Fridtjof Lund-Johansen

Plasmacytoid Dendritic Cells (Natural Interferon- α/β-Producing Cells) Accumulate in Cutaneous Lupus Erythematosus Lesions

Plasmacytoid dendritic cell (P-DC) precursors in peripheral blood produce large amounts of interferon (IFN)-alpha/beta when triggered by viruses. However, when incubated with interleukin-3 and CD40 ligand, the same precursors differentiate into mature DCs that stimulate naïve CD4(+) T cells to produce Th2 cytokines. We recently reported that P-DCs accumulate in nasal mucosa of experimentally induced allergic rhinitis, supporting a role for this DC subset in Th2-dominated inflammation. Here we examined whether P-DCs accumulate in cutaneous lesions of lupus erythematosus (LE), a disorder associated with increased IFN-alpha/beta production. Our results showed that P-DCs were present in 14 out of 15 tissue specimens of cutaneous LE lesions, but not in normal skin. Importantly, the density of P-DCs in affected skin correlated well (r(s) = 0.79,P < 0.0005) with the high number of cells expressing the IFN-alpha/beta-inducible protein MxA, suggesting that P-DCs produce IFN-alpha/beta locally. Accumulation of P-DCs coincided also with the expression of L-selectin ligand peripheral lymph node addressin on dermal vascular endothelium, adding further support to the notion that these adhesion molecules are important in P-DC extravasation to peripheral tissue sites. Together, our findings suggested that P-DCs are an important source of IFN-alpha/beta in cutaneous LE lesions and may therefore be of pathogenic importance.

Regular ArticlesPlasmacytoid Dendritic Cells (Natural Interferon- α/β-Producing Cells) Accumulate in Cutaneous Lupus Erythematosus Lesions

Plasmacytoid dendritic cell (P-DC) precursors in peripheral blood produce large amounts of interferon (IFN)-alpha/beta when triggered by viruses. However, when incubated with interleukin-3 and CD40 ligand, the same precursors differentiate into mature DCs that stimulate naïve CD4(+) T cells to produce Th2 cytokines. We recently reported that P-DCs accumulate in nasal mucosa of experimentally induced allergic rhinitis, supporting a role for this DC subset in Th2-dominated inflammation. Here we examined whether P-DCs accumulate in cutaneous lesions of lupus erythematosus (LE), a disorder associated with increased IFN-alpha/beta production. Our results showed that P-DCs were present in 14 out of 15 tissue specimens of cutaneous LE lesions, but not in normal skin. Importantly, the density of P-DCs in affected skin correlated well (r(s) = 0.79,P < 0.0005) with the high number of cells expressing the IFN-alpha/beta-inducible protein MxA, suggesting that P-DCs produce IFN-alpha/beta locally. Accumulation of P-DCs coincided also with the expression of L-selectin ligand peripheral lymph node addressin on dermal vascular endothelium, adding further support to the notion that these adhesion molecules are important in P-DC extravasation to peripheral tissue sites. Together, our findings suggested that P-DCs are an important source of IFN-alpha/beta in cutaneous LE lesions and may therefore be of pathogenic importance.

Experimentally Induced Recruitment of Plasmacytoid (CD123high) Dendritic Cells in Human Nasal Allergy

Recent evidence suggests that the previously enigmatic cell type designated plasmacytoid monocytes can function as dendritic cells and contribute substantially to both innate and adaptive immunity. This cell type has previously been described only in bone marrow, blood, and organized lymphoid tissue, but not at effector sites with direct Ag exposure such as the mucosae. Plasmacytoid dendritic cells (P-DCs) matured in vitro can induce T cells to produce allergy-promoting Th2 cytokines; therefore, their possible occurrence in nasal mucosa during experimentally elicited allergic rhinitis was examined. Patients with silent nasal allergy were challenged topically with relevant allergen daily for 7 days. Biopsy specimens as well as blood samples were obtained before and during such provocation, and P-DCs were identified by their high expression of CD123 (IL-3R α-chain), together with CD45RA. Our results showed that P-DCs were present in low and variable numbers in normal nasal mucosa but increased dramatically during the allergic reaction. This accumulation concurred with the expression of the L-selectin ligand peripheral lymph node addressin on the mucosal vascular endothelium. The latter observation was particularly interesting in view of the high levels of L-selectin on circulating P-DC precursors and of previous reports suggesting that these cells can enter organized lymphoid tissue via high endothelial venules (which express peripheral lymph node addressin constitutively). Together, our findings suggested that P-DCs are involved in the triggering of airway allergy and that they are directed to allergic lesions by adhesion molecules that normally mediate leukocyte extravasation in organized lymphoid tissue.

Differential surface expression of cell adhesion molecules during granulocyte maturation.

Individual steps of granulocyte maturation, such as lineage commitment, proliferation, maturation, and migration from the marrow to the peripheral blood, may be influenced by distinct interactions with the bone marrow stroma. To identify candidates of membrane components involved in maturational stage‐specific interactions, we studied changes in the expression of cell adhesion molecules along the granulocyte maturational pathway. Three‐color flow cytometric measurements were used to measure levels of cell adhesion molecules along this pathway. The α chains of VLA‐4 (CD49d) and VLA‐5, the inte‐ grin 01 chain (CD29), and CD31 (PECAM‐1) were expressed in high density on all early myeloid cells but down‐modulated during postproliferative maturation. CD44 and L‐selectin were expressed on CD34+ myeloid progenitor cells and mature granulocytes but down‐ modulated during the intermediate stages of maturation. The granulocyte receptor for endothelial selectins, sLex, was specifically expressed by myeloid progenitor cells. sLex was down‐modulated during the intermediate stages of granulocyte maturation but up‐regulated again during terminal maturation. In contrast, CD67, a putative granulocyte adhesion molecule, was negative on progenitors, transiently up‐regulated during the intermediate stages of maturation, and almost absent from the surface of mature granulocytes. These results show that each stage of granulocyte maturation is associated with the expression of a unique combination of cell adhesion molecules. L‐selectin, CD44, and 01 integrins were regulated as previously described for immature lymphopoietic cells and may therefore play general roles in the compartmen‐ talization and development of leukocytes. In contrast, sLex and CD67 were specifically expressed by myeloid cells and could be specifically important for compartmen‐ talization of distinct phases of granulocyte maturation.

Targeting of cancer neoantigens with donor-derived T cell receptor repertoires

Outsourcing cancer immunotherapy Successful cancer immunotherapy depends on a patients T cells recognizing tumor-specific mutations and then waging a lethal attack. Despite tumors harboring many mutations, most individuals have very few T cells that respond to these so-called “neo-antigens.” Strønen et al. isolated T cells from healthy donors that responded to predicted neo-antigens expressed by melanomas taken from three patients, sometimes including neo-antigens that the patients own T cells ignored (see the Perspective by Yadav and Delamarre). Testing whether such an outsourcing strategy could improve clinical outcomes will be an important next step. Science, this issue p. 1337; see also p. 1275 T cells from healthy human donors may be an important resource for outsourcing cancer immunotherapy. Accumulating evidence suggests that clinically efficacious cancer immunotherapies are driven by T cell reactivity against DNA mutation–derived neoantigens. However, among the large number of predicted neoantigens, only a minority is recognized by autologous patient T cells, and strategies to broaden neoantigen-specific T cell responses are therefore attractive. We found that naïve T cell repertoires of healthy blood donors provide a source of neoantigen-specific T cells, responding to 11 of 57 predicted human leukocyte antigen (HLA)– A*02:01–binding epitopes from three patients. Many of the T cell reactivities involved epitopes that in vivo were neglected by patient autologous tumor-infiltrating lymphocytes. Finally, T cells redirected with T cell receptors identified from donor-derived T cells efficiently recognized patient-derived melanoma cells harboring the relevant mutations, providing a rationale for the use of such “outsourced” immune responses in cancer immunotherapy.

Epidermal Growth Factor Receptor Inhibition Induces Trichomegaly

Case report. The epidermal growth factor receptor (EGFR) is important for normal skin development and function (1 /3). Binding of the ligands EGF or TGF-a induces dimerization of the receptor and activation of the intracellular tyrosine kinase, resulting in downstream activation of different signalling pathways including MAP-kinases. EGFR expression is upregulated in several malignancies and therapies targeting EGFR are now under development. Strategies include antibodies inhibiting ligand-binding and low molecular weight compounds inhibiting the intracellular tyrosine kinase. In this case report we describe growth of eyelashes (trichomegaly) in a 26 year-old female treated with irinotecan and Cetuximab (chimeric antibody against EGFR, Imclone) (see Fig. 1). The patient was diagnosed with colon cancer with liver metastases and received treatment with irinotecan (350 mg/m every 3 weeks) for several months. Owing to progressive disease, Cetuximab was added to the irinotecan treatment. As seen in most patients on irinotecan treatment, our patient developed alopecia grade II that was unaltered on the combined Cetuximab /irinotecan treatment. After the first week of treatment, the patient developed the typical acneiform erythematous rash commonly described in patients on Cetuximab and other EGFR inhibitors (4). She observed a marked increase in the length of her eyelashes and eyebrows after about 10 weeks of treatment. The photograph was taken after 20 weeks of treatment. Owing to the grade II acneiform erythematous rash, the patient began treatment with tetracycline (300 mg/day) starting in week 19. The patient had a clinical, radiological and biochemical response to Cetuximab treatment with a reduction in tumour size of 47% and plasma CEA levels were decreased from 432 to B/5 (normal range). A skin biopsy taken at 28 weeks after start of treatment showed histopathological findings as previously described in patients on EGFR inhibitors (4), with reduced Ki-67 expression compared to normal skin biopsies. There was no evidence of androgen-stimulated hair growth. Plasma sex hormone levels and elevated SHBG were in the range normally seen in young females taking contraceptive pills. The patient did not use any other medication. Similar trichomegaly was also observed in another female patient with metastatic colon cancer who was treated with Cetuximab monotherapy after progression on irinotecan treatment. Because of the marked and inconvenient increase in the length of her eyelashes, she resorted to using scissors to trim her eyelashes.

Involvement of plasmacytoid dendritic cells in human diseases.

In vitro studies have reported that plasmacytoid dendritic cells (PDCs) exert multiple functions, including production of interferon (IFN)-alpha as effector cells and regulation of T-cell responses as mature DCs. Here we review recent data obtained in situ showing that PDCs accumulate in lesions of type I IFN-related disorders (virus infections and lupus erythematosus), Th2 cell-dominated allergic reactions, and ovarian carcinoma. These results demonstrate that PDCs do migrate to peripheral tissues during inflammation, which lends further support to the view that PDCs most likely are important players in innate and adaptive immunity in vivo. Future research should aim at defining the exact pathogenic or defense roles of PDCs in such disorders and determine whether these cells are potential targets for therapeutic intervention in microbial infections, allergy, autoimmunity, or cancer.

Expression of leukocyte differentiation antigens during the differentiation of HL-60 cells induced by 1,25-dihydroxyvitamin D3: comparison with the maturation of normal monocytic and granulocytic bone marrow cells.

1,25‐Dihydroxyvitamin D3 [1,25‐(OH)2D3] induces monocytic differentiation of the HL‐60 leukemic cell line. The present study investigated whether and to what extent this differentiation resembles the normal maturation of monocytic cells in the bone marrow. Multidimensional flow cytometry was used to identify changes in antigen expression that occur in normal bone marrow cells at distinct stages of monocytic and granulocytic maturation. HL‐60 cells were analyzed in the same manner after exposure to 1,25‐(OH)2D3 to determine whether the hormone induces a similar sequence of phenotypic changes. In the leukemic cells, monocytic features were sequentially induced and several maturational steps could be resolved. CD14, CD32, CD53, CD15, CDw65, CD29, CD16, and CD66b were modulated in 1,25‐(OH)2D3‐induced HL‐60 cells as in the normal monocytic maturational pathway. Differences were observed for CD15s and CDw17. The expression pattern of CD44 during differentiation of HL‐60 cells resembled that in granulocytic cells. The results therefore suggest that 1,25‐(OH)2D3 induces a differentiation program in HL‐60 cells that in many ways resembles that of normal monocytic cells in the bone marrow but also carries elements of the granulocytic pathway.

Expression of cell surface antigens during the differentiation of HL-60 cells induced by 1,25-ihydroxyvitamin D3, retinoic acid and DMSO

HL-60 cells were induced to differentiate by 1,25-dihydroxyvitamin D3, retinoic acid or DMSO. In order to investigate to which extent this maturation mimics the in vivo monocytic or myeloid differentiation, we compared induced HL-60 cells with peripheral blood monocytes and granulocytes by using a panel of mAbs directed against myeloid cell surface antigens. Upon exposure to 1,25-(OH)2D3, HL-60 cells acquired a differentiation phenotype close to that of mature monocytes. The changes in myeloid cell surface antigens induced by retinoic acid or DMSO paralleled the expression pattern of these molecules in normal granulopoiesis, although maturation was not achieved and partially defective.

Rapid Generation of Rotavirus-Specific Human Monoclonal Antibodies from Small-Intestinal Mucosa

The gut mucosal surface is efficiently protected by Abs, and this site represents one of the richest compartments of Ab-secreting cells in the body. A simple and effective method to generate Ag-specific human monoclonal Abs (hmAbs) from such cells is lacking. In this paper, we describe a method to generate hmAbs from single Ag-specific IgA- or IgM-secreting cells of the intestinal mucosa. We found that CD138-positive plasma cells from the duodenum expressed surface IgA or IgM. Using eGFP-labeled virus-like particles, we harnessed the surface Ig expression to detect rotavirus-specific plasma cells at low frequency (0.03–0.35%) in 9 of 10 adult subjects. Single cells were isolated by FACS, and as they were viable, further testing of secreted Abs by ELISPOT and ELISA indicated a highly specific selection procedure. Ab genes from single cells of three donors were cloned, sequenced, and expressed as recombinant hmAbs. Of 26 cloned H chain Ab genes, 22 were IgA and 4 were IgM. The genes were highly mutated, and there was an overrepresentation of the VH4 family. Of 10 expressed hmAbs, 8 were rotavirus-reactive (6 with Kd < 1 × 10−10). Importantly, our method allows generation of hmAbs from cells implicated in the protection of mucosal surfaces, and it can potentially be used in passive vaccination efforts and for discovery of epitopes directly relevant to human immunity.