M.J.-Y.P., K.J.H., and G.K. contributed equally to this work. 2 Current address: Unité de Régulation des Infections Rétrovirales, Institut Pasteur, Paris, France. Received for publication April 18, Accepted for publication June 21, This work was supported by the Intramural Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health and by the U.K. Medical Research Council (U ). Address correspondence and reprint requests to Dr. Kim J. Hasenkrug or Dr. George Kassiotis, Rocky Mountain Laboratories, National Institutes of Health, 903 South 4th Street, RML, Hamilton, MT (K.J.H.) or Division of Immunoregulation, Medical Research Council National Institute for Medical Research, The Ridgeway, London NW7 1AA, U.K. (G.K.). addresses: khasenkrug@nih.gov (K.J.H.) or gkassio@nimr.mrc.ac.uk (G.K.) Abbreviations used in this article: B6, C57BL/6; F-MuLV, Friend murine leukemia virus; FV, Friend virus; GC, germinal center; LDV, lactate dehydrogenase-elevating virus; MHC I, MHC class I; NIMR, National Institute for Medical Research; pi, postinfection; RML, Rocky Mountain Laboratories; SFFV, spleen focus-forming virus; wt, wild-type. With a few notable exceptions, most scientific investigation of viral pathogenesis and immunology has been performed under highly controlled conditions using single pathogenic agents. However, animals (including humans) in the natural world carry an immunological history of previous infections and a multiplicity of chronic infections that can have profound consequences on the immune responses to subsequent infections (1, 2). Infection with a particular pathogen reshapes the T cell repertoire in a manner that can either enhance or diminish immune responses to heterologous virus challenges, even when the original infection is completely resolved (2). In situations in which viruses persist, the mechanisms used by viruses to evade elimination by the immune system can alter the reactivity to subsequent infectious insults (3). In addition to persistent infections, concomitant infections may occur, particularly in i.v. drug users who may become simultaneously infected with HIV and hepatitis B or C virus. Thus, it is of interest to study the immunological effects of coinfections, particularly in models in which the details of the isolated infections are well understood. In the current study, we investigate coinfection with two mouse viruses: Friend virus (FV) (4), and lactate dehydrogenase-elevating virus (LDV) (5). FV is an oncogenic retroviral complex consisting of a nonpathogenic, replication-competent Friend murine leukemia virus (F-MuLV) and a pathogenic, but replication-defective, virus called spleen focus-forming virus (SFFV) (6). Infected mice rapidly develop splenomegaly as a result of proliferation of erythroid progenitors from stimulation of erythropoietin receptors by the gp55 envelope protein of the SFFV component (7). This proliferation leads to lethal FV-induced erythroleukemia unless the mice mount complex immune responses, including B cell and CD4 + and CD8 + T cell responses (8, 9). LDV is a small, nonpathogenic, RNA virus of the Arteriviridae family (10) that replicates very rapidly in a subset of macrophages involved in scavenging extracellular lactate dehydrogenase. The lysis of these macrophages results in an excess of lactate dehydrogenase in the serum. Immune responses are relatively ineffective against LDV, which establishes life-long persistent infections with little pathological consequence to the host (5). LDV is endemic in many mouse populations, including the laboratory mice used in the early days to propagate FV (11), and FV/LDV coinfections were common in the laboratory infections until recently (12). Previous work demonstrated that coinfection of FV with LDV impaired adaptive immune responses to FV, including CD8 + T cell responses (12), CD4 + T cell responses (13), and FV-specific neutralizing Ab responses (14). The current study was initiated to determine the mechanism through which LDVmediated suppression of FV-specific immune responses occurred. One possible mechanism by which LDV could induce broad effects on early FV-specific immune responses is by alteration of the cytokine milieu induced by the innate immune response to LDV. We examined cytokine profiles of FVand FV/LDV coinfected mice in a kinetic manner to identify candidate cytokines and then investigated the effects of LDV-induced cytokines on FV-specific immune responses. Unexpectedly, the key cytokine determined

3 2522 NEGATIVE IMPACT OF IFN-g to be dampening FV-specific B cell and CD8 + T cell responses was IFN-g, a cytokine known to have potent antiviral properties and that generally promotes the types of immune responses that are suppressed in this scenario. Materials and Methods Mice Ten- to sixteen-week-old B6.A-Fv2 s mice [congenic to C57BL/6 (B6) but carrying the Fv2 susceptibility allele] were described previously (14). B6 mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and subsequently maintained at the Rocky Mountain Laboratories (RML) or the National Institute for Medical Research (NIMR) animal facilities. Where indicated, experiments were conducted with wkold female (C57BL/10 3 A.BY) F 1 mice bred at RML. The relevant FV resistance genotype of these mice is H2 b/b, Fv1 b/b, Fv2 r/s, and Rfv3 r/s. B6- backcrossed IFN-gR deficient mice (Ifngr1 2/2 ) maintained at RML and NIMR and B6-backcrossed IFN-abR deficient mice (Ifnar1 2/2 ) maintained at NIMR were described previously (15, 16). B6-backcrossed B cell-deficient (Ighm 2/2 ) mice (17) were also maintained at NIMR or purchased from The Jackson Laboratory. B6.PL-Thy1a/CyJ mice were purchased from The Jackson Laboratory. Mice were treated in accordance with the regulations and guidelines of the Animal Care and Use Committee of RML, the National Institutes of Health, or the U.K. Home Office regulations and NIMR guidelines. Viruses and infections The FV used in this study was a retroviral complex of B-tropic murine leukemia virus (F-MuLV-B) and polycythemia-inducing SFFV. The FV stock was free of LDV and was obtained as previously described (12). A stock of FV (6000 spleen focus-forming units) that contained LDV (10 5 ID 50 ) was also used (FV/LDV). Assessment of infection FV-infected cells were detected by flow cytometry using anti-fv gag mab 34 bound to secondary goat anti-mouse IgG2b labeled with allophycocyanin (Invitrogen) (18). On surfaces of infected cells mab 34 recognizes glycosylated gag (glyco gag). To determine the number of cells producing infectious virus, serial dilutions of spleen cells were plated onto susceptible Mus dunni cells and cocultured for 2 d, as previously described (18). Cells were fixed with ethanol and incubated sequentially with F-MuLV envelope-specific mab 720 and peroxidase-conjugated goat anti-mouse IgG (Cappel Laboratories). Foci of infection were identified following incubation with aminoethylcarbazole substrate. Flow cytometry Single-cell suspensions were prepared from the spleen or bone marrow by mechanical disruption on nylon mesh, followed by treatment with ammonium chloride-potassium to lyse erythrocytes. Blood samples were depleted of red cells by incubation in 2% dextran sulfate (1:1 ratio) for 30 min at 37 C prior to treatment with ammonium chloride-potassium. The expression of cell surface markers was analyzed using fluorochromeconjugated Abs to CD4 (clone RM4-5), CD8 (clone ), CD11a (2D7), CD11b (M1/70), CD11c (clone HL3), CD19 (clone 1D3), NK1.1 (clone PK136), and Ter119 (clone Ter119) (BD Pharmingen). FV-specific CD8 + T cells were identified by binding to a D b GagL MHC class I tetramer (Beckman Coulter), as previously described (19). For detection of intracellular IFN-g production, cells were cultured in DMEM supplemented with 10% FCS in the presence of 10 mg/ml brefeldin A for 5 h. The cells were then stained for surface expression of lineage markers, fixed with 2% formaldehyde, permeabilized with 0.1% saponin in PBS containing 0.1% sodium azide and 1% FBS, and incubated with anti IFN-g (clone XMG1.2) (BD Pharmingen). For detection of Foxp3 expression by intracellular staining, we used the anti-mouse/rat Foxp3 (FJK-16s) and the Foxp3 staining set (ebioscience). For analysis of B cell activation, cells were stained with directly conjugated Abs to B220, CD19, IgD, CD38, and GL7 (ebiosciences). Data were acquired using FACSCalibur, LSRII (BD Biosciences), or CyAn (Dako) flow cytometers and analyzed with FlowJo v8.7 (Tree Star) and Summit v4.3 (Dako) analysis software, for the BD Biosciences and Dako cytometers, respectively. Ab treatments B6.PL-Thy1a/CyJ mice were depleted of CD8 + T cells by i.p. injection of 100 mg anti-cd8 (clone 169.4) 7 and 5 d before infection (20). Ab treatment achieved.98% depletion, as determined by flow cytometric analysis of blood. Neutralization of IFN-g in (C57BL/10 3 A.BY) F 1 mice was performed by i.p. injection of 250 mg XMG1.2 Ab in 500 ml PBS at days 0, 2, and 4 postinfection (pi). Experiments with control Abs gave equivalent results to PBS treatments; therefore, PBS treatments were used for most experiments. Bone marrow chimeras and adoptive transfers Selective B cell reconstitution in B cell-deficient mice was achieved following bone marrow transplantation of nonirradiated recipients, as previously described (21). Briefly, one mouse-equivalent of bone marrow cells from either wild-type (wt) or Ifngr1 2/2 donor mice was injected into nonirradiated Ighm 2/2 recipient mice. Chimeras were used $8 wk postbone marrow transfer. For T cell-adoptive transfers, spleen cells were isolated from either B6 or Ifngr1 2/2 mice, and CD8 + cells were separated using the MidiMACS Separation System (MACS), as recommended by the manufacturer (Miltenyi Biotec). A total of CD8 + cells in 0.5 ml phosphate-buffered balanced salt solution containing 15 U/ml heparin sodium (Solo Pak Laboratories) was injected i.v. into B6.PL-Thy1a/CyJ congenic mice 1 wk post-cd8 T cell depletion. Mice were infected with virus 1 d postadoptive transfer. Cytokine assays Serum cytokines/chemokines were measured on a Luminex system (Bio- Plex 100) using the mouse cytokine kits (Bioplex Mouse cytokine group II and Bioplex Mouse Cytokine Standard; Bio-Rad Laboratories) following the manufacturer s instructions. Data were collected with a minimum of 100 beads/analyte (IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 [p40], IL-12 [p70], IL-13, IL-17, eotaxin, G-CSF, GM-CSF, IFN-g, KC, MCP-1 [MCAF], MIP-1a, MIP-1b, RANTES, TNF-a) using Bio- Plex Manager Software (Bio-Rad Laboratories). IFN-a and IFN-g concentrations in plasma samples were quantified with the Verikine Mouse IFN-a Elisa kit (PBL InterferonSource and the mouse IFN-g Femto-HS Elisa kit (ebioscience), respectively, according to the manufacturers instructions. FV-neutralizing Ab assay Serum titers of FV-neutralizing Abs were measured as previously described (14). The dilution of serum that resulted in 50% neutralization was taken as the neutralizing titer. Results Types I and II IFN responses are induced by FV/LDV coinfection, but not by FV infection LDV is a natural mouse virus that was a common contaminant of FV stocks that had been maintained by in vivo passage (12). Studies showed that mice infected with FV had delayed antiviral immune responses and diminished virus control when coinfected with LDV (12 14). To determine whether differences in cytokine or chemokine responses correlated with the delayed recovery of FV/LDV coinfected mice, we used a multiplex assay to measure plasma levels of 23 cytokines and chemokines in a kinetic manner. Experiments in B6.A-Fv2 s mice, which carry a nonimmunological FV susceptibility gene (14), showed upregulation of several cytokines, but only G-CSF was induced comparably at early time points in both FV infections and FV/LDV coinfections (Fig. 1A). All of the other factors that gave positive results were produced selectively in response to FV/LDV coinfection and were IFN inducible. These results suggested that FV/LDV coinfection, but not FV infection, was associated with a strong early IFN response. To directly quantify the IFN responses, mouse plasmas were analyzed by ELISA over a 2-d time course following FV infection and FV/LDV coinfection. Rapid and potent induction of IFN-a was associated with FV/LDV coinfection, but not with FV infection (Fig. 1B, left panel). These results were consistent with a previous study showing that IFN-a responses were due to the presence of LDV (22). Similarly, FV/LDV coinfection, but not FV infection, caused significant release of IFN-g, peaking at 24 h pi (Fig. 1B, right panel).

4 The Journal of Immunology 2523 FIGURE 1. Inflammatory and IFN responses to FV/LDV coinfection or FV infection. (A) Cytokine and chemokine levels in serum samples of B6.A-Fv2 s mice at indicated time points following FV infection or FV/LDV coinfection. (B) IFN-a (left panel) and IFN-g (right panel) levels in plasma samples of B6 mice at indicated time points following FV infection or FV/LDV coinfection. Data are the means of four to six mice/ group; statistical analysis was performed using the unpaired Student t test (*p, 0.05). Type I IFN responses during FV/LDV coinfection have minimal effect on FV titers The dramatic difference in types I and II IFN responses between FV infection and FV/LDV coinfection raised the possibility that LDV-induced IFNs may have affected the course of FV infection. To examine the potential impact of IFNs, the course of FV replication was compared between wt mice and mice lacking signaling through IFN type I or type II receptors. Mice infected with FV had largely recovered from acute infection by 2 wk pi, whereas coinfection with LDV caused significantly increased peak virus levels and delayed recovery by 1 wk (Fig. 2A, open symbols). For both FV infections and FV/LDV coinfections, mice deficient in type 1 IFN signaling (Ifnar1 2/2 ) had slightly increased peak levels of FV infection compared with wt mice, but the kinetics of recovery were very similar to wt mice, and virus control was maintained long-term (Fig. 2A). Thus, the lack of type I IFN signaling caused slightly worse FV infections, but recovery was not affected significantly. Type II IFN responses during FV/LDV coinfection enhance FV titers In striking contrast to IFN-abR deficiency, IFN-gR (type 2 IFNR) deficiency had a significant impact on acute FV replication during FV/LDV coinfection (Fig. 2B). Interestingly, although IFN-g is usually considered a potent antiviral cytokine, FV levels were significantly reduced in IFN-gR deficient mice compared with wt mice at 14 d pi when acute FV replication was peaking during coinfection with LDV (Fig. 2B). Thus, IFN-g signaling allowed for increased FV titers during acute coinfection. This finding suggested that LDV-specific induction of IFN-g could largely account for increased FV titers during coinfection. In line with previous findings (23), mice lacking IFN-gR failed to maintain long-term control of FV infection (Fig. 2B). Thus, IFN-g signaling appeared detrimental to control of acute infection but critical for control of chronic infection. To ensure that the results from IFN-gR deficient mice were not due to a developmental problem related to genetic inactivation of the Ifngr gene, the effect of blocking IFN-g signaling with anti IFN-g neutralizing Abs was tested in susceptible (C57BL/10 3 A.BY) F 1 mice coinfected with FV/LDV. IFN-g neutralization during the first week of infection reduced the day-14 FV infection to levels comparable to those seen in mice infected with FV alone, as measured by viral Ag + spleen cells (Fig 2C), virus-producing spleen cells (infectious centers) (Fig. 2D), and FV-induced splenomegaly (Fig 2E). These results confirmed that a major factor contributing to higher FV levels in LDV-coinfected mice was LDV induction of the IFN-g response. Multiple cell types produce IFN-g in response to FV/LDV coinfection Intracellular cytokine staining and flow cytometry were used next to investigate the sources of IFN-g from the major cellular subsets of the spleen, bone marrow, and blood at the peak of production (24 h pi). Consistent with the ELISA data, IFN-g was produced predominantly in response to FV/LDV coinfection rather than FV alone (Fig. 3). Multiple cell types responded, with strong responses by NK and NKT cells (Fig. 3). The best responses occurred in the bone marrow, an early site of FV replication (24, 25). IFN-gR signaling during acute coinfection is required for CD4 T cell expansion but negatively affects CD8 T cells Effective control of FV infection is mediated by complex immune responses, including CD4 + and CD8 + T cells and B cells (26), and it was shown that coinfection with LDV can negatively impact all three cell types (12 14). Therefore, we investigated whether LDV-

5 2524 NEGATIVE IMPACT OF IFN-g FIGURE 2. Effect of type I or II IFN response on the course of FV infection. B6 wt mice or B6-congenic Ifnar1 2/2 or Ifngr1 2/2 mice were infected with FV alone or coinfected with FV/LDV, and the course of FV infection was monitored at the indicated time points. (A) Percentage of erythroid (Ter119 + ) cells expressing FV viral Ag (glyco-gag, detected by mab34) in the spleens of wt or Ifnar1 2/2 mice. (B) Percentage of erythroid (Ter119 + ) cells expressing FV viral Ag (glyco-gag) in the spleens of wt or Ifngr1 2/2 mice. Data in (A) and (B) are the means of 4 16 mice/group; statistical analysis was performed using the nonparametric two-tailed Mann Whitney rank-sum test. (C) Percentage of erythroid (Ter119 + ) cells expressing FV viral Ag (glyco-gag) in the spleens of susceptible (C57BL/10 3 A.BY) F 1 mice at 14 d pi with FVor FV/LDV, which were treated or not with anti IFN-g.(D) Splenocytes in (C) were assayed for virus-producing cells by an infectious centers assay. (E) Spleens in (C) were weighed to determine FV-induced splenomegaly. Symbols represent individual mice. Data in (C E) are representative of five independent experiments using four to six mice/group; statistical analysis was performed using the unpaired Student t test. induced IFN-g signaling affected any of these lymphocyte subsets at 14 d pi, the peak of FV/LDV infection. To examine the intensity of the antiviral T cell responses, we first analyzed upregulation of the activation-induced isoform of CD43 (clone1b11), which, like CD11a (27, 28), has been used to follow virus-specific CD4 + and CD8 + T cell responses (29 31). Virtually all CD43 hi CD4 + T cells FIGURE 3. IFN-g induction in multiple cell types 24 h pi with FV/LDV. IFN-g was measured by intracellular cytokine staining of single-cell suspensions harvested from spleen, bone marrow, and blood obtained from susceptible (C57BL/10 3 A.BY) F 1 mice infected with FV or FV/LDV. Graphs display the percentage of IFN-g + cells, as defined by surface markers: NK1.1CD3 2 (NK cells), CD4 + CD3 + (CD4 Th cells), CD8 + CD3 + (CD8 cytotoxic T cells), B220 + (B cells), NK1.1 + CD3 + (NKT cells), Gr1 + (granulocytes), CD11b + (monocytes/granulocytes), and CD11c + (dendritic cells). Symbols represent individual mice. Data are representative of two independent experiments with four mice/group; statistical analysis was performed using the unpaired Student t test. *p, 0.05, ** p, 0.01.

6 The Journal of Immunology 2525 in FV/LDV coinfection also expressed CD11a and contained a large percentage of recently divided cells (Ki67 + ) (Fig. 4A). In FV/LDV coinfection, the percentage of activated (CD43 + ) CD4 + T cells was significantly reduced in the absence of IFN-gR signaling, as illustrated by representative CD43 graphs and mean percentages in bar graph form (Fig. 4B). Thus, IFN-g signaling was necessary for the full activation of CD4 + T cells. Interestingly, and in contrast to CD4 + T cells, FV/LDV coinfection induced more activation of CD8 + T cells in mice deficient in IFN-gR signaling than in wt mice (Fig. 4C). This unexpected increase in CD8 + T cell activation was associated with lower FV infection in IFN-gR deficient mice compared with wt mice (Fig. 4D). In addition to the overall CD8 + T cell response, we used tetramer staining to analyze the response to the immunodominant FV epitope (19, 32). At 14 d pi, the percentage of tetramer + cells in Ifngr1 2/2 mice was significantly greater than in wt mice (Fig. 4E). Using absolute numbers of CD8 + tetramer + T cells and infectious centers from the spleens at 2 wk pi, the E:T ratios were calculated for wt and Ifngr1 2/2 mice (Fig. 4F). The Ifngr1 2/2 mice averaged 100-fold more tetramer + CD8 + T cells for every infected cell than did the wt mice. To confirm the results from Ifngr1 2/2 mice, we used susceptible (C57BL/10 3 A.BY) F 1 mice and treated them with anti IFN-g Abs during FV/LDV coinfection to neutralize the LDV-induced IFN-g response. Anti IFN-g therapy during the first week of infection reduced FV levels by 100-fold at 2 wk pi (Fig. 4G), and it significantly increased both the proportions of CD8 + tetramer + T cells (Fig. 4H) and the E:T ratios (Fig. 4I). CD8 + T cells were shown to be required for the protective effect of anti IFN-g therapy, because the therapy was not successful in CD8-depleted mice (Fig. 4G). Thus, although IFN-g signaling appeared necessary for full activation of CD4 + T cells, it hampered the activation of CD8 + T cells in response to FV/LDV coinfection. To determine whether the lack of IFN-g signaling specifically in the CD8 + T cells affected their ability to expand and control virus levels, wt mice were partially depleted of endogenous CD8 + T cells to make room in the T cell niche and were adoptively transferred with either wt or Ifngr1 2/2 CD8 + T cells prior to infection with FV/LDV. The donor cells were identified by expression of Thy1.2 and IFN-gR (data not shown). Interestingly, wt and Ifngr1 2/2 CD8 + T cells (Fig. 4J), as well as tetramer + CD8 + T cells (Fig. 4K), expanded to similar levels in response to infection and activated to similar levels (Fig. 4L). However, no donor cell-specific differences in virus control were observed (Fig. 4M), suggesting that the inhibitory effect of LDV-induced IFN-g on CD8 + T cells was indirect. IFN-gR signaling enhances germinal center B cell formation, polyclonal B cell activation, and FV infection of B cells Finally, we examined the effect of IFN-gR deficiency on the B cell response to FV. FV infection drives expansion of B cells with a germinal center (GC) phenotype (B220 + IgD lo CD38 lo GL7 hi ), which is greatly elevated during FV/LDV coinfection as a result of LDV-induced polyclonal B cell activation (33, 34). Notably, the GC B cell response induced by either FV infection or FV/LDV coinfection was dramatically curtailed in IFN-gR deficient hosts, although FV/LDV coinfection was still able to induce a detectable response (Fig. 5A, 5B). Thus, IFN-g was a predominant factor in the GC B cell response and polyclonal B cell activation. FV is a type C retrovirus that infects actively dividing cells expressing the ecotropic virus receptor mcat-1 (35). Next to erythroblasts, which are the primary targets for FV infection, B cells are the most heavily infected cell type (36). Consequently, a reduction in GC formation and polyclonal B cell activation due to a lack of IFN-g signaling could have an overall effect on FV infection levels. Indeed, the levels of B cell infection in both IFNgR deficient mice (Fig. 5C) and anti IFN-g treated mice (Fig. 5D) were reduced compared with wt or untreated mice, respectively. Thus, lack of IFN-g signaling significantly reduced FV infection in B cells. The development of virus-neutralizing Abs, which is dependent on GC B cell responses (37), was shown to be critical for control of FV infections, especially after the first few weeks (38). Indeed, the defective GC B cell responses in Ifngr1 2/2 hosts were accompanied by reduced or absent FV-neutralizing Abs at 5 wk pi, particularly in FV infections but also in FV/LDV coinfections (Fig. 5E). This failure to generate virus-neutralizing Abs was likely responsible for the lack of long-term FV control in Ifngr1 2/2 hosts (Fig. 2B). Interestingly, a proportion of FV/LDV-coinfected, but not FVinfected, Ifngr1 2/2 mice produced FV-neutralizing Abs (Fig. 5E), indicating that the presence of LDV could partially compensate for the lack of IFN-g. However, in some of the FV/LDV-coinfected mice the presence of FV-neutralizing Abs was not associated with protection against high FV titers (Fig. 5F). This finding is consistent with previous results demonstrating that FV-neutralizing Abs are essential, but not sufficient, for virus control (8). The use of Ifngr1 2/2 mice clearly demonstrated a role for IFN-g in the CG B cell and FV-neutralizing Ab responses. However, given that the CD4 + T cell response depended on IFN-gR signaling (Fig. 4B), we examined whether the impaired GC B cell response was due to a B cell-intrinsic effect of IFN-gR deficiency or a secondary effect from compromised T cell help. To address this question, we generated bone marrow chimeras by reconstituting nonirradiated B cell-deficient Ighm 2/2 hosts with either wt or Ifngr1 2/2 bone marrow. The chimeras were then coinfected with FV/LDV, and their GC B cell responses were measured at 14 d pi. Although the frequencies of total B cells were similar in both types of chimeras (Fig. 6A, left panel), B cell-specific IFNgR deficiency resulted in a significant reduction in the frequency of GC B cells (Fig. 6A, right panel). Thus, the LDV-induced GC B cell response was dependent on intrinsic IFN-gR signaling in B cells. Furthermore, lack of IFN-gR signaling specifically in B cells resulted in reduced FV-induced splenomegaly (Fig. 6B) and infection levels, as measured by splenic infectious centers (Fig. 6C). Lack of intrinsic IFN-gR signaling specifically in B cells did not fully recapitulate the phenotype of mice totally deficient in IFNgR. Although the levels of infection were reduced compared with wt mice, they were still significantly higher than in mice fully deficient in IFN-gR signaling (compare Figs. 4D, 6C). This was likely because mice with IFN-gR deficiency solely in B cells did not have the restored CD8 + T cell responses observed in the fully deficient mice. In fact, the CD8 + T cell responses (known to be of host origin, as determined by flow cytometric analysis of IFN-gR expression; data not shown) in the mice reconstituted with B cells deficient in IFN-gR signaling were unexpectedly worse than in mice reconstituted with wt B cells. The frequencies of activated (CD43 + ) CD8 + T cells (Fig. 6D) and of tetramer + FV-specific CD8 + T cells (Fig. 6E) were significantly lower in chimeras reconstituted with Ifngr1 2/2 than with wt B cells. These results indicated that, although B cell-specific IFN-gR signaling increased FV replication in those cells, the signaling was beneficial for the CD8 + T cell response. Discussion IFNs are a diverse family of proteins that are typically induced very rapidly in the innate response to viral infections. In addition to their

7 2526 NEGATIVE IMPACT OF IFN-g FIGURE 4. IFN-gR signaling during FV/LDV coinfection is required for expansion of CD4 + T cells, but it indirectly suppresses expansion of FVspecific CD8 + T cells. (A) Gating strategy of spleen cells demonstrating that CD43 + (clone 1B11) CD4 + T cells are Ag experienced (CD11a + ). Foxp3 + cells were gated out to remove regulatory T cells, which do not display a naive phenotype. Bar graphs show the expansion of splenocytes from wt mice (average spleen weight, 390 mg) and Ifngr1 2/2 mice (average spleen weight, mg) that expressed the activation marker CD43 + at 14 d postcoinfection with FV/LDV for CD4 + (B) orcd8 + (C) cells. Data are mean 6 SEM of four to six mice/group; statistical analysis was performed using the unpaired Student t test. (D) Splenocytes in (B) and(c) were assayed for virus-producing cells by an infectious centers assay. (E) Percentage of MHC class I (MHC I) tetramer + cells (specific for FV glyco-gag) first gated on CD8 + T cells. The mean absolute numbers of tetramer + CD8 + T cells from wt mice was /spleen and from Ifngr1 2/2 mice was /spleen (n = 5/group). (F) NumberofCD8 + MHC I tetramer + cells divided by the number of virus-producing cells, as measured by an infectious centers assay. (G) Splenocytes from (C57BL/10 3 A.BY) F 1 mice at 14 d postcoinfection with FV/LDV, with and without anti IFN-g and anti-cd8 treatment, were assayed for virus-producing cells by an infectious centers assay (average spleen weight for untreated mice, 1211 mg; average spleen weight for anti IFN-g treated mice, 453 mg). (H) Percentage of CD8 + MHC I tetramer + cells from (C57BL/10 3 A.BY) F 1 mice first gated on CD8 + T cells at 14 d postcoinfection with FV/LDV and treated or not with anti IFN-g. Themean absolute numbers of tetramer + CD8 + T cells were /spleen from untreated mice and /spleen from anti IFN-g treated mice (n = 5 6/ group). (I) RatioofthenumberofCD8 + MHC I tetramer + cells divided by the number of virus-producing cells as measured by an infectious centers assay. wt or Ifngr1 2/2 CD8 + T cells were adoptively transferred into congenic Thy1.1 mice and coinfected with FV/LDV for 14 d. (J) Percentage of donor CD8 + T cells (Thy1.2 + ) from recipient mice coinfected with FV/LDV. (K) The number of donor CD8 + MHC I tetramer + cellsfirstgatedondonorspecific Thy1.2 + and CD8 +.(L) The number of donor cells in (J) that expressed CD43 + CD8 +.(M) Splenocytes in (J) were assayed for virus-producing cells by an infectious centers assay. Symbols represent individual mice. Data are representative of two to five independent experiments with four to six mice/group; statistical analysis was performed using the unpaired Student t test.

8 The Journal of Immunology 2527 FIGURE 5. IFN-g dependent B cell activation and Ab response to FV infection. (A) Flow cytometric example of CD38 lo GL7 + GC cells, in gated B220 + IgD lo B cells, in the spleens of wt or Ifngr1 2/2 mice 14 d after FV infection or FV/LDV coinfection. (B) Percentage of CD38 lo GL7 + GC cells, in gated B220 + IgD lo B cells, in the same mice described in (A). Data are the means of 8 10 mice/group; statistical analysis was performed using the unpaired Student t test. (C) The number of B cells (B220 + splenocytes) that express FV viral Ag (glyco-gag) on the cell surface from wt and Ifngr1 2/2 mice at 14 d postcoinfection with FV/LDV. Symbols represent individual mice. Data are representative of three independent experiments using four or five mice/group; statistical analysis was performed using the unpaired Student t test. (D) The number of B cells (B220 + splenocytes) that express FV viral Ag (glyco-gag) on the cell surface from (C57BL/10 3 A.BY) F 1 mice at 14 d postcoinfection with FV/LDV. Symbols represent individual mice. Data are representative of five independent experiments using four to six mice/group; statistical analysis was performed using the unpaired Student t test. (E) Titer of FV-neutralizing activity in the sera of FV-infected or FV/LDV-coinfected wt or Ifngr1 2/2 mice at 35 d pi. Data are mean 6 SEM of 8 12 mice/group; statistical analysis was performed using the nonparametric two-tailed Mann Whitney rank-sum test. (F) Association between the titer of FV-neutralizing activity and percentage of FV-infected (glyco-gag) erythroid (Ter119 + ) cells in the spleens of the same mice described in (E). The dashed lines depict the limits of detection. direct antiviral activity, IFNs can exert important immunomodulatory effects that can either promote or inhibit responses (39, 40). Interestingly, no type I IFN response was detectable from infection with FV only, although LDV elicited a strong and rapid type I IFN response (Fig. 1B). Mice deficient in receptors for type I IFN, and therefore incapable of responding to type I IFN, had slightly elevated peak virus titers but they showed control over FV that was very similar to wt mice. Likewise, FV infections did not elicit type II IFN responses but LDV did (Fig. 1B). However, in contrast to FIGURE 6. FV replication enhanced by direct IFN-g signaling in B cells. Nonirradiated B cell-deficient mice were reconstituted with either wt or Ifngr1 2/2 bone marrow, resulting in selective B cell reconstitution. The mice were coinfected with FV/LDV 8 wk postreconstitution. (A) Percentages of B220 + B cells (left panel) and CD38 lo GL7 + GC cells (right panel), in gated B220 + IgD lo B cells, in the spleens of reconstituted mice 14 d post-fv/ldv coinfection. Data are mean 6 SEM of 8 12 mice/ group; statistical analysis was performed using the unpaired Student t test. (B) Spleens from chimeric mice were weighed to determine FV-induced splenomegaly at 14 d postcoinfection with FV/LDV. (C) Splenocytes in (B) were assayed for virus-producing cells by an infectious centers assay. (D) Percentage of activated CD43 + cells first gated on CD8 + T cells from chimeric mice at 14 d postcoinfection with FV/LDV. (E) Percentage of CD8 + T cells from splenocytes in (D) staining with MHC I tetramer (specific for FV-glycosylated gag). Data in (B E) are representative of two independent experiments using four to five mice each; statistical analysis was performed using the unpaired Student t test. the lack of major effects from type I IFNs, LDV-induced IFN-g produced strong effects on FV-specific adaptive immunity and virus control. Somewhat surprisingly, however, the overall effect was not beneficial to the host in the early phase of infection. In fact, our results indicate that LDV-induced IFN-g production is the primary mechanism by which LDV coinfection enhances FV infection. By neutralizing the LDV-induced IFN-g response, either genetically or therapeutically, we could diminish FV titers to levels comparable to those observed in FV-infected mice without LDV coinfection. Although IFN-g is generally considered to promote Th1 responses critical for antiviral immunity, the finding that IFN-g can have inhibitory effects on Th1 adaptive immune responses is not unprecedented. IFN-g was shown to be capable of downmodulating Th1 responses by promoting T cell apoptosis (41) and

9 2528 NEGATIVE IMPACT OF IFN-g inhibiting T cell proliferation in LCMV infection (42). Using the OT-1 model to study CD8 + T cell responses, it was further shown that IFN-g can act to limit CD8 + T cell expansion and promote contraction (43). IFN-g mediated negative regulation of CD8 + T cells is generally thought to occur through direct effects on the cells, whereas indirect effects, such as increased MHC class I and immunoproteosome expression, enhance CD8 + T cell activity (44). However, the current data with regard to CD8 + T cells lacking IFN-gR (Fig. 4K, 4L) suggest that the suppressive effect of IFN-g on FV-specific CD8 + T cells was due to indirect, rather than direct, effects. Indirect effects are unlikely to come from either CD4 + Th or regulatory CD4 + T cells; previous studies showed that the acute CD8 + T cell response to FV/LDV infection is not CD4 + T cell dependent, and depletion of CD4 + T cells neither improved nor abrogated the CD8 + T cell response (12). IFN-g signaling in APCs, such as CD11b + cells, was shown to counter-regulate CD8 + T cell expansion (43), but we did not detect IFN-g associated changes in APCs when we compared untreated mice with mice receiving anti IFN-g (data not shown). The B cell is another type of APC that appears to be important in FV infection. Interestingly, however, lack of IFN-gR specifically in B cells was detrimental to the CD8 + T cell response. This suggests that, in the context of an IFN-gR deficient mouse, the B cells are probably not the primary APC and that another cell type is fulfilling the APC function for CD8 + T cells. Thus, although it is clear that IFN-g inhibits the CD8 + T cell response to FV infection during LDV coinfection, the downstream mediators of that inhibition are not known, but they do not appear to be from CD4 + T cells or B cells. It was also shown in vaccinia virus infections that IFN-g deficiency increases CD8 + T cell responses (45). In that situation, the increase in CD8 + T cells was attributed to increased virus titers, but a causal relationship was not proven, and the increase could have been due to similar effects as seen in our current study. Our results confirm a critical role for FV-specific CD8 + T cells in the control of acute infection (46). Depletion of CD8 + T cells during acute FV/LDV coinfection increased FV titers in the spleen by two logs 10, and the antiviral effect of anti IFN-g Ab treatment was ablated by depletion of CD8 + T cells (Fig. 4). The importance of the CD8 + T cell response in controlling acute FV indicates that the delay of this response by LDV-induced IFN-g was an important factor in the lack of early FV control in coinfected mice. In contrast, IFN-g appeared to be beneficial for the expansion and activation of the CD4 + T cell response, indicating that LDV-induced suppression of the CD4 + T cell response (13) occurs through an IFN-g independent mechanism. These results are consistent with a previous study showing that CD4 + T cells were affected by LDV coinfection through an acceleration of the contraction phase of the FV-specific CD4 + T cell response (13). It was interesting that a lack of IFN-g signaling specifically in B cells was sufficient to significantly reduce FV infection levels. Importantly, this reduction occurred even though the FV-specific CD8 + T cell response was not restored in those mice. Thus, the virus titer reduction was not due to an indirect effect on the CD8 + T cells, but it appeared to be intrinsic to B cells. We found that IFN-gR deficiency specifically in B cells diminished the GC response to FV infection, as well as the production of FVneutralizing Abs. These findings are consistent with an established role for IFN-g and GCs in the maturation of B cell responses, especially the IgG2a response to FV, as well as other viruses (16, 23, 47, 48). That being said, the virus-neutralizing response is known to be very important in the control of FV infection, not in the promotion of it. The promotion of FV infection was likely due to LDV-induced polyclonal activation of B cells, which was largely, although not wholly, dependent on IFN-g signaling. Because FV is a type C retrovirus that needs actively dividing cells for productive infection, LDV-induced polyclonal activation of B cells provides an additional source of target cells for FV infection and spread. Because B cells are the most heavily infected subset next to erythroblasts (36), the effect on overall infection is significant. We found that some FV/LDV-coinfected Ifngr1 2/2 mice were able to produce FV-neutralizing Abs at late stages of infection. The presence of Ab responses in some mice may have been due to the lack of complete dependence on IFN-g signaling for LDVinduced activation of B cells. In fact, it was reported that LDVinduced hyperproduction of polyclonal IgG2a is independent of IFN-g production (49), although our data suggest partial dependence rather than complete independence. Nevertheless, LDV induction of FV-neutralizing Abs did not correlate with improved FV control, likely because Ab could not compensate long-term for poor CD8 + T cell responses. Although our results demonstrate that IFN-g can have detrimental effects on a host during an acute viral infection, eventual control of the infection is lost without IFN-g. This was true for both FV infection and FV/LDV coinfections. Although early IFN-g responses in FV/LDV coinfection cause a worse and more prolonged acute infection, most of the mice go on to recover, albeit with chronic infections of both FV and LDV (12). Ultimately then, the IFN-g response must be considered beneficial, and it determines the difference between recovery from acute infection or succumbing to lethal disease. Thorough understanding of the pleiotropic effects of the IFN-g response in the context of specific infections could allow the development of immunotherapies to maximize both the antiviral and immunomodulatory effects of this potent cytokine in the treatment of life-threatening infections. However, the current study illustrates the importance of understanding the full consequences of IFN-g mediated effects in the context of an active infection. Acknowledgments We thank Anne O Garra for the Ifnar1 2/2 mice. We are grateful for assistance from the Medical Research Council Division of Biological Services and the Flow Cytometry Facilities at the National Institute of Allergy and Infectious Diseases, National Institutes of Health and the Medical Research Council National Institute for Medical Research. Disclosures The authors have no financial conflicts of interest. References 1. Virgin, H. W., E. J. Wherry, and R. Ahmed Redefining chronic viral infection. Cell 138: Welsh, R. M., J. W. 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