Kirsten Freitag

Clathrin- and Caveolin-Independent Entry of Human Papillomavirus Type 16—Involvement of Tetraspanin-Enriched Microdomains (TEMs)

Background Infectious 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 Findings Using 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/Significance Tetraspanins 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.

Inhibition of Transfer to Secondary Receptors by Heparan Sulfate-Binding Drug or Antibody Induces Noninfectious Uptake of Human Papillomavirus

ABSTRACT Infection with various human papillomaviruses (HPVs) induces cervical cancers. Cell surface heparan sulfates (HS) have been shown to serve as primary attachment receptors, and molecules with structural similarity to cell surface HS, like heparin, function as competitive inhibitors of HPV infection. Here we demonstrate that the N,N′-bisheteryl derivative of dispirotripiperazine, DSTP27, efficiently blocks papillomavirus infection by binding to HS moieties, with 50% inhibitory doses of up to 0.4 μg/ml. In contrast to short-term inhibitory effects of heparin, pretreatment of cells with DSTP27 significantly reduced HPV infection for more than 30 h. Using DSTP27 and heparinase, we furthermore demonstrate that HS moieties, rather than laminin 5, present in the extracellular matrix (ECM) secreted by keratinocytes are essential for infectious transfer of ECM-bound virions to cells. Prior binding to ECM components, especially HS, partially alleviated the requirement for cell surface HS. DSTP27 blocks infection by cell-bound virions by feeding into a noninfectious entry pathway. Under these conditions, virus colocalized with HS moieties in endocytic vesicles. Similarly, postattachment treatment of cells with heparinase, cytochalasin D, or neutralizing antibodies resulted in uptake of virions without infection, indicating that deviation into a noninfectious entry pathway is a major mode of postattachment neutralization. In untreated cells, initial colocalization of virions with HS on the cell surface and in endocytic vesicles was lost with time. Our data suggest that initial attachment of HPV to HS proteoglycans (HSPGs) must be followed by secondary interaction with additional HS side chains and transfer to a non-HSPG receptor for successful infection.

Highly Focused T Cell Responses in Latent Human Pulmonary Mycobacterium tuberculosis Infection

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.

Interleukin-7 or interleukin-15 enhances survival of Mycobacterium tuberculosis-infected mice.

ABSTRACT Both antigen-presenting cells and immune effector cells are required to effectively eradicate or contain Mycobacterium tuberculosis-infected cells. A variety of cytokines are involved to ensure productive “cross talk” between macrophages and T lymphocytes. For instance, infection of macrophages with mycobacteria leads to effective interleukin-7 (IL-7) and IL-15 secretion, and both cytokines are able to maintain strong cellular immune responses of α/β and γ/δ T cells. Here we show that either cytokine is able to enhance survival of M. tuberculosis-infected BALB/c mice significantly compared to application of IL-2, IL-4, or phosphate-buffered saline (as a control). Enhanced survival could be achieved only when IL-7 or IL-15 was delivered as a treatment (i.e., 3 weeks postinfection), not when it was administered at the time of infection. Increased survival of M. tuberculosis-infected animals was observed following passive transfer of spleen cells harvested from M. tuberculosis-infected, IL-7- or IL-15-treated animals, but not after transfer of spleen cells obtained from mice which received either cytokine alone. Histological examination revealed that IL-7 and IL-15 failed to significantly impact on the number and composition of granulomas formed or the bacterial load. Our data indicated that administration of IL-7 or IL-15 toM. tuberculosis-treated animals resulted in a qualitatively different cellular immune response in spleen cells as reflected by increased tumor necrosis factor alpha and decreased gamma interferon secretion in response to M. tuberculosis-infected antigen-presenting cells.

Peptide-specific CD8+ T-cell evolution in vivo: Response to peptide vaccination with Melan-A/MART-1

Monitoring of CD8+ T‐cell responses in cancer patients during peptide vaccination is essential to provide useful surrogate markers and to demonstrate vaccine efficacy. We have longitudinally followed CD8+ T‐cell responses in 3 melanoma patients who were immunized with peptides derived from Melan‐A/MART‐1. Recombinant HLA‐A2 tetramers loaded with the naturally presented Melan‐A/MART‐1 nonamer peptide (AAGIGILTV) and the Melan‐A/MART‐1 analog (ELAGIGILTV) were used in combination with phenotypical analysis for different T‐cell subsets including naive T cells, effector T cells, “true memory” T cells and “memory effector” T cells, based on CD45RA/RO and CCR7‐expression. At least in a single patient, T cells binding to the AAGIGILTV epitope were detected in naive, precursor (CD45RA+/CCR7+) CD8+ T cells, and CD8+ T cells binding to the analog ELAGIGILTV peptide were identified in the terminally differentiated (CD45RA+/CCR7−) T‐cell subset. Molecular and functional analysis of tetramer‐binding T cells revealed that the T‐cell receptor (TCR) repertoire was oligo/polyclonal in AAGIGILTV‐reactive T cells, but different and restricted to a few TCR clonotypes in ELAGIGILTV‐reactive T cells prior to vaccination. The TCR repertoire reactive with Melan‐A/MART‐1 peptide epitopes was broadened during vaccination and exhibited a different profile of cytokine release after specific stimulation: tetramer‐binding T cells from 2/3 patients secreted granulocyte/macrophage colony‐stimulating factor (GM‐CSF) and interferon‐γ but not interleukin‐2 (IL‐2) in response to Melan‐A/MART‐1 peptides. In the third patient, tetramer‐binding T cells secreted IL‐2 exclusively. Our results show that T‐cell responses to peptide vaccination consist of different T‐cell subsets associated with different effector functions. Complementary analysis for TCR CDR3 and cytokine profiles may be useful to define the most effective CD8+ T‐cell population induced by peptide vaccination.

Human Papillomavirus Type 16 E7 Peptide-Directed CD8+ T Cells from Patients with Cervical Cancer Are Cross-Reactive with the Coronavirus NS2 Protein

ABSTRACT Human papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are required for cellular transformation and represent candidate targets for HPV-specific and major histocompatibility complex class I-restricted CD8+-T-cell responses in patients with cervical cancer. Recent evidence suggests that cross-reactivity represents the inherent nature of the T-cell repertoire. We identified HLA-A2 binding HPV16 E7 variant peptides from human, bacterial, or viral origin which are able to drive CD8+-T-cell responses directed against wild-type HPV16 E7 amino acid 11 to 19/20 (E711-19/20) epitope YMLDLQPET(T) in vitro. CD8+ T cells reacting to the HLA-A2-presented peptide from HPV16 E711-19(20) recognized also the HLA-A2 binding peptide TMLDIQPED (amino acids 52 to 60) from the human coronavirus OC43 NS2 gene product. Establishment of coronavirus NS2-specific, HLA-A2-restricted CD8+-T-cell clones and ex vivo analysis of HPV16 E7 specific T cells obtained by HLA-A2 tetramer-guided sorting from PBL or tumor-infiltrating lymphocytes obtained from patients with cervical cancer showed that cross-reactivity with HPV16 E711-19(20) and coronavirus NS252-60 represents a common feature of this antiviral immune response defined by cytokine production. Zero of 10 patients with carcinoma in situ neoplasia and 3 of 18 patients with cervical cancer showed ≥0.1% 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 E711-19(20) CD8+-T-cell response may reflect cross-reactivity with a common pathogen and that variant peptides may be employed to drive an effective cellular immune response against HPV.

Mast Cells Expedite Control of Pulmonary Murine Cytomegalovirus Infection by Enhancing the Recruitment of Protective CD8 T Cells to the Lungs

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.

Longitudinal analysis of Mycobacterium tuberculosis 19-kDa antigen-specific T cells in patients with pulmonary tuberculosis: association with disease activity and cross-reactivity to a peptide from HIVenv gp120.

CD8+ T cells play a central role in immune protection against infection with Mycobacterium tuberculosis. One of the target epitopes for anti‐M. tuberculosis directedCD8+ T cells is the HLA‐A2‐restricted 19‐kDa lipoprotein peptide VLTDGNPPEV. T cell clones directed against this epitope recognized not only the nominal peptide ligand, but also a closely related peptide (VPTDPNPPEV) from the HIV envelope gp120 (HIVenv gp120) protein characterized by IFN‐γ release. This cross‐reactivity was confirmed in ex vivo in M. tuberculosis 19‐kDa tetramer‐sorted T cells from patients with tuberculosis and in HIVgp120 tetramer‐reactive T cells sorted from HIV+ patients. M. tuberculosis 19‐kDa antigen‐reactive T cells were present in HLA‐A2+ patients (10/10) with HIV infection with no evidence of M. tuberculosis infection, but they are absent in peripheral blood lymphocytes from healthy HLA‐A2+ individuals (10/10). M. tuberculosis 19‐kDa antigen‐reactive T cells were elevated in acute pulmonary tuberculosis, declined with response to therapy (7/10 patients) and resided in the terminally differentiated CD8+ T cell subset. CD8+ cross‐reactive T cells recognizing HIVenv or M. tuberculosis 19‐kDa antigens may contribute to pathogenesis in individuals co‐infected with both pathogens and may also present a marker for active tuberculosis.

Human Papillomavirus Type 33 E7 Peptides Presented by HLA-DR*0402 to Tumor-Infiltrating T Cells in Cervical Cancer

ABSTRACT Several characteristics make human papillomavirus (HPV) amenable to vaccination. Anti-HPV-directed vaccines are based on the observation that HPV E6 and E7 oncoproteins are constitutively expressed in HPV-positive cervical cancer and may serve as tumor rejection antigens. Five HPV types (16, 18, 31, 33, and 45) account for 80% of cervical cancer. Until now, the type of immune response capable of mediating an effective antitumor response has not been defined. In order to define the anticancer-directed immune response in situ, we characterized CD4+ and CD8+ sorted T cells from peripheral blood lymphocytes, freshly harvested tumor tissue, and tumor-infiltrating lymphocytes (TIL) from a patient with cervical cancer. The HLA-DR-restricted CD4+ T-cell receptor VB16-, VA10-, VA21-, and VA22-positive CD4+ T-cell line derived from TIL recognizes autologous HLA-DR*0402+(HPV33+) cervical cancer cells, as determined by gamma interferon secretion. Testing of different peptides spanning the E7 gene revealed that the HPV3373–87 peptide ASDLRTIQQLLMGTV represents the immunodominant epitope which can also be presented by the DR*0401 allele to TIL. Such major histocompatibility complex class II-presented peptides represent attractive candidates to augment T-cell responses directed against autologous tumor cells.

MHC class II Tetramer Guided Detection of Mycobacterium tuberculosis‐specific CD4+ T Cells in Peripheral Blood from Patients with Pulmonary Tuberculosis

Novel diagnostic tools are needed to diagnose latent infection and to provide biologically meaningful surrogate markers to define cellular immune responses against Mycobacterium tuberculosis (MTB). Interferon gamma‐based assays have recently been developed in addition to the more than 100‐year‐old tuberculin skin test (TST) for the immune diagnosis of MTB in blood. The advent of soluble MHC/peptide tetramer molecules allows to objectively enumerate antigen‐specific T cells. We identified novel MHC class II‐restricted MTB epitopes and used HLA‐DR4 tetrameric complexes to visualize ex vivo CD4+ T cells directed against the antigens Ag85B and the 19‐kDa lipoprotein, shared between MTB and other Mycobacterium species, and CD4+ T cells which recognize the MTB‐associated ESAT‐6 antigen. MTB‐reactive CD4+ T cells reside predominantly in the CD45RA+ CD28+ and CD45− CD28+ T‐cell subset and recognize naturally processed and presented MTB epitopes. HLA‐DR4‐restricted, Ag85B or ESAT‐6‐specific CD4+ T cells show similar dynamics over time in peripheral blood mononuclear cells (PBMC) when compared with CD8+ T cells directed against the corresponding HLA‐A2‐presented MTB epitopes in patients with pulmonary MTB infection and subsequent successful therapy. This was not found to be true for T‐cell responses directed against the 19‐kDa lipoprotein. The dissection of the cellular immune response in M. tuberculosis infection will enable novel strategies for monitoring MTB vaccine candidates and to gauge CD4+ T cells directed against MTB.