Konrad Reske

Dendritic cells from mouse bone marrow: In vitro differentiation using low doses of recombinant granulocyte-macrophage colony-stimulating factor☆

Dendritic cells (DC) are potent stimulator cells that are crucially involved in primary T cell responses. Since DC comprise a minor population in lymphoid cell suspensions tedious and time consuming procedures are required for their preparation. This work outlines an in vitro culture system that promotes the differentiation of DC from unfractionated mouse bone marrow (BM) cells in the presence of low doses of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). Unlike co-induced BM-macrophages the outgrowing BM-DC express high levels of MHC class II molecules; they are negative for specific and nonspecific esterase and are nonphagocytic. A rapid one step purification procedure employing immunomagnetic bead selection yielded up to 95% BM-DC enriched cell fractions. The bead-selected BM-DC were powerful inducers of the allogeneic mixed leukocyte reaction. Thus, our findings demonstrate that low dose rGM-CSF-driven in vitro culture of BM cells provides convenient access to substantial numbers of DC and will greatly facilitate their further exploration.

Direct binding of autoimmune disease related T cell epitopes to purified Lewis rat MHC class II molecules

Abstract New strategies applied in the treatment of experimental autoimmune disease models involve blocking or modulation of MHC–peptide–TCR interactions either at the level of peptide–MHC interaction or, alternatively, at the level of T cell recognition. In order to identify useful competitor peptldes one must be able to assess peptide–MHC interactions. Several well described autoimmune disease models exist in the Lewis rat and thus this particular rat strain provides a good model system to study the effect of competitor peptldes. So far no information has been available on the peptide binding characteristics of the Lewis rat MHC class II RT1.BI molecule. We have now developed a biochemical binding assay which enables competition studies in which the relative MHC binding affinity of a set of non-labelled peptldes can be assessed while employing detection of blotlnylated marker peptides by chemllumlnescence. The assay is sensitive and specific. We have used this assay to determine the binding characteristics of several disease associated T cell determinants and their sequence analogues in the Lewis rat. Notably, most of the autoimmune disease associated peptide sequences tested were found to be intermediate to poor binders. Single amino acid substitutions at defined positions were sufficient to turn certain peptldes into good binders. These results are relevant to the design of competitor peptldes in the treatment of experimental autoimmune diseases.

An in vitro test for endocytotic activation of murine epidermal Langerhans cells under the influence of contact allergens

Several in vivo and in vitro studies have shown that contact sensitizing agents induce enhanced internalization of cell membrane constituents by epidermal Langerhans cells (LC). However the intracellular distribution of the internalized material has not yet been clearly defined. For this reason we investigated the uptake of gold-labeled antibodies against MHC class II molecules by cultured murine LC under the influence of various contact sensitizing agents, non-sensitizing analogues, and irritants. Antigen-antibody complexes were visualized by light microscopy using the silver enhancement technique and by pre-embedding electron microscopy. Viability was monitored by staining dead cells with propidium iodide. For light-microscopic evaluation of the intracellular distribution pattern of gold particles, a stimulation index was defined and used for the assessment of endocytotic activation. Untreated and solvent treated (control) cells exhibited an accumulation of internalized gold complexes into large aggregates composed of few intracellular vesicles. Cytoplasmic staining was absent and few gold particles were detectable in the endocytotic organelles under these conditions. In contrast to the non-sensitizing compounds DCNB and DNBSO3, which had no effect at all, treatment with subtoxic concentrations of the contact sensitizing agents DNFB, DNCB, TNCB, K2Cr2O7, NISO4 and p-phenylenediamine resulted in diffuse intracellular staining which was most pronounced in the submembraneous region. This was due to the numerous endocytotic vesicles which were closely associated with the cell membrane. Consequently a significant increase in the stimulation index was noted for these compounds. An irritant such as sodium lauryl sulphate used in subtoxic concentrations did not influence the intracellular distribution of internalized gold particles whereas toxic amounts of this compound induced a diffuse intracellular staining pattern indicative of membrane destruction. This approach represents a practical and reliable test for endocytotic activation of murine LC and may be useful for in vitro tests of the activating and possibly sensitizing properties of new chemical compounds.

Uptake of bead-adsorbed versus soluble antigen by bone marrow derived dendritic cells triggers their activation and increases their antigen presentation capacity.

The property to internalize particles has for long time been ascribed primarily to macrophages. DC in contrast were considered generally as phagocytosis negative. Fully mature DC which can be isolated from various tissues of the body do indeed not take up particulate material; however immature DC which arise in differentiating bone marrow cultures do exhibit phagocytic capacity1. Consistent with their immature phenotype epidermal Langerhans cells were also described to possess phagocytic potential2.

Isolation of the I-Ak core complex and its associated γ-polypeptide by affinity chromatography on monoclonal immuno-adsorbent 10-2.16 Sepharose CL-4B☆

Spleen cell-derived I-Ak antigens have previously been shown by us to be comprised of four noncovalently associated polypeptide chains (alpha, gamma, beta 1, beta 2). Here we report that pretreatment of the detergent solubilized four-chain structure with chaotropic ions yielded an alloantigenically reactive core complex (alpha/beta 1, beta 2) and a dissociated polypeptide gamma-chain which was not recognized by monoclonal antibody 10-2.16 (anti-I-Ak). The lack of alloantigenic determinants on gamma and the observation that gamma did not reassociate with alpha/beta 1, beta 2 upon removal of the chaotropic reagent suggested a rapid purification procedure for both moieties. A pure preparation of the serologically intact core complex was achieved by repeated affinity chromatography on hybridoma immunoadsorbent 10-2.16 Sepharose CL-4B. Concurrently its pertinent gamma-polypeptide was obtained in a highly enriched form and was purified to homogeneity by an additional gel electrophoretic step.

Complete coding nucleotide sequence of cDNA for the class II RT1.B βI chain of the Lewis rat

We have established the first full length cDNA clone for the beta light chain of the MHC class II alpha, beta heterodimer (isotype RT1.B) of the rat. Clone pLR beta 118 was obtained from a self-primed lambda gt10 cDNA library of IFN-tau treated bone marrow-derived macrophages of the Lewis rat. Subcloning of pLR beta 118 into a transcription vector with subsequent in vitro transcription and translation using the reticulocyte lysate system in the presence of microsomes followed by immunoprecipitation with mAb OX6 and two-dimensional gel electrophoresis revealed the intact RT1.B beta I-chain.

Processing Requirements for the Recognition of Insulin Fragments by Murine T cells

In this study we investigated aspects of antigen processing using insulin and insulin A chain-derived fragments as model antigens in Ab alpha Ak beta-restricted T-cell stimulation. Similarly to other proteins, the immunodominant region of insulin recognized by these T cells is limited in size. It is located on the insulin A chain and encompasses a portion of the molecule that is represented faithfully by peptide A1-14(SSO3-)3. Efficient presentation of intact insulin and its entire A chain is dependent on uptake and processing by APC. Whereas peptides stemming from various globular proteins are known to be presented to T cells by APC without requiring processing, this is not the case with A-chain fragment A1-14 (SSO3-)3. This observation suggested that, in addition to proteolytic degradation, other mechanisms might play a role in the processing of these antigens. Three cys-residues are located in close proximity to those amino acid residues of the insulin A chain that are inferred to participate in the specific interaction with MHC class II molecules and the TcR. In A-chain derivatives that are stimulatory for the T cells or in intact insulin these cys residues are engaged in disulfide bonds or are S-sulfonated. Both linkages can be reversibly modified by reaction with thiols. Functional data indicate that from intact insulin and from structurally distinct A-chain derivatives a closely similar or identical peptide is formed and bound to class II molecules for recognition by the T cells. The question arises as to whether, in this processed peptide, the cys residues are present in reduced form, engaged in disulfide bonds, or are modified in some other way. Taken together, these findings suggest that modification of cys residues or isomerization of disulfide bonds may play a role in insulin processing. It can be expected that other proteins carrying cys residues in their immunodominant peptides may show similar processing requirements. The inhibition of N-glycosylation of proteins by tunicamycin in APC blocked the processing and presentation of insulin and OvA whereas, under the same conditions, the presentation of a processing-independent peptide was not affected. Furthermore, an autoreactive T-cell clone was capable of recognizing tunicamycin-treated APC. Since the expression of class II molecules was found to be unaltered as demonstrated by cytofluorometric analysis the deficient N-glycosylation appears to have little influence on class II antigen-mediated T-cell recognition but interferes with uptake of antigen and/or its processing by APC.

Identification of major histocompatibility complex class II-associated peptides derived from freshly prepared rat Langerhans cells using MALDI-PSD and Edman degradation

The isolation and identification is described of MHC class II-bound peptides derived from Langerhans cells. A combination of preparative micro-HPLC, MALDI-MS, Edman degradation was used for determining the amino acid sequence of MHC-associated peptides. Sample handling was crucial because fractions containing trace amounts of material require immediate storage at -80 degrees C to prevent peptide losses.

Development of rat DC by in vitro culture of bone marrow cells.

Dendritic cells (DC) represent a subpopulation of leukocytes of bone marrow (BM) origin, involved in crucial immunological reactions. DC play a fundamental role in the primary immune response by stimulating quiescent T cells. In this study we describe an in vitro culture system to raise DC from unfractionated bone marrow (BM) cells of LEWIS rats in the presence of low doses of mouse recombinant GM-CSF, that was successfully used in previous work to culture mouse DC1,2,3.

Modulation of accessory cell function of immortalized bone marrow-derived macrophages by granulocyte/macrophage colony-stimulating factor

To generate cloned macrophage populations with sensitivity towards granulocyte/macrophage colony-stimulating factor (GM-CSF), bone marrow-derived macrophages (BMMΦ) were immortalized by transformation with SV40. A panel of transformed clones was established. The majority of clones represented independently derived transformants, as evidenced by restriction fragment length polymorphism using genomic DNA digested with EcoRI and TaqI and the 5.2 kb SV40 DNA for hybridization analysis. The cells belong to the macrophage lineage according to several criteria, e.g. the presence of nonspecific esterase, their phagocytic capacity and their morphology. Many clones were potent antigen-presenting cells (APC), without exogenous stimulation. Two clones, which did not act efficiently as APC when used untreated, were positively responsive to GM-CSF. GM-CSF stimulation of both clones resulted in potent APC capacity. I-Aα, I-Aβ and γ chain-specific transcripts were observed upon stimulation with GM-CSF, corresponding to detectable levels of class II surface display as revealed by cytofluorometric analysis. Thus the macrophage clones established will allow dissection of the differential effects of GM-CSF on the parameters of antigen presentation.