Mogens H. Claesson

Vaccination with p53-peptide-pulsed dendritic cells, of patients with advanced breast cancer: report from a phase I study.

Peptides derived from over-expressed p53 protein are presented by class I MHC molecules and may act as tumour-associated epitopes. Due to the diversity of p53 mutations, immunogenic peptides representing wild-type sequences are preferable as a basis for a broad-spectrum p53-targeting cancer vaccine. Our preclinical studies have shown that wild-type p53-derived HLA-A2–binding peptides are able to activate human T cells and that the generated effector T cells are cytotoxic to human HLA-A2+, p53+ tumour cells. In this phase I pilot study, the toxicity and efficacy of autologous dendritic cells (DCs) loaded with a cocktail of three wild-type and three modified p53 peptides are being analysed in six HLA-A2+ patients with progressive advanced breast cancer. Vaccinations were well tolerated and no toxicity was observed. Disease stabilisation was seen in two of six patients, one patient had a transient regression of a single lymph node and one had a mixed response. ELISpot analyses showed that the p53-peptide–loaded DCs were able to induce specific T-cell responses against modified and unmodified p53 peptides in three patients, including two of the patients with a possible clinical benefit from the treatment. In conclusion, the strategy for p53-DC vaccination seems safe and without toxicity. Furthermore, indications of both immunologic and clinical effect were found in heavily pretreated patients with advanced breast cancer. An independent clinical effect of repeated administration of DCs and IL-2 can not of course be excluded; further studies are necessary to answer these questions.

The Role of Up-Regulated Serine Proteases and Matrix Metalloproteinases in the Pathogenesis of a Murine Model of Colitis

Proteinases are important at several phases of physiological and pathological inflammation, mediating cellular infiltration, cytokine activation, tissue damage, remodeling, and repair. However, little is known of their role in the pathogenesis of inflammatory bowel disease. The aim of this study was to assess the role of tissue proteases in a mouse model of colitis. Proteolytic activity was analyzed, using gel and in situ zymography, in colonic tissues from severe combined immunodeficient mice with colitis induced by transfer of CD4(+) T lymphocytes. Serine proteinase levels increased in colitic tissue, with major species of 23 kd, 30 kd, and 45 kd. Co-migration and inhibition studies indicated that the 23-kd proteinase was pancreatic trypsin and that the 30-kd species was neutrophil elastase. Matrix metalloproteinase (MMP)-9 expression, and MMP-2 and MMP-9 activation, was elevated in colitic tissues. Proteinase levels followed a decreasing concentration gradient from proximal to distal colon. Proteolysis was localized to infiltrating leukocytes in diseased severe combined immunodeficient mice. Transmural inflammation was associated with serine proteinase and MMP activity in overlying epithelium and with marked subepithelial proteolytic activity. The results demonstrate a clear elevation in the levels and activation of proteases in colitis, potentially contributing to disease progression through loss of epithelial barrier function.

One-Pot, Mix-and-Read Peptide-MHC Tetramers

Background Cytotoxic T Lymphocytes (CTL) recognize complexes of peptide ligands and Major Histocompatibility Complex (MHC) class I molecules presented at the surface of Antigen Presenting Cells (APC). Detection and isolation of CTLs are of importance for research on CTL immunity, and development of vaccines and adoptive immune therapy. Peptide-MHC tetramers have become important reagents for detection and enumeration of specific CTLs. Conventional peptide-MHC-tetramer production involves recombinant MHC production, in vitro refolding, biotinylation and tetramerization; each step followed by various biochemical steps such as chromatographic purification, concentration etc. Such cumbersome production protocols have limited dissemination and restricted availability of peptide-MHC tetramers effectively precluding large-scale screening strategies involving many different peptide-MHC tetramers. Methodology/Principal Findings We have developed an approach whereby any given tetramer specificity can be produced within 2 days with very limited effort and hands-on time. The strategy is based on the isolation of correctly oxidized, in vivo biotinylated recombinant MHC I heavy chain (HC). Such biotinylated MHC I HC molecules can be refolded in vitro, tetramerized with streptavidin, and used for specific T cell staining-all in a one-pot reaction without any intervening purification steps. Conclusions/Significance We have developed an efficient “one-pot, mix-and-read” strategy for peptide-MHC tetramer generation, and demonstrated specific T cell straining comparable to a commercially available MHC-tetramer. Here, seven peptide-MHC tetramers representing four different human MHC (HLA) class I proteins have been generated. The technique should be readily extendable to any binding peptide and pre-biotinylated MHC (at this time we have over 40 different pre-biotinylated HLA proteins). It is simple, robust, and versatile technique with a very broad application potential as it can be adapted both to small- and large-scale production of one or many different peptide-MHC tetramers for T cell isolation, or epitope screening.

Enteric bacterial antigens activate CD4+ T cells from scid mice with inflammatory bowel disease

Scid mice transplanted with CD4+ T cells from congenic donor mice develop a chronic and lethal inflammatory bowel disease (IBD) 2–3 months post‐transplantation. In the present study we have investigated the response of CD4+ T cells from scid mice with colitis against fecal extracts. Our results show that in contrast to CD4+ T cells from normal BALB/c mice, CD4+ T cells from scid mice with colitis proliferate strongly in response to antigen‐presenting cells (APC) pulsed with fecal extracts. The IBD‐associated T cells did not respond to either extracts from food antigens or fecal extracts from germ‐free mice, which indicates that they recognize bacterial antigens in the fecal extracts. CD4+ T cells isolated from the colonic lamina propria of scid mice 3 weeks post transplantation also responded vigorously to fecal extracts, demonstrating that reactive CD4+ T cells are present in the gut mucosa of transplanted scid mice prior to clinical manifestations of IBD. CD4+ T cells activated by fecal extracts produced high amounts of IL‐2 and IFN‐γ, intermediate amounts of IL‐4 and low amounts of IL‐10, consistent with a Th1 profile. The proliferative reactivity towards fecal extracts was restricted by MHC class II molecules and dependent on antigen processing, as the response could be blocked by anti‐MHC class II antibodies or a short fixation of the APC. This study demonstrates that class II‐restricted CD4+ Th1 cells, which recognize enteric bacterial antigens, infiltrate the gut mucosa and spleen of transplanted scid mice prior to and during the course of colitis.

Therapeutic dendritic cell vaccination of patients with metastatic renal cell carcinoma: a clinical phase 1/2 trial.

Therapeutic dendritic cell (DC) vaccination against cancer is a strategy aimed at activating the immune system to recognize and destroy tumor cells. In this nonrandomized phase 1/2 trial, we investigated the safety, feasibility, induction of T-cell response, and clinical response after treatment with a DC-based vaccine in patients with metastatic renal cell carcinoma. Twenty-seven patients with progressive cytokine-refractory metastatic renal cell carcinoma were vaccinated with DCs loaded with either a cocktail of survivin and telomerase peptides or tumor lysate depending on their HLA-A2 haplotype, and low-dose IL-2 was administered concomitantly. Tumor response, immune response, and serum IL-6 and YKL-40 were measured during treatment. Vaccine generation was successful in all patients and no serious adverse events were observed. None of the patients had an objective response but 13/27 patients obtained disease stabilization (SD) for more than 8 weeks. An antigen-specific immune response was demonstrated in 6/6 patients tested. Furthermore, significant alterations in serum YKL-40 and IL-6 were found during treatment. In conclusion, DC vaccination in our setting is feasible and without severe toxicity. Almost half of the patients obtained SD, and in more than 1/3 of the patients, SD persisted for more than 6 months. However, the evaluation of SD is difficult to interpret in the absence of a randomized trial and, therefore, these results should be interpreted with caution. Antigen-specific immune responses were observed in a subset of the treated patients.

Phenotypic and functional markers for 1α,25-dihydroxyvitamin D3-modified regulatory dendritic cells

The clinical use of dendritic cells (DCs) to induce antigen‐specific immune tolerance has been hampered by the lack of a widely acknowledged method for generating human regulatory DCs but even more so by the non‐existence of reliable markers. Thus, we set out to find reliable markers that can be measured with simple methods to identify regulatory DCs that are applicable for future clinical studies. Human DCs were generated from peripheral blood monocytes in the presence of 1α,25‐dihydroxyvitamin D3 (VD3), which gave rise to a phenotype that resembles immature DCs, with the exception of high CD14 and reduced CD1a on the cell surface. These VD3‐treated DCs exert a long‐lasting inefficient T cell stimulation and induce T cell hyporesponsiveness with regulatory potential. Importantly, such VD3‐treated DCs were readily distinguishable from untreated DCs by low levels of interleukin‐23 secretion and low expression of miR‐155 upon exposure to maturation stimuli. Furthermore, VD3‐treated DCs showed over‐expression of miR‐378. All these features can be used as robust markers for quality control of VD3‐treated regulatory DCs in future clinical studies.

Immunotoxicity of the pyrethroid insecticides deltametrin and α-cypermetrin

Abstract The synthetic pyrethroids deltametrin and α-cypermetrin were studied for effects on the immune system in 28-day studies in F344 male rats. Sixteen rats per group were dosed with either deltametrin 0, 1, 5, or 10 mg/kg body wt./day or α-cypermetrin 0, 4, 8, or 12 mg/kg body wt./day in soy bean oil by gavage. Haematology, bone marrow cell counts, tests for natural killer (NK) cell activity and mitogen response (Con A and STM) as well as quantitation of lymphocyte subpopulations were performed. Spleen cells from immunized animals (six animals/group) were tested for antibody production (SRBC-PFC). Volumes of lymphoid compartments of mesenteric lymph nodes and thymus were estimated using stereological methods. In the deltametrin study an effect was found in the groups receiving 5 or 10 mg/kg body wt. The effects were: increased weight of mesenterial lymph nodes, decreased thymus weight in immunized animals and an increase in numbers of SRBC-PFC and splenic NK cell activity. An effect on relative adrenal weight was seen in the 10 mg/kg body wt. group. No severe effects on the immune system was found. The lowest effect level of α-cypermetrin was 12 mg/kg body wt./day based on increased relative adrenal weight.

Increased intracellular Th1 cytokines in scid mice with inflammatory bowel disease

Severe combined immunodeficient (scid) mice engrafted with small pieces of full thickness gut wall from immunocompetent syngenic donors develop a chronic and lethal colitis. Lymphocytes from the lamina propria of engrafted mice were analyzed for phorbol ester/ionomycin‐induced cytokine production by intracellular staining. A 4 – 5‐fold increase in the fraction of IFN‐γ‐producing CD4+ lamina propria T cells was found in moderately and severely diseased mice when compared to healthy congenic C.B‐17 control mice. The number of IL‐2‐producing T cells was increased by approximately 2‐fold when comparing mice suffering from severe disease to healthy control mice. The fraction of TNF‐α positive CD4+ T cells was increased by a factor of two in both moderately and severely diseased mice. When analyzing Th2 cytokines, it was found that the levels of IL‐4‐producing CD4+ T cells was not altered in diseased animals, whereas the fraction IL‐10‐producing CD4+ T cells was reduced by a factor of 20. The combined data showed a 15 – 25‐fold increase in the Th1/Th2 ratio of diseased mice when compared to healthy control mice. No intracellular cytokines could be detected in lymphocytes not treated with phorbol ester/ionomycin. The present data identify a prominent role for Th1‐type T helper cells in the immunopathogenesis of gut wall graft‐induced inflammatory bowel disease in scid mice.

CD4+ T lymphocytes injected into severe combined immunodeficient (SCID) mice lead to an inflammatory and lethal bowel disease

Transfer of 2 × 105 congenic or semiallogenic purified TCRαβ+ CD4+ T cells to SCID mice leads to an infiltration of the recipient gut lamina propria and epithelium with a donor‐derived CD4+ T cell subset which induces a lethal inflammatory bowel disease (IBD) in the recipients. In contrast, IBD was not observed in SCID mice transplanted with unfractionated splenic cells. The earliest detectable pathological changes after CD4+ T cell transfer were proliferation and hypertrophy of the entire colonic epithelial layer, including increased mitotic activity, increased expression of epithelial nuclear proliferation antigen, and elongation of the crypts. Later on, massive mononuclear cell infiltration, hypertrophy of all layers of the colon and occasional epithelial ulcerations were observed. At this stage, accumulations of IgA, IgM and small numbers of IgG1‐, IgG2‐and IgG3‐secreting plasma cells were present in the lamina propria of both the small and large intestine. We conclude that low numbers of intraveneously transferred CD4+ T cells induce IBD in SCID mice. In the late stages of CD4+ T cell‐induced IBD, the colonic lamina propria becomes infiltrated with macrophages, neutrophils and plasma cells secreting IgA, IgM, and to a lesser degree IgG antibodies which might play an accessory role in the pathogenesis of IBD.

Dendritic cells in peripheral tolerance and immunity

Dendritic cells capable of influencing immunity exist as functionally distinct subsets, T cell‐tolerizing and T cell‐immunizing subsets. The present paper reviews how these subsets of DCs develop, differentiate and function in vivo and in vitro at the cellular and molecular level. In particular, the role of DCs in the generation of regulatory T cells is highlighted.