Immune evasion in the tumor microenvironment (TME) is a crucial barrier for effective cancer therapy, and plasticity of innate immune cells may contribute to failures of targeted immunotherapies. Here, we show that rivaroxaban, a direct inhibitor of activated coagulation factor X (FX), promotes antitumor immunity by enhancing infiltration of dendritic cells and cytotoxic T cells at the tumor site. Profiling FX expression in the TME identifies monocytes and macrophages as crucial sources of extravascular FX. By generating mice with immune cells lacking the ability to produce FX, we show that myeloid cell–derived FX plays a pivotal role in promoting tumor immune evasion. In mouse models of cancer, we report that the efficacy of rivaroxaban is comparable with anti–programmed cell death ligand 1 (PD-L1) therapy and that rivaroxaban synergizes with anti–PD-L1 in improving antitumor immunity. Mechanistically, we demonstrate that FXa promotes immune evasion by signaling through protease-activated receptor 2 and that rivaroxaban specifically targets this cell-autonomous signaling pathway to reprogram tumor-associated macrophages. Collectively, our results have uncovered the importance of FX produced in the TME as a regulator of immune cell activation and suggest translational potential of direct oral anticoagulants to remove persisting roadblocks for immunotherapy and provide extravascular benefits in other diseases.

Drug lead compound identification increasingly uses high‐throughput screening in conjunction with computational methods. Hits found in this way are generally of low affinity (typically about 10−5 M) and require further development. Such low affinity can result in significant changes in binding mode upon change of experimental conditions: at pH 7, the Factor Xa inhibitor 1 (see picture) binds in the specificity pocket of trypsin through its pyridinyl group (yellow); at pH 8, the same pocket is occupied by the chloronaphthyl moiety (magenta), with a significant reorganization of the ligand binding site (circled).

The mechanisms that lead to the tegumentation of herpesviral particles are only poorly defined. The phosphoprotein 65 (pp65) is the most abundant constituent of the virion tegument of human cytomegalovirus (HCMV). It is, however, nonessential for virion formation. This seeming discrepancy has not met with a satisfactory explanation regarding the role of pp65 in HCMV particle morphogenesis. Here, we addressed the question of how the overall tegument composition of the HCMV virion depended on pp65 and how the lack of pp65 influenced the packaging of particular tegument proteins. To investigate this, we analyzed the proteomes of pp65-positive (pp65pos) and pp65-negative (pp65neg) virions by label-free quantitative mass spectrometry and determined the relative abundances of tegument proteins. Surprisingly, only pUL35 was elevated in pp65neg virions. As the abundance of pUL35 in the HCMV tegument is low, it is unlikely that it replaced pp65 as a structural component in pp65neg virions. A subset of proteins, including the third most abundant tegument protein, pUL25, as well as pUL43, pUL45, and pUL71, were reduced in pp65neg or pp65low virions, indicating that the packaging of these proteins was related to pp65. The levels of tegument components, like pp28 and the capsid-associated tegument proteins pp150, pUL48, and pUL47, were unaffected by the lack of pp65. Our analyses demonstrate that deletion of pp65 is not compensated for by other viral proteins in the process of virion tegumentation. The results are concordant with a model of pp65 serving as an optional scaffold protein that facilitates protein upload into the outer tegument of HCMV particles. IMPORTANCEThe assembly of the tegument of herpesviruses is only poorly understood. Particular proteins, like HCMV pp65, are abundant tegument constituents. pp65 is thus considered to play a major role in tegument assembly in the process of virion morphogenesis. We show here that deletion of the pp65 gene leads to reduced packaging of a subset of viral proteins, indicating that pp65 acts as an optional scaffold protein mediating protein upload into the tegument.

The tegument protein pp65 of human cytomegalovirus (HCMV) is abundant in lytically infected human foreskin fibroblasts (HFF), as well as in virions and subviral dense bodies (DB). Despite this, we showed previously that pp65 is dispensable for growth in HFF. In the process of refining a DB-based vaccine candidate, different HCMV mutants were generated, expressing a dominant HLA-A2-presented peptide of the IE1 protein fused to pp65. One of the mutant viruses (RV-VM1) surprisingly showed marked impairment in virus release from HFF. We hypothesized that analysis of the phenotypic alterations of RV-VM1 would provide insight into the functions of pp65, poorly defined thus far. RV-VM1 infection resulted in nuclear retention of the fusion protein and reorganization of nuclear inclusion bodies. Coimmunoprecipitation experiments suggested that wild-type (wt) pp65 and pp65-VM1 were substrates of the viral pUL97 kinase in vitro and formed a complex with the viral RNA-export protein pUL69 and with pUL97 in lysates of infected cells. No evidence for an impairment of pUL97 within this complex was found. However, RV-VM1 replication in infected cells was resistant to a pUL97 inhibitor, and pUL97 inhibitors mimicked the mutant in terms of pp65 being retained in the nucleus. The results suggest that the life cycle of RV-VM1 was impeded at the stages of early-late transcription, RNA export or capsid maturation. wt-pp65 may play a role at these stages of infection, and complex formation with pUL69 and pUL97 may be important for that function. INTRODUCTIONHuman cytomegalovirus (HCMV) is a ubiquitous pathogen that severely affects individuals with impaired or immature immune-defence functions (Griffiths et al., 2009;Mocarski et al., 2007). The components of the HCMV tegument have recently attracted considerable attention, as these proteins carry functions important for various stages of the virus replication cycle (reviewed by Kalejta, 2008). The phosphoprotein pp65 (ppUL83) has long been known as an abundant tegument constituent (Roby & Gibson, 1986), yet it is dispensable for HCMV growth in human foreskin fibroblast (HFF) cell cultures (Schmolke et al., 1995b).pp65 is an important target antigen of cellular and humoral immune responses (Beninga et al., 1995; McLaughlinTaylor et al., 1994; Plachter et al., 1990;Wills et al., 1996). It would thus be reasonable to assume that modification or deletion of the protein would be favourable for the virus. However, the sequence of pp65 is highly conserved in laboratory strains and clinical isolates (Dolan et al., 2004;Pande et al., 1991) and lack of pp65 expression in such strains has never been reported. Indeed, detection of pp65 has been used as a reliable diagnostic marker for acute HCMV infection in transplant recipients for over 15 years (Grefte et al., 1992a, b Initial studies described an association of pp65 with serine/ threonine kinase activity (Britt & Auger, 1986;Somogyi et al., 1990). Subsequent analyses identified the cellular Polo-like kinase 1 (Gallina et al., 1999) and the vi...

Exogenous introduction of particle-associated proteins of human cytomegalovirus (HCMV) into the major histocompatibility complex (MHC) class I presentation pathway by subviral dense bodies (DB) is an effective way to sensitize cells against CD8 T-cell (CTL) recognition and killing. Consequently, these particles have been proposed as a platform for vaccine development. We have developed a strategy to refine the antigenic composition of DB. For proof of principle, an HCMV recombinant (RV-VM3) was generated that encoded the immunodominant CTL determinant IE1 TMY from the IE1 protein in fusion with the major constituent of DB, the tegument protein pp65. To generate RV-VM3, a bacterial artificial chromosome containing the HCMV genome was modified by applying positive/negative selection based on the expression of the bacterial galactokinase in conjunction with l Red-mediated homologous recombination. This method allowed the efficient and seamless insertion of the DNA sequence encoding IE1 TMY in frame into the pp65 open reading frame (UL83) of the viral genome. RV-VM3 expressed its fusion protein to high levels. The fusion protein was packaged into DB and into virions. Its delivery into fibroblasts by these viral particles led to the loading of the MHC class I presentation pathway with IE1 TMY and to efficient killing by specific CTLs. This demonstrated that a heterologous peptide, not naturally present in HCMV particles, can be processed from a recombinant, DB-derived protein to be subsequently presented by MHC class I. The results presented here provide a rationale for the optimization of a vaccine based on recombinant DB. INTRODUCTIONInfection with human cytomegalovirus (HCMV; family Herpesviridae, subfamily Betaherpesvirinae) may cause significant morbidity and mortality in individuals with immature or compromised immune-defence functions, but proceeds asymptomatically in most healthy adults (Pass, 2001). Prevention of HCMV infection or disease by a vaccine has been ranked as a high-priority goal (Stratton et al., 2001). Although several vaccine approaches have been developed, none have yet entered routine clinical practice (Pepperl-Klindworth & Plachter, 2006;Plotkin, 2004;Schleiss & Heineman, 2005;Zhong & Khanna, 2007). We have demonstrated that HCMV dense bodies (DB) provide a promising basis for vaccine development (Pepperl et al., 2000;Pepperl-Klindworth et al., 2002). DB are enveloped, subviral particles devoid of capsids and viral DNA. They are released from infected fibroblast cultures and enter cells presumably via the normal HCMV entry processes. The major constituent of DB is pp65 (ppUL83) (Varnum et al., 2004), which is a prominent target of the CD4 and CD8 T-cell (CTL) responses following natural infection (Beninga et al., 1995;McLaughlin-Taylor et al., 1994;Wills et al., 1996). Although pp65 by itself appears to be an attractive antigen for the design of a vaccine, other HCMV proteins may also be important and should thus be considered (Reddehase, 2002). One of these, as far as the CTL response is co...

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Human cytomegalovirus (HCMV) particle morphogenesis in infected cells is an orchestrated process that eventually results in the release of enveloped virions. Proteomic analysis has been employed to reveal the complexity in the protein composition of these extracellular particles. Only limited information is however available regarding the proteome of infected cells preceding the release of HCMV virions. We used quantitative mass spectrometry to address the pattern of viral and cellular proteins in cells, infected with derivatives of the AD169 laboratory strain. Our analyses revealed a remarkable conservation in the patterns of viral and of abundant cellular proteins in cells, infected for 2 hours, 2 days, or 4 days. Most viral proteins increased in abundance as the infection progressed over time. Of the proteins that were reliably detectable by mass spectrometry, only IE1 (pUL123), pTRS1, and pIRS1 were downregulated at 4 days after infection. In addition, little variation of viral proteins in the virions of the different viruses was detectable, independent of the expression of the major tegument protein pp65. Taken together these data suggest that there is little variation in the expression program of viral and cellular proteins in cells infected with related HCMVs, resulting in a conserved pattern of viral proteins ultimately associated with extracellular virions.