Blockade of T Cell Activation Using a Surface-Linked Single-Chain Antibody to CTLA-4 (CD152)

Abstract

CTLA-4 (CD152) engagement can down-regulate T cell activation and promote the induction of immune tolerance. However, the strategy of attenuating T cell activation by engaging CTLA-4 has been limited by sharing of its natural ligands with the costimulatory protein CD28. In the present study, a CTLA-4-specific single-chain Ab (scFv) was developed and expressed on the cell surface to promote selective engagement of this regulatory molecule. Transfectants expressing anti-CTLA-4 scFv at their surface bound soluble CTLA-4 but not soluble CD28. Coexpression of anti-CTLA-4 scFv with anti-CD3ε and anti-CD28 scFvs on artificial APCs reduced the proliferation and IL-2 production by resting and preactivated bulk T cells as well as CD4+ and CD8+ T cell subsets. Importantly, expression of anti-CTLA-4 scFv on the same cell surface as the TCR ligand was essential for the inhibitory effects of CTLA-4-specific ligation. CTLA-4-mediated inhibition of tyrosine phosphorylation of components of the proximal TCR signaling apparatus was similarly dependent on coexpression of TCR and CTLA-4 ligands on the same surface. These findings support a predominant role for CTLA-4 function in the modification of the proximal TCR signal. Using T cells from DO11.10 and 2C TCR transgenic mice, negative regulatory effects of selective CTLA-4 ligation were also demonstrated during the stimulation of Ag-specific CD4+ and CD8+ T cells by MHC/peptide complexes. Together these studies demonstrate that selective ligation of CTLA-4 using a membrane-bound scFv results in attenuated T cell responses only when coengaged with the TCR during T cell/APC interaction and define an approach to harnessing the immunomodulatory potential of CTLA-4-specific ligation.

Lack of a role for transforming growth factor-β in cytotoxic T lymphocyte antigen-4-mediated inhibition of T cell activation

“…CTLA-4 ligation on previously activated T cell blasts has been shown to rapidly inhibit the phosphorylation of the CD3 chain and the Src kinase ZAP-70 (28,29). A similar study demonstrated that ligation of CTLA-4 regulates the phosphorylation and activation of the downstream-signaling molecules ERK and JNK (30).…”

The soluble isoform of CTLA‐4 as a regulator of T‐cell responses

“…The rationale behind anti-CTLA-4 Ab therapy is that it enhances immune responses against tumor Ags primarily by enhancing tumor-specific effector T-cell responses. With regard to boosting effector T-cell responses, however, blockade of CTLA-4 is surprisingly inconsistent; with several groups reporting that blockade of mCTLA-4 interaction with B7 ligands in the presence of TCR coactivation can actually inhibit T-cell activation [39][40][41][42][43][44]. In particular, experiments in which cell surface cross-linking of mCTLA-4 occurs demonstrate the capacity of anti-CTLA-4 antibodies to inhibit T-cell responses.…”

Ligation of Cytotoxic T Lymphocyte Antigen-4 to T Cell Receptor Inhibits T Cell Activation and Directs Differentiation into Foxp3+ Regulatory T Cells

“…One mechanism by which CTLA-4 may induce T cell inhibition involves recruitment of the Src homology-2 domain-containing protein tyrosine phosphatase-1 (12) and protein phosphatase 2A (13,14) to the vicinity of the T cell receptor (TCR) in the immune synapse (15,16). This recruitment may result in dephosphorylation of the signaling molecules within the TCR complex (17) and consequent termination of T cell activation.…”

“…These ligands must be provided in the cis configuration and expressed on the same APC that activates the T cell (17). Hence, effective CTLA-4-mediated inhibition of T cells requires both proper configuration of the effector molecules and correct timing of their interactions.…”

“…However, early engagement of CTLA-4 by its ligand and its subsequent cross-linking to the TCR can prematurely dampen TCR signaling, causing T cell inhibition and hyporesponsiveness, or anergy. This concept has been validated experimentally using a variety of methods, including the following: (i) cross-linking T cell-activating antibodies (anti-CD3/anti-CD28) using an agonistic anti-CTLA-4 antibody by co-immobilization on a bead or via a secondary antibody (19 -21); (ii) molecularly engineering a surfacelinked agonistic scFv against CTLA-4 on an APC (17,22,23); and (iii) chemically cross-linking antibodies that recognize specific antigens on an APC to an agonistic anti-CTLA-4 antibody (24 -26).…”