Human volunteers receiving TGN1412, a humanized CD28-specific monoclonal antibody, experienced a life-threatening cytokine release syndrome during a recent trial. Preclinical tests using human PBMCs had failed to announce the rapid release of TNF, IFN-␥, and other toxic cytokines in response to this CD28 "superagonist" (CD28SA). CD28SA activate T-lymphocytes by ligating CD28 without overt engagement of the TCR. They do, however, depend on "tonic" TCR signals, which they amplify. Here we show that short-term preculture of PBMCs at high, but not at low, cell density results in massive cytokine release during subsequent stimulation with soluble TGN1412. Restoration of reactivity was cell-contact dependent, involved functional maturation of both monocytes and T cells, was sensitive to blockade by HLA-specific mAb, and was associated with TCR polarization and tyrosine phosphorylation. CD4 effector memory T cells were identified as the main source of proinflammatory cytokines. Importantly, responses to other T-cell activating agents, including microbial antigens, were also enhanced if PBMCs were first allowed to interact under tissue-like conditions. We provide a protocol, which strongly improves reactivity of circulating T cells to soluble stimulants, thereby allowing for more reliable preclinical testing of both activating and inhibitory immunomodulatory drugs. (Blood. 2011;118(26):6772-6782)

CD28 superagonists (CD28SAs) are potent T-cell-activating monoclonal antibodies (mAbs).

Antibodies specific for TNFRSF receptors that bind soluble ligands without getting properly activated generally act as strong agonists upon FcγR binding. Systematic analyses revealed that the FcγR dependency of such antibodies to act as potent agonists is largely independent from isotype, FcγR type, and of the epitope recognized. This suggests that the sole cellular attachment, achieved by Fc domain-FcγR interaction, dominantly determines the agonistic activity of antibodies recognizing TNFRSF receptors poorly responsive to soluble ligands. In accordance with this hypothesis, we demonstrated that antibody fusion proteins harboring domains allowing FcγR-independent cell surface anchoring also act as strong agonist provided they have access to their target. This finding defines a general possibility to generate anti-TNFRSF receptor antibodies with FcγR-independent agonism. Moreover, anti-TNFRSF receptor antibody fusion proteins with an anchoring domain promise superior applicability to conventional systemically active agonists when an anchoring target with localized disease associated expression can be addressed.

The identification of regulatory T cells (Treg cells) in human peripheral blood is an important tool in diagnosis, research, and therapeutic intervention. As compared to lymphoid tissues, the frequencies of circulating Treg cells identified as CD4 CD25 Foxp3 are, however, low. We here show that many of these cells remain undetected due to transient down regulation of Foxp3, which rapidly decays in the absence of cytokine-mediated STAT5 signals. Short-term incubation of PBMCs or isolated CD4 T cells, but not of lymph node cells, with IL-2, -7, or -15 more than doubles the frequency of Foxp3 CD25 among CD4 T cells detectable by flow cytometry. This increase is not due to cell division but to upregulation of both proteins. At the same time, the uncovered Treg cells up-regulate CD25 and down-regulate CD127, making them accessible to viable cell sorting. "Latent" Treg cells have a demethylated FOXP3 TSDR sequence, are enriched in naïve, non-cycling cells, and are functional. The confirmation of our findings in RA and SLE patients shows the feasibility of uncovering latent Treg cells for immune monitoring in clinical settings. Finally, our results suggest that unmasking of latent Treg cells contributes to the increase in circulating CD4 CD25 Foxp3 cells reported in IL-2 treated patients.

In rodents, low doses of CD28-specific superagonistic monoclonal antibodies (CD28 superagonists, CD28SA) selectively activate regulatory T cells (Treg). This observation has recently been extended to humans, suggesting an option for the treatment of autoimmune and inflammatory diseases. However, a mechanistic explanation for this phenomenon is still lacking. Given that CD28SA amplify T cell receptor (TCR) signals, we tested the hypothesis that the weak tonic TCR signals received by conventional CD4+ T cells (Tconv) in the absence of cognate antigen require more CD28 signaling input for full activation than the stronger TCR signals received by self-reactive Treg. We report that in vitro, the response of mouse Treg and Tconv to CD28SA strongly depends on MHC class II expression by antigen-presenting cells. To separate the effect of tonic TCR signals from self-peptide recognition, we compared the response of wild-type Treg and Tconv to low and high CD28SA doses upon transfer into wild-type or H-2M knockout mice, which lack a self-peptide repertoire. We found that the superior response of Treg to low CD28SA doses was lost in the absence of self-peptide presentation. We also tested if potentially pathogenic autoreactive Tconv would benefit from self-recognition-induced sensitivity to CD28SA stimulation by transferring TCR transgenic OVA-specific Tconv into OVA-expressing mice and found that low-dose CD28SA application inhibited, rather than supported, their expansion, presumably due to the massive concomitant activation of Treg. Finally, we report that also in the in vitro response of human peripheral blood mononuclear cells to CD28SA, HLA II blockade interferes with the expansion of Treg by low-dose CD28SA stimulation. These results provide a rational basis for the further development of low-dose CD28SA therapy for the improvement of Treg activity.