CD80 and CD86 differentially regulate mechanical interactions of T-cells with antigen-presenting dendritic cells and B-cells

Abstract

Functional T-cell responses are initiated by physical interactions between T-cells and antigen-presenting cells (APCs), including dendritic cells (DCs) and B-cells. T-cells are activated more effectively by DCs than by B-cells, but little is known about the key molecular mechanisms that underpin the particular potency of DC in triggering T-cell responses. To better understand the influence of physical intercellular interactions on APC efficacy in activating T-cells, we used single cell force spectroscopy to characterize and compare the mechanical forces of interactions between DC:T-cells and B:T-cells. Following antigen stimulation, intercellular interactions of DC:T-cell conjugates were stronger than B:T-cell interactions. DCs induced higher levels of T-cell calcium mobilization and production of IL-2 and IFNγ than were elicited by B-cells, thus suggesting that tight intercellular contacts are important in providing mechanically stable environment to initiate T-cell activation. Blocking antibodies targeting surface co-stimulatory molecules CD80 or CD86 weakened intercellular interactions and dampen T-cell activation, highlighting the amplificatory roles of CD80/86 in regulating APC:T-cell interactions and T-cell functional activation. The variable strength of mechanical forces between DC:T-cells and B:T-cell interactions were not solely dependent on differential APC expression of CD80/86, since DCs were superior to B-cells in promoting strong interactions with T-cells even when CD80 and CD86 were inhibited. These data provide mechanical insights into the effects of co-stimulatory molecules in regulating APC:T-cell interactions.

Figure 1. B-cells and DCs exhibit a similar phenotype.

B-cells (Red) and DCs (Black) were stained with antibodies against H-2K^b (MHC class I), H-2K^b/Ova (pMHC), CD11c, CD19, CD54 (ICAM-1), CD80 (B7-1) and CD86 (B7-2). Filled histograms: isotype controls; Unfilled histograms: staining with antibody. Data are representative of three independent experiments.

Figure 2. Cytokine secretion by DC:T-cell and B:T-cell co-cultures.

(A) and (B): secretion of IL-2 (left) and IFN-γ (right) after 1 day co-culture of CD8+ OT-I T-cells with DCs or B-cells pre-pulsed or not with Ova peptide (10 pg–10 µg/ml). *p<0.01; **p<0.001, unpaired t-test. Bars indicate mean ± s.e.m. Data are representative of three independent experiments.

Figure 3. Calcium response of T-cells bound to APCs.

(A) Representative differential interference contrast (DIC) images of the DC:T-cell or B:T-cell conjugates overlaid with the Fluo-4 (green) and Fura-red (red) fluorescent signals (loaded in T-cells only) at the indicated time points. DCs or B-cells were pre-pulsed or not with Ova peptides (10 ng/ml) for 4 h prior to co-culture with T-cells. (B) Time course of intracellular calcium concentrations in the responding T-cells, as measured by Fluo-4/Fura-red ratio. Each plot represents data from a pair of cell:cell conjugates. (C) Average calcium response of T-cells bound to DC (black) or B-cell (red) pre-pulsed with Ova peptides at different concentrations (0.1 ng–1 µg/ml). To quantify early calcium response in T-cells, Fluo-4/Fura-red ratios were measured every 5s in responding T-cells and were then integrated for 3 min from the time of the initial calcium increase (grey line, Fig 4B). For each condition, the average calcium response was measured by pooling data of APC:T-cell conjugates (n>15 pairs of cells) from >3 independent experiments. Bars indicate mean ± s.e.m. *p<0.01, unpaired t-test.

Figure 4. Differential Strength of Mechanical Forces between APC:T-cell Conjugates.

(A) Schematic illustration of AFM experiments. A T-cell-mounted AFM cantilever was placed above a DC or B-cell that was firmly attached to a glass cover slip. The T-cell was then brought into contact with the target cell. Interaction forces were measured by the deflection of the cantilever after a pre-defined contact time. (B) Corresponding force-distance curve of DC:T-cell (black) or B:T-cell (red) interactions. F represents maximal interaction force (double arrow). (C) Interaction forces of DC:T-cell (black) or B:T-cell conjugates (red) in the absence (-Ova) or presence of Ova peptide (+Ova) for contact time of ∼1–3 sec and 3 min. *p<0.01, unpaired t-test. Bars indicate mean ± s.e.m (n>10 pairs of cells). For each condition, OT-I T-cells were isolated from >3 independent experiments.

Figure 5. Contribution of CD80 and CD86 to Interaction Forces and Cytokine Secretion.

(A) Interaction forces; (B) IL-2 and (C) IFN-γ production of DC:T-cell or B:T-cell conjugates in the presence of blocking antibodies against CD80 and/or CD86. DCs or B-cells were pre-pulsed with Ova peptides (+Ova, 10 ng/ml) before antibody blocking. All force measurements were conducted with contact time of 3 min. *p<0.01; unpaired t-test. For each condition, OT-I T-cells were isolated from >3 independent experiments. αCD80: blocking antibody targeting CD80; αCD86: blocking antibody targeting CD86; αCD80/86: αCD80 and αCD86 simultaneously; IgG: isotype controls for both αCD80 and αCD86.