Intact CD100-CD72 Interaction Necessary for TCR-Induced T Cell Proliferation

Abstract

Targeting CD100 by antibody blockade is a potential therapeutic strategy for cancers, but the functional effects on T cells following blockade of this immune activating molecule are rarely considered. Indeed, CD100 is highly expressed in T cells and anti-CD100 antibodies play a role during T cell proliferation; however, the outcome varies from different studies and the underlying mechanism is still unclear. To address this, monoclonal antibody clones directed against CD100 were evaluated. In their soluble form, four of these antibodies significantly reduced the expansion of T cells in the presence of bead-bound anti-CD3/CD28, either in total peripheral blood mononuclear cell or purified T cell culture systems. Similar inhibition was seen when blocking CD100-CD72 interaction by soluble anti-CD72 instead of anti-CD100 antibodies. Conversely, restoring the interaction by CD72-Fc eliminated the soluble anti-CD100-induced inhibitory effect. Taken together, these results reveal that T cell proliferation is regulated by CD100 via interaction with CD72. They further establish an in vitro system to evaluate the inhibitory effect of anti-CD100 antibodies on T cells, to which attention should be paid in clinical trials in order to avoid potential side effects.

Keywords: CD72; T cell proliferation; antibody blockade; semaphorin-4D/CD100; soluble anti-CD100.

Figure 1

Blocking CD100 inhibits T cell proliferation. (A) Representative histograms of total T cells (top), CD4 T cells (middle), and CD8 T cells stained with carboxyfluorescein 6 succinimidyl ester (CFSE) and cultured with Dynabeads anti-CD3/CD28 and soluble anti-CD100 (clone 133-1C6, 10 µg/ml) or isotype control (mouse IgM with azide) after 5 days of culture. The first column shows unstimulated cells. The CFSE^low population indicates the proliferating cells. (B) Soluble anti-CD100 (clone 133-1C6) at indicated concentrations (10, 1, and 0.1 µg/ml) were added into the culture system (n = 4). Inhibition ratio: (proliferated T cells % in isotype control−proliferated T cells % in current group)/proliferated T cells % in isotype control. Titration experiments were repeated twice with peripheral blood mononuclear cells from four different donors.

Figure 2

Four out of six anti-CD100 monoclonal antibody clones inhibit T cell proliferation. (A) Staining of activated peripheral blood mononuclear cells (PBMCs) with isotype control purified (gray) or different clones of anti-human CD100 purified (red) followed by anti-mouse IgG or IgM APC. PBMCs were stimulated by anti-CD3/CD28. CD3⁺ T cells were used for analysis. Experiments were repeated twice with two different PBMC preparations. (B) Total PBMCs pre-stained with Carboxyfluorescein 6 succinimidyl ester (CFSE) and cultured with Dynabeads anti-CD3/CD28 in the presence of indicated soluble anti-CD100 clones, respectively (10 µg/ml). Representative histograms of CFSE fluorescence on T cells after 5 days of culture were shown. Isotype indicates mouse IgM with azide. (C) Summary of data showing the inhibition ratio of each clone (n = 2–6). Experiments were repeated three times with six PBMC preparations in total.

Figure 3

Inhibitory effect of soluble anti-CD100 in purified T cell culture system. (A) Peripheral blood mononuclear cells (PBMCs) were stained with anti-CD3 and dead cell marker (DCM). CD3⁺DCM⁻ alive T cells were purified by FACS sorting. (B) Purified T cells were stained with Carboxyfluorescein 6 succinimidyl ester (CFSE) and cultured with Dynabeads anti-CD3/CD28 and soluble anti-CD100 clone 30/CD100 or 133-1C6 (10 µg/ml), respectively. CFSE fluorescence on CD3 T cells was analyzed 5 days later. Isotype indicates mouse IgM with azide. (C) Inhibition ratio was calculated (n = 3, ***P < 0.001). (D) FSC/SSC of cultured cells was compared at day 5 in indicated groups. Data were collected from three experiments. Three different individuals were included.

Figure 4

CD100–CD72 interaction is required for T cell proliferation. (A) CD72 expression increased on T cells after anti-CD3/CD28 stimulation. Gated on CD3⁺DCM⁻ T cells, gray shadow represents isotype controls. CD72⁺ % in T cells and CD72 MFI (Geom. Mean) of total T cells were subjected to statistical analysis (n = 6, **P < 0.01, ****P < 0.0001). (B) 5 µg/ml soluble anti-CD72 was added into total peripheral blood mononuclear cell (PBMC) plus Dynabeads anti-CD3/CD28 culture system, Carboxyfluorescein 6 succinimidyl ester (CFSE) fluorescence on CD3 T cells was analyzed 5 days later. (C) The inhibiting effect induced by anti-CD100 or anti-CD72 (final conc. 5 µg/ml) was partially eliminated by CD72-Fc (n = 4, ***P < 0.001, ****P < 0.0001). The histograms are representative of three independent experiments. PBMCs from six individuals were included in the statistical comparisons.