Keld Kaltoft

Establishment of two continuous T-cell strains from a single plaque of a patient with mycosis fungoides.

SummaryFrom a plaque biopsy of a patient with mycosis fungoides, two different continuous cell lines were established by including both IL-2 and IL-4 in culture medium. Both continuous cell lines appeared with characteristic chromosome markers after approximately 40 cell population doublings. The initial karyotype recognized in T cells from the skin biopsy was 46,XY and the karyotypes of the continuous cell strains were 46,XY, -18, + i(18q) and another with multiple chromosome aberrations as described in Sezary T-cell leukemia. Phenotyping with monoclonal antibodies and T-cell receptor analysis indicates that the latter cell strain represents a minority of T-cells in the plaque. Due to its many chromosomal aberrations it probably represents the malignant cell, which may be a central cell in the immune stimulation taking place in the skin.

Treatment of mycosis fungoides with recombinant interferon‐αza2 alone and in combination with etretinate

Eleven patients with mycosis fungoides (MF) were treated with recombinant alpha‐intcrfcron (rIFN‐α2a2) in combination with etretinate (seven patients) or alone. One patient, who also received etretinate, went into complete remission and remained without signs of MF after 18 months. Six patients experienced partial remission; one of these was treated with rIFN‐α2a alone and was clinically in complete remission, but had still a pteomorphic skin infiltrate. Two patients were non‐evaluable, and two stopped therapy due to progressive disease. Five patients discontinued therapy due to side‐effects although three had partial remission of their disease. Only four patients received 12 months therapy. The study shows that rIFN‐α2a in combination with etretinate or alone can induce remission of MF.

Adoptive transfer of allogeneic cytotoxic T lymphocytes equipped with a HLA-A2 restricted MART-1 T-cell receptor: a phase I trial in metastatic melanoma.

PURPOSE: We did a phase I dose-escalation trial to evaluate the feasibility and safety of intratumoral injections of C Cure 709, an allogeneic, continuous CTL cell line that, restricted by HLA-A2, recognizes MART-1-positive tumor cells through transduction with a T-cell receptor encoding gene. EXPERIMENTAL DESIGN: Cells were administered intratumorally in four dose levels ranging from 10(8) to 10(9) cells/d on days 1, 4, 7, 10, 14, and 28 of each treatment cycle to patients with metastatic melanoma. Main inclusion criteria were HLA-A2 tissue type, MART-1-positive tumor cells, and metastases suitable for ultrasound-guided injections. Patients were assessed for toxicity and response. Three to six patients were treated per dose level. Patients without progressive disease were offered up to three treatment cycles. RESULTS: Fifteen patients received a total of 24 treatment cycles with a total of 266 injections of C Cure 709. Toxicity was minor to moderate and most common injection site reactions were fever, fatigue, nausea/vomiting, and arthralgia/myalgia. Side effects disappeared in general within 24 hours. Toxicity was not dose dependent. One patient obtained a partial response, encompassing both metastases used and not used for intratumoral injections. Remaining patients did not achieve an overall response. In addition, we observed local regression of metastases used for injection in two patients and of metastases not used for injection in one patient. CONCLUSION: Intratumoral injections of C Cure 709 are feasible, safe, and capable of inducing tumor regression. Further investigation in a phase II setting is warranted.

The effect of etanercept and infliximab on the production of tumour necrosis factor α, interferon-γ and GM-CSF in in vivo activated intestinal T lymphocyte cultures

Abstract Background/aims: Infliximab (Ifx) is effective in the treatment of Crohns disease (CD) and rheumatoid arthritis (RA), etanercept (Eta) in RA but not in CD. The mechanisms underlying these clinical differences are not fully understood, but this knowledge could be valuable to identify responders and develop new treatments. This study compares Eta and Ifx in vitro regarding transmembrane tumour necrosis factor α (tmb-TNF-α) expression and interferon-γ (IFN-γ), TNF-α and granulocyte macrophage colony stimulating factor (GM-CSF) production in intestinal T lymphocytes. Methods: T lymphocyte cultures were established from biopsies from 10 CD patients and three healthy controls. The cytokine production and the expression of tmb-TNF-α were measured in the presence of Ifx/Eta. Results: Eta and Ifx downregulated the production of IFN-γ and GM-CSF in colonic T lymphocytes from CD patients and healthy controls. Both drugs bound tmb-TNF-α on activated T lymphocytes besides neutralising TNF-α, Eta less efficiently than Ifx (406 pg/ml (337–475); 133 pg/ml (119–147); p =0.004). TNF-α was detectable with the present assay in cell lines cultured in the presence of excess Eta. Conclusions: We have established that Eta is just as efficient as Ifx in downregulating IFN-γ and GM-CSF production in vitro and Eta bound to tmb-TNF-α. However, Eta bound the TNF-α molecule, important in CD, less efficiently than Ifx.

Immunocytochemical identification of the human α2-macrogiobulin receptor in monocytes and fibroblasts: Monoclonal antibodies define the receptor as a monocyte differentiation antigen☆

The alpha 2-macroglobulin receptor was recently purified from rat liver and human placenta. Three different monoclonal antibodies have now been raised against the human receptor and expression of the 440-kDa receptor protein is demonstrated in human placenta, fibroblasts, liver, and monocytes by immunoblot analysis. Flow cytometric studies showed that anti-alpha 2-macroglobulin receptor monoclonal antibodies bind to 90-100% of the blood monocyte population and not to other blood cells. This defines the alpha 2-macroglobulin receptor as a monocyte differentiation antigen, different from any of the classified leucocyte cluster determinants. Electron microscopic gold immunocytochemistry revealed the subcellular distribution of the receptor in human cultured monocytes and fibroblasts. In these cells, 18-33% of the gold particles were found on the outside of the plasma membrane, and in fibroblasts, especially, in coated invaginations. The intracellular receptors were mainly distributed in vesicles and tubular structures.

Mimotopes for tumor-specific T lymphocytes in human cancer determined with combinatorial peptide libraries

Mimotopes of a tumor‐associated T cell epitope were determined using randomized and combinatorial peptide libraries and a CD8+ T cell clone specific for the cutaneous T cell lymphoma cell line MyLa. Antigen recognition by this clone was found to be HLA‐B8 restricted. More than 80 % of HLA‐matched patients with cutaneous T cell lymphoma had mimotope‐specific CD8+ T cells in their peripheral blood. Mimotope‐specific T cells isolated and expanded from a patient lysed MyLa cells in in vitro assays thus demonstrating their cytolytic capacity and tumor specificity. Mimotope vaccination of a patient without detectable mimotope‐specific T cells induced frequencies of these cells of up to 1.82 % of the peripheral blood CD8+ T cells.

Nonsense suppressors of Saccharomyces cerevisiae can be generated by mutation of the tyrosine tRNA anticodon.

The yeast amber suppressor, SUP5-a, was previously shown to cause the insertion of tyrosine at the sites of UAG nonsense codons. Nucleotide sequencing established that this SUP5-a suppressor specifies a mutant tyrosine transfer RNA (tRNA) which has the anticodon CΨA instead of the normal GΨA, an alteration identical to that found in the tyrosine-inserting amber suppressor from Escherichia coli.

Common clonal chromosome aberrations in cytokine-dependent continuous human T-lymphocyte cell lines

Antigen-mediated T-cell proliferation is a transient phenomenon. Like other somatic cells, T lymphocytes generally show replicative senescence in vitro. However, we here show that cytokine-dependent continuous (immortal) T-cell lines can be established from skin biopsy specimens of inflammatory skin diseases. Continuous growth can be obtained by culturing T cells in medium supplemented with interleukin-2 and interleukin-4, but without antigen or antigen-presenting cells added. Loss of the T-cell antigen receptor complex is observed in some of the continuous T-cell lines. Most T-cell lines develop clonal chromosome aberrations during continuous growth. Aberrations for chromosomes 1, 2, 8, 16, and 18 are most commonly observed.

Development of Cutaneous Pseudolymphoma Following Ciclosporin Therapy of Actinic Reticuloid

The patient is a 57-year-old man with actinic reticuloid, who despite systemic prednisone, azathioprine, topical steroid, and sun-protective cream had to stay indoors in the summer of 1986. In February 1987 he was started on ciclosporin (CS), 5.5 mg per kg body weight, and skin symptoms did not develop as usual in spring and summer 1987. Following 4 months of successful therapy, he developed an indolent tumor on his right chin and parapsoriasis en plaque on the lower arms and legs. Histological examinations of the tumor showed an intense lymphoid infiltrate of a pseudolymphomatous type. The tumor regressed partly following discontinuation of CS, but additional radiation therapy had to be administered. His clinical symptoms of actinic reticuloid reappeared. Eight months after CS treatment he developed a malignant T cell lymphoma with metastasis in the regional lymph nodes on the neck. CS should not be used in diseases with potential premalignant features even though its therapeutic efficacy in actinic reticuloid was impressive.

Cytokine-Driven Immortalization of in vitro Activated Human T Lymphocytes

Like other normal human somatic cells, T lymphocytes are believed to have a finite in vitro life span. However, continuous T lymphocyte cell lines can often be established from chronic inflammatory skin diseases when the culture medium is supplemented with IL-2 and IL-4 but without antigen and accessory cells added. Based on the assumption that these continuous T lymphocyte cell lines were activated by antigen during the chronic inflammation taking place in vivo, I investigated whether peripheral blood T lymphocytes could be induced to cytokine-dependent continuous growth following antigen activation. Upon allostimulation, peripheral blood CD4+ T lymphocytes reproducibly escape from cellular senescence. These IL-2- and IL-4-dependent continuous T cell lines show high telomerase activity. Withdrawal of either IL-2 or IL-4 results in cell growth arrest concomitant with down-regulation of telomerase activity. When cultured continuously, these CD4+ human T lymphocytes gradually lose expression of CD28.