CD1d structure and regulation on human thymocytes, peripheral blood T cells, B cells and monocytes

Abstract

Human T cells expressing CD161 and an invariant T-cell receptor (TCR) alpha-chain (Valpha24invt T cells) specifically recognize CD1d and appear to have immunoregulatory functions. However, the physiological target cells for this T-cell population, and whether alterations in CD1d expression contribute to the regulation of Valpha24invt T-cell responses, remain to be determined. A series of antibodies were generated to assess CD1d expression, structure and regulation on human lymphoid and myeloid cells. CD1d was expressed at high levels by human cortical thymocytes and immunoprecipitation analyses showed it to be a 48 000-MW glycosylated protein. However, after solubilization, the majority of the thymocyte CD1d protein, but not CD1d expressed by transfected cells, lost reactivity with monoclonal antibodies (mAbs) against native CD1d, indicating that it was alternatively processed. Moreover, thymocytes were not recognized by CD1d-reactive Valpha24invt T-cell clones. Medullary thymocytes and resting peripheral blood T cells were CD1d-, but low-level CD1d expression was induced on activated T cells. CD1d was expressed by B cells in peripheral blood and lymph node mantle zones, but germinal centres were CD1d-. Resting monocytes were CD1d+ but, in contrast to CD1a, b and c, their surface expression of CD1d was not up-regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) activation. These results demonstrate constitutive CD1d expression by human professional antigen-presenting cells and that post-translational processing of CD1d may contribute to regulation of the activity of CD1d-specific T cells.

Figure 1

Specificity of anti-CD1d antibodies and thymocyte CD1d expression by indirect immunofluorescence. (a) Polyclonal rabbit anti-CD1d and mouse monoclonal 27.1, 42.1 and 51.1 anti-CD1d antibodies were tested by indirect immunofluorescence against C1R cells stably transfected with CD1a, b, c or d, as indicated. The top three panels (C1R CD1a, C1R CD1b and C1R CD1c) are the CD1a, b and c transfectants stained with anti-CD1a (OKT6), anti-CD1b (4A76) and anti-CD1c (M241) monoclonal antibodies (mAbs), respectively. The thick lines represent specific antibodies and the thin lines are control antibodies (normal rabbit or mouse immunoglobulin G [IgG]). (b) Freshly isolated human thymocytes were stained by indirect immunofluorescence with the indicated specific (thick lines) or control (thin lines) antibodies.

Figure 2

CD1d expression by peripheral blood lymphocytes and monocytes. Peripheral blood mononuclear cells from two representative normal donors were analysed by two-colour immunofluorescence with the indicated directly fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated specific and control antibodies. (a) Anti-T-cell receptor αβ (TCR αβ) versus control or 42.1 anti-CD1d analysis of cells in the resting lymphocyte gate. (b) Anti-CD19 versus control or 42.1 anti-CD1d analysis of cells in the resting lymphocyte gate. (c) Anti-CD14 versus control or 42.1 anti-CD1d analysis of cells in the monocyte gate. (d) Histogram of CD1d expression on B cells from two additional donors, examined as described above, gating of the B-cell (CD19⁺) population.

Figure 3

CD1d expression by activated monocytes. (a) Freshly isolated peripheral blood mononuclear cells in the monocyte gate were analysed by indirect immunofluorescence with rabbit anti-CD1d or 27.1 anti-CD1d antibodies (thick lines) versus control non-immune rabbit or mouse serum (thin lines). (b) Granulocyte–macrophage colony-stimulating factor (GM-CSF)- and interleukin-4 (IL-4)-cultured monocytes were analysed by indirect immunofluorescence with anti-CD1a, b, c or d monoclonal antibodies (mAbs) (thick lines) versus control Abs (thin lines). The CD1d Ab shown was 27.1, but identical results were obtained with the rabbit anti-CD1d antibody. (c) Monocytes cultured in GM-CSF, IL-4 and lipopolysaccharide (LPS) were analysed as described above in (b). FITC, fluorescein isothiocyanate; FL1-H, FITC channel.

Figure 4

CD1d expression by activated T cells. (a) Freshly isolated peripheral blood mononuclear cells (PBMC) from two donors were cultured for 24 hr with or without stimulation and then analysed by indirect immunofluorescence with 42.1 anti-CD1d monoclonal antibody (mAb) (42.1) (thick lines) or isotype-matched control Ab (thin lines). Similar results were obtained with the other anti-CD1d mAbs and the rabbit anti-CD1d Ab (not shown). The very weak staining in the cells cultured without stimulation was not seen in the freshly isolated resting T cells (results not shown). (b) Freshly isolated peripheral blood mononuclear cells isolated 1 week apart from the same donor were analysed by indirect immunofluorescence with the indicated specific mAbs (thick lines) versus control antibodies (thin lines). FL1-H, FITC channel.

Figure 5

Immunoperoxidase analysis of CD1 expression in thymus and lymph node. (a) Thymus stained with the 51.1 anti-CD1d monoclonal antibody (mAb). (b) Thymus stained with the OKT6 anti-CD1a mAb. (c) Lymph node stained with 51.1 anti-CD1d mAb. Comparable results were obtained with the 27.1 and 42.1 anti-CD1d mAbs (not shown).

Figure 6

CD1d immunoprecipitations from thymocytes and specificity of D5 monoclonal antibody (mAb). (a) Streptavidin blot of immunoprecipitates from biotinylated thymocyte lysates: lane 1, normal mouse serum; lanes 2 and 3, sequential 42.1 anti-CD1d mAb precipitations; lane 4, D5 anti-CD1d mAb; lane 5, OKT6 anti-CD1a precipitation; lane 6, 42.1 precipitation from CD1d-transfected C1R cells. (b) Deglycosylation of CD1d from surface-iodinated thymocyte lysates: lane 1, normal mouse serum immunoprecipitation; lanes 2 and 3, D5 anti-CD1d mAb immunoprecipitates untreated (lane 2) or treated lane 3 with N-glycanase (N-glc). (c) D5 immuno-precipitation from CD1a precleared biotinylated thymocyte lysate: lanes 1–4, lysate immunoprecipitated sequentially with OKT6 (anti-CD1a) beads (lanes 1–3) followed by reimmunoprecipitation with D5 (anti-CD1d) beads (lane 4); lane 5, D5 precipitation from an equal amount of lysate without a CD1a preclear. (d) Immunoprecipitates from unlabelled thymocyte lysates immunoblotted with BBM.1 anti-β₂m mAb: lanes 1–4, fresh thymocyte lysate; lanes 5–7, equivalent amounts of D5 anti-CD1d mAb-depleted thymocyte lysate. Immunoprecipitations were as follows: lane 1, normal mouse serum; lanes 2 and 5, anti-CD1a (OKT6) mAb; lanes 3 and 6, anti-CD1b (4A76) mAb; lanes 4 and 7, anti-CD1c (M241) mAb. Samples were analysed on a 15% gel under non-reducing conditions.

Figure 7

CD1d immunoprecipitations from thymocytes and transfected cells immunoblotted with rabbit anti-CD1d. (a) Immuno-precipitates from unlabelled thymocytes immunoblotted with affinity-purified rabbit anti-CD1d: lanes 1 and 2, primary and secondary 42.1 monoclonal antibody (mAb) precipitations, respectively; lanes 4 and 5, primary and secondary 51.1 mAb precipitations, respectively; lanes 3 and 6, D5 anti-CD1d precipitations from 42.1 and 51.1 precleared lysates, respectively. (b) Lysates from C1R cells expressing wild-type CD1d (lanes 1–6) or a CD1d/a chimeric protein (lanes 7–12) immunoprecipitated and immunoblotted with affinity-purified rabbit anti-CD1d: lanes 1 and 7, normal mouse serum; lanes 2–4 and 8–10, three sequential immunoprecipitations with 42.1 mAb; lanes 5, 6 and 11, 12, sequential D5 mAb immunoprecipitations of the 42.1-cleared lysates. All samples were analysed on 12% reducing gels.

Figure 8

Lack of thymocyte CD1d recognition by Vα24^invt T cells. A CD1d-reactive Vα24^invt clone, DN2.B9, was cultured with the indicated stimulator cells in the presence of phorbol 12-myristate 13-acetate (PMA). Proliferation was determined by ³H-thymidine incorporation. The stimulator cells or DN2.B9 T cells were omitted from the top and bottom experiments, respectively. The response to the C1R CD1d transfectants was set at 100%. fix, light glutaraldehyde fixation; Mock, transfected with empty expression vector.