Induction of dendritic cell costimulator molecule expression is suppressed by T cells in the absence of antigen-specific signalling: role of cluster formation, CD40 and HLA-class II for dendritic cell activation

Abstract

Full activation of T lymphocytes by dendritic cells (DC) during antigen presentation is known to require the interaction of several inducible receptor-ligand pairs. We have postulated that the reciprocal activation of DC by T lymphocytes is also important. Potential signalling molecules that might increase the stimulatory capacity of DC during antigen presentation to T lymphocytes were tested using an in vitro model. Fresh human blood DC were cocultured with CD4+ and CD8+ allogeneic or with autologous T lymphocytes plus Staphylococcus superantigen A (SEA). Surprisingly, costimulator expression on DC cocultured with T lymphocytes was reduced in comparison to DC cultured alone. However, the minority (10-30%) of DC clustering with T lymphocytes showed antigen-specific up-regulation of the CD40, CD80 and CD86 costimulator molecules, whereas the non-clustered DC (70-90%) had less up-regulation than control DC cultured alone and did not respond to antigen-specific triggering. Monoclonal antibodies (mAb) to CD40 ligand (CD40L) and human leucocyte antigen (HLA)-DR, but not lymphocyte function-associated antigen-1 (LFA-1), LFA-3 or HLA-class I, significantly inhibited the T-lymphocyte induction of DC costimulator expression. Since HLA-class II, but not HLA-class I mAb, inhibited allogeneic T-lymphocyte-mediated activation of DC, CD4 T lymphocytes appear to be the main subset activating DC in the mixed lymphocyte reaction. Cross-linking of CD40, but not HLA-class II, up-regulated DC or B-cell costimulator expression. Although direct class II signalling does not appear to play a role in DC activation, antigen-specific T-cell recognition contributes via other mechanisms to regulate DC activation.

Figure 1

Expression of costimulator molecules on DC and T lymphocytes following DC–T-lymphocyte coculture. (a) DC were cultured as indicated; either alone, or with CMFDA-labelled autologous or allogeneic T lymphocytes in a 1:3 ratio. Negative control mAb or CD25 (top panel) and CD86 (lower panel) staining on DC and T lymphocytes in each of the coculture systems after 36 hr are shown as contour plots. Upper panel: the increases in CD25 MFI between T lymphocytes cultured alone (not shown) or T lymphocytes stimulated by autologous or allogeneic DC are given. Lower panel: Decreases in CD86 MFI values between DC cultured alone and with T lymphocytes (CMFDA⁻ events only) are shown. Representative of at least four experiments performed. (b) Differences in MFI are shown for the indicated costimulator molecules between DC cultured either alone (dotted line) or with a tetanus toxoid-specific line (TCL; solid line) plus 5 μg/ml tetanus toxoid. T lymphocytes (CD3⁺ events) were electronically excluded from this analysis. The level of isotype control staining is shown in grey. Representative of two experiments performed.

Figure 2

Expression of costimulator molecules on DC following coculture with paraformaldehyde-fixed or live T lymphocytes. Fresh blood DC were incubated with either paraformaldehyde-fixed or live autologous, CMFDA-labelled T lymphocytes for 16 hr then labelled for the indicated costimulator molecules. Results are plotted as MFI values normalized to DC cultured in absence of T lymphocytes (100%; dotted line). The results of two experiments are plotted.

Figure 3

Antigen-specific regulation of DC costimulator molecule expression by allo- or superantigen. Fresh blood DC were incubated with (a) autologous T lymphocytes and SEA at the indicated concentrations, or with (b) allogeneic or autologous T lymphocytes. Co-cultures were harvested after 16 hr incubation and separated into unclustered (‘loose’) and clustered populations using 50% FCS gradients. Cells were then labelled with CD86 and PE-SAM and DC were analysed by electronically gating out the CMFDA⁺ (green) T-lymphocyte population. The mean fluorescent intensities of clustered DC were normalized to control DC populations cultured without T lymphocytes (100%; dotted line). (a) is pooled data of three or four independent experiments shown as means±SEM and (b) is a representative experiment of two performed (see also Table 1).

Figure 4

Inhibition of DC–T-lymphocyte cluster formation by mAb to DC and T-lymphocyte surface molecules. Clusters of fresh blood DC and CMFDA-labelled T lymphocytes were allowed to form for 16 hr in the presence of 10 ng/SEA and the indicated mAb or mouse immunoglobuin control (all at 10 μg/ml). DC–T-lymphocyte clusters were then harvested by FCS gradients, disrupted into single cells and the relative numbers of clustered DC and T lymphocytes were determined by flow cytometry. Plots show the means of four experiments (±SEM) with the number of clustered DC or T lymphocytes normalized to the mouse immunoglobulin control cultures for each experiment (100%; dotted line).

Figure 5

Inhibition of T lymphocyte-mediated activation of DC by mAb. DC were incubated for 16 hr with (a) autologous T lymphocytes with 10 ng/ml SEA or (b) allogeneic T lymphocytes in the presence of isotype-matched mAb controls or mAb to DC or T lymphocyte surface antigens. DC were identified in these cocultures with labelling with HLA-DR-PE-Cy5. The levels of CD86 (a,b) and CD80 (b) expression were determined on the DR⁺⁺/CMFDA⁻ DC by labelling in a third colour. Plots show the means of four individual experiments (±SEM) with the CD80 and CD86 MFI values of cocultured DC normalized for each experiment to the control cocultures without mAb (100%; dotted line). Statistically significant differences (by the two-tailed unpaired Student’s t-test) are indicated for: **IgG1 versus CD40L, P<0·01 and *IgG2a versus HLA-DR, P<0·02 and (b); IgG2a versus HLA-DR; **P<0·01, *P<0·02 for CD80 and CD86 levels, respectively).

Figure 6

Effect of class II and CD40 cross-linking on DC costimulator expression. (a) DC were incubated with SEA (1 μg/ml), CD40 ligand trimer (CD40LT; 10 μg/ml) or in medium alone (MED) for the indicated times, then labelled with CD86 mAb plus FITC-SAM. Data are from one of two similar time–courses. (b) the biological activity of SEA was checked by incubating 5×10⁴ PBMC per well with the indicated concentrations of SEA or phytohaemagglutinin (5 μg/ml) for 3 days prior to pulsing with 0·5 μCi/well of thymidine for 16 hr. Results are plotted as means of triplicate wells±SD. Representative of two experiments performed.

Figure 7

Influence of CD40 and class II mAb cross-linking on B-cell costimulator expression. Tonsil B cells were cross-linked with CD40, HLA-DR, or Sal5 control mAb, as described in the Materials and Methods, for 18 hr and the levels of CD80 and CD86 expression were determined. Bracketed values show changes in mean fluorescence intensity and percentage positive cells, respectively, as compared to control mAb cross-linking (one of four representative experiments).