Attenuation of innate immunity by cytomegalovirus IL-10 establishes a long-term deﬁcit of adaptive antiviral immunity W. L. William Changa and Peter A. Barrya,b,c,1 a Center for Comparative Medicine, bDepartment of Pathology and Laboratory Medicine, and cCalifornia National Primate Research Center, University of California, Davis, CA 95616
Edited* by Thomas E. Shenk, Princeton University, Princeton, NJ, and approved November 16, 2010 (received for review September 15, 2010)
| monkey model
uman cytomegalovirus (HCMV) establishes a lifelong persistence in immune-competent hosts in the presence of antiviral immune responses that effectively limit viral pathogenesis. The general absence of HCMV disease imposes an extraordinarily large immunological burden on its infected hosts because almost 10% of memory CD4+ and CD8+ T cells are speciﬁc to HCMV antigens (1). The ability to establish and maintain a persistent infection in the presence of such antiviral immunity requires a commensurate dedication of the HCMV coding capacity to immune evasive/modulating proteins that alter cellular activation, signaling, trafﬁcking, and apoptosis. Up to 60% of the HCMV ORFs can be deleted without affecting viral replication in cultured ﬁbroblasts (2). A sizeable number of these HCMV proteins subvert the immune surveillance of the hosts (3), including those that (i) disrupt natural killer (NK) cell and antigen-speciﬁc CD8+ T-cell functions, (ii) are high afﬁnity receptors for β-chemokines and the B and T lymphocyte attenuator, (iii) modulate the cell cycle, and (iv) stimulate innate effector cell trafﬁcking and/or alter the functionality of multiple immune cell types. One example of this latter group is the viral interleukin-10 ortholog (vIL-10) of cellular IL-10 (cIL-10) encoded by the UL111A ORF in primate CMV, including HCMV, rhesus CMV (RhCMV), African green monkey CMV, and baboon CMV (4). The sequence of HCMVencoded vIL-10 (hcmvIL-10) is highly divergent from cIL-10 (5), yet in vitro studies have demonstrated that hcmvIL-10 binds to the ligand-binding subunit of cellular IL-10 receptor (IL-10R1) with binding afﬁnity comparable to that of cIL-10 (6). cIL-10 is a well-characterized cytokine that suppresses cell-mediated immune responses while enhancing humoral immune responses (7). www.pnas.org/cgi/doi/10.1073/pnas.1013794108
Despite the considerable genetic drift between viral and host IL10 proteins, the functionality of hcmvIL-10 is exceedingly conserved with cIL-10 in multiple immune effector cells (8–13). With pleiotropic and cell type-dependent modulatory properties in vitro, expression of hcmvIL-10 could inﬂuence virus–host interactions, and contribute to the establishment and/or maintenance of persistence in an immune-competent host. RhCMV infection in rhesus macaques strongly recapitulates HCMV infection in both immune-competent individuals and those lacking a functional immune system (14). The combination of RhCMV encoding its own vIL-10 (rhcmvIL-10) and the availability of tools to engineer the RhCMV genome enabled direct comparison of the in vivo phenotype of a rhcmvIL-10-deleted RhCMV with its parental virus. The results of this study show that the absence of rhcmvIL-10 is associated with profound changes in both innate and adaptive immune responses after s.c. inoculation of RhCMV in naïve rhesus monkeys. These included changes in the (i) magnitude and constitution of innate immune cells at the site of experimental inoculation, (ii) inﬂux of dendritic cells (DC) to the draining lymph nodes (LN) and induction of adaptive immune responses, (iii) kinetics of antibody maturation and magnitude of antiviral antibody titers, and (iv) quality and quantity of RhCMV-speciﬁc T-cell responses. Together with the recent study demonstrating the critical role of the viral inhibitors of MHC-I antigen presentation during primary and nonprimary RhCMV infection (15), the results of this study highlight the complexity of the multilayered mechanisms of HCMV immune evasion. Results Construction and In Vitro Properties of rhcmvIL-10–Deleted Mutant of RhCMV. Both vIL-10 of HCMV and RhCMV share low sequence
homology to their host cellular counterparts (27% and 25%, respectively) (4). The expression kinetics (Fig. S1) of the RhCMV UL111A ORF are very similar to those of its HCMV ortholog (9). This work took advantage of the full-length genome of the 68-1 strain of RhCMV (referred to as WT) engineered into a BAC (16) and the Red/ET mutagenesis system (17). Using homologous recombination, the ﬁrst two coding exons of the UL111A gene were replaced with a zeocin expression cassette containing a terminal stop codon to yield pRhCMV/BAC-ΔUL111A (Fig. S2A). Deletion of the ﬁrst two exons eliminated most of the region of rhcmvIL-10 necessary for binding to IL-10R1 (6). Mutated RhCMV BAC plasmids were screened by zeocin resistance and diagnostic PCR (Fig. S2B). PCR amplicons from mutant clones were cloned and sequenced to verify the ﬁdelity of homologous recombination. Comprehensive restriction digestions were performed to conﬁrm that no major alterations were introduced
Author contributions: W.L.W.C. and P.A.B. designed research; W.L.W.C. performed research; W.L.W.C. and P.A.B. analyzed data; and W.L.W.C. and P.A.B. wrote the paper. The authors declare no conﬂict of interest. *This Direct Submission article had a prearranged editor. 1
To whom correspondence should be addressed. E-mail: [email protected]
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1013794108/-/DCSupplemental.
PNAS | December 28, 2010 | vol. 107 | no. 52 | 22647–22652
Human cytomegalovirus (HCMV) and many other pathogens exploit the IL-10 pathway, as part of their infectious cycle, either through their own encoded IL-10 (hcmvIL-10 for HCMV) or manipulation of the cellular IL-10 signaling cascade. Based on the in vitro demonstrations of its pleiotropic and cell type-dependent modulatory nature, hcmvIL-10 could profoundly attenuate host immunity, facilitating the establishment and maintenance of a persistent infection in an immune-competent host. To investigate the impact of extrinsic IL-10 on the induction and maintenance of antiviral immune responses in vivo, rhesus macaques were inoculated with variants of rhesus cytomegalovirus (RhCMV) either expressing or lacking the RhCMV ortholog of hcmvIL-10 (rhcmvIL-10). The results show that rhcmvIL-10 alters the earliest host responses to viral antigens by dampening the magnitude and speciﬁcity of innate effector cells to primary RhCMV infection. In addition, there is a commensurate reduction in the quality and quantity of early and long-term, RhCMV-speciﬁc adaptive immune responses. These ﬁndings provide a mechanistic basis of how early interactions between a newly infected host and HCMV could shape the long-term virus–host balance, which may facilitate the development of new prevention and intervention strategies for HCMV.
anywhere else within the genome during mutagenesis of the UL111A gene (Fig. S2C; EcoRI data shown). The in vitro growth parameters of RhCMV-ΔUL111A, derived by transfection of ﬁbroblasts with pRhCMV/BAC-ΔUL111A plasmid DNA, were equivalent to those of RhCMV-WT (Fig. 1A) and similar to the lack of in vitro growth defects of HCMVΔUL111A compared with its parental HCMV strain AD169 (9). A bioassay was performed to conﬁrm the loss of rhcmvIL-10 coding capacity in RhCMV-ΔUL111A. Rhesus monocyte-derived DC (MoDC) were treated for 24 h with conditioned medium from ﬁbroblasts infected with either WT or ΔUL111A in the presence or absence of LPS. The maturation status of MoDC after incubation was assessed by FACS for surface expression of CD86. Whereas WT supernatant inhibited CD86 expression in unstimulated (i.e., no LPS) and especially LPS-activated MoDC (Fig. 1B Upper), cells incubated with ΔUL111A-conditioned medium expressed a signiﬁcantly higher level of CD86 than those treated with WT-conditioned medium (Fig. 1B Lower, unstimulated). The difference between WT and ΔUL111A on modulation of CD86 expression was more substantial when cells were activated with LPS (Fig. 1B Lower, LPS). These data also suggested that there were factors in ΔUL111A-conditioned medium, other than rhcmvIL-10, suppressing the maturation of MoDC, because the level of CD86 expression in ΔUL111A-treated group did not reach that of the control group (cells incubated with DMEM). In sum, these results demonstrated that one of the RhCMV proteins inhibiting MoDC maturation, rhcmvIL-10, was deleted from the genome of RhCMV-ΔUL111A. Viral Parameters of RhCMV-ΔUL111A Infection. To maximize the potential for detecting possible subtle effects on host immune responses by viral IL-10, seronegative rhesus monkeys were challenged with a low, biologically relevant titer of RhCMV (400 plaque forming units). Like WT, ΔUL111A infection was subclinical. The absence of rhcmvIL-10 in monkeys inoculated with RhCMV-ΔUL111A did not elicit any changes in peripheral lymphoid subsets from those observed after inoculation with RhCMV-WT (Fig. S3). The virological parameters were similar between the WT and ΔUL111A groups. Low levels of viral DNA (