Different Epitopes on the Dendritic Cell-Associated NLDC-145 Molecule during Ontogeny

Kraal, Georg; Avis, Linda; Wijffels, J.F.A.M.; Hoeben, Kees A.; Hart, Hennie Ter

doi:10.1016/s0171-2985(11)80507-3

“…2). Parallel treatment with antibody 6D2, prepared to a different epitope on the NLDC-145 antigen (16), had no effect (Fig. 2).…”

Section: Resultsmentioning

confidence: 94%

“…NLDC-145/anti-DEC-205 antibody was prepared as described (15) and used as culture supernatant or Protein G puri®ed from Ig-free supernatant. 6D2 antibody binds a different epitope on the NLDC-145 antigen (16) anti-rat antibody, Vector ABC peroxidase reagents and 3amino-9-ethylcarbazole (AEC) were from Vector (Burlingame, CA). Anti-active caspase-3 (clone C92-605), FITC±anti-mouse Thy-1.2 (CD 90.2/53-2.1), FITC±anti-mouse TCR b chain (H57-597), FITC±anti-mouse CD8a (53-6.7) and phycoerythrinconjugated anti-mouse CD4 (GK 1.5) were obtained from PharMingen (San Diego CA).…”

Section: Reagentsmentioning

confidence: 99%

In vitro evidence for participation of DEC-205 expressed by thymic cortical epithelial cells in clearance of apoptotic thymocytes

Small

¹

,

Kraal²

2003

International Immunology

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Binding of apoptotic cells was compared after incubation of thymocytes with two clones of murine thymic stromal cells to which CD4(+)/CD8(+) thymocytes attach. With the BA/10, but not the BA/2, clone, thymocytes with apoptotic morphology were bound irreversibly. These tightly bound thymocytes were further identified as apoptotic in terms of active caspase-3 and DNA fragmentation assayed in situ. FACS analysis indicated that the apoptotic thymocytes are at an early double-positive stage and results with mice mutant for the Fas gene showed that the Fas-Fas ligand system is not involved. Comparison of BA/10 and BA/2 cells showed that the former, but not the latter, can be induced to express CDR-1 antigen which is characteristic of cortical epithelial thymic stroma and constitutively express DEC-205, a surface protein common to cortical thymic epithelium and dendritic cells. Antibody NLDC-145 that is specific for the DEC-205 protein strongly reduced the number of stromal cells with bound apoptotic thymocytes. Preincubation of thymocytes in dexamethasone dramatically increased the number of bound apoptotic cells, indicating that the thymic cortical epithelial cells can participate in clearance of apoptotic thymocytes through involvement of DEC-205.

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“…Nonadherent spleen cells (5-8 ml at around 5 × 10 6 cells per ml) were layered onto 2 ml of metrizamide [Nygaard, Oslo, Norway; analytical grade 13.7 % (w/v)], centrifuged for 10 min at 600 × g and the mononuclear cells at the interface collected, washed once and resuspended in medium. These separated cells from specific pathogen-free normal mice were greater than 75 % DC [3], positive for NLDC 145 labeling [25] and sensitive to lysis with a specific antibody for DC (33D1) plus complement [26]. Less than 3 % were macrophages which could be labeled with the F4/80 mAb [27] and the remaining cells were T lymphocytes.…”

Section: Cell Suspensionsmentioning

confidence: 97%

Transfer of antigen between dendritic cells in the stimulation of primary T cell proliferation

Knight¹,

Iqball²,

Roberts³

et al. 1998

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Primary proliferative T cell responses require stimulation with antigen-pulsed dendritic cells (Ag-DC). Here we show that for optimal stimulation, dendritic cells (DC) not exposed directly to antigen are also required. Ag-DC added to DC-depleted T cells caused negligible primary stimulation; adding back DC resulted in stimulation. These effects were seen using the contact sensitizer fluorescein isothiocyanate (FITC), FITC conjugated to ovalbumin (FITC-OVA) or influenza virus as antigens. DC co-cultured with Ag-DC (using FITC or FITC-OVA) acquired antigen indicating that antigen was transferred between DC. DC that acquired antigen secondarily were separated by cell sorting and stimulated primary T cell proliferation directly. DC were also pulsed with FITC, washed thoroughly and incubated overnight. Supernatants contained shed antigen since DC incubated in these supernatants acquired antigen as indicated by flow cytometry. DC acquiring the shed antigen also stimulated T cell proliferation although the stimulation was not as effective as that seen when cell contact between DC and antigen-bearing DC occurred. Thus, in primary stimulation, activation of T cells may occur when there is an antigen gradient between Ag-DC and DC and the mechanisms underlying these effects are now being sought. We propose that this unique interaction between antigen-presenting cells may be a paradigm for self/non-self discrimination.

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“…These separated cells from specific pathogen-free normal mice were greater than 75 % DC [3], positive for NLDC 145 labeling [25] and sensitive to lysis with a specific antibody for DC (33D1) plus complement [26]. g/ml streptomycin, 5 × 10 -5 M 2-ME and 10 % FCS.…”

Section: Cell Suspensionsmentioning

confidence: 99%

“…Lysis with antibody plus complement was used to deplete cell preparations of DC using 33D1 [25] or of T cells using anti-thy 1.2 (AT83a). Cells at 5 × 10 5 -5 × 10 6 /ml were mixed with antibody and rabbit serum as a source of complement (Buxted Rabbit Co., Buxted, GB) or with complement alone as a control for cytotoxicity, incubated for 60 min at 37°C and washed twice in medium.…”

Section: Antibody Depletionmentioning

confidence: 99%

Transfer of antigen between dendritic cells in the stimulation of primary T cell proliferation

Knight

¹

,

Iqball

²

,

Roberts

³

et al. 1998

View full text Add to dashboard Cite

Primary proliferative T cell responses require stimulation with antigen-pulsed dendritic cells (Ag-DC). Here we show that for optimal stimulation, dendritic cells (DC) not exposed directly to antigen are also required. Ag-DC added to DC-depleted T cells caused negligible primary stimulation; adding back DC resulted in stimulation. These effects were seen using the contact sensitizer fluorescein isothiocyanate (FITC), FITC conjugated to ovalbumin (FITC-OVA) or influenza virus as antigens. DC co-cultured with Ag-DC (using FITC or FITC-OVA) acquired antigen indicating that antigen was transferred between DC. DC that acquired antigen secondarily were separated by cell sorting and stimulated primary T cell proliferation directly. DC were also pulsed with FITC, washed thoroughly and incubated overnight. Supernatants contained shed antigen since DC incubated in these supernatants acquired antigen as indicated by flow cytometry. DC acquiring the shed antigen also stimulated T cell proliferation although the stimulation was not as effective as that seen when cell contact between DC and antigen-bearing DC occurred. Thus, in primary stimulation, activation of T cells may occur when there is an antigen gradient between Ag-DC and DC and the mechanisms underlying these effects are now being sought. We propose that this unique interaction between antigen-presenting cells may be a paradigm for self/non-self discrimination.

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Different Epitopes on the Dendritic Cell-Associated NLDC-145 Molecule during Ontogeny

Cited by 11 publications

References 13 publications

In vitro evidence for participation of DEC-205 expressed by thymic cortical epithelial cells in clearance of apoptotic thymocytes

In vitro evidence for participation of DEC-205 expressed by thymic cortical epithelial cells in clearance of apoptotic thymocytes

Transfer of antigen between dendritic cells in the stimulation of primary T cell proliferation

Transfer of antigen between dendritic cells in the stimulation of primary T cell proliferation

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