Hanni Höhn

Clonal expansion of melan a-specific cytotoxic T lymphocytes in a melanoma patient responding to continued immunization with melanoma-associated peptides

Peptides derived from human tumor antigens have been used in a number of clinical trials to induce specific immune responses against autologous tumors in cancer patients. Although favorable clinical results were observed in single patients, immune responses correlating with tumor regression were either not detected or in case of responses, the T‐cell specificity was difficult to demonstrate. In this study, we analyzed antigen‐specific T‐cell responses induced in the skin and in peripheral blood lymphocytes (PBL) in an HLA‐A2‐positive melanoma patient. The patient showed major regression of metastatic melanoma under continued immunization with peptides derived from the melanocyte differentiation antigens Melan A/MART‐1, tyrosinase and gp100/Pmel17. Based on the identification of different T‐cell receptor (TCR) families reactive with Melan A/MART‐1, we have demonstrated that i.d. immunization with peptides alone leads to oligoclonal expansion of Melan A/MART‐1‐specific cytotoxic T lymphocytes (CTL), detectable in local delayed‐type hypersensitivity (DTH) reactions and PBL. A monoclonal expansion of a Melan A/MART‐1‐specific TCR VB 16 CTL was reproducibly observed after in vitro stimulation with Melan A/MART‐1 peptides. The same TCR VB 16 CTL clone was detected in skin biopsies taken from vitiligo areas. Our findings provide strong evidence for the effective induction of specific T‐cell responses to Melan A/MART‐1 by i.d. immunization with peptide alone, which accounts for dermal depigmentation, specific cytotoxicity against Melan A/MART‐1‐expressing melanoma cells and clinical tumor regression. Int. J. Cancer 86:538–547, 2000.

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.

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.

Reduced T‐cell receptor CD3ζ‐chain protein and sustained CD3ε expression at the site of mycobacterial infection

Control of mycobacterial infection by the cellular immune system relies both on antigen‐presenting cells and on T lymphocytes. The quality of an effective cellular immune response is dependent on functional signal transduction residing in the cytoplasmic tails of the T‐cell receptor CD3 components. In order to investigate potential effects of mycobacteria on T‐cell receptor signalling, we examined the protein expression of T‐cell signal transduction molecules (CD3ζ, ZAP‐70, p59fyn, p56l ck). In Western blots of peripheral blood mononuclear cells of Mycobacterium tuberculosis infected patients, only the CD3ζ‐chain showed a marked reduction in protein expression. To investigate the situation in situ, immunoenzymatic and immunofluorescence stainings for CD3ε and CD3ζ expression were performed on sections of normal lymphoid tissue, M. leprae infected and sarcoid tissue. CD3ε and CD3ζ expression were similar with respect to intensity, localization and the number of cells stained in normal lymphoid tissue and in sarcoid granulomas. In contrast, the granulomas of M. leprae infected tissues showed a significantly reduced expression of CD3ζ compared to CD3ε. Using double immunofluorescence analysis, virtually no CD3ζ expression could be detected in comparison to the CD3ε expression in the lesions. Apparently, mycobacteria are capable of significantly reducing CD3ζ‐chain expression, which may be restored by cytokines. IL‐2‐enhanced ζ‐chain expression and T‐cell effector functions, defined by interferon‐γ release, in M. tuberculosis‐specific and human leucocyte antigen‐DR restricted CD4+ T cells isolated from granuloma lesions from patients with pulmonary tuberculosis. Because CD3ζ is essential for CD3 signalling and for eliciting T‐cell effector functions, reduced CD3ζ protein expression could result in altered signal transduction and inefficient T‐cell effector functions. Alternatively, reduced CD3ζ‐chain expression may protect T cells from repetitive TCR stimulation associated with anergy or apoptosis.

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.

Naturally processed and HLA-B8-presented HPV16 E7 epitope recognized by T cells from patients with cervical cancer

Several major histocompatibility complex (MHC) alleles have been reported to present peptides derived from the HPV16 E7 oncoprotein to T cells. We describe an overrepresentation of the HLA‐B8 allele (28.44%) in cervical cancer patients as compared to the MHC class I allele frequency in a local healthy control population (18.80%) and the identification of an HLA‐B8‐binding peptide TLHEYMLDL (HPV16 E77–15), which is able to drive HPV16 E7‐specific and MHC class I‐restricted T‐cell responses in peripheral blood lymphocytes from healthy individuals. TLHEYMLDL‐specific T cells recognize the naturally processed and presented peptide on HPV16+ cervical cancer cells transfected with the HLA‐B8 gene defined by IFN‐γ production. This peptide epitope is also recognized by freshly harvested tumor‐infiltrating T cells or T cells from tumor‐draining lymph nodes from patients with cervical cancer determined by flow cytometry as well as by tetramer in situ staining. HLA‐B8‐restricted HPV E77–15‐specific T cells reside predominantly in the CD8+ CD45RA+ CCR7+ precursor or in the differentiated CD8+ CD45RA+ CCR7− T‐cell population.

Differential MHC class II component expression in HPV-positive cervical cancer cells: Implication for immune surveillance

Effective eradication of human papillomavirus (HPV)‐positive tumors may require CD8+ and CD4+ T‐cell‐mediated immune responses. Ectopic expression of MHC class II surface molecules has been described in the context of cervical cancer, but coexpression with other components of the MHC class II antigen presentation pathway has not been addressed. We have evaluated the MHC class II antigen presentation pathway in malignant squamous epithelium of HPV+ cervical cancer lesions by in situ costaining HLA‐DR with CLIP or DMA/DMB. Cervical cancer cells exhibit 3 MHC class II phenotypes: (i) DR+/CLIP+ or DM+; (ii) DR+/CLIP‐ or DM‐; and (iii) DR‐/CLIP+ or DM+. The identical profile has been identified in HPV+ ME180 cells, which serve as a target for HLA‐DR4‐restricted and HPV68, E7‐specific CD4+ T cells. IFN‐γ pretreatment of ME180 cells, associated with differential trafficking of MHC class II molecules, is necessary for effective T‐cell recognition. Although proinflammatory cytokines may facilitate MHC class II‐restricted antigen recognition in tumor cells, different phenotypes of the MHC class II antigen presentation pathway may be associated with evasion from CD4+‐mediated cellular immune responses.

Improved detection of melanoma antigen-specific T cells expressing low or high levels of CD8 by HLA-A2 tetramers presenting a Melan-A/Mart-1 peptide analogue

MHC class I tetramers containing peptide epitopes are sensitive tools for detecting antigen‐specific CD8+ T‐cell responses. We demonstrate here that binding of HLA‐A2 tetramers to CD8+ T cells specific for the melanoma‐associated antigen Melan‐A/MART‐1 can be fine‐tuned by altering either the bound peptide epitope or residues in the α3 domain of HLA‐A2, which is important for CD8 binding. Antigen‐specific T cells expressing high levels of CD8 could be detected using HLA‐A2 tetramers containing the peptide AAGIGILTV, an epitope which is naturally processed and presented from Melan‐A/MART‐1. In contrast, low CD8‐expressing, antigen‐specific T cells could be detected efficiently only by using a mutated HLA‐A2 tetramer with an altered CD8 binding site or, less efficiently, using the wild‐type HLA‐A2 tetramer loaded with the peptide analogue ELAGIGILTV, which is superior in stimulating antigen‐specific T‐cell responses. Our results suggest ways to optimize the identification and expansion of antigen‐specific T cells with different requirements for the costimulatory CD8 molecule in facilitating T‐cell receptor binding to peptide variants.