BackgroundInfectious entry of human papillomaviruses into their host cells is an important step in the viral life cycle. For cell binding these viruses use proteoglycans as initial attachment sites. Subsequent transfer to a secondary receptor molecule seems to be involved in virus uptake. Depending on the papillomavirus subtype, it has been reported that entry occurs by clathrin- or caveolin-mediated mechanisms. Regarding human papillomavirus type 16 (HPV16), the primary etiologic agent for development of cervical cancer, clathrin-mediated endocytosis was described as infectious entry pathway.Methodology/Principal FindingsUsing immunofluorescence and infection studies we show in contrast to published data that infectious entry of HPV16 occurs in a clathrin- and caveolin-independent manner. Inhibition of clathrin- and caveolin/raft-dependent endocytic pathways by dominant-negative mutants and siRNA-mediated knockdown, as well as inhibition of dynamin function, did not impair infection. Rather, we provide evidence for involvement of tetraspanin-enriched microdomains (TEMs) in HPV16 endocytosis. Following cell attachment, HPV16 particles colocalized with the tetraspanins CD63 and CD151 on the cell surface. Notably, tetraspanin-specific antibodies and siRNA inhibited HPV16 cell entry and infection, confirming the importance of TEMs for infectious endocytosis of HPV16.Conclusions/SignificanceTetraspanins fulfill various roles in the life cycle of a number of important viral pathogens, including human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, their involvement in endocytosis of viral particles has not been proven. Our data indicate TEMs as a novel clathrin- and caveolin-independent invasion route for viral pathogens and especially HPV16.

Due to the association of human papillomaviruses (HPV) with development of multiple carcinomas, especially cervical carcinomas, early diagnosis and prevention of infection with HPV are of great medical and economic importance. Knowledge of the early steps of papillomavirus infection, which results in infectious entry, will help develop means to prevent HPV-induced lesions. Since HPV are difficult to propagate in cell culture, surrogate infection systems with marker-encoding viral capsids, called HPV pseudovirions, have been developed and successfully used in investigating the HPV entry pathway as well as in testing of substances interfering with HPV infection (2, 33). These studies have led to the identification of specifically modified heparan sulfate proteoglycans (HSPGs) as primary attachment receptors for papillomaviruses (13,15) and to heparin and other sulfated polysaccharides as inhibitors of HPV infection (1, 7, 13). Recently, carrageenan, an unbranched sulfated polysaccharide from algae with saccharide linkages reminiscent of galactosaminoglycans, has been reported to inhibit HPV infection primarily by preventing the binding of virions to the cell (4). Dispirotripiperazine (DSTP) derivatives represent another substance class with proven antiviral potential. DSTP27 (an N,NЈ-bisheteryl derivative of DSTP), one of the most active derivatives of this new class of low-molecular-weight antiherpetic compounds, interacts with specific forms of cell surface HSPGs (26). In addition to the inhibition of herpes virus attachment and infection, DSTP27 efficiently blocks the attachment and uptake of members from other virus families that depend on HSPGs as primary attachment molecules (25). In contrast to the HS analogs such as heparin and pentosan polysulfate that have short-lived effects, pretreatment of cells with DSTP27 induces a longlasting antiviral effect. Based on computer modeling, DSTP27 possibly interacts with two O-sulfate groups located on neighboring saccharides of the HS chain (27). Using the octosaccharide essential for HS-mediated entry of herpes simplex virus type 1 (HSV-1) into host cells (20), these computational studies further show that DSTP27 may additionally interact with a carbonyl group, thus increasing the strength of compound binding.Since HPV bind specifically to sulfated polysaccharide residues of cell surface HSPGs, particularly 2-O-and 6-O-sulfated HS chains in addition to N-sulfated residues (27), DSTP27 was predicted to work as a potent inhibitor of HPV infection. In this report we demonstrate that DSTP27 efficiently prevents HPV infection when applied several hours pre-or postinfection of cells. This is achieved by two putatively different mech-* Corresponding author. Mailing address:

The lungs are a noted predilection site of acute, latent, and reactivated cytomegalovirus (CMV) infections. Interstitial pneumonia is the most dreaded manifestation of CMV disease in the immunocompromised host, whereas in the immunocompetent host lung-infiltrating CD8 T cells confine the infection in nodular inflammatory foci and prevent viral pathology. By using murine CMV infection as a model, we provide evidence for a critical role of mast cells (MC) in the recruitment of protective CD8 T cells to the lungs. Systemic infection triggered degranulation selectively in infected MC. The viral activation of MC was associated with a wave of CC chemokine ligand 5 (CCL5) in the serum of C57BL/6 mice that was MC-derived as verified by infection of MC-deficient KitW-sh/W-sh “sash” mutants. In these mutants, CD8 T cells were recruited less efficiently to the lungs, correlating with enhanced viral replication and delayed virus clearance. A causative role for MC was verified by MC reconstitution of “sash” mice restoring both, efficient CD8 T-cell recruitment and infection control. These results reveal a novel crosstalk axis between innate and adaptive immune defense against CMV, and identify MC as a hitherto unconsidered player in the immune surveillance at a relevant site of CMV disease.

The elucidation of the molecular and immunological mechanisms mediating maintenance of latency in human tuberculosis aids to develop more effective vaccines and to define biologically meaningful markers for immune protection. We analyzed granuloma-associated lymphocytes (GALs) from human lung biopsies of five patients with latent Mycobacterium tuberculosis (MTB) infection. MTB CD4+ and CD8+ T cell response was highly focused in the lung, distinct from PBL, as assessed by TCR-CDR3 spectratyping coupled with a quantitative analysis of TCR VB frequencies. GALs produced IFN-γ in response to autologous macrophages infected with MTB and to defined MTB-derived HLA-A2-presented peptides Ag85a242–250, Ag85b199–207, early secreted antigenic target 6 (ESAT-6)28–36, 19-kDa Ag88–97, or the HLA-DR-presented ESAT-61–20 epitope. Immune recognition of naturally processed and presented MTB epitopes or the peptide ESAT-61–20 could be linked to specific TCR VB families, and in two patients to unique T cell clones that constituted 19 and 27%, respectively, of the CD4+ and 17% of the CD8+ GAL population. In situ examination of MTB-reactive GALs by tetramer in situ staining and confocal laser-scanning microscopy consolidates the presence of MHC class I-restricted CD8+ T cells in MTB granuloma lesions and supports the notion that clonally expanded T cells are crucial in immune surveillance against MTB.

Expansion of the CD8 T-cell memory pool, also known as 'memory inflation', for certain but not all viral epitopes in latently infected host tissues is a special feature of the immune response to cytomegalovirus. The L d -presented murine cytomegalovirus (mCMV) immediate-early (IE) 1 peptide is the prototype of an epitope that is associated with memory inflation. Based on the detection of IE1 transcripts in latently infected lungs it was previously proposed that episodes of viral gene expression and antigenic activity due to desilencing of a limited number of viral genes may drive epitope-specific memory inflation. This would imply direct antigen presentation through latently infected host tissue cells rather than cell death-associated cross-presentation of viral antigens derived from productively infected cells through uninfected, professional antigenpresenting cells (profAPCs). To address the role of bone marrow-derived profAPCs in CD8 T-cell priming and memory to mCMV, we have used here a combined sex-mismatched and MHC class-I mismatched dual-marker bone marrow chimera model in which presentation of the IE1 epitope is restricted to donor-derived sry + L d+ cells of haematopoietic differentiation lineages. Successful CD8 T-cell priming specific for the L d -and D d -presented inflationary epitopes IE1 and m164, respectively, but selective failure in IE1 epitope-specific memory inflation in these chimeras indicates different modes of antigen presentation involved in CD8 T-cell priming and memory inflation. These data suggest that memory inflation during mCMV latency requires expression of the epitope-presenting MHC class-I molecule by latently infected non-haematopoietic host tissue cells and thus predicts a role for direct antigen presentation in memory inflation. INTRODUCTIONIt is current opinion that priming of CD8 T-cells during acute viral infections is accomplished mainly by uninfected professional antigen-presenting cells (profAPCs) that take up and 'cross-present' viral antigens derived from infected cells undergoing infection-associated cell death (den Haan & Bevan, 2001; Heath & Carbone, 2001;Kurts et al., 2010;Shen & Rock, 2006). For the infection with murine cytomegalovirus (mCMV), comparably efficient priming of CD8 T-cells in the presence and absence of 'viral inhibitors of direct antigen presentation', known as immunoevasins (for reviews, see Doom & Hill, 2008; Hansen & Bouvier, 2009;Lemmermann et al., 2011a;Reddehase, 2002), provided a reasonable argument for priming by cross-presentation (Böhm et al., 2008;Gold et al., 2002;Munks et al., 2007). More direct evidence for cross-presentation of mCMV epitopes was provided only recently by in vivo priming of CD8 T-cells with MHC class-I (MHC-I)-deficient fibroblasts infected with a spreaddefective virus mutant, mCMV-DgL, conditions which plausibly prevent any direct antigen presentation (Snyder et al., 2010). A potent cross-presenting cell type is the CD8 + CD11c + subset of dendritic cells (DCs) (Allan et al., 2003;Belz et al., 2004;Schnorrer et al...

Reactivation of latent cytomegalovirus (CMV) in the transient state of immunodeficiency after hematopoietic cell transplantation (HCT) is the most frequent and severe viral complication endangering leukemia therapy success. By infecting the bone marrow (BM) stroma of the transplantation recipient, CMV can directly interfere with BM repopulation by the transplanted donor-derived hematopoietic cells and thus delay immune reconstitution of the recipient. Cytopathogenic virus spread in tissues can result in CMV disease with multiple organ manifestations of which interstitial pneumonia is the most feared. There exists a 'window of risk' between hematoablative treatment and reconstitution of antiviral immunity after HCT, whereby timely reconstitution of antiviral CD8 T cells is a recognized positive prognostic parameter for the control of reactivated CMV infection and prevention of CMV disease. Supplementation of endogenous reconstitution by adoptive cell transfer of 'ready-to-go' effector and/or memory virus epitope-specific CD8 T cells is a therapeutic option to bridge the 'window of risk.' Preclinical research in murine models of CMV disease has been pivotal by providing 'proof of concept' for a benefit from CD8 T-cell therapy of HCT-associated CMV disease (reviewed in Holtappels et al. Med Microbiol Immunol 197:125-134, 2008). Here, we give an update of our previous review with focus on parameters that determine the efficacy of adoptive immunotherapy of CMV infection by antiviral CD8 T cells in the murine model.

1% HPV16 E7-reactive T cells in CD8؉ peripheral blood lymphocytes. In vivo priming with HPV16 was confirmed in patients with cervical cancer or preinvasive HPV16-positive lesions using HLA-A2 tetramer complexes loaded with the E6-derived epitope KLPQLCTEL. In contrast, we could not detect E6-reactive T cells in healthy individuals. These data imply that the measurement of the HPV16 E7 11-19(20)

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