Cell contact-dependent priming and Fc interaction with CD32+ immune cells contribute to the TGN1412-triggered cytokine response.

Following inconspicuous preclinical testing, the superagonistic anti-CD28 mAb TGN1412 was applied to six study participants who all developed a devastating cytokine storm. We verified that TGN1412 treatment of fresh PBMCs induced only moderate responses, whereas restoration of tissue-like conditions by high-density preculture (HDC) allowed vigorous cytokine production. TGN1412 treatment of T cells isolated from HDC-PBMCs induced moderate cytokine responses, which upon additional anti-IgG crosslinking were significantly boosted. Moreover, coincubation of TGN1412-treated T cells with B cells expressing the intermediate affinity Fcγ receptor IIB (CD32B), or coincubation with CD32B(+) transfectants, resulted in robust T cell activation. This was surprising because TGN1412 was expressed as an Ig of the subclass 4 (IgG4), which was shown before to exhibit only minor affinity to FcγRs. Transcriptome analysis of TGN1412-treated T cells revealed that similar gene signatures were induced irrespective of whether T cells derived from fresh or HDC-PBMCs were studied. Collectively, these data indicate that HDC-PBMCs and HDC-PBMC-derived T cells mount rapid TGN1412 responses, which are massively boosted by FcγR crosslinking, in particular by CD32-expressing B cells. These results qualify HDC-PBMCs as a valuable in vitro test system for the analysis of complex mAb functions.

ICOS-LICOS interaction is critically involved in TGN1412-mediated T-cell activation.

TGN1412, a superagonistic CD28-specific antibody, was shown to require Fc-cross-linking or immobilization as a prerequisite to mediate T-cell proliferation and cytokine release in vitro. We used primary human umbilical vein endothelial cells (HUVECs) to study their ability to induce activation of TGN1412-treated T cells. We confirmed that peripheral primary human T cells do not show activation upon stimulation with soluble TGN1412 alone. Nevertheless, cocultivation of TGN1412-treated T cells with HUVECs induced T-cell activation that was further enhanced using cytokine prestimulated HUVECs. Unexpectedly, Fc-FcγR interaction was dispensable for endothelial cell-mediated proliferation of TGN1412-treated T cells. Transwell-culture assays showed that TGN1412-treated T cells need direct cell-to-cell contact to HUVECs to induce proliferation. We found that costimulatory ICOS-LICOS interaction between T cells and endothelial cells is critically involved in TGN1412-mediated effects. Blocking LICOS reduced TGN1412-mediated T-cell proliferation significantly, whereas recombinant LICOS fully conferred TGN1412-mediated T-cell proliferation. Of note, cytokine stimulation enhanced LICOS expression on HUVECs and ICOS-LICOS interaction up-regulated ICOS expression on TGN1412-treated T cells. Hence, we provide a model of positive feedback conferred by ICOS-LICOS interaction between TGN1412-treated T cells and endothelial cells.