Catherine A. Forbes

Murine cytomegalovirus m157 mutation and variation leads to immune evasion of natural killer cells

Effective natural killer (NK) cell recognition of murine cytomegalovirus (MCMV)-infected cells depends on binding of the Ly49H NK cell activation receptor to the m157 viral glycoprotein. Here we addressed the immunological consequences of variation in m157 sequence and function. We found that most strains of MCMV possess forms of m157 that evade Ly49H-dependent NK cell activation. Importantly, repeated passage of MCMV through resistant Ly49H+ mice resulted in the rapid emergence of m157 mutants that elude Ly49H-dependent NK cell responses. These data provide the first molecular evidence that NK cells can exert sufficient immunological pressure on a DNA virus, such that it undergoes rapid and specific mutation in an NK cell ligand enabling it to evade efficient NK cell surveillance.

The BALB.B6-Cmv1 r mouse: a strain congenic for Cmv1 and the NK gene complex

Genetically determined resistance to murine cytomegalovirus (MCMV) in mice is controlled by both H2 and non-H2 genes. We have identified a phenotypically-defined, autosomal dominant, non-H2 gene, designated Cmvl, that regulates MCMV replication in the spleens of inbred mice. C57BL/6J mice possess the Cmvl{sup r} and exhibit low titers of MCMV replication, whereas the BALB/c strain, which possesses the Cmvl{sup s} allele, exhibits high virus titers in the spleen. 19 refs., 4 figs.

Functional Consequences of Natural Sequence Variation of Murine Cytomegalovirus m157 for Ly49 Receptor Specificity and NK Cell Activation

The Ly49H activating receptor on C57BL/6 (B6) NK cells plays a key role in early resistance to murine cytomegalovirus (MCMV) infection through specific recognition of the MCMV-encoded MHC class I-like molecule m157 expressed on infected cells. The m157 molecule is also recognized by the Ly49I inhibitory receptor from the 129/J mouse strain. The m157 gene is highly sequence variable among MCMV isolates, with many m157 variants unable to bind Ly49HB6. In this study, we have sought to define if m157 variability leads to a wider spectrum of interactions with other Ly49 molecules and if this modifies host susceptibility to MCMV. We have identified novel m157–Ly49 receptor interactions, involving Ly49C inhibitory receptors from B6, BALB/c, and NZB mice, as well as the Ly49HNZB activation receptor. Using an MCMV recombinant virus in which m157K181 was replaced with m157G1F, which interacts with both Ly49HB6 and Ly49CB6, we show that the m157G1F–Ly49C interactions cause no apparent attenuating effect on viral clearance in B6 mice. Hence, when m157 can bind both inhibitory and activation NK cell receptors, the outcome is still activation. Thus, these data indicate that whereas m157 variants predominately interact with inhibitory Ly49 receptors, these interactions do not profoundly interfere with early NK cell responses.

NK gene complex haplotype variability and host resistance alleles to murine cytomegalovirus in wild mouse populations

The NK gene complex (NKC) on mouse chromosome 6 encodes receptors that are expressed on NK cells, such as Ly49H, and is involved in regulating NK cell control of virus infections, such as murine cytomegalovirus (MCMV). In the present study, we investigated the level of allelic heterogeneity in NKC loci in populations of outbred wild mice. This work revealed extensive levels of heterogeneity within two wild mouse populations. Analysis of MCMV replication in a population of specific pathogen‐free outbred wild mice revealed that low viral titres, which are normally associated with the Cmv1 r allele of the Cmv1 host resistance locus, were not prevalent in the mice tested. Hence, NKC‐mediated resistance associated with Cmv1 r/Ly49H‐like effects was rare in this population. Overall, these data indicate that the NKC region is highly polymorphic and thus it is very likely that it confers on mice sufficient variability to cope with infection by a range of pathogens.

Peptide based cytotoxic T-cell vaccines; delivery of multiple epitopes, help, memory and problems

Synthetic CD8+ cytotoxic T-lymphocyte (CTL) peptide epitope based vaccines are being developed against a number of human diseases. Here we describe extensive preclinical testing of peptide epitope vaccines formulated with a protein as a source of CD4 help and Montanide ISA 720, an adjuvant currently in human clinical trials. Such water-in-oil formulations could effectively co-deliver several peptide epitopes and simultaneously induce multiple independent CTL responses. The efficiency of CTL induction by some peptides was, however, dependent on the aqueous buffer conditions, with poor performance correlating with non-covalent peptide oligomerisation. Any of a number of proteins currently used in human vaccines could supply CD4 help and no difference in CTL induction was obtained if the CD4 response was amnestic or a primary. Peptide immunisation was found to induce long term CTL memory and the recall of protective responses did not depend on an amnestic CD4 response. Slow pyroglutamic acid formation and rapid oxidation of methionine residues was observed in water-in-oil formulations, however, the latter had no effect on CTL induction. These data highlight the need to monitor for potential deleterious chemical events and interpeptide interactions, but illustrate that peptide based vaccination can effectively deliver multiple epitopes, in conjunction with any protein, and induce protective memory.

Ly49C-Dependent Control of MCMV Infection by NK Cells Is Cis-Regulated by MHC Class I Molecules

Natural Killer (NK) cells are crucial in early resistance to murine cytomegalovirus (MCMV) infection. In B6 mice, the activating Ly49H receptor recognizes the viral m157 glycoprotein on infected cells. We previously identified a mutant strain (MCMVG1F) whose variant m157 also binds the inhibitory Ly49C receptor. Here we show that simultaneous binding of m157 to the two receptors hampers Ly49H-dependent NK cell activation as Ly49C-mediated inhibition destabilizes NK cell conjugation with their targets and prevents the cytoskeleton reorganization that precedes killing. In B6 mice, as most Ly49H+ NK cells do not co-express Ly49C, the overall NK cell response remains able to control MCMVm157G1F infection. However, in B6 Ly49C transgenic mice where all NK cells express the inhibitory receptor, MCMV infection results in altered NK cell activation associated with increased viral replication. Ly49C-mediated inhibition also regulates Ly49H-independent NK cell activation. Most interestingly, MHC class I regulates Ly49C function through cis-interactions that mask the receptor and restricts m157 binding. B6 Ly49C Tg, β2m ko mice, whose Ly49C receptors are unmasked due to MHC class I deficient expression, are highly susceptible to MCMVm157G1F and are unable to control a low-dose infection. Our study provides novel insights into the mechanisms that regulate NK cell activation during viral infection.

Transcriptional analysis of human cytomegalovirus and rat cytomegalovirus homologues of the M73/M73.5 spliced gene family

Previous analysis of the M73-to-m74/M75 intergenic region of murine cytomegalovirus (MCMV) identified a family of 3′-co-terminal spliced transcripts that includes M73 and M73.5. The current study investigated whether similar families of spliced genes also exist in the human CMV (HCMV) and rat CMV (RCMV) genomes. Northern blot, RT-PCR and RACE-PCR analysis of HCMV transcripts showed that while mRNAs from HCMV UL73 and a putative UL73.5 homologue were spliced and 3′-co-terminal, they were not 5′-co-terminal. In contrast, the spliced RCMV R73 and R73.5 transcripts were arranged in a similar manner to those in MCMV and found to be both 5′ and 3′-co-terminal. In both the HCMV and RCMV genomes, additional non-coding spliced transcripts were found to originate from these regions. These results highlight that families of spliced transcripts coding for structural glycoproteins are likely to be a conserved feature of this region of betaherpesviral genomes.

Glycosylation contributes to variability in expression of murine cytomegalovirus m157 and enhances stability of interaction with the NK-cell receptor Ly49H

NK cell‐mediated resistance to murine cytomegalovirus (MCMV) is controlled by allelic Ly49 receptors, including activating Ly49H (C57BL/6 strain) and inhibitory Ly49I (129 strain), which specifically recognize MCMV m157, a glycosylphosphatidylinositol‐linked protein with homology to MHC class I. Although the Ly49 receptors retain significant homology to classic carbohydrate‐binding lectins, the role of glycosylation in ligand binding is unclear. Herein, we show that m157 is expressed in multiple, differentially N‐glycosylated isoforms in m157‐transduced or MCMV‐infected cells. We used site‐directed mutagenesis to express single and combinatorial asparagine (N)‐to‐glutamine (Q) mutations at N178, N187, N213, and N267 in myeloid and fibroblast cell lines. Progressive loss of N‐linked glycans led to a significant reduction of total cellular m157 abundance, although all variably glycosylated m157 isoforms were expressed at the cell surface and retained the capacity to activate Ly49HB6 and Ly49I129 reporter cells and Ly49H+ NK cells. However, the complete lack of N‐linked glycans on m157 destabilized the m157‐Ly49H interaction and prevented physical transfer of m157 to Ly49H‐expressing cells. Thus, glycosylation on m157 enhances expression and binding to Ly49H, factors that may impact the interaction between NK cells and MCMV in vivo where receptor–ligand interactions are more limiting.

Ly49C Impairs NK Cell Memory in Mouse Cytomegalovirus Infection

NK cells possess inhibitory receptors that are responsible for self-MHC class I recognition; beyond their inhibitory function, accumulating evidence indicates that such receptors confer NK cell functional competence through an unclear process termed “licensing.” Ly49C is the main self-specific inhibitory Ly49 receptor in H-2b C57BL/6 (B6) mice. We used B6 Ly49C-transgenic and B6 β2 microglobulin (β2m)-knockout Ly49C-transgenic mice to investigate the impact of licensing through this inhibitory receptor in precursor and mature NK cells. We found that self-specific inhibitory receptors affected NK cell precursor survival and proliferation at particular developmental stages in an MHC class I–dependent manner. The presence of Ly49C impacted the NK cell repertoire in a β2m-dependent manner, with reduced Ly49A+, Ly49G2+, and Ly49D+ subsets, an increased DNAM-1+ subset, and higher NKG2D expression. Licensed NK cells displayed a skewed distribution of the maturation stages, which was characterized by differential CD27 and CD11b expression, toward the mature phenotypes. We found that Ly49C-mediated licensing induced a split effect on NK cell functions, with increased cytokine-production capabilities following engagement of various activating receptors while cytotoxicity remained unchanged. Analysis of licensed NK cell functions in vivo, in a system of mouse CMV infection, indicated that licensing did not play a major role in the NK cell antiviral response during acute infection, but it strongly impaired the generation and/or persistence of memory NK cells. This study unravels multifaceted effects of licensing on NK cell populations and their functions.

Combination immune checkpoint blockade as an effective therapy for mesothelioma

ABSTRACT Mesothelioma is an aggressive asbestos induced cancer with extremely poor prognosis and limited treatment options. Immune checkpoint blockade (ICPB) has demonstrated effective therapy in melanoma and is now being applied to other cancers, including mesothelioma. However, the efficacy of ICPB and which immune checkpoint combinations constitute the best therapeutic option for mesothelioma have yet to be fully elucidated. Here, we used our well characterised mesothelioma tumour model to investigate the efficacy of different ICBP treatments to generate effective therapy for mesothelioma. We show that tumour resident regulatory T cell co-express high levels of CTLA-4, OX40 and GITR relative to T effector subsets and that these receptors are co-expressed on a large proportion of cells. Targeting any of CTLA-4, OX40 or GITR individually generated effective responses against mesothelioma. Furthermore, the combination of αCTLA-4 and αOX40 was synergistic, with an increase in complete tumour regressions from 20% to 80%. Other combinations did not synergise to enhance treatment outcomes. Finally, an early pattern in T cell response was predictive of response, with activation status and ICP receptor expression profile of T effector cells harvested from tumour and dLN correlating with response to immunotherapy. Taken together, these data demonstrate that combination ICPB can work synergistically to induce strong, durable immunity against mesothelioma in an animal model.