Ralf Agger

Two populations of splenic dendritic cells detected with M342, a new monoclonal to an intracellular antigen of interdigitating dendritic cells and some B lymphocytes

A monoclonal has been isolated that labels an intracellular antigen in dendritic cells and some B cells. The M342 hamster immunoglobulin was selected because it stained cells in the periarterial sheaths of spleen, the deep cortex of lymph node, and the thymic medulla –‐ the same regions in which one finds interdigitating cells, the presumptive in situ counterparts of isolated lymphoid dendritic cells. M342 labeled an antigen within granules of isolated dendritic cells, but only in cells that had been cultured for a day and not in fresh isolates. This extends recent findings that most freshly isolated spleen dendritic cells are located in the periphery of the white pulp nodule and may serve as precursors for the periarterial pool of interdigitating cells, the site for M342 staining in situ. By electron microscopic immunolabeling, the M342 antigen was found exclusively in a type of multivesicular body. M342 staining was not found in mononuclear phagocytes from blood and peritoneal cavity. Peritoneal B cells expressed M342+ granules, and upon appropriate stimulation splenic B cells developed reactive granules as well. We conclude that M342 is a strong marker for interdigitating cells. Its existence reveals intracellular specializations in the vacuolar system of antigen‐presenting cells including subsets of dendritic cells.

Human dendritic cell antigen presentation and chemotaxis are inhibited by intrinsic 25-hydroxy vitamin D activation

The immunomodulatory effects of vitamin D have primarily been investigated using the biologically active form 1,25-dihydroxy vitamin D3 (1,25-D3). It was recently demonstrated that dendritic cells (DC) are able to convert the inactive 25-hydroxy vitamin D3 (25-D3) into the active form via 1 alpha-hydroxylase. In this study, we set out to examine the possible consequences of this conversion on adaptive immune functions. Human monocyte-derived DC were matured by lipopolysaccharide (LPS) in the presence or absence of 25-D3. Subsequently, the conversion of 25-D3 into 1,25-D3, and the effects on surface marker expression, cytokine production, antigen-presenting capacity and chemotaxis of the DC were examined. 25-D3 was clearly converted into 1,25-D3 in the DC cultures and the process was accompanied by a reduced expression of CD80 (p<0.01), CD83 (p<0.01), CD86 (p=0.02), and HLA-DR (p=0.02). Also, the levels of the pro-inflammatory cytokines tumour necrosis factor (TNF) alpha (p=0.02) and interleukin (IL) 12 (p<0.01) were reduced. Interestingly, however, the CD14 expression (p<0.01) and the production of IL-1 beta (p<0.01) and IL-6 (p<0.01) increased. Thus, 25-D3 affected the delicate interplay between anti- and pro-inflammatory cytokines produced by the DC. Concurrently, 25-D3 reduced DC capacity to induce proliferation of antigen-specific T cells and DC chemotaxis towards chemokine (CC) ligand 21. This indicates that 25-D3 has a regulating function following intrinsic 1 alpha-hydroxylation, a mechanism that potentially has an immunomodulatory effect in vivo.

Potent Influence of Bovine Serum Proteins in Experimental Dendritic Cell-Based Vaccination Protocols

Typically autologous dendritic cells (DCs) intended for vaccination are generated from bone marrow derived stem cells or blood monocytes, loaded with antigen and introduced into the organism. However, addition of serum to DC culture medium is often necessary. Thus, serum proteins will be taken up and presented by the DCs together with other antigens. If heterologous serum is used, some of the serum proteins might be antigenic and thus induce a strong immune response when introduced in the recipient. We used the murine model of malignant melanoma, B16, to investigate the consequences of addition of fetal calf serum (FCS) to the medium for culturing murine DCs. The results showed that vaccination of mice with DCs cultured in vitro in the presence of FCS but in the absence of extraneous tumour antigens, protected the mice from challenge with B16 tumour cells similarly cultured in FCS. This protection could not be elicited by vaccination with FCS alone. Interestingly, the protective effect of DC vaccination was abolished when the challenging B16 tumour cells were free of serum proteins. Thus, these results show that DCs grown in the presence of FCS are able to induce immunity, which may be mistaken to be tumour immunity.

25-Hydroxy vitamin D3 modulates dendritic cell phenotype and function in Crohn’s disease

BackgroundIn Crohn’s disease (CrD), vitamin D may help to balance an exaggerated immune response and thereby improve the disease course. The immunomodulating effects depend on the activation of 25-hydroxy vitamin D3 (25-D3), into 1,25-dihydroxy vitamin D3 (1,25-D3). This activation has previously been shown to take place in dendritic cells (DC) from healthy individuals. We hypothesised that DC from CrD patients are able to regulate and control inflammatory responses through 25-D3 activation.MethodsDuring differentiation, monocyte-derived DC from 20 CrD patients were cultured with either 25-D3 or 1,25-D3 and matured with lipopolysaccharide (LPS). We examined DC surface marker expression, cytokine production, and the ability to induce cell proliferation in an allogeneic mixed leukocyte reaction.ResultsFollowing stimulation with LPS, DC exposed to either 25-D3 or 1,25-D3 exhibited lower expression levels of CD80, CD83, CD86, and HLA-DR and diminished TNF-α production compared with DC cultured with LPS alone. In contrast, CD14 expression and IL-6 production were higher following 25-D3 or 1,25-D3 treatment. Compared with LPS alone, both forms of vitamin D3 reduced the ability of DC to activate lymphocytes.ConclusionsFollowing stimulation with 25-D3, DC from CrD patients displayed a reduced response to LPS with a diminished capability to activate T cells compared with DC stimulated with LPS alone. These data indicate that intrinsic activation of 25-D3 occurs in DC from CrD patients and show that 25-D3 can modulate DC function in CrD. Our data suggest that vitamin D deficiency may contribute to the uncontrolled inflammatory process seen in CrD.

Targeted antiepidermal growth factor receptor (cetuximab) immunoliposomes enhance cellular uptake in vitro and exhibit increased accumulation in an intracranial model of glioblastoma multiforme.

Therapeutic advances do not circumvent the devastating fact that the survival rate in glioblastoma multiforme (GBM) is less than 5%. Nanoparticles consisting of liposome-based therapeutics are provided against a variety of cancer types including GBM, but available liposomal formulations are provided without targeting moieties, which increases the dosing demands to reach therapeutic concentrations with risks of side effects. We prepared PEGylated immunoliposomes (ILs) conjugated with anti-human epidermal growth factor receptor (EGFR) antibodies Cetuximab (α-hEGFR-ILs). The affinity of the α-hEGFR-ILs for the EGF receptor was evaluated in vitro using U87 mg and U251 mg cells and in vivo using an intracranial U87 mg xenograft model. The xenograft model was additionally analyzed with respect to permeability to endogenous albumin, tumor size, and vascularization. The in vitro studies revealed significantly higher binding of α-hEGFR-ILs when compared with liposomes conjugated with isotypic nonimmune immunoglobulin. The uptake and internalization of the α-hEGFR-ILs by U87 mg cells were further confirmed by 3D deconvolution analyses. In vivo, the α-hEGFR-ILs accumulated to a higher extent inside the tumor when compared to nonimmune liposomes. The data show that α-hEGFR-ILs significantly enhance the uptake and accumulation of liposomes in this experimental model of GBM suggestive of improved specific nanoparticle-based delivery.

Biodistribution and tumor localization of lymphokine-activated killer T cells following different routes of administration into tumor-bearing animals.

Abstract Purpose: The efficiency of adoptive cellular immunotherapy of cancer might depend on the number of effector cells that reach the malignant tissues. In the present study, the biodistribution and tumor localization of ex vivo lymphokine-activated T killer (T-LAK) cells was investigated. Methods: T-LAK cells were labeled with 125I-dU or the fluorescent dye tetramethylrhodamine isothiocyanate (TRITC) and transferred by intravenous, -cardiac, -portal or -peritoneal injection into normal (C57BL/6) mice or mice with syngeneic day-7 to day-12 B16 melanoma metastases established in various organs. The overall biodistribution of the T-LAK cells was measured by gamma counting and their tumor localization by fluorescence microscopy. Results: At 16 h after intravenous injection, the organ distribution of 125I-dU-labeled T-LAK cells was identical in normal and tumor-bearing animals. Fluorescence microscopy of lung tissue from animals receiving TRITC-labeled T-LAK cells revealed, however, a fivefold higher accumulation of T-LAK cells in lung metastases than in the surrounding normal lung tissue (1174 and 226 cells/mm2 respectively). Some pulmonary metastases were, however, resistant to infiltration. Very few intravenously injected cells redistributed to other organs or to tumors in these, since only 60 and 30 T-LAK cells/mm2 were found within metastases of the adrenal glands and the liver respectively. However, following injection of T-LAK cells via the left ventricle of the heart, a threefold increase (from 60 to 169 cells/mm2) in the number of transferred cells in metastases of the adrenal glands was observed. Moreover, following locoregional administration of T-LAK cells into the portal vein, tenfold higher numbers (from 30 to 400 cells/mm2) were found in hepatic metastases than were observed following intravenous or intracardiac injection. In the liver, a surprisingly large number of intraportally injected T-LAK cells (approx. 1300/mm2) were observed to accumulate in the perivascular spaces of the portal, but not the central veins. Even though some superficial ovarian and liver metastases were separated from the peritoneal cavity by only the peritoneal lining, no localization into these metastases was seen following intraperitoneal injection of the T-LAK cells. While treatment of tumor-bearing animals with T-LAK cells plus IL-2 reduced lung metastases by 76% as compared to treatment with IL-2 alone (P < 0.03), no significant reduction of liver metastases was seen. Conclusions: T-LAK cells are able to localize substantially into tumor metastases in various anatomical locations, but mainly following locoregional injection. This finding might have important implications for the design of future clinical protocols of adoptive immunotherapy based on T cells.

Production and secretion of immunoglobulins by in vitro-activated human B lymphocytes.

Activation induced by pokeweed mitogen in cultures of mononuclear cells from human blood was followed sequentially by simultaneous quantitation of live cells, thymidine incorporation, cells displaying cytoplasmic IgM, IgG, IgA or IgD, cells secreting IgM, IgG or IgA and cumulated IgM secretion. Maximal cellular activity was found after 7 days of culture, with means of 16000 IgM‐, 20700 IgG‐ and 9900 IgA‐secreting cells per 106 originally cultured cells. The cumulated IgM secretion after 21 days of culture averaged 10400 ng per 106 originally cultured cells. A close correlation was found between the number of IgM‐secreting cells and the cumulated IgM secretion.

T cell homing to tumors detected by 3D-coordinated positron emission tomography and magnetic resonance imaging.

A general hindrance to progress in adoptive cellular therapy is the lack of detailed knowledge of the fate of transferred cells in the body of the recipient. In this study, we present a novel technique for tracking of 124I-labeled cells in situ, which combines the high spatial resolution of magnetic resonance imaging with the high sensitivity and spatial accuracy of positron emission tomography. We have used this technique, together with determination of tissue radioactivity, flow cytometry, and microscopy, to characterize and quantitate the specific accumulation of transferred CD8+ T cells in tumor tissue in a mouse model. Transgenic CD8+ T cells, specific for the ovalbumin peptide SIINFEKL, were adoptively transferred to recipients carrying a subcutaneous tumor of the ovalbumin-expressing malignant melanoma cell line B16-OVA. The number of SIINFEKL-specific CD8+ cells in the tumor tissue was determined by flow cytometry each day for 8 consecutive days after adoptive transfer. From low levels 1 day after injection, their number gradually increased until day 5 when an average of 3.3×106 SIINFEKL-specific cells per gram tumor tissue was found. By applying the combined positron emission tomography/magnetic resonance imaging technique we were able to determine the position of the transferred, 124I-labeled SIINFEKL-specific T cells in 3 dimensions in recipient mice, and could demonstrate a highly significant accumulation of the 124I label in and around the subcutaneous B16-OVA tumors compared with normal tissue. Accumulation of 124I was significantly higher in B16-OVA than in B16 tumors not expressing the OVA antigen.

Comparison of membrane antigens of mouse dendritic cell types

The surface antigens of veiled cells (VC) isolated from the thoracic duct of mesenteric lymphadenectomized (MLNX) mice have been analyzed by means of monoclonal antibodies and compared with those of dendritic cells (DC) from the spleen, lymph node dendritic cells (LNDC) and peritoneal macrophages (PMO). All dendritic cell types were intensely stained with anti-Ia whereas only 11% PMO were labelled. Neither VC, DC or LNDC expressed the two antigens Mac-1 or F4/80 which are present on macrophages. 63% VC and 11-14% DC and LNDC expressed Mac-2, which is a macrophage subpopulation marker. From 11-23% of the dendritic cell types reacted with anti-Mac-3 which recognizes an antigen Mac-3 found on the surface of macrophages and interdigitating cells. Anti-33D1 which reacts with an antigen on DC was also cytotoxic towards a proportion of VC and DC. Anti-NLDC-145, which recognizes an antigen on interdigitating cells and VC from lymph nodes, reacted with 67% isolated LNDC and to a lesser extent with VC from the thoracic duct and DC from the spleen. The results are discussed in the light of possible relationships between these non-lymphoid cells.

Accumulation in tumor tissue of adoptively transferred T cells: A comparison between intravenous and intraperitoneal injection

Accumulation of T cells at the tumor is essential in cancer immunotherapy based on adoptive transfer of tumor-specific T cells. To gain further insight into the accumulation process and to evaluate the effect of using different routes of cell transfer, we investigated the accumulation of ovalbumin-specific CD8+ T cells (OT-I) injected either intravenously (IV) or intraperitoneally (IP) into mice carrying a subcutaneous tumor of the ovalbumin-expressing melanoma cell line B16-OVA. Maximal accumulation of the adoptively transferred cells in tumor tissue was observed 5 days after injection, irrespective of the injection route. The route of injection affected neither the total number of adoptively transferred cells found in tumor tissue nor the kinetics of this accumulation. In the spleen, however, the accumulation of adoptively transferred cells was clearly dependent on the injection route. IP injections resulted in a large number of adoptively transferred cells in the spleen on all days analyzed. In comparison, IV injection resulted in significantly fewer adoptively transferred cells in the spleen, and this number decreased over time. The route of injection affected neither the activation status of the adoptively transferred T cells that accumulated at the tumor site, nor the ability of these cells to control tumor growth. Two cell populations, SIINFEKL-tetramerLow(TetLow)CD69+CD25+ and TetHighCD69−CD25−, were present in tumor samples, whereas only TetHighCD69−CD25− cells accumulated in the spleen. In tumors, IV injection resulted in a higher fraction of adoptively transferred cells with an activated phenotype (TetLowCD69+CD25+) compared with IP injection.