This process leads to the onset of T-cell-mediated ocular inflammation, with cellular features resembling those of human AIU. proof of concept studies for CD28 antagonists in autoimmunity, with a special focus on the mechanisms of action. in mice leads to a lethal lymphoproliferative syndrome [6,7], whilst in humans some immune dysregulation (such as Graves disease, autoimmune hypothyroidism, and type 1 diabetes)  and lymphoproliferative diseases  result from genetic alterations that cause CTLA-4 deficiency. Open in a separate window Physique 1 While many T cell co-stimulatory systems have been described, the main molecules driving de novo T cell responses are CD28 and CTLA-4. CD28 binds to CD80/86 Rutaecarpine (Rutecarpine) expressed on antigen-presenting cells and warms up T cells (through the activation of pro-inflammatory, pro-survival, and activating and proliferation factors), whereas CTLA-4 cools it down by counteracting CD28-mediated events, by eliminating CD80 molecules on antigen-presenting cells and by activating unfavorable regulators. The first biologics used to interfere with CD28-mediated signals were wild-type or high-affinity, recombinant-soluble domains of CTLA-4 fused with an immunoglobulin Fc domain name (e.g., many forms of labscale CTLA4-Ig, abatacept, and belatacept). CTLA4-Ig molecules dock onto CD80 and CD86 and therefore inhibit binding of CD28. By doing so, CTLA4-Ig molecules have the capacity to also inhibit binding of CTLA-4 to CD80/86 (and also of CD80 to PD-L1 ), which might perturb the co-inhibitory function of membrane-bound CTLA-4. Indeed, a reduced accessibility of CTLA-4 for CD80/86, with CTLA4-Ig or other reagents, led experimentally in vitro [11,12] and in vivo [13,14,15] to a reduction of the suppressive functions of Treg cells. Therefore, we and others proposed [16,17] that selectively targeting CD28 might share the benefit of CTLA4-Ig (blockade of CD28-mediated signals) without perturbing the co-inhibitory Rutaecarpine (Rutecarpine) CD80/86-CTLA4 axis required for the control of Treg cell functions and for the control of effector T cells, particularly for highly differentiated effector memory T cells (such as Th17 cells), which are tightly controlled by CTLA-4 . Since then, the selective CD28 blockade proof of concept has been tested in experimental transplantation and autoimmune settings and has also begun evaluation in humans. Blocking the CD28-CD80/CD86 pathway using anti-CD28 monoclonal antibodies (mAbs) has been challenging due to the inadvertent stimulatory activity of most conventional anti-CD28 mAbs (for review ). More precisely, because CD28 is expressed on cell membranes as homodimers, anti-CD28 antibodies, which are also homodimeric molecules, induce a clustering CXCR7 of CD28 molecules, which results in the phosphorylation of PI3K, a molecular signal also induced by engagement of CD80/86 . This occurs independently of the binding epitope, so that a given anti-CD28 antibody can be an antagonist of CD80/86 (if it binds to the MYPPPY domain name recognized by CD80/86) while still presenting agonistic properties. To date, all antibodies directed against CD28 were found to activate the receptor instead of only blocking access to its ligand. An exception is the anti-rat JJ319 mAb, which in vivo rapidly induces internalization of CD28 and presents functional antagonist properties . To our knowledge, an antibody-inducing CD28 downmodulation has not been identified in another species. Superagonistic anti-CD28 antibodies (such as the TGN1412 antibody ) bind to the laterally uncovered CD loop of CD28 and induce a non-physiological engagement of CD28 Rutaecarpine (Rutecarpine) resulting in polyclonal T cell activation and cytokine release even in the absence of TCR stimulation . To develop antagonist-only anti-CD28 antibodies, mutations in the Fc domain name have been introduced to prevent Rutaecarpine (Rutecarpine) cross-linking of CD28 through Fc/FcR conversation. However, while this strategy was efficient in vivo in rodents [24,25], Fc-silenced anti-CD28 mAb still co-stimulated human T cell proliferation and cytokine release in vitro , which halted their clinical development. In our first studies specifically targeting CD28, we also introduced a strategy that.