To date, only a single Friend virus (FV) peptide recognized by CD4+ T cells in FV-infected mice of the resistant H-2b haplotype has been described. To more thoroughly examine the repertoire of CD4+ T cell responses in H-2b mice infected with this retrovirus, 18mer peptides spanning the FV gag, pol, and env coding regions with 11mer overlaps were synthesized. The peptides were then used to stimulate whole splenocytes and purified CD4+ T cells from FV-infected mice in an IFN ELISPOT assay. Nine new CD4+ T cell epitopes were identified, 3 encoded by gag, 1 by pol, and 5 by env. The high resistance of H-2b mice could be related to this very broad CD4+ T cell response against multiple peptides during FV infection.
C57BL/6 Rag2−/−γc−/− CD47−/− triple-knockout mice engrafted with fetal human bone marrow, liver, thymus (TKO-BLT) not only develop high levels of multi-lineage hematopoiesis but also organized lymphoid tissues including mesenteric lymph nodes, splenic follicles and gut-associated lymphoid tissues of human origin. A unique advantage of these mice is that they sustain human cell and tissue engraftment long-term without the development of graft versus host disease. Thus they can be used for long-term studies not previously feasible with other models. The production of TKO-BLT mice to obtain healthy mice with high level reconstitution of human cells and tissues requires specialized methods that are presented in detail.
Using an experimental evolution approach, we recently demonstrated that the mouse-specific pathogen Friend virus complex adapted to specific MHC genotypes, which resulted in fitness tradeoffs when viruses were exposed to hosts possessing novel MHC polymorphisms. Here we report the analysis of patterns of pathogen adaptation and virulence evolution from viruses adapting to one of three hosts that differ across the entire genome (A/WySn, DBA/2J and BALB/c). We found that serial passage of Friend virus complex through these mouse genotypes resulted in significant increases in pathogen fitness (156-fold) and virulence (11-fold). Adaptive responses by post-passage viruses also resulted in host-genotype-specific patterns of adaptation. To evaluate the relative importance of MHC versus non-MHC polymorphisms as factors influencing pathogen adaptation and virulence, we compared the magnitude of fitness tradeoffs incurred by post-passage viruses when infecting hosts possessing either novel MHC polymorphisms alone or hosts possessing novel MHC and non-MHC polymorphisms. MHC polymorphisms alone accounted for 71% and 83% of the total observed reductions in viral fitness and virulence in unfamiliar host genotypes, respectively. Strikingly, these data suggest that genetic polymorphisms within the MHC, a gene region representing only ~0.1% of the genome, are major host factors influencing pathogen adaptation and virulence evolution.
MHC; experimental evolution; serial passage; host-pathogen
Friend virus (FV) infection of mice induces the expansion and activation of regulatory T cells (Tregs) that dampen acute immune responses and promote the establishment and maintenance of chronic infection. Adoptive transfer experiments and the expression of Neuropilin 1 indicate that these cells are predominantly natural Tregs rather than virus-specific conventional CD4+ T cells that converted into induced Tregs. Analysis of Treg TCR Vβ chain usage revealed a broadly distributed polyclonal response with a high proportionate expansion of the Vβ5+ Treg subset, which are known to be responsive to endogenous retrovirus-encoded superantigens. In contrast to the major population of Tregs, the Vβ5+ subset expressed markers of terminally differentiated effector cells, and their expansion was associated with the level of the antiviral CD8+ T cell response rather than the level of FV infection. Surprisingly, the expansion and accumulation of the Vβ5+ Tregs was IL-2 independent but dependent upon TNFα. These experiments reveal a subset-specific Treg induction by a new pathway.
Friend virus is a murine retrovirus that causes acute disease leading to lethal erythroleukemia in most strains of mice. Strains of mice that mount the proper immune responses can recover from acute infection, but develop life-long chronic infections. The study of this infection has revealed the types of immune responses required for both recovery from the acute phase and the control of the chronic phase of infection. This knowledge has led to vaccines and therapeutics to prevent and treat infections and associated disease states. The FV model has provided insights into immunological mechanisms found to be relevant to human infections with viruses such as HIV-1 and HTLV-1.
Regulatory T cells; suppression; retrovirus; immunotherapy; CD8+ T cells; chronic viral infection; Friend virus
The mechanisms whereby different vaccines may expand distinct antigen-specific T cell clonotypes or induce disparate degrees of protection are incompletely understood. We found that several delivery modes of a model retroviral antigen, including natural infection, preferentially expanded initially rare high-avidity CD4+ T cell clonotypes, known to mediate protection. In contrast, the same antigen vectored by human Adenovirus serotype 5 (Ad5) induced clonotypic expansion irrespective of avidity, eliciting a predominantly low-avidity response. Non-selective clonotypic expansion was caused by relatively weak Ad5-vectored antigen presentation and was reproduced by replication-attenuated retroviral vaccines. Mechanistically, the potency of antigen presentation determined the speed and, consequently, completion of the CD4+ T cell response. Whereas faster completion retained the initial advantage of high-avidity clonotypes, slower completion permitted uninhibited accumulation of low-avidity clonotypes. These results highlighted the importance of antigen presentation patterns in determining the clonotypic composition of vaccine-induced T cell responses and ultimately the efficacy of vaccination.
Tetherin/BST-2 is a host restriction factor that could directly inhibit retroviral particle release by tethering nascent virions to the plasma membrane. However, the immunological impact of Tetherin during retrovirus infection remains unknown. We now show that Tetherin influences antiretroviral cell-mediated immune responses. In contrast to the direct antiviral effects of Tetherin, which are dependent on cell surface expression, the immunomodulatory effects are linked to the endocytosis of the molecule. Mice encoding endocytosis-competent C57BL/6 Tetherin exhibited lower viremia and pathology at 7 days post-infection with Friend retrovirus (FV) compared to mice encoding endocytosis-defective NZW/LacJ Tetherin. Notably, antiretroviral protection correlated with stronger NK cell responses. In addition, FV infection levels were significantly lower in wild-type C57BL/6 mice than in Tetherin knock-out mice at 2 weeks post-infection, and antiretroviral protection correlated with stronger NK cell and virus-specific CD8+ T cell responses. The results demonstrate that Tetherin acts as a modulator of the cell-mediated immune response against retrovirus infection in vivo.
Tetherin; Bst-2; PDCA-1; H-2; NK cell; Friend retrovirus
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 co-infection with another microbe. It has been previously shown that infection of mice with lactate dehydrogenase-elevating virus (LDV) impairs early adaptive immune responses to Friend virus (FV) co-infection. To investigate the mechanism of this impairment we examined LDV-induced innate immune responses and found LDV- specific induction of IFNα and IFNγ. LDV-induced IFNα had little effect on FV infection or immune responses, but unexpectedly, LDV-induced IFNγ production dampened Th1 adaptive immune responses and enhanced FV infection. Two distinct effects were identified. First, LDV-induced IFNγ signaling indirectly modulated FV- specific CD8+ T cell responses. Second, intrinsic IFNγ signaling in B cells promoted polyclonal B cell activation and enhanced early FV infection, despite promotion of germinal center formation and neutralizing antibody production. Results from this model reveal that IFNγ production can have detrimental impacts on early adaptive immune responses and virus control.
The mechanisms whereby different vaccines may expand distinct Ag-specific T cell clonotypes or induce disparate degrees of protection are incompletely understood. We found that several delivery modes of a model retroviral Ag, including natural infection, preferentially expanded initially rare high-avidity CD4+ T cell clonotypes, known to mediate protection. In contrast, the same Ag vectored by human adenovirus serotype 5 induced clonotypic expansion irrespective of avidity, eliciting a predominantly low-avidity response. Nonselective clonotypic expansion was caused by relatively weak adenovirus serotype 5–vectored Ag presentation and was reproduced by replication-attenuated retroviral vaccines. Mechanistically, the potency of Ag presentation determined the speed and, consequently, completion of the CD4+ T cell response. Whereas faster completion retained the initial advantage of high-avidity clonotypes, slower completion permitted uninhibited accumulation of low-avidity clonotypes. These results highlighted the importance of Ag presentation patterns in determining the clonotypic composition of vaccine-induced T cell responses and ultimately the efficacy of vaccination.
Therapeutic administration of IFN-α in clinical trials significantly reduced HIV-1 plasma viral load and HTLV-I proviral load in infected patients. The mechanism may involve the concerted action of multiple antiretroviral effectors collectively known as ‘restriction factors’, which could vary in relative importance according to the magnitude of transcriptional induction. However, direct genetic approaches to identify the relevant IFN-α restriction factors will not be feasible in humans in vivo. On the other hand, mice encode an analogous set of restriction factor genes and could be used to obtain insights on how IFN-α could inhibit retroviruses in vivo. As expected, IFN-α treatment of mice significantly upregulated the transcription of multiple restriction factors including Tetherin/BST2, SAMHD1, Viperin, ISG15, OAS1 and IFITM3. However, a dominant antiretroviral factor, Apobec3, was only minimally induced. To determine whether Apobec3 was necessary for direct IFN-α antiretroviral action in vivo, wild-type and Apobec3-deficient mice were infected with Friend retrovirus then treated with IFN-α. Treatment of infected wild-type mice with IFN-α significantly reduced acute plasma viral load 28-fold, splenic proviral load 5-fold, bone marrow proviral load 14-fold and infected bone marrow cells 7-fold, but no inhibition was observed in Apobec3-deficient mice. These findings reveal that IFN-α inhibits acute Friend retrovirus infection primarily through the antiviral effector Apobec3 in vivo, demonstrate that transcriptional induction levels did not predict the mechanism of IFN-α-mediated control, and highlight the potential of the human APOBEC3 proteins as therapeutic targets against pathogenic retrovirus infections.
Recovery from Friend Virus 3 (Rfv3) is a single autosomal gene encoding a resistance trait that influences retroviral neutralizing antibody responses and viremia. Despite extensive research for 30 years, the molecular identity of Rfv3 has remained elusive. Here we demonstrate that Rfv3 is encoded by Apobec3. Apobec3 maps to the same chromosome region as Rfv3 and has broad inhibitory activity against retroviruses including HIV. Not only did genetic inactivation of Apobec3 convert Rfv3-resistant mice to a susceptible phenotype, but Apobec3 was found to be naturally disabled by aberrant mRNA splicing in Rfv3-susceptible strains. The link between Apobec3 and neutralizing antibody responses highlights an Apobec3-dependent mechanism of host protection that might extend to HIV and other human retroviral infections.
Conventional CD4+ T cells play an important role in viral immunity. In most virus infections, they provide essential help for antiviral B and T cell responses. In chronic infections, including HIV infection, an expansion of regulatory T cells (Tregs) has been demonstrated, which can suppress virus-specific CD4+ T cell responses in vitro. However, the suppressive activity of Tregs on effector CD4+ T cells in retroviral infection is less well documented in vivo. We took advantage of a transgenic mouse in which Tregs can be selectively depleted to determine the influence of such cells on retrovirus-specific CD4+ T cell responses during an ongoing infection. Mice were infected with Friend retrovirus (FV), and Tregs were depleted during the acute phase of the infection. In nondepleted mice, activated CD4+ T cells produced Th1-type cytokines but did not exhibit any antiviral cytotoxicity as determined in a major histocompatibility complex (MHC) class II-restricted in vivo cytotoxic T lymphocyte (CTL) assay. Depletion of Tregs significantly increased the numbers of virus-specific CD4+ T cells and improved their cytokine production, whereas it induced only very little CD4+ T cell cytotoxicity. However, after dual depletion of Tregs and CD8+ T cells, conventional CD4+ T cells developed significant cytotoxic activity against FV epitope-labeled target cells in vivo and contributed to the control of virus replication. Thus, both Tregs and CD8+ T cells influence the cytotoxic activity of conventional CD4+ T cells during an acute retroviral infection.
The study of regulatory T cells (Treg) requires methods for both in vivo and in vitro analyses, both of which have different limitations, but which complement each other to give a more complete picture of physiological function. Our analyses have focused on Treg-mediated suppression of CD8+ T cells, and in particular Tregs induced by viral infection. One of the unique characteristics of virus-induced Tregs is that they can suppress in vitro without the requirement for additional stimulation. This ability correlates with their activated status in vivo. Furthermore, in vivo activated Tregs can suppress the function of CD8+ T cells both in vitro and in vivo, while leaving proliferation intact. Interestingly, further in vitro stimulation of these Tregs confers to them the ability to suppress both the function and proliferative ability of CD8+ T cell targets.
regulatory T cells; CD8+ T cells
It was recently reported that inhibitory molecules such as PD-1 were up-regulated on CD8+ T cells during acute Friend retrovirus infection, and that the cells were prematurely exhausted and dysfunctional in vitro. The current study confirms that most activated CD8+ T cells up-regulated expression of PD-1 during acute infection and revealed a dichotomy of function between PD-1hi and PD-1lo subsets. More PD-1lo cells produced anti-viral cytokines such as IFNγ and TNFα, while more PD-1hi cells displayed characteristics of cytotoxic effectors such as production of granzymes and surface expression of CD107a. Importantly, CD8+ T cells mediated rapid in vivo cytotoxicity and were critical for control of acute Friend virus replication. Thus direct ex vivo analyses and in vivo experiments revealed high CD8+ T cell functionality and indicate that PD-1 expression during acute infection is not a marker of T cell exhaustion.
Tetherin is a membrane protein of unusual topology expressed from rodents to humans that accumulates enveloped virus particles on the surface of infected cells. However, whether this ‘tethering’ activity promotes or restricts retroviral spread during acute retrovirus infection in vivo is controversial. We report here the identification of a single nucleotide polymorphism in the Tetherin gene of NZW/LacJ (NZW) mice that mutated the canonical ATG start site to GTG. Translation of NZW Tetherin from downstream ATGs deleted a conserved dual-tyrosine endosomal sorting motif, resulting in higher cell surface expression and more potent inhibition of Friend retrovirus release compared to C57BL/6 (B6) Tetherin in vitro. Analysis of (B6×NZW)F1 hybrid mice revealed that increased Tetherin cell surface expression in NZW mice is a recessive trait in vivo. Using a classical genetic backcrossing approach, NZW Tetherin expression strongly correlated with decreased Friend retrovirus replication and pathogenesis. However, the protective effect of NZW Tetherin was not observed in the context of B6 Apobec3/Rfv3 resistance. These findings identify the first functional Tetherin polymorphism within a mammalian host, demonstrate that Tetherin cell surface expression is a key parameter for retroviral restriction, and suggest the existence of a restriction factor hierarchy to counteract pathogenic retrovirus infections in vivo.
Significant portions of the human and mouse genomes are comprised of retroviral sequences, revealing the long history of conflict between mammalian hosts and retroviruses that led to the evolution of host restriction factors. Nucleotide mutations in restriction factor genes provide a glimpse of this ongoing evolutionary process, but studies that directly probe the impact of restriction factor mutations during retrovirus infection are limited. In this study, we identified a single nucleotide mutation in the Tetherin host restriction gene that resulted in retention of Tetherin on the cell surface. In cell culture, Tetherin accumulates virions on the infected cell surface and prevents virion release, but some studies suggested that Tetherin might facilitate cell-to-cell virus spread. Our studies reveal that the Tetherin polymorphism inhibits retrovirus replication and disease. Thus, increased Tetherin cell surface expression enhanced the antiretroviral function of Tetherin. These results could have important implications in harnessing the biology of Tetherin for controlling pathogenic retroviruses such as HIV-1.
Members of the APOBEC3 family of deoxycytidine deaminases counteract a broad range of retroviruses in vitro through an indirect mechanism that requires virion incorporation and inhibition of reverse transcription and/or hypermutation of minus strand transcripts in the next target cell. The selective advantage to the host of this indirect restriction mechanism remains unclear, but valuable insights may be gained by studying APOBEC3 function in vivo. Apobec3 was previously shown to encode Rfv3, a classical resistance gene that controls the recovery of mice from pathogenic Friend retrovirus (FV) infection by promoting a more potent neutralizing antibody (NAb) response. The underlying mechanism does not involve a direct effect of Apobec3 on B cell function. Here we show that while Apobec3 decreased titers of infectious virus during acute FV infection, plasma viral RNA loads were maintained, indicating substantial release of noninfectious particles in vivo. The lack of plasma virion infectivity was associated with a significant post-entry block during early reverse transcription rather than G-to-A hypermutation. The Apobec3-dependent NAb response correlated with IgG binding titers against native, but not detergent-lysed virions. These findings indicate that innate Apobec3 restriction promotes NAb responses by maintaining high concentrations of virions with native B cell epitopes, but in the context of low virion infectivity. Finally, Apobec3 restriction was found to be saturable in vivo, since increasing FV inoculum doses resulted in decreased Apobec3 inhibition. By analogy, maximizing the release of noninfectious particles by modulating APOBEC3 expression may improve humoral immunity against pathogenic human retroviral infections.
Members of the APOBEC3 gene family can potently inhibit a broad range of retroviruses, including HIV-1. In cell culture, APOBEC3 counteracts retroviruses by: (1) reducing the infectivity of virions; and (2) inducing lethal G-to-A hypermutation in the next target cell. The selective advantage to the host of an ‘indirect’ restriction factor that is incorporated into virions and acts in the next target cell remains mysterious. We previously showed that Apobec3 encodes Rfv3, a classical resistance gene that controls the neutralizing antibody response against Friend retrovirus infection in mice. Here we demonstrate that Apobec3 promotes the release of substantial levels of noninfectious virions in the plasma during acute FV infection, resulting in a more potent antibody response directed against intact virions. Thus, we propose that APOBEC3 evolved as an innate mechanism to promote high concentrations of retrovirus antigen in a native but noninfectious form to effectively prime the neutralizing antibody response. These findings could have important implications for improving HIV-1 specific antibody responses.
It is well established that CD4+ T cells play an important role in immunity to infections with retroviruses such as HIV. However, in recent years CD4+ T cells have been subdivided into several distinct populations that are differentially regulated and perform widely varying functions. Thus, it is important to delineate the separate roles of these subsets, which range from direct antiviral activities to potent immunosuppression. In this review, we discuss contributions from the major CD4+ T cell subpopulations to retroviral immunity. Fundamental concepts obtained from studies on numerous viral infections are presented along with a more detailed analysis of studies on murine Friend virus. The relevance of these studies to HIV immunology and immunotherapy is reviewed.
Rfv3 is an autosomal dominant gene that influences the recovery of resistant mice from Friend retrovirus (FV) infection by limiting viremia and promoting a more potent neutralizing antibody response. We previously reported that Rfv3 is encoded by Apobec3, an innate retrovirus restriction factor. However, it was recently suggested that the Rfv3 susceptible phenotype of high viremia at 28 days postinfection (dpi) was more dominantly controlled by the B-cell-activating factor receptor (BAFF-R), a gene that is linked to but located outside the genetically mapped region containing Rfv3. Although one prototypical Rfv3 susceptible mouse strain, A/WySn, indeed contains a dysfunctional BAFF-R, two other Rfv3 susceptible strains, BALB/c and A.BY, express functional BAFF-R genes, determined on the basis of genotyping and B-cell immunophenotyping. Furthermore, transcomplementation studies in (C57BL/6 [B6] × BALB/c)F1 and (B6 × A.BY)F1 mice revealed that the B6 Apobec3 gene significantly influences recovery from FV viremia, cellular infection, and disease at 28 dpi. Finally, the Rfv3 phenotypes of prototypic B6, A.BY, A/WySn, and BALB/c mouse strains correlate with reported Apobec3 mRNA expression levels. Overall, these findings argue against the generality of BAFF-R polymorphisms as a dominant mechanism to explain the Rfv3 recovery phenotype and further strengthen the evidence that Apobec3 encodes Rfv3.
B cells are one of the targets of Friend virus (FV) infection, a well-established mouse model often used to study retroviral infections in vivo. Although B cells may be effective in stimulating cytotoxic T lymphocyte responses, studies involving their role in FV infection have mainly focused on neutralizing antibody production. Here we show that polyclonal activation of B cells promotes their infection with FV both in vitro and in vivo. Furthermore, we demonstrate that complement opsonization of Friend murine leukemia virus (F-MuLV) enhances infection of B cells, which correlates with increased potency of B cells to activate FV-specific CD8+ T cells.
APOBEC proteins have evolved as innate defenses against retroviral infections. Human immunodeficiency virus (HIV) encodes the Vif protein to evade human APOBEC3G; however, mouse retroviruses do not encode a Vif homologue, and it has not been understood how they evade mouse APOBEC3. We report here a murine leukemia virus (MuLV) that utilizes its glycosylated Gag protein (gGag) to evade APOBEC3. gGag is critical for infection of in vitro cell lines in the presence of APOBEC3. Furthermore, a gGag-deficient virus restricted for replication in wild-type mice replicates efficiently in APOBEC3 knockout mice, implying a novel role of gGag in circumventing the action of APOBEC3 in vivo.
Apobec3/Rfv3 is an innate immune factor that promotes the neutralizing antibody response against Friend retrovirus (FV) in infected mice. Based on its evolutionary relationship to activation-induced deaminase (AID), Apobec3 might directly influence antibody class switching and affinity maturation independently of viral infection. Alternatively, the antiviral activity of Apobec3 may indirectly influence neutralizing antibody responses by reducing early FV-induced pathology in critical immune compartments. To distinguish between these possibilities, we immunized wild-type and Apobec3-deficient C57BL/6 (B6) mice with (4-hydroxy-3-nitrophenyl) acetyl (NP) hapten and evaluated the binding affinity of the resultant NP-specific antibodies. These studies revealed similar affinity maturation of NP-specific IgG1 antibodies between wild-type and Apobec3 deficient mice in the absence of FV infection. In contrast, hapten-specific antibody affinity maturation was significantly compromised in Apobec3-deficient mice infected with FV. In highly susceptible (B6 x A.BY)F1 mice, the B6 Apobec3 gene protected multiple cell types in the bone marrow and spleen from acute FV infection including erythroid, B, T and myeloid cells. In addition, B6 Apobec3 deficiency was associated with elevated immunoglobulin levels but decreased induction of splenic germinal center B cells and plasmablasts during acute FV infection. These data suggest that Apobec3 indirectly influences FV-specific neutralizing antibody responses by reducing virus-induced immune dysfunction. These findings raise the possibility that enabling Apobec3 activity during acute infection with human pathogenic retroviruses such as HIV-1 may similarly facilitate stronger virus-specific neutralizing antibody responses.
Infection of mice with Friend virus induces the activation of CD4+ regulatory T cells (Tregs) that suppress virus-specific CD8+ T cells. This suppression leads to incomplete virus clearance and the establishment of virus persistence. We now show that Treg-mediated suppression of CD8+ T cells is tissue-specific, occurring in the spleen but not the liver. Regardless of infection status there was a five fold lower proportion of Tregs in the liver compared to the spleen, much lower absolute cell numbers, and the relatively few Tregs present expressed less CD25. Results indicated that reduced expression of CD25 on liver Tregs was due to microenvironmental factors including low levels of IL-2 production by CD4+ T helper cells in that tissue. Low CD25 expression on liver Tregs did not impair their ability to suppress CD8+ T cells in vitro. Correlating with the decreased proportion of Tregs in the liver was a significantly increased proportion of virus-specific CD8+ T cells compared to the spleen. Importantly, the virus-specific CD8+ T cells from the liver did not appear suppressed as they produced both IFNγ and granzyme B, and they also showed evidence of recent cytolytic activity (CD107a+). The functional phenotype of the virus-specific CD8+ T cells correlated with a ten-fold reduction of chronic Friend virus levels in the liver compared to the spleen. Thus, suppression of CD8+ T cells by virus-induced regulatory T cells occurs in a tissue-specific manner and correlates with profound effects on localized levels of chronic infection.
Murine norovirus (MNV) is a highly infectious but generally nonpathogenic agent that is commonly found in research mouse colonies in both North America and Europe. In the present study, the effects of acute and chronic infections with MNV on immune responses and recovery from concurrent Friend virus (FV) infections were investigated. No significant differences in T-cell or NK-cell responses, FV-neutralizing antibody responses, or long-term recovery from FV infection were observed. We conclude that concurrent MNV infections had no major impacts on FV infections.