Markus J. Maeurer

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.

Host immune response in renal cell cancer: Interleukin-4 (IL-4) and IL-10 mRNA are frequently detected in freshly collected tumor-infiltrating lymphocytes

Human renal cell cancer (RCC) is clearly responsive to immunotherapy. Clinical responses may be mediated by “non-specific” (e. g. natural killer, NK, cells) or “specific” MHC-class-I-restricted tumor-specific CD8+ T lymphocytes. Typically RCC progresses, however, despite significant infiltration of various lymphoid cells. We examined freshly isolated RCC tumor-infiltrating lymphocytes (TIL) derived from seven RCC patients for cytokine expression by the polymerase chain reaction (PCR). Established RCC tumor cell lines derived from these RCC patients were negative for interleukin-2 (IL-2), IL-4, IL-10, and interferon γ and found to be positive for tumor necrosis factor α (TNFα), IL-6, IL-1β, granulocyte/macrophage-colony-stimulating factor (GM-CSF), and transforming growth factor β1 (TGFβ1) message as detected by PCR. An identical pattern of cytokine mRNA expression was identified in other long-term RCC lines and in normal human kidney cells upon culture, but not in two Wilms tumor cell lines tested. Short-term-, and long-term-established RCC lines, but not Wilms tumor lines, secreted substantial levels of GM-CSF, TNFα, IL-1β, and IL-6 as detected by enzyme-linked immunosorbent assay. Both RCC lines and Wilms tumor lines secreted TGFβ1. In comparison, normal kidney cells secreted IL-6 and GM-CSF, but not IL-1β, or TFGβ1 under identical in vitro cell culture conditions. We applied PCR-based methods to characterize the cytokine mRNA expression pattern in immune cells infiltrating into renal cell cancer without the need for expansion of such effector cells in vitro. Examining freshly collected RCC TIL by PCR from patients with primary cell cell cancer, we could demonstrate that such cells, but not lympho-mononuclear cells harvested from normal human kidney tissue, typically exhibit IL-4 and IL-10 mRNA expression.

Interleukin-7 (IL-7) in colorectal cancer: IL-7 is produced by tissues from colorectal cancer and promotes preferential expansion of tumour infiltrating lymphocytes.

Despite increasing survival rates for patients with colorectal cancer, additional treatment options are required, including active or passive immunotherapy for patients with metastatic disease. Freshly harvested colorectal cancer specimens and in vitro cultured colorectal cancer cell lines were examined for IL–7 protein secretion in order to examine the potential role of this cytokine in the interaction between tumour cells and the host immune system. Freshly harvested colorectal cancer specimens (21/21), or normal adjacent mucosa (3/3), as well as long‐term established colorectal cancer cell lines (3/4) exhibited IL‐7 mRNA expression as detected by RT‐PCR and confirmed by Southern Blot analysis. Freshly harvested colorectal cancer tissue (16/18), or long‐term established colorectal cancer cell lines (2/4) secreted in vitro IL‐7 as detected by ELISA. In contrast, breast, pancreatic, or lung cancer cell lines, as well as several haematopoietic cancer cells lines, tested negative for IL‐7 mRNA and protein. The authors tested different cytokines (IL‐1β, IL‐2, IL‐7, or a combination of IL‐1β/IL‐7) in vitro for the ability to expand tumour ‐ infiltrating T lymphocytes (TIL) from individual patients (n=9) with colorectal cancer. TIL populations were tested at day 14 after in vitro propagation for phenotypic analysis by FACS and for reactivity directed against NK and LAK sensitive target cells and autologous cancer cells as measured by cytotoxicity and cytokine release. TIL obtained from colorectal cancer lesions can be efficiently expanded in the presence of IL‐7, some (3/9) of which appear to exhibit autologous tumour recognition as measured by cytolytic effector functions and by detection of IFNγ and TNFα release. Detection of IL‐7 mRNA expression in colorectal cancer, in normal mucosa adjacent to tumour, as well as the ability of colorectal cancer tissue to secrete IL‐7, raises new questions about the biology of the host / tumour interactions in colorectal cancer.

Autologous human dendriphages pulsed with synthetic or natural tumor peptides elicit tumor-specific CTLs in vitro

The recent identification of tumor-associated antigens and tumor-associated antigen-derived peptide epitopes recognized by cytolytic T lymphocytes (CTLs) in the context of major hislocompatibility complex (MHC) class I molecules has prompted the development of peptide-based vaccines for the treatment of human cancers, particularly melanoma. The design of such clinical protocols requires an understanding of the inherent immunogenicity of the peptide(s) and a choice of a facilitating adjuvant promoting cellular immunity against these peptidcs. We have evaluated the abilities of a series of defined synthetic peptide epitopes derived from MART-1/Melan-A, gp100, tyrosinase, and MAGE-3 or unfractionatcd peptides natu-rally presented by melanoma MHC molecules to elicit HLA-A2-restricted and melanoma-reactive CTLs from the peripheral blood of normal donors or patients with metastatic melanoma. Autologous peripheral blood dendritic cells (DCs), which were easily generated from all donors when cultured in the presence of recombinant human interleukin-4 and recombinant human granulocyte-macrophage colony-stimulating factor were pulsed with melanoma peptides and used to “prime” and/or “boost” CTL cultures in vitro. Our results suggest that antimelanoma CTLs may be reproducibly generated in short-term in vitro cultures in this manner using either a subset of the defined synthetic peptides (MART-1/Melan-A27–35, MART-1/Melan-A32–40, gp100280–288, tyrosinase368–376 and MAGE-3271–279) or unfractionated peptides (containing both idiotypic and shared melanoma epitopes) derived from freshly isolated autologous melanoma lesions. These in vitro data support the use of autologous DCs prepulsed with such peptides as an appropriate antigen adjuvant delivery system in melanoma peptide-based vaccines.

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.

Human Papillomavirus 16 E6-specific CD45RA+ CCR7+ High Avidity CD8+ T Cells Fail to Control Tumor Growth Despite Interferon-?? Production in Patients With Cervical Cancer

We defined the nature of the cellular immune response in 5 women with human papillomavirus (HPV) 16+cervical carcinoma at a single time point when surgery was performed for treatment. To monitor the differences in T-cell recognition, 2 approaches of tetramer-guided technology were employed: (i) the in situ localization of major histocompatibility complex class I peptide complexes in the tumor lesions and (ii) the ex vivo sorting of HLA-A*0201–restricted and HPV16 E6-reactive T cells. CD8+ T cells from the periphery (peripheral blood lymphocytes), the tumor (tumor-infiltrating lymphocytes), and T cells harvested from draining lymph nodes (T-LN) were analyzed. HPV16 E6 tetramer-sorted lymphocytes from the different anatomic sites recognized an HLA-A*0201–restricted E6 peptide irrespective of the type of antigen-presenting cells used for stimulation as determined by interferon-γ production: autologous tumor cells, HLA-A*0201 surrogate antigen-presenting cells pulsed with the nominal peptide, and an HLA-A*0201–matched human dendritic cell line transgenic for HPV16 E6. Further analysis showed that the HPV16 E6-reactive CD8+ T cells were of high avidity defined by blocking with an anti-CD8-α specific monoclonal antibody. We found that HPV16 E6-reactive T cells reside preferentially within the CD45RA+ CCR7+ T-cell subpopulation of tumor-infiltrating lymphocyte, peripheral blood lymphocyte, and T-LN in cervical cancer patients, suggesting that successful immune surveillance of HPV16+ tumor cells in cervical cancer patients is impaired. The CD45RA+/CCR7+ phenotype of HPV antigen-reactive T cells may serve as an indicator of dysfunctional T cells, despite effective interferon-γ production in response to HPV antigens.

Tumor Antigen-specific T-cells are Present in the CD8αα+ T-cell Effector-memory Pool

CD8+ T-cell memory formation has recently been demonstrated to be associated with CD8αα homodimer expression by T-cells in mice. Up to now, the knowledge about the clinical significance of CD8αα+ T-cells in humans is limited. We assessed in longitudinally collected blood samples from patients with melanoma, who underwent a peptide-based vaccination, the role of CD8αα+ T-cells in tumor-specific cellular immune responses. Phenotypic analysis showed that the expression of CD8αα+ by T-cells was stable over time and associated with a CD45RA+/−CCR7− effector-memory profile. Melan-A/MART-1–specific T-cells were identified in the CD8αα+ T-cell compartment by tetramer technology. Detection of intracellular cytokine production (interleukin-2, interferon-γ, and tumor necrosis factor-α) upon phorbol 12-myristate 13-acetate-ionomycin stimulation in CD8αα+ and CD8αβ+ T-cells revealed that CD8αα+ T-cells show a unique cytokine production pattern (tumor necrosis factor-α and interferon-γ production) as compared with CD8αβ+ T-cells. T-cell receptor-CDR3 length analysis revealed that Melan-A/MART-1–specific CD8αα+ T-cells showed a similar T-cell receptor-repertoire as compared with Melan-A/MART-1–specific CD8αβ+ T-cells. Our results show that CD8αα+ T-cells represent a compartment of CD45RA+/− effector-memory cells in the peripheral circulation of patients with melanoma and suggest that CD8αα+ T-cells may originate from CD8+ T-cells that have down-regulated the expression of the CD8β chain. CD8αα+ and tetramer-specific T-cells may represent a valuable marker to gauge long-term antigen-specific T-cell memory.

Identification of neoepitopes recognized by tumor-infiltrating lymphocytes (TILs) from patients with glioma

Neoepitope-specific T-cell responses have been shown to induce durable clinical responses in patients with advanced cancers. We explored the recognition patterns of tumor-infiltrating T lymphocytes (TILs) from patients with glioblastoma multiforme (GBM), the most fatal form of tumors of the central nervous system. Whole-genome sequencing was used for generating DNA sequences representing the entire spectrum of ‘private’ somatic mutations in GBM tumors from five patients, followed by 15-mer peptide prediction and subsequent peptide synthesis. For each mutated peptide sequence, the wildtype sequence was also synthesized and individually co-cultured with autologous GBM TILs, which had been expanded in vitro with a combination of interleukin (IL)-2, IL-15 and IL-21. After seven days of culture, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and/or IL-17A production was measured by ELISA in culture supernatants, and used as an epitope-specific immune response readout. Mutated peptides that induced a strong cytokine response were considered to contain legitimate neoepitopes. TILs from 5/5 patients with GBM exhibited specific immune reactivity profiles to the nominal target peptides, defined by IFN-γ and/or TNF-α production, as well as IL-17A. Neoepitopes, defined by mutated peptides inducing IFN-γ and/or TNF-α production without or only minimal reactivity to the wildtype sequences, were found for each individual patient. CD8+ TILs dominated the patients’ responses to private neoepitopes. The present study shows that neoepitope-specific TIL reactivity constitutes an important arm of anti-tumor immune responses in patients with GBM, and thus a powerful tool for developing next-generation personalized immunotherapies.

Expression of TAAs in glioblastoma and expansion of anti-TAA -reactive T cells

Meeting abstracts Active cellular therapy (ACT) using ex-vivo expanded T cells from patients with cancer, obtained by apheresis, can represent a viable source for anti-cancer directed cellular therapy. TAAs expressed in glioblastoma may represent attractive targets for i) CARS, ii) transgenic T

Mycobacterium tuberculosis Vaccination Imprints on T-Cell Dynamics Associated with Mycobacterium tuberculosis Challenge in Rhesus

Non-human primate models aid to design novel vaccine strategies. We analyzed therefore the dynamics of cellular immune responses induced by a bacille Calmette-Guerin (BCG) / recombinant BCG (rBCG, perfringolysin plus Ag85A/B, TB10.4) prime, subsequent adenoviral (Ad35 expressing Ag85A/B and TB10.4) boosts followed by a challenge with M. tuberculosis (Mtb, Erdman strain) in peripheral blood mononuclear cells from rhesus macaques. T-cell compartment mobilization was defined by CD45RA/CCR7/CD27/CD28 expression on CD4+, CD8αα+ and CD8αβ+ T-cells and functional analysis (STAT-5 phosphorylation) was carried out using multicolor flow cytometry. CD4+ as well as CD8+ T-cells showed a marked decrease in IL-7 receptor alpha-chain (IL-7Rα) expression in vaccinated animals (n=12/12) after the first adenoviral boost, but not in control animals (who received saline). BCG- and rBCG- vaccinated animals showed a dramatic decrease of precursor (CD45RA+CCR7+) and subsequent increase of activated central memory (CD45RA-CCR7+) T-cells in the CD4+, CD8αα+ and CD8αβ+ T-cell compartments 2 weeks after Mtb challenge. In contrast, this pattern was observed in control animals only 7 weeks after Mtb challenge. Expression of IL-7Rα was markedly reduced in CD4+ T-cell subsets two weeks after Mtb challenge in BCG-vaccinated animals, yet not in rBCG-vaccinated animals or control animals. These data show that vaccination profoundly shapes the dynamics of immune memory T-cells associated with Mtb infection.