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. 2012 Mar 30;287(14):11098-107.
doi: 10.1074/jbc.M111.283705. Epub 2012 Feb 15.

Ligation of cytotoxic T lymphocyte antigen-4 to T cell receptor inhibits T cell activation and directs differentiation into Foxp3+ regulatory T cells

Jozsef Karman  1 , Ji-Lei Jiang, Nathan Gumlaw, Hongmei Zhao, Juanita Campos-Rivera, Jose Sancho, Jinhua Zhang, Canwen Jiang, Seng H Cheng, Yunxiang Zhu
Affiliations

Ligation of cytotoxic T lymphocyte antigen-4 to T cell receptor inhibits T cell activation and directs differentiation into Foxp3+ regulatory T cells

Jozsef Karman et al. J Biol Chem. .

Abstract

Cross-linking of ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) during the early phase of T cell activation attenuates TCR signaling, leading to T cell inhibition. To promote this event, a bispecific fusion protein comprising a mutant mouse CD80 (CD80w88a) and lymphocyte activation antigen-3 was engineered to concurrently engage CTLA-4 and cross-link it to the TCR. Cross-linking is expected to be attained via ligation of CTLA-4 first to MHCII and then indirectly to the TCR, generating a CTLA-4-MHCII-TCR trimolecular complex that forms between T cells and antigen-presenting cells during T cell activation. Treating T cells with this bispecific fusion protein inhibited T cell activation. In addition, it induced the production of IL-10 and TGF-β and attenuated AKT and mTOR signaling. Intriguingly, treatment with the bispecific fusion protein also directed early T cell differentiation into Foxp3-positive regulatory T cells (Tregs). This process was dependent on the endogenous production of TGF-β. Thus, bispecific fusion proteins that engage CTLA-4 and co-ligate it to the TCR during the early phase of T cell activation can negatively regulate the T cell response. Bispecific biologics with such dual functions may therefore represent a novel class of therapeutics for immune modulation. These findings presented here also reveal a potential new role for CTLA-4 in Treg differentiation.

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Figures

FIGURE 1.
FIGURE 1.
Designs of BsB and BsBΔ. A, schematic drawings of the BsB and BsBΔ fusion proteins. B, schematic drawing of pMHCII, the TCR and co-stimulatory molecules in the immune synapse, as well as the proposed scheme for BsB-mediated cross-linking of CTLA-4 to the TCR via the CTLA-4-MHC II-TCR trimolecular complex. The fusion protein engages CTLA-4 and indirectly ligates the TCR via binding to MHCII in the immune synapse. The two solid sides of the triangle denote cross-linking of MHCII and CTLA-4 as well as MHCII and TCR; the dashed side depicts ligation of CTLA-4 to TCR. The dotted line indicates inhibition of TCR signaling by BSB-engaged CTLA-4. C, schematic drawing showing that the action of BsBΔ is similar to that of BsB except it is unable to ligate the TCR.
FIGURE 2.
FIGURE 2.
Inhibition of IL-2 production in allogenic T cell activation and T cell proliferation by BsB in a mixed lymphocyte reaction. A, naïve T cells from C57BL/6 mice and LPS-treated and irradiated BALB/c APCs were mixed with the test constructs for 2 days. Culture media were then harvested and assayed for IL-2. Only BsB and CTLA-4Ig inhibited T cell activation, as indicated by a decreased amount of IL-2 in the media. B, T cell proliferation as assessed by CFSE dilution. Mixed lymphocyte reactions were set up as described in A; however, naive T cells were prelabeled, and the cells cultured for 5 days to allow cell divisions to occur as indicated by CFSE dilution. mCTLA-4Ig inhibited both IL-2 production and cell proliferation, whereas BsB inhibited IL-2 production but increased T cell proliferation. The figure is representative of more than five independent but similar studies.
FIGURE 3.
FIGURE 3.
Induction of Foxp3+ Tregs and IL-10 and TGF-β production by BsB and involvement of CTLA-4 and TGF-β in Treg induction. A, allogenic mixed lymphocyte reactions were set up as described in the legend to Fig. 2, using naïve CD4+CD62LhiCD25−GFP− cells that had been isolated from Foxp3-EGFP knock-in mice in the presence of the test constructs. Five days post-activation, CD4+ T cells were analyzed for GFP expression by flow cytometry. Tregs were gated as GFP+ and CD25+ cells. Only BsB treatment led to GFP expression, indicating induction of Foxp3+ Tregs (middle left panel). Culture media were collected for cytokine analysis (right panels), which revealed elevated IL-10 and TGF-β levels in the presence of BsB. B, mCTLA-4Ig, mLAG-3Ig, BsBΔ + mLAG-3Ig failed to induced Tregs. C, CTLA-4 engagement is required for Treg induction. Inclusion of a blocking antibody to CTLA-4 reduced Tregs by ∼50%. D, requirement of autocrine TGF-β for Treg induction is indicated by the complete blockade of Treg induction in the presence of a blocking antibody to TGF-β. The data are representative of numerous independent but similar studies. In both C and D, naïve T cells from wildtype C57BL/6 mice were used as these studies were performed to concurrently evaluate Treg proliferation using CFSE labeling, which was not compatible with EGFP.
FIGURE 4.
FIGURE 4.
Suppressive function of BsB-induced Tregs. A, BsB- or TGF-β-induced Tregs were purified by flow cytometry and mixed with CFSE-labeled naïve Tresp prepared from C57BL/6 mice at the indicated ratios in Transwells (hatched columns) or regular culture wells (filled columns). LPS-treated allogenic BALB/c APCs were added to stimulate T cell activation. The results (mean + S.E.) indicate the percentage of proliferating Tresp, based on a CFSE dilution without Tregs (Tresp + APC only) set to 100%. B, anti-IL-10 and anti-TGF-β antibodies were added to cells in regular culture wells at a Tresp:Treg ratio of 1:1 to determine the contribution of cytokines to T cell proliferation. The anti-TGF-β antibody partially inhibited the suppressive function of TGF-β-induced Tregs (left panel) but did not affect BsB-induced Tregs (right panel). The figure is representative of more than three independent but similar studies.
FIGURE 5.
FIGURE 5.
Down-regulation of AKT and mTOR phosphorylation by BsB. Naïve T cells were cultured in round-bottomed 96-well plates co-coated with anti-CD3, anti-CD28, and BsB, mouse IgG (mIgG), or mouse PD-L1 (mPD-L1) for 18 h. Cells deemed not activated were cultured in wells coated with IgG only. The phosphorylation status of AKT and mTOR was then monitored by flow cytometry after staining with fluorescently labeled antibodies to phosphorylated AKT and mTOR. MFI denotes mean fluorescent intensity. This figure represents one of three independent experiments.
FIGURE 6.
FIGURE 6.
Sustained Foxp3+ expression in Tregs in response to continuous stimulation with BsB. Round-bottomed 96-well plates were co-coated with anti-CD3, anti-CD28 and BsB or mouse IgG. Naïve T cells from Foxp3-EGFP knock-in mice were cultured for 5 days to induce Tregs (left panels), which were then purified from the BsB-treated cells (red square) and restimulated in another round of culture in co-coated wells, as above, for 5 days, before analysis by flow cytometry for GFP+ cells. Reculturing of purified Tregs with the mouse IgG control for 5 days resulted in a loss of Foxp3+ expression in ∼60% of cells (upper right quadrant of upper right panel), whereas <7% of the Tregs recultured with BsB had lost Foxp3+ expression (upper right quadrant of bottom right panel). This figure represents one of three independent experiments.
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