Selective-CD28 blockade breaks Teff/APC immunological synapses (IS) and enhances Treg/APC immunological synapses.

<p>Human Teff and Treg were introduced into Labtech coated with poly-L-lysine and containing a pool of 3 B-EBV cell lines (APC). After 15 min incubation at 37°C, cells were fixed, stained and imaged by confocal microscopy. (<b>A</b>) Localization of CD3 (red) and PKC-θ (green) in the IS formed by Teff and APCs. (<b>B</b>) Expression of CD3 and PKC-θ on whole cell after CD28-blockade. (<b>C</b>) Distribution of CD3 (red) and PKC-θ (green) on Treg cells in control condition. (<b>D</b>) Polarization of CD3 and PKC-θ in the IS formed by Treg and APCs after CD28-blockade. Ten to 15 microscope fields were examined and the experiment has been repeated 3 times independently Therefore data are representative of more than 90 events. Histograms represent CD3 (red) and PKC-θ (green) intensity at the interaction between Teff (A, B) and Treg (C, D) with APCs and indicate the distribution of these 2 molecules at the interaction level with or without CD28 blocking antibody.</p

Enhanced suppressive activity of Treg with selective CD28-blockade.

<p>Treg cells were first primed against alloantigens, washed out and added to CD4⁺CD25⁻ effector T cells stimulated with allogeneic irradiated mDC at the indicated ratio. Priming was performed with or without CD28 antagonists. Round symbols: no antagonist. Squares: addition of a CD28 antagonist. CD4⁺CD25⁻ effector T cells proliferation (A, B) and IL-2 synthesis (C, D) after addition of clonal Treg (empty symbols; A, C) or natural Treg (filled symbols; B, D). Addition of Treg to APC in the absence of CD4⁺CD25⁻ effector T cells resulted in less than 1000 CPM and undetectable IL-2 synthesis (data not shown). Results are mean cpm ± SD or concentration in supernatants measured by CBA of one representative assay out of 3. *p<0.05.</p

Live-cell dynamic analysis of human Teff and Treg cells in presence of human B-EBV APCs.

<p>(<b>A, C</b>) Screenshots from movies representing Teff or Treg cells stained in green (non-activated cells) or red (activated cells) by Fura2-AM calcium probe in the presence of unstained B-EBV lymphocytes (APCs) in poly-L-lysine coated Labtech. Teff and Treg were pre-incubated with anti-CD28 (FR104) or anti-CD28+ anti-CTLA-4 or nothing (control). Images were analyzed over a period of 20 min. (<b>B, D</b>) Teff and Treg cell motility over 20 minutes period in indicated conditions. (<b>E</b>) Mean of contact times between Teff cells and APCs in the presence or not of various indicated antibodies. (<b>F</b>) Mean of traveled distances by Teff over 20 minutes in the same conditions as above. (<b>G</b>) Mean of Teff cell velocity over 20 minutes in the same conditions as above. (<b>H</b>) Mean of contact times between Treg cells and APCs in the presence or not of various indicated antibodies. (<b>I</b>) Mean of traveled distances by Treg cells over 20 minutes in the same conditions as above. (<b>J</b>) Measurement of Teff cell velocity over 20 minute period in the same conditions as above. All indicated antibodies were used at 10 µg/ml. (n≥30 cells for each condition) ***P<0.001; **P<0.01 and *P<0.05.</p

Calcium flux profiles and quantification of calcium responses in Teff and Treg.

<p>The calcium flux of Teff (<b>A</b>) and Treg (<b>C</b>) cells, which established contacts with APCs, were analyzed by measuring the fluorescence of calcium probe (Fura2-AM) over a period of 20 minutes. All antibodies were used at 10 µg/ml. The profile of one representative cell for each condition is shown. Quantification of Teff cell (<b>B</b>) and Treg (<b>D</b>) activation was set by the number of calcium flux peaks/min. The number of calcium peaks analyzed in each condition was >30. *P<0.05 and ***p<0.001.</p

Proposed model for the role of CD28, CTLA-4 and PD-L1 in human Teff and Treg-APCs interactions.

<p>In control conditions, CD80/86 expressed by APCs interact with CD28, CTLA-4 and PD-L1 on Teff and Treg leading to long contacts and activation of Teff and to short contacts and absence of activation of Treg (A). In the presence of a selective CD28 antagonist, CD80/86 interact with CTLA-4 and PD-L1, leading to enhanced motility, reduced contacts and absence of activation in Teff, and to enhanced contacts and activation in Treg. Interestingly Treg motility is not affected, showing absence of modulation by CTLA-4 of the TCR-Stop signal in human Treg (B). If CD28 and CTLA-4 are simultaneously blocked, CD80 interacts with PD-L1. In these conditions Teff make long contacts with APCs but do not activate. Treg make short contacts and do not activate either, like in control conditions, and show enhanced velocity but not motility (C). If CD28, CTLA-4 and PD-L1 are simultaneously blocked, the only additional difference is that motility is increased in Treg, which shows that PD-L1 controls motility of human Treg (D). PD-1 and CD86 are also expressed by T cells and this adds a layer of complexity if T-T interactions had to be also addressed in addition to T-APCs interactions.</p

Phenotypic and functional analyses of human regulatory T cells (Treg#1) and allogeneic human B-EBV cells phenotype.

<p>(<b>A</b>) Proliferation of CD4⁺CD25⁻ T cells stimulated with irradiated allogeneic PBMC at day 5 in presence or not of autologous natural CD4⁺CD25^HighCD127^Low regulatory T cells (nTreg) or clone Treg#1 at a 1∶1 ratio. **P<0.01. (<b>B</b>) The phenotype of Treg#1 clone is compared to effector T cell (Teff) by Flow Cytometry. Control: filled gray, Teff: black line and Treg#1: red line. (<b>C</b>) Costimulatory molecule expression analysis on CD20⁺ B-EBV lymphocytes (pool of 3 cell lines). Control: filled gray and B-EBV cells: green line.</p

LFA-1 activation analyses by Flow Cytometry.

<p>(<b>A</b>) Profiles of CD11a, CD18 activation epitope (CD18^act, representing “high affinity” conformation) and CD18 (CD18^tot) expression by Teff and Treg#1 cells pre-incubated or not with indicated antibodies. Anti-CD28 and anti-CTLA-4 were used at 10 µg/ml. (<b>B</b>) Histograms of Mean Fluorescent Intensity (MFI) of CD11a, CD18^act and CD18^tot expressed on Teff and Treg#1 cells. Ratio CD18^act MFI: CD18^tot was established to analyze LFA-1 high affinity conformation in indicated conditions. Data are representative of more than three different experiments. Filled gray, negative control; Red line, Teff and Treg cells alone; Green line, cells with APC; Blue line, cells with APC and anti-CD28; and Black line: cells with APC, anti-CD28 and anti-CTLA-4.</p

Novel CD28 antagonist mPEG PV1-Fab’ mitigates experimental autoimmune uveitis by suppressing CD4+ T lymphocyte activation and IFN-γ production

<div><p>Autoimmune Uveitis is an important chronic inflammatory disease and a leading cause of impaired vision and blindness. This ocular autoimmune disorder is mainly mediated by T CD4⁺ lymphocytes poising a T_H1 phenotype. Costimulatory molecules are known to play an important role on T cell activation and therefore represent interesting therapeutical targets for autoimmune disorders. CD28 is the prototypical costimulatory molecule for T lymphocytes, and plays a crucial role in the initiation, and maintenance of immune responses. However, previous attempts to use this molecule in clinical practice achieved no success. Thus, we evaluated the efficacy of mPEG PV1-Fab’ (PV1), a novel selective CD28 antagonist monovalent Fab fragment in the treatment of Experimental Autoimmune Uveitis (EAU). Here, we showed that PV1 treatment decreases both average disease score and incidence of EAU. A decrease in the activation profile of both T CD4⁺ and T CD8⁺ eye-infiltrating lymphocytes was evidenced. In the periphery, T CD4⁺ cells from PV1-treated mice also showed a decrease in their activation status, with reduced expression of CD69, CD25, and PD-1 molecules. This suppression was not dependent on Treg cells, as both their frequency and absolute number were lower in PV1-treated mice. In addition, frequency of CD4⁺IFN-γ⁺ T cells was significantly lower in PV1-treated group, but not of IL-17-producing T cells. Moreover, after specific restimulation, PV1 blockade selectively blocked IFN-γ production by CD4⁺ lymphocytes Taken together, our data suggest that mPEG PV1-Fab’ acts mainly on IFN-γ-producing CD4⁺ T cells and emphasize that this specific CD28 blockade strategy is a potential specific and alternative tool for the treatment of autoimmune disorders in the eye.</p></div

mPEG PV1-Fab’ treatment decreases T_reg population.

<p>Female B10.RIII mice were immunized with 50 μg/animal of 161–180 IRBP in CFA, plus 500 ng/animal of PTx boost. Starting on day 9, mice were treated every 4 days with CD28 antagonist, PV1 (10mg/Kg; ip), or left untreated. On day 14 mice were sacrificed and dLN were collected for immunophenotyping of regulatory T cells. (A) Representative plot showing gate strategy for defining T_reg population. Foxp3 expression was defined by using a Fluorescence Minus One (FMO) control. (B) Frequency and (C) total number of CD3⁺CD4⁺CD25⁺Foxp3⁺ cells in dLN of B10.RIII mice. (D) Frequency and (E) total numbers of CD3⁺CD4⁺CD25⁺Foxp3⁺ cells in spleen of B10.RIII mice. Data are representative of three independent experiments; 5-mice per group. Median and range are depicted. **, p<0.01, two-tailed Mann-Whitney test.</p

PV1-treated mice exhibited less activated eye-infiltrating lymphocytes.

<p>B10.RIII mice were immunized with 50 μg/animal of 161–180 IRBP in CFA, plus 500 ng/animal of PTx boost. Starting on day 9, mice were treated every 4 days with CD28 antagonist, PV1 (10mg/Kg; ip), or left untreated. On day 14 mice were sacrificed and eyes were collected for immunophenotyping of eye-infiltrating leukocytes. (A) Total count of eye-infiltrating leukocytes. (B) Frequency of CD4⁺ and CD8⁺ T lymphocytes infiltrating the eyes of B10.RIII mice. (C) Total number of CD4⁺ T lymphocytes and (D) CD8⁺ T lymphocytes. (E) Representative plots display CD44 and CD62L expression by CD4⁺ and CD8⁺ cells. (F) T_effector/T_naïve ratio (as defined by CD44 and CD62L expression) for CD4⁺ and (G) CD8⁺ T lymphocytes. (H) Frequency of CD4⁺CD25⁺ T cells in uveitic eyes. (I) Frequency of CD4⁺PD-1⁺ T cells uveitic eyes. Data combined from three independent experiments; 5–10 mice per group. In (A), (C), (D), (F) and (G), median and range are depicted. In (B), (H) and (I) Mean ± SD are depicted.*, p<0.05, two-tailed Mann-Whitney test.</p