Major histocompatibility complex class II invariant chain expression in non-antigen-presenting cells

Abstract

In contrast to the generally accepted belief, the major histocompatibility complex (MHC) class II invariant chain (Ii) is commonly expressed intracellularly in cells that do not present exogenous antigens. Such cells include resting peripheral blood T cells and natural killer (NK) cells. In T cells, the Ii is associated with a 77 000 molecular-weight molecule (p77) that has yet to be identified. This molecule is co-precipitated with the anti-Ii monoclonal antibody (mAb) VCD-1, but not with mAb BU-45. This suggests that in the p77-Ii complex, the extracellular epitope of Ii recognized by BU-45 is hidden, whereas the Ii epitope for VCD-1 remains exposed. In antigen-presenting cells (APCs), p77 association with the Ii was minimal, if detectable. The p77-Ii association in non-professional APCs suggests that the Ii may have another, more general, function other than the one accepted in antigen presentation.

Figure 1

Cell surface and intracellular labelling of the invariant chain (Ii) and class II molecules in normal peripheral blood lymphocytes (PBL). Normal peripheral blood mononuclear cells (PBMC), separated on Ficoll–Hypaque, were labelled sequentially with the indicated monoclonal antibodies (mAbs) and goat anti-mouse immunoglobulin-conjugated fluorescein isothiocyanate (FITC) in the absence (left column) or presence (right column) of saponin, as described in Materials and methods. The cells were analysed with a Becton-Dickinson fluorescence-activated cell sorter (FACScan flow cytometer) and CELLQuest software using a light scatter gate for the lymphocytes. The shaded histograms represent cells labelled with control monoclonal antibody (mAb).

Figure 2

Normal peripheral blood lymphocytes (PBL) double-labelled with monoclonal antibodies (mAbs) to T- or natural killer (NK) cell markers and VCD-1. Normal peripheral blood mononuclear cells (PBMC) were labelled with control, anti-CD4, -CD8 or -CD16 mAbs and goat anti-mouse immunoglobulin-conjugated fluorescein isothiocyanate (FITC) (FL1, as indicated). This was followed by labelling with either biotinylated control antibody or biotinylated VCD-1 and Streptavidin-RPE in the presence of saponin (FL2, as indicated). The cells were analysed with a Becton-Dickinson fluorescence-activated cell sorter (FACScan flow cytometer) using Lysys II software. The lymphocyte gate, verified by CD45/CD14 backgating, contained at least 95% lymphocytes.

Figure 3

Immunoprecipitation of the invariant chain (Ii) from metabolically labelled peripheral blood T cells. T cells were purified from normal peripheral blood mononuclear cells (PBMC) by labelling the PBMC with anti-class II and anti-CD16 monoclonal antibodies (mAbs), mixing them with goat anti-mouse immunoglobulin G (IgG) magnetic cell sorting (MACS) beads coated with goat anti-mouse immunoglobulin and passing them through a Mini-MACS column. The purified T cells were metabolically labelled with ³⁵S-methionine at 100 µCi/ml for 2 hr, lysed and immunoprecipitated with VCD-1. The precipitate was subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), using an 11% gel under reducing conditions, and fluorographed. M_r, relative molecular mass.

Figure 4

(a) Immunoprecipitation of the invariant chain (Ii) from metabolically labelled T- and B-cell lines. HPB-ALL cells and Epstein–Barr virus (EBV)-transformed lymphocytes were labelled metabolically for 2 hr with ³⁵S-methionine. The cells were lysed with detergent and the lysate subjected to immunoprecipitation with either VCD-1 monoclonal antibody (mAb) or control mAb. The precipitated proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), using an 11% acrylamide gel under reducing conditions, and fluorographed. (b) Immunoblot of unlabelled HPB-ALL lysate with VCD-1. Cell lysate was subjected to SDS–PAGE, using an 11% acrylamide gel under reducing conditions, electroblotted onto polyvinylidene difluoride (PVDF) membrane and then probed with VCD-1 and peroxidase-labelled goat anti-mouse immunoglobulin. IP, immunoprecipitate; M_r, relative molecular mass.

Figure 5

Immunoprecipitation of the invariant chain (Ii) with VCD-1 and BU-45 monoclonal antibodies (mAbs) from metabolically labelled Epstein–Barr virus-transformed lymphocytes (EBVL). EBVL were labelled metabolically for 2 hr with ³⁵S-methionine. The cells were lysed with detergent and the lysate was subjected to immunoprecipitation with either VCD-1, BU-45 or control mAb. The precipitated proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), using an 11% acrylamide gel under reducing conditions, and fluorographed. M_r, relative molecular mass.

Figure 6

(a) Intracellular labelling of the invariant chain (Ii) and class II molecules in K562 cells. Untreated cells of the K562 cell line (left column) and cells treated with Lymphocult-T cytokine mixture for 3 days (right column) were labelled sequentially with the indicated monoclonal antibodies (mAbs) and goat anti-mouse-conjugated fluorescein isothiocyanate (FITC) in the presence of saponin. The shaded histograms in the fluorescence-activated cell sorter (FACS) analysis represent cells labelled with control mAb. (b) Immunoprecipitation of Ii with VCD-1 from metabolically labelled K562 cells. Untreated K562 cells and K562 cells treated with Lymphocult-T cytokine mixture for 3 days were labelled metabolically for 2 hr with ³⁵S-methionine. The cells were lysed with detergent and the lysate was subjected to immunoprecipitation with either VCD-1 or control mAb. The precipitated proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), using an 11% acrylamide gel under reducing conditions, and fluorographed. M_r, relative molecular mass.

Figure 7

Intracellular labelling of the invariant chain (Ii) and class II molecules in hepatocarcinoma cells. Cells of the hepatocarcinoma cell line HEP-G2 were labelled sequentially with the indicated monoclonal antibodies (mAbs) and goat anti-mouse-conjugated fluorescein isothiocyanate (FITC) in the presence of saponin. The shaded histograms in the fluorescence-activated cell sorter (FACS) analysis represent cells labelled with control mAb.