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M Assenmacher

Publications and source records attributed to M Assenmacher.

At least 19 recordsLinked to original sources
PubMed2001

Ex vivo IFN-gamma secretion by circulating CD8 T lymphocytes: implications of a novel approach for T cell monitoring in infectious and malignant diseases.

To elucidate the functional heterogeneity of Ag-specific T lymphocyte populations, we combined labeling of lymphocytes with MHC/peptide tetramers and a cell surface affinity matrix for IFN-gamma. Magnetic cell sorting of IFN-gamma-positive lymphocytes allowed the selective enrichment and identification of live Ag-specific cytokine-secreting cells by flow cytometry. Naive, memory, and effector Ag-specific populations were evaluated in healthy HLA-A2 individuals. Significant fractions of influenza- and CMV-specific cells secreted IFN-gamma upon challenge with cognate peptide, consistent with an effector/memory status. The sensitivity of the approach allowed the detection of significant numbers of CMV-specific IFN-gamma-secreting cells ex vivo (i.e., without Ag stimulation). This was not apparent when using previously described assays, namely, ELISPOT or intracellular IFN-gamma staining (cytospot). CD8+ T cells specific for the melamoma-associated Ag Melan-A/MART-1 did not produce IFN-gamma upon challenge with cognate peptide, reminiscent with their naive functional state in healthy individuals. In contrast, CD45RA(low) Melan-A/MART-1 tumor-specific cells from three of three melanoma patients presented levels of activity similar to those found for influenza- or CMV virus-specific lymphocytes, compatible with a functional differentiation into competent effector/memory T lymphocytes in vivo. Notably, a sizable fraction of Melan-A/MART-1-specific cells from a patient secreted IFN-gamma ex vivo following peptide-based vaccination. Thus, the high sensitivity of the assay provides a valuable tool to monitor effector T cell responses in different clinical situations.

Antigens, Neoplasm↗
PubMed2001

An instructive component in T helper cell type 2 (Th2) development mediated by GATA-3.

Although interleukin (IL)-12 and IL-4 polarize naive CD4(+) T cells toward T helper cell type 1 (Th1) or Th2 phenotypes, it is not known whether cytokines instruct the developmental fate in uncommitted progenitors or select for outgrowth of cells that have stochastically committed to a particular fate. To distinguish these instructive and selective models, we used surface affinity matrix technology to isolate committed progenitors based on cytokine secretion phenotype and developed retroviral-based tagging approaches to directly monitor individual progenitor fate decisions at the clonal and population levels. We observe IL-4-dependent redirection of phenotype in cells that have already committed to a non-IL-4-producing fate, inconsistent with predictions of the selective model. Further, retroviral tagging of naive progenitors with the Th2-specific transcription factor GATA-3 provided direct evidence for instructive differentiation, and no evidence for the selective outgrowth of cells committed to either the Th1 or Th2 fate. These data would seem to exclude selection as an exclusive mechanism in Th1/Th2 differentiation, and support an instructive model of cytokine-driven transcriptional programming of cell fate decisions.

Animals↗
PubMed2001

Rapid enrichment and detection of melanoma cells from peripheral blood mononuclear cells by a new assay combining immunomagnetic cell sorting and immunocytochemical staining.

Commonly used methods for detection of melanoma cells in blood, including RT-PCR and immunocytochemistry, display only a limited sensitivity and specificity. Reliable detection of less than one melanoma cell per ml of blood is hardly possible using these methods. To obtain greater sensitivity so that a single melanoma cell in up to 25 ml of blood can be detected (5 x 10(7) peripheral blood mononuclear cells, or PBMC), we developed a new assay for combined enrichment and immunocytochemical detection of disseminated melanoma cells from PBMC of patients with malignant melanomas. Melanoma cells are directly magnetically labeled using colloidal superparamagnetic microparticles approximately 60 nm in diameter conjugated to the anti-melanoma monoclonal antibody 9.2.27, with no reactivity to normal cells in blood. Magnetically labeled melanoma cells are enriched from PBMC by magnetic cell separation and detected by a new approach for immunocytochemical staining with monoclonal mouse anti-melanoma antibodies (anti-MelanA and HMB-45). The efficiency of this assay was demonstrated in a model system in which 5-500 tumor cells from the melanoma cell line SK-MEL-28 were seeded into PBMC samples from healthy donors containing 5 x 10(7) leukocytes. Mean recovery of the seeded tumor cells was 47.4 +/- 13.99% (n = 15). Applying the assay to 20-50 ml blood samples of patients with stage III-IV malignant melanomas, we were able to detect melanoma cells in two of eight patients (25%).

Antibodies, Monoclonal↗
PubMed2001

Regulation of expression of IL-4 alleles: analysis using a chimeric GFP/IL-4 gene.

CD4 cells from mice heterozygous for an IL-4 and a GFP/IL-4 gene frequently express a single allele. Analysis of IL-4 or GFP production by cells from recently primed Th2 cells indicates that essentially all are competent to transcribe either allele but have a low probability of doing so. By contrast, long-term Th2 clones show distinct and heritable ratios in the proportion of cells that express IL-4 or GFP. We conclude that in the course of Th2 priming an early efficient event renders both alleles capable of being inefficiently transcribed; a second, less frequent event occurs that renders one allele more competent, accounting for the differential expression of IL-4 and GFP in different clones.

Alleles↗
PubMed2001

Adoptive tumor therapy with T lymphocytes enriched through an IFN-gamma capture assay.

Successful adoptive T-cell therapy has been demonstrated in viral disease and selected forms of cancer. However, it is limited by the difficulty to efficiently isolate and amplify autologous tumor-reactive T-cell clones. Tetramers of major histocompatibility complex (MHC) class I and peptide have facilitated the characterization of CD8+ T cells specific for tumor-associated antigens. However, for adoptive T-cell therapy, MHC-tetramers have limitations: they require knowledge of tumor antigens, which is often not available; they select T cells with a single specificity, thereby posing risk for selection of tumor escape variants; they do not select for function, so that T cells may be anergic when isolated from cancer patients; and they do not allow the isolation of CD4+ T cells that can be essential for tumor rejection. Because interferon (IFN)-gamma is essential for tumor rejection, we isolated live T cells based on their IFN-gamma production. IFN-gamma secreted by previously activated T cells is retained on the cell surface, allowing their specific isolation and expansion. We show here that IFN-gamma+ but not IFN-gamma- T cells from tumor-immunized mice are cytolytic and mediate tumor rejection upon adoptive transfer. Importantly, tumor-specific T cells can be enriched from lymphocytes infiltrating human renal cell carcinoma by the IFN-gamma capture assay.

Animals↗
PubMed2001

Purification of cytomegalovirus-specific CD8 T cells from peripheral blood using HLA-peptide tetramers.

Cytomegalovirus (CMV) reactivation and disease remains an important clinical problem for patients after allogeneic stem cell transplantation. Impaired cellular immune control of viral replication is responsible for viral reactivation, and transfer of CMV-specific T cells from transplant donors can be effective in providing protection. Recent reports have indicated that the frequency of CMV-specific CD8(+) T cells in the peripheral blood of healthy donors is surprisingly high. Here we demonstrate that by using a combination of human leucocyte antigen (HLA) Class I-peptide tetramers and magnetic selection it is possible to select CMV-specific T cells from CMV antibody-positive individuals to high purity. Reliable purification of CMV-specific T cells up to 99.8% of CD8(+) cells was possible within hours, even when starting with a precursor frequency of < 0.1% of peripheral blood CD8(+) T cells. CMV-specific T cells remained functional after the selection process. This novel form of antigen-specific T-cell selection should facilitate the selection of T cells for cellular immunotherapy to treat or prevent CMV disease after transplantation. In addition, this technique could potentially be applied to many antigens including against other infective agents and tumour-specific antigens.

Adoptive Transfer↗
PubMed2000

Stat6-independent GATA-3 autoactivation directs IL-4-independent Th2 development and commitment.

The initial source of IL-4-inducing Th2 development and the mechanism of stable Th2 commitment remain obscure. We found the reduced level of IL-4 production in Stat6-deficient T cells to be significantly higher than in Th1 controls. Using a novel cell surface affinity matrix technique, we found that IL-4-secreting Stat6-deficient T cells stably expressed GATA-3 and Th2 phenotype. Introducing GATA-3 into Stat6-deficient T cells completely restored Th2 development, inducing c-Maf, Th2-specific DNase I hypersensitive sites in the IL-4 locus, and Th2 cytokine expression. The fact that GATA-3 fully reconstitutes Th2 development in Stat6-deficient T cells indicates it is a master switch in Th2 development. Finally, GATA-3 exerts Stat6-independent autoactivation, creating a feedback pathway stabilizing Th2 commitment.

Animals↗
PubMed1999

Correlation analysis between frequencies of circulating antigen-specific IgG-bearing memory B cells and serum titers of antigen-specific IgG.

Recent studies in mice have indicated that the long-lasting specific antibody responses seen after vaccination are probably due to the existence of long-lived plasma cells. Therefore, because the maintenance of humoral immunity does not necessarily reflect continuous restimulation of long-lived memory B cells, the question arises as to what degree antibody immunity, as determined by measuring serum immunoglobulin titers against a particular antigen, and memory B cell immunity, as determined by counting circulating memory B cells with specificity for that same antigen, correlate. Here, using a new assay combining two-step immunomagnetic enrichment with multiparameter flow cytometry to detect, enumerate and characterize antigen-specific memory B cells, we show for tetanus toxin C-fragment in blood of normal tetanus toxoid vaccinized donors, and for wasp venom phospholipase A1B in blood of wasp venom-allergic donors undergoing an immune therapy with wasp venom, that there is no statistically significant linear correlation between the frequencies of circulating antigen-specific IgG-bearing memory B cells and the serum titers of antigen-specific IgG. This lack of a statistically significant linear correlation is in accordance with the idea that B memory cells and plasma cells represent independently controlled forms of immunological memory.

B-Lymphocytes↗
PubMed1999

Enrichment and detection of live antigen-specific CD4(+) and CD8(+) T cells based on cytokine secretion.

Following appropriate antigen-specific stimulation, CD4(+) and CD8(+) T lymphocytes rapidly express cytokines. Based on this stimulation-induced cytokine secretion and using cell surface affinity matrix technology we have developed a new method that permits specific, rapid and efficient detection, isolation and characterization of live antigen-specific CD4(+) and CD8(+) T lymphocytes. The power of this technique is demonstrated here for HLA-A0201-restricted influenza matrix protein peptide 58-66-specific CD8(+) cytotoxic T lymphocytes, influenza A virus- and recombinant tetanus toxin C fragment-specific Th1 cells and tetanus toxoid-specific Th2 cells.

Antigen Presentation↗
PubMed1998

Commitment of individual Th1-like lymphocytes to expression of IFN-gamma versus IL-4 and IL-10: selective induction of IL-10 by sequential stimulation of naive Th cells with IL-12 and IL-4.

Commitment of Th lymphocytes to the Th1 phenotype, as characterized by the expression of the major proinflammatory cytokine IFN-gamma, may be critically involved in the establishment of chronic inflammation and inflammatory autoimmune disease. To date, it has been shown that in IL-12-stimulated murine Th cell lines containing a major fraction of Th1 cells, Th2 cells can be induced by IL-4 until about 2 wk after initial activation, but not later. Here we analyze, based on the magnetic isolation of viable Th1 cells according to their specific expression of IFN-gamma, the cytokine commitment of individual Th1 cells. After activation of naive Th cells with Ag and IL-12 for up to 5 wk, isolated IFN-gamma-producing cells were restimulated with Ag and IL-4. Within the first 3 to 4 wk of IL-12 stimulation, some IFN-gamma+ cells stopped expression of IFN-gamma when restimulated with IL-4. However, within only 1 to 2 wk of IL-12 stimulation, few IFN-gamma+ cells could be converted to produce IL-4. Others continued to express IFN-gamma and thus were already committed to a proinflammatory, Th1-like phenotype. Surprisingly, within 3 wk of IL-12 stimulation, many of the IFN-gamma-producing cells responded to IL-4 restimulation by expression of IL-10, but neither IFN-gamma nor IL-4, i.e., by conversion to a suppressive, anti-inflammatory phenotype.

Animals↗
PubMed1998

Sequential production of IL-2, IFN-gamma and IL-10 by individual staphylococcal enterotoxin B-activated T helper lymphocytes.

Upon primary activation, T helper (Th) cell populations express different cytokines transiently and with different kinetics. Stimulation of naive murine splenic Th cells with the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) in vitro results in expression of IL-2, IFN-gamma and IL-10 with fast, intermediate and slow kinetics, respectively. This first report of a functional analysis of cells separated alive according to cytokine expression shows that these cytokines are not produced by different Th cell subpopulations, but can be expressed sequentially by individual Th cells. Th cells, activated with SEB for 1 day and isolated according to expression of IL-2, using the cellular affinity matrix technology, upon continued stimulation with SEB later secrete most of the IFN-gamma and IL-10. Likewise, after 2 days of SEB culture, cells expressing IFN-gamma, separated according to specific surface-associated IFN-gamma as detected by magnetofluorescent liposomes, 1 day later secrete IL-10. Thus, individual Th1 cells can contribute to the control of their own IFN-gamma expression by sequential expression of first IL-2, supporting their proliferation, and later IL-10, down-regulating the production of IFN-gamma-inducing monokines and limiting the pro-inflammatory effects of IFN-gamma.

Animals↗
PubMed1998

Systemic T-cell unresponsiveness during rush bee-venom immunotherapy.

By rush bee-venom immunotherapy, subjects reacting allergically to the venom can be effectively anergized, although the mechanism of action is not known. Here we analyzed the systemic effects of rush desensitization on the T cells of allergic patients. In most patients, we found reduced frequencies of T cells recalled to express CD69 and the cytokines interleukin (IL)-4 and interferon-gamma (IFN-gamma) after stimulation of peripheral blood mononuclear cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin, as compared with normal donors. These frequencies are progressively reduced during immunotherapy. The frequency of cells expressing IL-2 does not change. A few patients show a different response to immunotherapy: frequencies of cells expressing CD69, IL-4, or IFN-gamma do not change, and remain similar to those of normal donors. However, the frequency of cells able to express IL-2 is increased. The analysis of cytokine expression in CD45RO+ vs CD45RO- T-cell populations revealed differences between normal and allergic donors. In allergic patients, higher frequencies of IL-4- and IFN-gamma-expressing cells among the CD45RO- subpopulation were found than in normal donors. This situation is not modified by immunotherapy. The results reveal a certain degree of heterogeneity in the response of allergic patients to bee-venom rush immunotherapy; however, all are clearly differentiated from normal controls as judged by cytokine expression of CD45RO- T cells. In most allergic patients, a considerable percentage of Th cells become unresponsive to mitogenic stimulation, and may be responsible for the desensitization itself.

Adult↗
PubMed1998

Analysis and sorting of T cells according to cytokine expression.

Functional distinct populations of T helper cells can be defined according to the expression of cytokines. A remarkable diversity of cytokine expression has been demonstrated in single T cells even from clonal populations and up to now no stable surface markers have been described which on the single-cell level directly correlate with the secretion of a certain cytokine. Since cytokines are the major parameters of T cell effector function we have developed strategies which now allow to separate cells according to the specific cytokines they secrete. The "affinity matrix technology"--secreted molecules are relocated to the cell surface by an artificially created antibody matrix--allows to isolate cells according to a distinct secreted product. In addition to this universal, but laborious technology, we could demonstrate by high-sensitivity immunofluorescence that IFN-gamma and IL-10 but not IL-2 and IL-4 are specifically expressed in low copy number on the surface of cells secreting these cytokines. Surface IFN-gamma and IL-10 are the first unambiguous surface markers for pro-inflammatory IFN-gamma-secreting Th 0/1 cells and IL-10-producing anti-inflammatory Th2/3 cells. We have used purified cytokine-secreting T cells to study in individual T cells the sequential production of IL-2, IFN-gamma, and IL-10, the stability of IFN-gamma expression and the selective homing of IFN-gamma-producing cells into inflamed tissues.

Animals↗
PubMed1997

Surgery in patients with coronary artery disease--silent ischaemia during transurethral resection of tumors of prostate or bladder.

BACKGROUND: Asymptomatic episodes of myocardial ischemia in clinically stable patients seem to occur frequently and may hint at a worse prognosis. HYPOTHESIS: This study was undertaken to determine whether surgical patients with coronary artery disease (CAD) have a higher risk of cardiac ischemia during the perioperative period compared with the late postoperative period and compared with patients without CAD. METHODS: In all, 14 patients with and 14 patients without CAD were examined by Holter monitoring during the perioperative and three days later during the postoperative periods for the presence of ST-segment depression as a marker of silent myocardial ischemia. RESULTS: While patients without CAD did not show ST-segment depression, patients with CAD were found to have had 143 episodes of ST-segment depression, 49% in the perioperative and 51% in postoperative recordings. CONCLUSION: Though patients were asymptomatic with antianginal therapy, there were episodes of ST-segment depression indicating silent myocardial ischemia in patients with CAD. Surgical interventions such as transurethral resection of tumors of prostate or bladder did not produce an increase of ischemic burden registered by Holter monitoring.

Aged↗
PubMed1996

Specific expression of surface interferon-gamma on interferon-gamma producing T cells from mouse and man.

Interferon (IFN)-gamma is a potent immunoregulatory protein secreted by CD4+ and CD8+ T cells and by natural killer cells. Here, we show that IFN-gamma is specifically displayed at a low concentration on the cell surface of those activated T cells from mouse and man which express IFN-gamma. It is transiently expressed on the cell surface with kinetics similar to those of intracellular IFN-gamma expression. Detectable surface IFN-gamma is not expressed by activated T helper (Th) cells producing other cytokines but which do not express IFN-gamma. Thus, surface IFN-gamma is the first available marker for live T lymphocytes expressing IFN-gamma, e.g. Th1 cells.

Animals↗
PubMed1995

Analysis and sorting of live cells according to secreted molecules, relocated to a cell-surface affinity matrix.

We have developed a technology for analysis and sorting of live cells according to secreted molecules. An artificial affinity matrix, specific for the secreted product of interest, is created on the cell surface, and the cells are allowed to secrete for a defined time period. The secreted molecules bind to the affinity matrix on the secreting cell and are subsequently labeled with specific fluorescent or magnetic staining reagents for cytometric analysis and cell sorting. Crossfeeding of the secreted products to other cells is prevented by decreasing the permeability of the incubation medium. This approach will have a wide range of applications in biotechnology and biomedical research. Here, we describe analysis and sorting of hybridoma cells, according to secreted antibodies, and of activated T lymphocytes, according to secreted cytokines.

Animals↗