Infection Prevents Immune Control of Friend Virus B Lymphocyte Activation by Coinfection

B Lymphocyte Activation by Coinfection Prevents Immune Control of Friend Virus Infection

This information is current as Rute Marques, Inês Antunes, Urszula Eksmond, Jonathan of September 28, 2021. Stoye, Kim Hasenkrug and George Kassiotis J Immunol 2008; 181:3432-3440; ; doi: 10.4049/jimmunol.181.5.3432 http://www.jimmunol.org/content/181/5/3432 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

B Lymphocyte Activation by Coinfection Prevents Immune Control of Friend Virus Infection1

Rute Marques,* Ineˆs Antunes,* Urszula Eksmond,* Jonathan Stoye,† Kim Hasenkrug,‡ and George Kassiotis2*

Although the adaptive immune response almost invariably fails to completely eliminate retroviral infections, it can exert significant protection from disease and long-term control of viral replication. Friend virus (FV), a mouse retrovirus, causes persistent infection in all strains of mice and erythroleukaemia in susceptible strains, the course of which can be strongly influenced by both genetic and extrinsic factors. In this study we examine the impact of coinfection on the requirements for immune control of FV infection. We show that congenic C57BL/6 mice, in which the introduction of an allele of the Friend virus susceptibility 2 gene provides the potential for FV-induced leukemia development, effectively resist FV infection, and both T cell- and Ab-dependent mechanisms contribute to their resistance. However, we further demonstrate that coinfection with lactate dehydrogenase-elevating Downloaded from virus (LDV) renders these otherwise immunocompetent mice highly susceptible to FV infection and subsequent disease. The presence of LDV delays induction of FV-specific neutralizing Abs and counteracts the protective contribution of adaptive immunity. Importantly, the disease-enhancing effect of LDV coinfection requires the presence of a polyclonal B cell repertoire and is reproduced by direct polyclonal B cell activation. Thus, immune activation by coinfecting pathogens or their products can contribute to the pathogenicity of retroviral infection. The Journal of Immunology, 2008, 181: 3432–3440. http://www.jimmunol.org/ he immune system has evolved in the face of simulta- tious pathogens, in a natural setting, are rarely encountered in iso- neous exposure to multiple microorganisms with variable lation and almost all humans are chronically infected by one or T pathogenicity and strategies to evade or divert the im- more persistent viruses. It is therefore also important to establish mune response. At the same time, the host’s response needs to be and study animal models for coinfection to define the mechanisms regulated at levels proportional to the infection, such that a balance of immunological control during complex infections. between efficient pathogen clearance and excessive immune pa- Infection of mice with Friend virus (FV)3, a mouse retrovirus, thology is achieved. A heightened state of immune activation in- results in viral persistence, the outcome of which is influenced by duced by one infecting pathogen can be beneficial for the control a number of genetic and extrinsic factors (11, 12). FV is a retro- of another, and coinfection with certain persistent herpesviruses viral complex, consisting of a replication-competent Friend helper by guest on September 28, 2021 has been recently shown to confer resistance to bacterial infection murine leukemia virus (F-MuLV) and a replication-defective (1). In contrast, resistance to other infections may be compromised spleen focus-forming virus (SFFV). SFFV encodes a truncated by concurrent infection with heterologous pathogens. Such exam- form of a retroviral envelope protein, gp55, which can bind to and ples include resistance to retroviral infection, and HIV in particu- activate the erythropoietin receptor (13). As a result of constitutive lar, in which activation of the immune system in response to viral, erythropoietin receptor activation by gp55, the initial phase of FV bacterial, or parasitic coinfections has been implicated in the in- infection in susceptible mice is characterized by massive splenic crease of both the risk of transmission and rate of disease progres- enlargement due to polyclonal proliferation of splenic erythro- sion in HIV-infected individuals (2–8). HIV infection is charac- blasts (14). Susceptibility to FV-induced splenomegaly is geneti- terized by systemic immune hyperactivation, the extent of which cally determined and controlled by the Friend virus susceptibility correlates with more rapid disease progression (2, 5, 9), whereas 2(Fv2) locus (14, 15). Fv2 encodes the Stk receptor tyrosine ki- low levels of T cell activation are associated with reduced suscep- nase, and a naturally expressed truncated form of Stk (Sf-stk) de- tibility to infection (10). Although experimental models for infec- termines the potential of FV-infected erythroblasts to proliferate in tion with a single infectious agent have been and will continue to response to viral gp55, in a cell-autonomous manner (16). Sf- be an invaluable tool in the study of immunity to infection, infec- stk is the most abundant form of Stk in Fv2s mice and is not expressed in Fv2r mice and thus susceptibility is dominant. Most inbred mouse strains are Fv2s, except C57BL/6 (B6) and *Division of Immunoregulation and †Division of Virology, Medical Research Council related strains, in which Sf-stk expression is decreased or ab- ‡ National Institute for Medical Research, London, United Kingdom; Laboratory of sent, and the strains are thus Fv2r (16). In Fv2s strains, recovery Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840 from acute FV-induced splenomegaly is largely immune medi- Received for publication January 16, 2008. Accepted for publication June 20, 2008. ated and depends on the host’s MHC haplotype (12, 17). Re- b The costs of publication of this article were defrayed in part by the payment of page covery is associated with an H2 haplotype, whereas mice of an charges. This article must therefore be hereby marked advertisement in accordance H2a or H2d haplotype, such as A or BALB/c (C), respectively, with 18 U.S.C. Section 1734 solely to indicate this fact. readily succumb to the disease (12, 17). 1 This work was supported by the U.K.’s Medical Research Council and the Portu- guese Foundation of Science and Technology (SFRH/BD/15208/2004 to I.A.). 2 Address correspondence and reprint requests to Dr. George Kassiotis, Division of 3 Abbreviations used in this paper: FV, Friend virus; F-MuLV, Friend helper murine Immunoregulation, Medical Research Council National Institute for Medical Re- leukemia virus; SFFV, spleen focus-forming virus; Fv2, Friend virus susceptibility 2; search, The Ridgeway, Mill Hill, London NW7 1AA, U.K. E-mail address: B6, C57BL/6; C, BALB/c; LDV, lactate dehydrogenase-elevating virus; HEL, hen- [email protected] egg lysozyme; iu, infectious units; MA, matrix; SI, spleen index. www.jimmunol.org The Journal of Immunology 3433

It has long been observed that resistance to FV-induced spleno- (16). Mice were then rendered homozygous for the Fv2s allele by inter- megaly can be modified by concurrent infections with unrelated cross-ing. All animal experiments were conducted according to U.K. Home viruses or microbial products. Although coinfection with some vi- Office regulations and local guidelines. ruses, such as Sendai virus, results in IFN-mediated suppression of Viruses and infections FV infection and subsequent disease (18, 19), preinfection or coinfection with other viruses, such as Newcastle disease virus, Guaroa The FV used in this study is a retroviral complex of a replication-competent B-tropic helper murine leukemia virus and a replication-defective virus or lactate dehydrogenase-elevating virus (LDV), enhances polycythemia-inducing spleen focus-forming virus (SFFVp), referred to as the pathogenicity of FV infection (20–22). Historical consider- FV. The FV stock was free of LDV and was obtained as previously de- ations suggest that LDV has, in fact, been present in many FV scribed (23). FV stocks were propagated in vivo and prepared as 10% stocks for more than 30 years (21, 23). As there is no selective weight to volume ratio homogenate from the spleens of LDV-free C mice pressure to maintain the replication-defective SFFV component infected with FV 12 days previously. A stock of FV, which historically contained LDV (23), was also used, and is referred to as FV/LDV. FV/ of FV in vitro, stocks of FV have been traditionally prepared by LDV stocks were also propagated in vivo and prepared as 10% weight to in vivo passages in mice of susceptible strains. Recent findings volume ratio homogenate from the spleen of LDV-free C mice coinfected suggest that LDV has also been propagated during these pas- with FV/LDV 12 days previously. A pool of 20 LDV-free C mice was used sages and has been maintained as an unsuspected passenger in for the preparation of FV or FV/LDV stocks. Infectious stocks of FV-free LDV were prepared as previously described (23). Mice received an inoc- most FV stocks to date (23). It has also been demonstrated that ulum of FV complex containing ϳ2,000 infectious units (iu) of F-MuLV the presence of LDV in FV stocks delays the FV-specific cy- (assayed in vitro) and between 1,000 and 2,000 spleen focus-forming units totoxic T cell response, thus increasing FV replication and sub- (assayed in vivo). In addition to FV, the FV/LDV inoculum also contained sequent disease (23). 10–100 iu of LDV, based on RT-PCR assays for viral RNA (23) and Downloaded from Despite the important influence of coinfecting viruses on the published data (24). Alternatively, mice received 10–100 iu of LDV alone. All viruses were injected via the tail vein in 0.1 ml of PBS. Cell-associated course of retroviral infection, our knowledge of the precise mech- FV in infected mice was estimated by flow cytometric detection of FV- anisms by which coinfections mediate their effects on retrovirus infected cells using surface staining for the glycosylated product of the infection and the specific immune response to it remains incom- viral gag gene (glyco-Gag), using the matrix (MA)-specific mAb 34 (mAb plete. We have used the FV/LDV system in an effort to understand 34). Splenomegaly is expressed as spleen index (SI), which is calculated as the weight of the spleen (in mg) divided by the weight of the rest of the the impact of coinfection on the requirements for immune control body (in g), to control for the effect of the size of the animal on spleen size. http://www.jimmunol.org/ of a retroviral infection. LDV is a fast-replicating cytopathic virus LDV infection was confirmed by serum lactate dehydrogenase assays which nevertheless establishes persistence in immunocompetent (QuantiChrom lactate dehydrogenase kit; BioAssay Systems) according mice by infecting a small subset of macrophages with limited re- the manufacturer’s instructions. newal capacity specialized in scavenging metabolic enzymes such Flow cytometric analysis as lactate dehydrogenase (24). Although ineffective in controlling the virus, a relatively weak LDV-specific T cell response is Single-cell suspensions were prepared from the spleen of donor mice fol- induced during infection (25, 26). In contrast, LDV infection lowing mechanical disruption of the organs on nylon mesh and were treated with ammonium chloride for erythrocyte lysis. For analysis of B triggers a potent polyclonal activation of B cells leading to cell activation cells were stained with directly conjugated Abs to B220, hypergammaglobulinaemia, particularly of the IgG2 subtypes CD19, IgD, CD38, and GL7 (eBioscience). FV-infected cells erythroid by guest on September 28, 2021 (27–33). However, only a small proportion of these Abs are cells were detected by direct staining with PE-conjugated Abs to Ter119 LDV-specific. Instead, many of these Abs are directed against (eBioscience) in conjunction with the anti-MA mAb 34 (mouse IgG2b). self-Ags and can form immune complexes in the absence of Anti-MA mAb 34 was prepared from the supernatant of hybridoma cultures with ammonium sulfate precipitation. Binding of mAb 34 to infected LDV-specificity (28, 29, 33). cells was visualized by an anti-mouse IgG2b FITC-conjugated secondary In this study we have used a newly generated erythroleukaemia- reagent (BD Biosciences). Four- and five-color cytometry was performed susceptible B6 congenic strain to reveal a strong dependency on on FACSCalibur (BD Biosciences) and CyAn (DakoCytomation) flow cy- LDV for efficient infection with FV. We show that, when these tometers, respectively, and analyzed with FlowJo v8.7 (Tree Star) or Sum- mit v4.3 (DakoCytomation) analysis software, respectively. mice are infected with FV alone, a highly effective immune response contains acute FV replication and prevents the develop- Neutralizing Ab assay ment of splenomegaly. In contrast, LDV coinfection renders these otherwise immunocompetent mice unable to mount an FV-specific In vitro FV-neutralizing Abs in the sera of infected mice were measured using a modification of a previously described viral titer assay (36). Mus response and thus highly susceptible to acute splenomegaly. Im- dunni cells (37) were transduced with the XG7 replication-defective ret- portantly, this effect of LDV on FV pathogenicity appears crucially roviral vector, expressing GFP from a human cytomegalovirus promoter dependent on its ability to activate B cells in a polyclonal manner, and a neomycin-resistance gene under the control of the long terminal because it is negated in mice with a monoclonal B cell population repeat (36). Maintenance of GFP expression was ensured by constant se- lection with 1 mg/ml G418. M. dunni-XG7 cells were then infected with of an unrelated specificity or in mice lacking B cells. Furthermore, B-tropic helper murine leukemia virus and the culture supernatant, which the disease-enhancing effect of LDV coinfection can be mimicked contained the pseudotyped XG7 vector, was harvested. Serial dilutions of by specific immune stimulation of B cells at the time of FV in- sera from infected mice were mixed with ϳ1,500 iu/ml pseudotyped XG7 fection. Thus, a heightened activation state of the immune system vector and allowed to incubate for 30 min at 37°C in IMDM cell culture medium containing 5% FCS. Mixtures were then added to untransduced M. is detrimental to the control of acute retroviral infection. ϩ dunni cells and incubated for 3 days. The percentage of GFP M. dunni cells at the end of the incubation period was assessed by flow cytometry, Materials and Methods using a FACSCalibur (BD Biosciences), and the dilution of serum which Mice resulted in 75% neutralization (i.e., 75% reduction in the percentage of GFPϩ M. dunni cells) was taken as the neutralizing titer. Inbred B6, C, and B6-back-crossed Rag1-deficient mice (B6.129S7- tm1Mom Ϫ/Ϫ Rag1 /J or B6-Rag1 ) were originally obtained from The Jackson Treatment of mice with FV-neutralizing antibodies Laboratory and were subsequently maintained at National Institute of Med- ical Research. Hen-egg lysozyme (HEL)-specific BCR-transgenic MD4 FV and FV/LDV infected were administered with the anti-gp70 of (34) and TCR-transgenic TCR7 mice (35) have been described previously. F-MuLV mAb 48 (38) at indicated time points postinfection. The anti-gp70 Fv2s-congenic B6 mice (B6.A-Fv2s) were generated by 10 consecutive mAb 48 was prepared from the supernatant of hybridoma cultures with back-crosses of A strain (Fv2s) mice onto the B6 background, selecting for ammonium sulfate precipitation and was diluted in PBS at a titer, which the Fv2s gene in each generation by PCR based on published sequence had in vitro FV-neutralizing activity equivalent to sera obtained from FV 3434 LYMPHOCYTE ACTIVATION PREVENTS RETROVIRUS IMMUNE CONTROL

FIGURE 1. An Fv2s allele restores susceptibility of B6 mice to splenomegaly induced by FV/LDV coinfection. A, Spleen weights of B6, B6.A-Fv2s, and C mice 14 days post-FV/LDV coinfection. The mean values Ϯ SEM of at least 10 mice per group, pooled from three experiments, are shown. B, H&E staining of spleen sections (top) and flow cytometric analysis of glyco-Gagϩ Ter119ϩ erythroid precursors (bottom) in the spleens of B6, B6.A-Fv2s, and C mice, 9 or 14 days post-FV/LDV coinfection, respectively. Results are representative of five mice per group from a single experiment. Numbers within the quadrants represent the percentage of cells in that quadrant in all splenocytes; numbers in parentheses represent the percentage of infected cells Downloaded from (glyco-Gagϩ) in erythroid precursors (Ter119ϩ) only. C, Course of FV/LDV coinfection, depicted as SI, in FV/LDV coinfected B6.A-Fv2s and C mice. The mean values Ϯ SEM of four to five mice per group per time point pooled from three experiments are shown. infected mice at day 21 of infection. Mice received 0.3 ml of mAb 48 period (Fig. 1C), as expected for a high-recovery strain (39). Thus, dilution i.p. Fv2s

the congenic B6.A- strain of mice combines the high-recovery http://www.jimmunol.org/ b s Histology phenotype of H2 mice with the susceptibility of Fv2 mice, as seen in hybrids, such as between C57BL/10 (B10) and A.BY mice Spleens from infected mice were removed, fixed with Bouin’s fixative, and (H2b/b, Fv2r/s), when FV/LDV coinfection is used (12, 39). embedded in paraffin. Sections were then stained with H&E, and photomi- crographs were taken with a stereo-microscope (Carl Zeiss) equipped with a digital camera. B6.A-Fv2s mice control FV infection but not FV/LDV In vivo lymphocyte activation coinfection For in vivo polyclonal lymphocyte activation mice were first infected with We next examined the impact of LDV coinfection on the course

FV and then received stimuli 30 min later. A single dose of 10 ␮g LPS by guest on September 28, 2021 (from Salmonella minnesota R595; Axxora) or 130 ␮g activating anti- of FV infection, by comparing the original LDV-containing FV mouse IgM (polyclonal goat anti-mouse IgM; Jackson ImmunoResearch stock with an LDV-free FV stock. Both FV infection alone and Europe) were injected i.v. in 0.1 ml PBS. These doses of LPS or anti-IgM FV/LDV coinfection resulted in rapid splenomegaly in fully were found to induce no splenomegaly when given alone to FV-uninfected susceptible C mice (Fig. 2, A and B), although the presence of control mice. LDV delayed its onset. In contrast, B6 mice remained resistant Results to both FV and FV/LDV (Fig. 2, C and D), with only marginal ϭ Fv2s renders B6 mice susceptible to FV-induced splenomegaly splenic enlargement seen with FV/LDV (SI 8.2). Surpris- s r ingly, even though B6.A-Fv2 mice were susceptible to severe To overcome the Fv2 -mediated genetic resistance of B6 mice splenomegaly following FV/LDV infection (Fig. 2F), they were (H2b, Fv2r), which would allow the study of FV infection in a relatively resistant to FV alone (Fig. 2E), with a maximum SI of series of commonly used B6-back-crossed gene-targeted strains, 11.7 on day 10 postinfection, which represents an enlargement we generated a congenic B6 strain (B6.A-Fv2s) carrying the Fv2s of ϳ3 times the normal spleen size. Splenomegaly induction in allele from the A strain. As the vast majority of previous studies of FV/LDV coinfected B6.A-Fv2s mice was not an independent FV infection have been performed using LDV-containing FV effect of infection with LDV, as it was dependent on the pres- stocks, we initially examined the susceptibility of B6.A-Fv2s mice ence of the Fv2s allele (Fig. 2, D and F) and infection of B6.A- to erythroleukaemia using an LDV-containing FV stock, such that Fv2s mice with LDV alone did not induce signiﬁcant spleno- comparisons with published data were possible. In contrast to B6 s mice, B6.A-Fv2s mice exhibited signiﬁcant splenic enlargement megaly (Fig. 2F). Resistance of B6.A-Fv2 mice to FV-induced (ϳ18 times the normal spleen size) 2 wk postinfection (Fig. 1A), splenomegaly was not due to failure of FV to infect mice of this particular genetic background, as the percentage and absolute which was similar but not identical to fully susceptible C mice ϩ (H2d, Fv2s) (Fig. 1A). The splenomegaly induced by FV infection number of infected Ter119 cells on day 7 of FV infection was in B6.A-Fv2s mice displayed all the hallmarks of the early FV- either the same in resistant B6 mice (Fig. 3A) or even higher in s induced disease in C mice, such as expansion of the splenic red susceptible B6.A-Fv2 mice (Fig. 3B) than those in FV/LDV pulp and disruption of normal splenic architecture (Fig. 1B, top), coinfection. Instead, resistance correlated with rapid reduction and dramatic increase in nucleated Ter119ϩ erythroid precursors, of FV loads in the following 2 wk of FV infection (particularly most of which expressed on their membranes retroviral glyco-Gag between day 7 and 14). In contrast, FV loads in FV/LDV coin- (Fig. 1B, bottom), an indicator for FV infection. In contrast to C fection continued to rise beyond day 7 and peaked on day 14 mice, which subsequently succumbed to infection due to an in- (Fig. 3, A and B). This pattern is consistent with rapid immune- ability to mount an effective cell-mediated antiviral response (12), mediated control of FV infection in B6 and B6.A-Fv2s mice and B6.A-Fv2s mice recovered from severe splenomegaly within 3 wk delayed clearance in FV/LDV coinfection, in agreement with ϫ and remained splenomegaly-free throughouta7moobservation similar observations is B10 A.BY F1 mice (12, 23). The Journal of Immunology 3435

FIGURE 2. Effect of LDV coinfection on the immune requirements for containment of FV infection. Spleen size, depicted as SI, of C (A and B), B6 (C and D), B6.A-Fv2s (E and F), B6.A-Fv2s MD4 (G and H), B6.A-Fv2s TCR7 (I and J), and B6.A- Fv2s Rag1Ϫ/Ϫ mice (K and L) over the course of FV infection (top)or LDV infection and FV/LDV coinfection (bottom). Data are the mean values Ϯ SEM of 5–12 mice per group per time point, pooled from three to ﬁve experiments. Downloaded from

Enhancement of FV-induced disease by LDV coinfection FV-infected B6.A-Fv2s mice (Fig. 3B), percentages of infected requires lymphocytes Ter119ϩ cells in the spleen were signiﬁcantly elevated in FV-

s http://www.jimmunol.org/ As resistance of B6.A-Fv2s mice to FV infection was consistent infected B6.A-Fv2 MD4 mice (Fig. 3C), in line with increased with immune-mediated protection, we next investigated in more peak splenomegaly in the latter (Fig. 2, E and G). Furthermore, FV-infected B6.A-Fv2s MD4 mice harbored readily detectable detail the immune response against FV infection and FV/LDV ϩ coinfection. To assess the contribution of humoral and cellular numbers of infected Ter119 cells throughout the course of infec- immunity to FV control, B6.A-Fv2s mice were rendered selec- tion (Fig. 3C), despite transient control of splenomegaly by day 21 s tively immunodeficient in either virus-specific Ab or T cell re- of infection (Fig. 2G). Therefore, inability of B6.A-Fv2 MD4 sponses, or both. B6.A-Fv2s mice were crossed to BCR transgenic mice to mount a neutralizing Ab response exacerbated FV infec- s mice, in which B cells were present in normal numbers, but ex- tion. B6.A-Fv2 MD4 mice coinfected with FV/LDV progressed to press the MD4 BCR (34), instead of endogenous ones, and there- splenomegaly with the same rate as FV/LDV coinfected B6.A- by guest on September 28, 2021 s fore can respond only to an unrelated epitope from HEL. In the Fv2 mice (Fig. 2H). Surprisingly, the lack of neutralizing Ab re- absence of an FV-specific B cell response, FV infection caused sponse did not accelerate or exacerbate splenomegaly in FV/LDV s significant early splenomegaly in B6.A-Fv2s MD4 mice (SI ϭ 32.2 coinfected B6.A-Fv2 MD4 mice (Fig. 2H). Indeed, its severity at the peak on day 10 postinfection) (Fig. 2G), compared with was significantly reduced in these mice compared with FV/LDV B6.A-Fv2s mice ( p ϭ 0.0007, Student’s t test on day 10). Spleno- coinfected B6.A-Fv2s mice ( p ϭ 0.012, Student’s t test on day 14). ϩ megaly in FV-infected B6.A-Fv2s MD4 mice was not, however, as Moreover, percentages of infected Ter119 cells in the spleen of severe as in fully susceptible C mice, and was also followed by FV/LDV coinfected B6.A-Fv2s MD4 mice were not increased in partial recovery until day 21 postinfection, after which time B6.A- comparison with those in the spleen of FV/LDV coinfected B6.A- Fv2s MD4 mice failed to control FV infection and displayed Fv2s mice (Fig. 3, B and C). Thus, a functional B cell response was chronic and progressive splenomegaly (Fig. 2G). Compared with beneficial in early control of FV infection but not in FV/LDV coinfection. As the absence of a functional B cell response in B6.A-Fv2s MD4 mice only partially increased susceptibility to FV infection, we examined whether protection was mediated by virus-specific T cells. B6.A-Fv2s mice were crossed to TCR transgenic mice, in which T cells were present in normal numbers, but express the HEL-specific TCR7 TCR (35). B6.A-Fv2s TCR7 mice fail to mount a T cell response to FV due to lack of appropriate TCR, and would also be defective in T cell-dependent neutralizing Ab production due to lack of T cell help. As a control, B6.A-Fv2s mice were also crossed to lymphocyte-deficient Rag1Ϫ/Ϫ mice. The lack of virus-specific T cells in B6.A-Fv2s TCR7 mice rendered them fully susceptible to FV-induced splenomegaly at levels and rate of progression comparable to C mice (Fig. 2I), from which they did FIGURE 3. Effect of LDV coinfection on the kinetics of expansion and not recover, demonstrating that resistance of B6.A-Fv2s mice to control of FV-infected erythroid precursors. The percentage of glyco-Gagϩ ϩ infection with FV alone is immune mediated. Progression to severe Ter119 cells, determined by flow cytometry, in splenocytes from B6 (A), B6.A-Fv2s (B), B6.A-Fv2s MD4 (C), and B6.A-Fv2s Rag1Ϫ/Ϫ mice (D) splenomegaly caused by infection with FV alone was also similar s infected with FV alone or coinfected with FV and LDV is shown. Data are between B6.A-Fv2 TCR7 mice and lymphocyte-deficient B6.A- s Ϫ/Ϫ s the mean Ϯ SEM of 7–12 mice per group per time point, from two ex- Fv2 Rag1 mice (Fig. 2K), indicating that B6.A-Fv2 TCR7 periments. The dashed line represents the limit of detection (0.2%). mice exhibit a complete lack of adaptive immunity to FV infection. 3436 LYMPHOCYTE ACTIVATION PREVENTS RETROVIRUS IMMUNE CONTROL

FIGURE 4. Effect of FV-neutralizing Ab administration on control of FV infection and FV/LDV coinfection. Spleen size, depicted as SI (A and C) and percentage of glyco-Gagϩ Ter119ϩ cells (B and D) in the spleen of B6.A-Fv2s MD4 mice infected with FV alone (A and B) or coinfected with FV and LDV (C and D), with (mAb 48) or without (Ϫ) administration of mAb 48. Abs were administered on day 3 and 6 and mice analyzed on day 10 of infection with FV alone, or on day 3, 6, and 9 and mice analyzed on day 10 of coinfection with FV and LDV. Data are the mean Ϯ SEM of three to four mice per group from one experiment. Downloaded from

However, neither B6.A-Fv2s TCR7 nor B6.A-Fv2s Rag1Ϫ/Ϫ mice or controlled by timely induction of FV-neutralizing Abs. We thus showed any enhancement or acceleration of splenomegaly, in com- hypothesized that a possible explanation for the observed contri- parison to fully immune competent B6.A-Fv2s mice when coin- bution of naturally induced virus-specific Abs in protection during fected with FV and LDV (Fig. 2, J and L). Again, induction of FV infection, but not FV/LDV coinfection, was that these Abs s Ϫ/Ϫ splenomegaly in FV/LDV coinfected B6.A-Fv2 Rag1 mice were only induced in the former. Contributing factors could be http://www.jimmunol.org/ was caused by FV, rather than LDV, as infection with LDV alone polyclonal B cell activation induced by LDV and the disruption of did not result in significant splenomegaly (Fig. 2L). Furthermore, normal splenic architecture caused by FV/LDV-induced spleno- following FV/LDV coinfection, percentages of infected Ter119ϩ megaly. Therefore, we used B6 mice, which do not experience cells in the spleen of immunodeficient B6.A-Fv2s Rag1Ϫ/Ϫ mice substantial splenomegaly or alterations in splenic structure with were comparable to those in the spleen of immunocompetent either FV alone or in combination with LDV, to examine the effect B6.A-Fv2s mice (Fig. 3, B and D). Together, these results sug- of LDV coinfection on induction of FV-specific Abs (Fig. 1B and gested that adaptive immunity does not contribute appreciably to Fig. 2, E and F). Sera from FV-infected B6 mice had detectable in the control of FV replication in the first 2 wk of this coinfection. vitro FV-neutralizing activity from day 14 postinfection, which increased dramatically only by day 21 and approached a plateau by by guest on September 28, 2021 Delayed induction of FV-neutralizing Abs in FV/LDV day 28 (Fig. 5). FV/LDV coinfection in B6 mice transiently coinfection The use of B cell-deficient mice has previously demonstrated an indispensable role for B cells and neutralizing Abs in the recovery from FV-induced splenomegaly during FV/LDV coinfection (40– 43). However, no positive contribution of virus-neutralizing Abs can be seen in FV/LDV coinfection of B6.A-Fv2s mice before day 21 of infection (Fig. 2, F and H), suggesting either that the Ab response did not provide any protection against acute FV/LDV coinfection or that it was not yet induced. In contrast, virus-specific Abs appeared to play an essential role in controlling infection with FV alone already from day 7 postinfection. Indeed, percentages of FV-infected Ter119ϩ cells in the spleen were increased in the absence of an FV-neutralizing Ab response when mice were infected with FV alone, but were unchanged or even decreased at later time points when mice were coinfected with FV and LDV (Fig. 3, B and C). To confirm that enhanced infection with FV in B6.A-Fv2s MD4 mice, compared with B6.A-Fv2s mice, was due to lack of FV-specific Ab response, FV-infected B6.A-Fv2s MD4 mice were administered with FV-neutralizing Abs. Injection of the anti-gp70 of F-MuLV mAb 48 (38), completely prevented induction of splenomegaly (Fig. 4A) and dramatically reduced the percentages of FV-infected Ter119ϩ cells (Fig. 4B) in the spleen of B6.A-Fv2s MD4 mice infected with FV. Thus, administration of FV-neutralizing Abs restored resistance of B6.A-Fv2s MD4 mice to FV infection. Similarly, injection of the mAb 48 significantly FIGURE 5. Effect of LDV coinfection on induction of FV-neutralizing reduced the degree of splenomegaly and percentages of FV-in- Abs. Titer of FV-neutralizing activity in the sera of FV infected or FV/ ϩ s fected Ter119 cells in the spleen of B6.A-Fv2 MD4 mice coin- LDV coinfected B6 mice. Data are the mean Ϯ SEM of four mice per fected with FV and LDV (Fig. 4, C and D). These results suggested group per time point. One representative of two experiments in shown. The that both FV infection and FV/LDV coinfection can be prevented dashed line represents the limit of detection (1:50). The Journal of Immunology 3437

delayed class-switching (44), and only started rising between days 21 and 28 (Fig. 5), without, however, reaching the levels seen in FV-infected B6 mice. Thus, coinfection with LDV delayed the neutralizing Ab response to FV.

Polyclonal B cell stimulation overcomes resistance of B6.A-Fv2s mice to FV infection LDV infection of immunocompetent mice causes polyclonal B cell activation and hypergammaglobulinemia (27, 30, 31), which might prevent induction of Ab responses to new infections (45). Inhibi- tion of the FV-neutralizing Ab response could be the mechanism underlying the enhancement of FV-induced splenomegaly by LDV coinfection. Alternatively, polyclonal activation of B cells per se could be directly responsible for the effect of LDV coinfection on FV-induced splenomegaly, independently of their ability produce FIGURE 6. Activation of polyclonal or monoclonal B cells by FV in- FV-specific Abs. We first addressed whether FV/LDV coinfection fection or FV/LDV coinfection. Percentages of CD38low GL7ϩ cells in was associated with higher levels of B cell activation than infec- Downloaded from gated B220ϩ CD19ϩ IgDϪ B cells in the spleen of B6.A-Fv2s or B6.A- tion with FV alone and whether or not LDV could cause activation Fv2s MD4 mice 14 days postinfection with FV alone or coinfection with of monoclonal B cells expressing the MD4 BCR transgene. Infec- FV and LDV. Numbers within the plots represent the mean percentage of tion of B6.A-Fv2s mice with FV alone gave rise to significant low ϩ CD38 GL7 cells of two to three mice per group. The absolute number numbers of B cells with the CD38low GL7ϩ germinal center-phe- of CD38low GL7ϩ B cells was on average 5.0 ϫ 106 and 0.1 ϫ 106 in the s s notype in the spleen 14 days postinfection (Fig. 6). In line with the spleen of FV-infected B6.A-Fv2 and B6.A-Fv2 MD4 mice, respectively; s ϫ 6 ϫ 6 B cell stimulatory effect of LDV, the spleens of B6.A-Fv2 mice and 17.6 10 and 2.5 10 in the spleen of FV/LDV-coinfected B6.A- http://www.jimmunol.org/ low Fv2s and B6.A-Fv2s MD4 mice, respectively. coinfected with FV and LDV contained twice as many CD38 GL7ϩ B cells at the same time-point (Fig. 6). Importantly, restriction of the BCR repertoire in B6.A-Fv2s MD4 mice severely re- induced serum FV-neutralizing activity in a proportion of mice on strained activation of B cells during either FV infection or FV/ day 14 postinfection (Fig. 5), which corresponds to an early IgM LDV coinfection (Fig. 6). The residual population of CD38low response (44). In comparison to FV infection, FV-neutralizing ac- GL7ϩ B cells observed in FV/LDV coinfected B6.A-Fv2s MD4 tivity in sera from FV/LDV coinfected B6 mice was homoge- mice likely represent B cells expressing endogenous BCRs, which neously below the detection limit (1:50) on day 21, suggestive of were still present at a frequency of ϳ5% in these mice (34). These by guest on September 28, 2021

FIGURE 7. Effect of polyclonal B cell stimuli on immune control of FV infection. A, Percentages of CD38low GL7ϩ cells in gated B220ϩ CD19ϩ IgDϪ B cells in the spleen of control B6.A-Fv2s mice or 10 days postadministration of LPS. B, Splenomegaly, depicted as SI, in B6.A-Fv2s mice infected with FV with or without concomitant administration of LPS or in mice given LPS alone. C, Flow cytometric detection of FV-infected erythroid precursors in the spleen of FV-infected B6.A- Fv2s mice without (top) or with (bottom) LPS administration. D, Percentages of CD38low GL7ϩ cells in gated B220ϩ CD19ϩ IgDϪ B cells in the spleen of control B6.A-Fv2s mice or 10 days postadministration of anti-IgM Ab. E, Splenomegaly in B6.A-Fv2s mice infected with FV with or without concomitant administration of anti-IgM Ab or in mice given anti-IgM Ab alone. F, Flow cytometric detection of FV-infected erythroid precursors in the spleen of FV-infected B6.A- Fv2s mice without (top) or with (bottom) anti-IgM Ab administration. Data in A and D represent the mean percentage of CD38low GL7ϩ cells of two to three mice per group. Data in B and E are the mean Ϯ SEM of 4–11 mice per group per time point, pooled from two to three independent experiments. Numbers within the quadrants in C and F represent the percentage of cells in that quadrant in all splenocytes; numbers in parentheses represent the percentage of infected cells (glyco-Gagϩ)in erythroid precursors (Ter119ϩ) only. Results in C and F are representative of three to four mice per group. No FV-infected cells were detected in mice given LPS or anti-IgM Ab alone (not shown). 3438 LYMPHOCYTE ACTIVATION PREVENTS RETROVIRUS IMMUNE CONTROL results indicated that BCR specificity is a crucial factor in B cell difference in susceptibility could be due to differences in the FV- stimulation during these infections. infecting dose used in different studies. To assess whether B cell activation by LDV at the time of FV/ Our analysis of FV infection in B6.A-Fv2s mice with selective LDV coinfection contributes to the severity of splenomegaly, we immunodeficiencies demonstrates that both T cell- and Ab-depen- substituted LDV with a polyclonal B cell stimulus. LPS is a potent dent mechanisms contributed to their resistance. The absence of an polyclonal activator of murine B cells, which induces cellular pro- FV-specific B cell response in B6.A-Fv2s MD4 mice revealed a liferation and Ab secretion. Indeed, injection of a small dose of biphasic effect in the control of FV-induced splenomegaly. FV- LPS in B6.A-Fv2s mice led to the emergence of CD38low GL7ϩ infected B6.A-Fv2s MD4 mice developed early (ϳday 10) splenic germinal center B cells 10 days later (Fig. 7A). When LPS was enlargement, from which they partially recovered until day 21. injected at the time of infection with FV alone, B6.A-Fv2s mice, Recovery was mediated by an FV-specific T cell response as which are otherwise resistant, rapidly developed severe spleno- B6.A-Fv2s TCR7 mice failed to recover. Nevertheless, T cell-me- megaly (Fig. 7B), with kinetics similar to immune-deficient B6.A- diated protection was incomplete as B6.A-Fv2s MD4 mice failed Fv2s TCR7 or B6.A-Fv2s Rag1Ϫ/Ϫ mice. Thus, LPS administra- to clear FV-infected cells and progressed to splenomegaly at later tion, which in itself did not cause any splenic enlargement (Fig. time points. Enhanced susceptibility of B6.A-Fv2s MD4 mice to 7B), was sufficient to overcome the immune-mediated resistance of early FV-induced splenomegaly due to lack of an FV-specific Ab B6.A-Fv2s mice. As observed in FV/LDV coinfected mice, the response is consistent with restoration of resistance in these mice, splenomegaly in FV-infected B6.A-Fv2s mice given LPS was pre- upon administration of FV-neutralizing mAbs. However, an early dominantly due to an uncontrolled expansion of Ter119ϩ cells, requirement for the FV-specific Ab response revealed in B6.A- most of which were expressing FV Ags (Fig. 7C). Although LPS Fv2s MD4 mice seems to be at odds with relatively late induction Downloaded from activates B cells polyclonally, it also activates other immune and of FV-neutralizing Abs, even in infection with FV alone. Indeed, non-immune cell types, most notably macrophages, which can also neither the assay used in this study, nor previously used assays (23, become infected with FV (46). To confirm that polyclonal B cell 47, 48), detected significant FV-neutralizing activity before day 21 activation was the primary cause of the failure to control acute FV of infection. However, non-neutralizing Abs may also contribute infection, B6.A-Fv2s mice were injected with an activating anti- to protection, and it has been previously shown that administration

IgM Ab at the time of FV infection, which would activate B cells of an FV p15 Gag-specific non-neutralizing mAb had a protective, http://www.jimmunol.org/ specifically. Administration of anti-IgM Ab was followed by the albeit weak, effect against FV infection (43). Furthermore, the im- appearance of CD38low GL7ϩ germinal center B cells 10 days later portance of Ab activity against infected cells, in addition to free (Fig. 7D). Similar to LPS injection, polyclonal B cell activation virions, has been recently documented in a macaque model for induced by anti-IgM Ab injection, which alone had no effect of HIV infection, by removal of the Fc-mediated effector function of splenic size, overcame the resistance of B6.A-Fv2s mice to spleno- the Abs (49). Such activity would not be detected by a classical megaly (Fig. 7E). Again, splenomegaly caused by anti-IgM Ab neutralization assay. Alternatively, an early effect of Abs on FV administration in FV-infected B6.A-Fv2s mice was due to expan- infection, before measurable FV-neutralizing Abs are induced, sion of infected Ter119ϩ cells (Fig. 7F). Thus, B cell activation by could reflect the action of natural, as opposed to induced, Abs. The either LPS or anti-IgM Ab treatment prevents the control of acute presence and protective function against infection of virus-specific by guest on September 28, 2021 FV infection by otherwise resistant mice. Abs naturally occurring in the sera of virus-naive animals has been previously documented (50). These natural Abs are present at low Discussion levels, and although they may be virus-specific they do not detect- For this study we developed a congenic B6 strain, B6.A-Fv2s, which ably neutralize virus infectivity in vitro (50). combines genetic susceptibility to FV-induced erythroid precursor ex- In contrast to their protective effect during infection with FV pansion with immune-mediated resistance to FV infection. Experi- alone, neither T cell- nor Ab-mediated immunity appeared to have ments with this mouse strain allowed us to reveal a fundamental role any impact on disease progression during the first 2 wk of FV/ for LDV coinfection-induced activation of the immune system in fail- LDV coinfection. Indeed, no enhancement of FV-induced disease ure to control acute FV infection. B6.A-Fv2s mice effectively con- could be demonstrated by partial or full adaptive immunodefi- trolled acute splenomegaly when challenged with FV alone, and their ciency during this time. Although the role of both T cell- and resistance was immune-mediated. In contrast, polyclonal B cell acti- Ab-mediated protection in recovery from FV-induced splenomeg- vation by concurrent infection with LDV or stimulation with B cell aly has been established (12, 17, 46), our findings are in line with stimuli counteracted the immune response to FV, and no evidence for previous studies with genetic or Ab-mediated lymphocyte deple- immune-mediated protection could be demonstrated during the early tion experiments, which also failed to reveal a role for either CD4ϩ acute phase of FV/LDV coinfection. or CD8ϩ T cells in protection from early splenomegaly in suscep- The introduction of an Fv2s allele onto the B6 genetic back- tible strains of mice following infection with, presumably, LDV- ground fully imparted susceptibility to FV-induced splenomegaly. contaminated FV stocks (39). Although concurrent infection with Disease progression and recovery upon FV/LDV coinfection of LDV could potentially interfere with subsequent effector mech- B6.A-Fv2s (H2b/b, Fv2s/s, Rfv3r/r) mice followed kinetics similar to anisms of FV-specific immunity, recent data showed that the ϫ b/b what has been previously described for B10 A.BY F1 mice (H2 , most likely explanation for the apparent lack of immune pro- r/s r/s ϫ Fv2 , Rfv3 ) mice (12, 39). In contrast to B10 A.BY F1 mice, tection during early FV/LDV coinfection is that FV-specific which still developed splenomegaly when infected with FV alone immune response is prevented or delayed. Indeed, induction of (23), our results suggest that B6.A-Fv2s mice are highly resistant to FV-specific CD8ϩ T cells was recently found to be delayed by FV-induced disease in the absence of LDV coinfection. The compo- LDV coinfection (23). Similarly, our results showed that induc- sition of resistance and susceptibility alleles is similar in the two tion of FV-neutralizing Abs was significantly delayed in B6 strains at all the described loci affecting FV infection, such as the H2, mice by LDV coinfection and that lack of FV-specific Abs in Fv1, Fv2, and Rfv3 loci (12). It could be that heterozygosity at one or B6.A-Fv2s MD4 mice had no detrimental effect in the first 3 wk more such loci or other alleles on the A strain genetic contribution in of the disease course when LDV was present. Thus, concurrent ϫ B10 A.BY F1 mice contributes to their enhanced susceptibility to infection with LDV prevents early induction of both cytotoxic FV infection, in comparison with B6.A-Fv2s mice. Alternatively, the (23) and, in the present study, Ab, responses to FV, suggesting The Journal of Immunology 3439 that LDV may act by inhibiting the provision of a common precise reasons for the delay in FV-induced splenomegaly are not requirement for both these arms of adaptive immunity, such as clear but they could be mediated by an antiviral response, through virus-specific CD4ϩ T cell help. the action of type I IFN, induced by LDV, similarly to coinfection Coinfection of partially or fully immunodeficient B6.A-Fv2s with other viruses (19). In fact, it has recently been shown that mice with FV and LDV demonstrated that not only did the adap- plasma type I IFN measured during the acute phase of FV/LDV tive immune response fail to afford any noticeable protection dur- coinfection was induced by LDV, whereas no measurable type I ing acute coinfection, surprisingly it contributed to the disease- IFN was induced by FV alone (54). Alternatively, LDV could be enhancing effect of LDV. Mice with a monoclonal B cell delaying the progression of FV-induced splenomegaly at the level population of unrelated specificity or mice lacking B cells had of FV-infected cell expansion and survival. Although LDV has a significantly milder disease, compared with fully immunocompe- strict cell-tropism for functionally specialized macrophages in tent mice coinfected with FV/LDV. Furthermore, the presence of vivo, it has been observed that retroviral infection renders non- LDV enhanced FV-induced splenomegaly in fully immunocom- permissive cells susceptible to LDV infection in vitro (55, 56) and petent mice, but had no effect or even inhibited FV-induced endogenous retroviral activity is required for successful infection splenomegaly in immunodeficient mice. Therefore, coinfection of neural tissue by LDV and development of neurological disease with LDV reverses the role of the adaptive immune response in FV in vivo (57). Thus, it could be envisaged that FV-infected erythroid infection from disease-inhibiting to disease-promoting. Impor- precursors, the expansion of which would normally lead to spleno- tantly, this effect of LDV correlated with its ability to cause poly- megaly following infection with FV alone, become targets for in- clonal B cell activation and could be mimicked by generic B cell fection and subsequent lysis by the cytopathic LDV in FV/LDV stimuli, such as LPS and anti-IgM Ab treatment. It has long been coinfection. Such a mechanism would be compatible with our ob- Downloaded from observed that microbial products, such as LPS and CpG oligonu- servation that the delay in accumulation of FV-infected erythroid cleotides (21, 51–53), or stimulation of hematopoiesis by erythro- cells caused by LDV is influenced by the presence of an Fv2s allele poietin injection or hypoxia (21), can exacerbate FV-induced and is noticeable in B6.A-Fv2s, but not in B6 mice (Fv2r). In splenomegaly. This enhancement has previously been thought to contrast, any effect of type I IFN would be expected to be the same be the result of target cell expansion (21). Although expansion of in both strains, regardless of the Fv2 status.

erythroid precursor cells by these treatments would certainly con- In conclusion, our results indicate that under certain conditions http://www.jimmunol.org/ tribute to enhancement of FV replication, this would have been in the immune response to retroviral infection can be highly effective addition to the immune-hyperactivation effect of LDV, most likely in the prevention of acute disease and subsequent containment of present in these previous studies (21). Our results suggest that infection. However, the effectiveness of the antiviral immunity can enhancement of FV pathogenicity induced by LDV coinfection is be severely undermined by concomitant immune activation in re- largely due to immune dysfunction. Firstly, resistance to infection sponse to coinfecting pathogens or their products. Thus, although with FV alone is immune-mediated and coinfection with LDV has strong activation of the immune system is beneficial when focused a marked inhibitory effect on the induction FV-specific immune on the infecting retrovirus, it can also be detrimental when diverted response. Secondly, the disease-enhancing effect of LDV coinfec- to other targets, especially in the case of retroviruses, which fre- tion depended on the presence of a polyclonal B cell repertoire and quently target the immune system for their replication. Polyclonal by guest on September 28, 2021 it could be reproduced by direct polyclonal B cell activation with activation of B cells might therefore represent an immune-evasion anti-IgM Abs. It must be noted, however, that delay of the FV- strategy, used both by retroviruses, such as HIV (58) and other neutralizing Ab response may not be the only mechanism under- persistent viruses, such as LCMV (59). As coinfecting pathogens lying the impact of B cell activation on FV infection. Treatment of modify both susceptibility to and immune requirement for control B6.A-Fv2s mice with anti-IgM Abs at the time of FV infection of retroviral infection, our data also highlight the need to extend resulted in more severe splenomegaly than lack of FV-specific Abs our understanding of the factors determining the outcome of ret- in B6.A-Fv2s MD4 mice. Moreover, B6.A-Fv2s mice experience roviral infection to include the influence of coinfection and any more severe acute disease than B6.A-Fv2s MD4 mice following host factors, which in turn modulate immune reactivity in general. FV/LDV coinfection, despite the lack of an Ab response common in both strains. Although our results clearly implicate B cell acti- Acknowledgments vation in LDV-mediated disease enhancement, it is still unclear We thank Drs. Anne O’Garra for the TCR7 mice, Facundo Batista for the what proportion or subset of B cells is involved. Although LPS and MD4 mice, and Elena Grigorieva for histology. anti-IgM Ab administration had similar effects on splenomegaly and induced comparable numbers of germinal center-phenotype B Disclosures cells, anti-IgM Abs target fewer B cells than LPS, owing to the wide- The authors have no financial conflict of interest. ranging expression of IgM on B cells. Furthermore, polyclonal B cell activation by LDV is selective to B cells with autoreactive BCRs (28, References 29, 33). It is therefore likely that activation of only a fraction of B cells 1. Barton, E. S., D. W. White, J. S. Cathelyn, K. A. Brett-McClellan, M. Engle, is responsible for the disease enhancement. Thus, it will be important M. S. Diamond, V. L. Miller, and H. W. Virgin. 2007. 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