WO2012127240A2

WO2012127240A2 - Immune assay

Info

Publication number: WO2012127240A2
Application number: PCT/GB2012/050635
Authority: WO
Inventors: Omar Saleem QURESHI; David Michael SANSOM
Original assignee: University of Birmingham
Current assignee: University of Birmingham
Priority date: 2011-03-24
Filing date: 2012-03-22
Publication date: 2012-09-27
Anticipated expiration: 2013-09-24
Also published as: GB201104950D0; WO2012127240A3

Abstract

The invention relates to a method of identifying a modulator of the immune response system, comprising assaying for the effect of a compound on the internalisation and recycling of CTLA-4 in a cell, wherein an increase in cycling of CTLA-4 compared to a control without the compound indicates that the compound suppresses the immune response and a decrease in the cycling of CTLA-4 compared to a control without the compound, indicates that the compound increases the immune response. The cycling of CTLA-4 may be determined by monitoring CTLA-4 or monitoring uptake of a CTLA-4 label into a cell.

Description

Immune Assay

The invention relates to a method of assaying for modulators of the immune system by monitoring the cycling of CTLA-4 in cells.

CTLA-4 is an essential regulator of immune responses whose mechanism of action is the subject of debate. The inventors demonstrate herein that a central molecular feature of CTLA-4 is its ability to capture and degrade co-stimulatory ligands from dendritic cells by a process of trans-endocytosis. Accordingly, all cells expressing CTLA-4, including activated and regulatory T cells, can remove and degrade costimulatory molecules resulting in impaired T cell costimulation. Moreover, acquisition of CD86 from antigen presenting cells can be demonstrated in vivo and is dependent on TCR engagement and CTLA-4 expression. These results provide a novel mechanism of immune regulation whereby CTLA-4 acts as an effector molecule to inhibit CD28 costimulation by the cell-extrinsic depletion of ligands. This model accounts for many of the key features of the CD28-CTLA-4 system providing an explanation for the sharing of ligands between CTLA-4 and CD28, the phenomenon of CTLA-4 endocytosis and the constitutive expression of CTLA-4 on regulatory T cells. CD80 is another costimulatory molecule that acts in the same way as CD86. The binding of CD86 and CD80 to CD28 act to stimulate the cell.

The information presented herein shows that the cycling of CTLA-4 is critical to the activation or suppression of the immune system and therefore provides a new way of identifying compounds that modulate the immune system. CTLA-4 (Cytotoxic T- Lymphocyte Antigen 4) is also known as CD 152 and in humans is encoded by the human CTLA-4 gene. It comprises an extracellular V domain, a transmembrane domain and cytoplasmic tail. Mutations in the gene have been associated with diseases such as Grave's disease, Hashimoto's disease, celiac and systemic lupus erythematosis.

The inventors have now identified that CTLA-4 binding to, for example, CD80 and CD86 ligands, takes the ligands into the cell where they are destroyed, and is then returned to the surface of the cell. By monitoring the recycling of CTLA-4 it is possible to identify compounds that will modulate the immune system by affecting the CTLA-4 cycling system. The invention provides a method of identifying a modulator of the immune response system, comprising assaying for the effect of a compound on the intemalisation and recycling of CTLA-4 in a cell, wherein an increase in cycling of CTLA-4 compared to a control without the compound indicates that the compound suppresses the immune response and a decrease in the cycling of CTLA-4 compared to a control without the compound, indicates that the compound increases the immune response.

Typically the cycling of the CTLA-4 is determined by monitoring the uptake of a CTLA-4 dependent label into the cell. The CTLA-4 dependency may be tested, for example by deleting CTLA-4 from the cell or blocking CTLA-4 with anti-CTLA-4 antibodies which are generally known in the art. For example, Ipilimumab is a monoclonal antibody which is produced by Bristol-Myers Squibb and binds to CTLA-4.

CTLA-4 uptake may be dependent on CTLA-4 binding to CD80 or CD86, especially CD86. CD80 and CD86 are generally known in the art and are found on dendritic cells activated B cells and monocytes.

The CD80 or CD86 may be directly or indirectly labelled. The label may be, for example, a radiolabel such as ³⁵S or ¹¹¹ In and the uptake is measured by increased radioactivity due to the CD80 or CD86 binding CTLA-4 and being taken into the cell. The CD80 or CD86 may be a fusion protein of the whole protein, or the CTLA-4 binding region of the protein with a detectable label, such as a colourimetric label. The colourimetric label may be, for example, green fluorescent protein (GFP) or aquorin. Alternatively, it may be detected using a bound fluorescent label such as FITC. It may also be labelled with biotin and the presence of the CD80 or CD86 may be detected using labelled avidin which binds to the biotin. The uptake of CD80 or CD86 may also be detected using labelled anti-CD80 or anti-CD86 antibodies, which are commercially available. The uptake of coloured labels may be detected using flow cytometry or confocal microscopy to detect the presence or amount of label in the cells. Such labels are generally known in the art.

Western blotting may be used to detect the uptake of CD80 or CD86 by the CTLA-4 expressing cell. The cell typically expressing CTLA-4 is typically a eukaryotic cell such as a mammalian, such as mouse or hamster, or fish (such as zebra fish) and insect cell. Preferably the cell is a human cell. It may be a T cell, for example an activated T cell, such as a regulatory T cell, It may be CD4⁺ CD25^" or CD4⁺ CD25⁺. It may naturally express CTLA-4. Alternatively, CTLA-4 may be expressed recombinantly in the cell. Methods of expressing proteins such as CTLA-4 are generally known in the art. The cell may be a Jurkat cell of a Chinese Hamster Ovary cell (CHO cell). The cells are preferably in vitro.

The CTLA-4-expressing cell may act as a recipient cell. The cell may receive CD86 or CD80 from a donor cell expressing the ligands. The donor cell may be a dendritic cell or CHO cell expressing CD86 or CD80.

The cells may be part of a whole organism such as a non-human mammal, fish or insect.

For example, the organism may be a rodent such as a mouse. The mouse may be, for example a CTLA-4^+/+D011 x ripmOV A/Rag-/- mouse.

The modulator may be a lysosomal inhibitor which inhibits CTLA-4 cycling. Such inhibitors include bafilomycin A.

The modulator may also be a compound that blocks binding of CD80 or CD86 to CTLA-4. These include antibodies or fragments of antibodies against CTLA-4.

Other inhibitors include inhibitors of CTLA-4 transendocytosis which disrupt removal of ligands. These will prevent CTLA-4 from suppressing immune responses thereby increasing immune responses. Example inhibitors will include those targeting actin recruitment to the CTLA-4 containing vesicles such as Latrunculin, a naturally occuring toxin obtained from sponges.

Blocking delivery of CTLA-4 to the plasma membrane by interfering with vesicle trafficking pathways will promote immune activation by disrupting CTLA-4 function. Example compounds for this purpose include inhibitors of phospholipase D (butan-l-ol) or inhibitors of ARF-1 such as brefeldin A. Compounds inhibiting delivery of CTLA-4 or its bound ligands to lysosomes will be effective immunomodulatory compounds. Such compounds will include lysosome neutralising compounds (ammonium chloride), vacuolar ATPase inhibitors (Bafilomycin A) and inhibitors of ubiquitylation.

The applicants have discovered that CTLA-4 degradation is controlled by modification of CTLA-4 with ubiquitin. Ubiquitination of CTLA-4 itself directly modifies the stability of CTLA-4, its surface expression and re-cycling pattern, all of which impact on its ability to function by removal and destruction of the ligand. Hence manipulation of the ubiquitin enzyme system has potential therapeutic benefit.

Ubiquitin contains 76 amino acid residues, two of which have particular importance (Gly-78 and Lys-48). Following activation of Gly-78 in an ATP-dependent manner ubiquitin is attached to the ε-amino group of lysine on the protein substrate.

Lys-45 is located on the surface of a Type III reverse turn and can generate multi-ubiquitin chains since its ε-amino group is exposed such that it can form an additional amide isopeptide bond (Myung, J., et al Med. Res. Rev. (2001) 21, 245-273).

Several cellular proteins use ubiquitination modification as their targeting signal. However, polyubiquitination and proteasomal degradation is not the only pathway followed by a membrane protein. Mono-ubiquitination, which involves the addition of an ubiquitin molecule to one lysine, or multi-ubiquitination, which involves the addition of an ubiquitin molecule to several lysine residues, can regulate the internalisation and sorting of target receptors to endocytic compartments (d'Azzo et al 2005 Traffic, 6, 429-441). ATP used by the ubiquitin- activating enzyme charges ubiquitin and forms an ubiquitin C-terminal adenylate. The activated ubiquitin molecule is then passed onto the ubiquitin-conjugating enzymes. Finally, ubiquitin-protein isopeptide ligases (E3) bind to the target protein and attach the C-terminus of ubiquitin to lysine residues of the target. Following the mono- ubiquitination of the protein substrate, a polyubiquitin chain can be formed through the same ubiquitination conjugation cascade (Myung et al, Supra and Hueng et al, Oncogene (2004) 23, 1958-1971).

The ubiquitin pathway provides a number of sites to target to adjust CTLA-4 recycling. The modulator being tested may be a modulator of ubiquitylation. The modulator may be:

(a) a ubiquitin ligase inhibitor. These typically conjugate to CTLA-4 thereby increasing CTLA-4 expression and immune regulation;

(b) a ubiquitin ligase enhancer that enhances interaction or activity of the CTLA-4 specific ligase, promoting CTLA-4 degeneration and imposing immune suppression;

(c) an inhibitor of de ubiquitinating enzymes (DuBs) that remove ubiquitin, resulting in increased CTLA-4 destruction and less immune suppression; and

(d) enhancing the activity of DuBs to protect CTLA-4 expression and increase immune suppression.

The use of such inhibitors or enhancers to modulate an immune response in vitro or in vivo is also provided.

The inhibitor may be MG132 (CAS number 133407-82-6) or UBEI-41 (available from Biogenova).

Compounds capable of keeping CTLA-4 and its bound ligand together in a "locked" state will downregulate CTLA-4 function. Such compounds could include antibodies recognising the complex of CTLA-4 and its bound ligands.

Compounds capable of increasing the rate of CTLA-4 recycling to the cell surface will be capable of enhancing CTLA-4 function.

Compounds capable of enhancing CTLA-4 delivery to lysosomes will promote immune activation.

The invention will now be described by way of example only with reference to the following figures:

Fig. 1. CTLA-4 mediated acquisition of co-stimulatory molecules. (A) Flow cytometric analysis of CD86-GFP transfer into CTLA-4 expressing cells. CHO cells expressing CD86- GFP (Far Red labeled) were co-cultured with CHO controls or with CTLA-4⁺ CHO cells in the presence or absence of 10 nM Bafilomycin A. Singlet CTLA-4 expressing cells were analyzed for GFP acquisition by excluding Far Red-i- donor cells from analysis (see fig. 5) (B) Projection of a confocal z-stack showing a CTLA-4⁺ CHO cell (blue) in contact with a CD86 GFP-expressing CHO cell (stains green) in the presence of Bafilomycin A (BafA). GFP inside the CTLA-4 cell appears as cyan puncta. (C) Confocal micrographs of adherent CD86- expressing CHO cells and CTLA-4⁺ CHO cells after overnight incubation. CD86 (stains green) and CTLA-4 (stains red) were detected by antibody staining. Co-localization of CD86 and CTLA-4 is shown in yellow. Lower panels show an enlargement of the boxed area, with single color images shown in white for equal contrast. CHO-CD86 cultured alone are shown in fig. S7A. (D) Confocal images of cells expressing wild type (wt) CTLA-4 or CTLA-4 lacking the cytoplasmic domain (del36) (glows red) incubated for 2 hours with CD86-GFP (glows green) expressing cells. (E) Flow cytometric analysis of CD86 surface expression on CHO-CD86 cells co-incubated with increasing numbers of untransfected (control upper line), wild-type CTLA-4 (lower line) or CTLA-4 del36 cells - middle line at right (expressed as %

+

CTLA-4 cells in the co-culture). Surface CD86 was detected by antibody staining. (F)

+

Response of CFSE-labeled CD4 CD25 T cells stimulated in the presence of anti-CD3 antibody with CD86-expressing cells fixed after co-culture with CTLA-4 WT (lower line) or CTLA-4 del36 (upper line). All data are single representatives of 3 or more independent experiments, Scale bars are 10 μιη.

Fig. 2. Human T cells use CTLA-4 to remove CD86 from dendritic cells. (A) Typical CD86 expression on a human monocyte-derived dendritic cell (DC) cultured in the absence of T

+

cells. (B) DC cultured for 72h with anti-CD3-activated CD4 CD25 T cells (outlined in white) stained with anti-CD86 (glows green) and anti-CTLA-4 (glows red). Single staining is shown as white for equal contrast. Cells were co-cultured in the absence or presence of blocking anti-CTLA-4. (C) Quantitation of surface CD80 and CD86 expression on DCs after co- culture with T cells in the presence or absence of anti-CTLA-4 determined by flow cytometry. Data show MFI change pooled from >5 experiments with SEM. (D) Confocal micrographs of allogeneic DCs co-cultured overnight with CTLA-4-transfected (CTLA-4 TF) or control resting CD4 CD25 human T cells. Cultures were fixed and stained with anti-CD86 (glows green) or anti-HLA-DR (glows red). White arrowsheads highlight position of T cells; green arrowsheads highlight DCs. White images show single color staining for contrast. (E) Mean fluorescence intensity of surface CD86 on DCs after incubation with either CTLA-4- transfected or control T cells as determined by flow cytometry. All data are representative of at least 3 independent experiments. Error bars represent the SEM. Scale bars = 10 μΜ.

Fig. 3. TCR stimulation promotes CTLA-4 trafficking and trans-endocytosis of CD86. (A)

+

Acquisition of CD86-GFP from CHO cells by human CD4 T cell blasts in the presence or absence of anti-CD3 stimulation. Right hand panels show the effect of anti-CTLA-4 antibody

+

on GFP uptake. (B) SEB-specific CD4 T cell blasts were incubated with either unpulsed or SEB-pulsed DCs. Cells were fixed and stained with anti-CD86 (glows green) and anti- CTLA-4 (glows red). Yellow indicates colocalization. Single colors are shown in white for equal contrast. (C) Surface levels of CD86 on DCs incubated with SEB-specific T cell blasts

+ + +

for 16h as determined by flow cytometry. (D) CD4 CD25 (Treg) or CD4 CD25 T cells were incubated with DCs and anti-CD3 overnight, fixed, stained using anti-CD86 (glows green), anti-CD3 for T cells (glows blue), and visualized by confocal microscopy. Arrow indicates

CD86 puncta within T cells. (E) Surface levels of CD86 on DCs incubated with CD4⁺CD25⁺

+

(Treg) and CD4 CD25 T cells overnight determined by flow cytometry. All data are representative of at least 3 independent experiments. Error bars represent the SEM. Scale bars = 10 μΜ.

Fig. 4. In vivo capture of CD86 by CTLA-4. Balbc Rag2^~/~ mice were reconstituted with CD86-GFP transduced Balbc Rag2^~/~ bone marrow to permit the development of APC expressing CD86-GFP. 3wk later mice were injected with DO11.10 CD4⁺ T cells and immunized as described in figure S16. (A) 6h after i.v. OVA peptide re-challenge, splenocytes were harvested, labeled at 4°C for CD4 (stains blue) and CD25 (stains red) and immediately imaged by confocal microscopy. Representative images of T cells from OVA peptide challenged or unchallenged mice are shown. (B) Representative images of CD4⁺ T cells purified from spleen after treatment in vivo with peptide showing CD4 and CD25 staining. (C) CD4⁺ T cells from either Ctla-4^+/+ or Ctla-4 (Rag2 DO11.10 Rip-mOVA) mice were injected into mice that previously received CD86-GFP Rag2 bone marrow cells. Cells were re-challenged with OVA in vivo as above. Splenocytes were isolated and immediately analyzed for CD86-GFP (glows green), CD25 (glows red) CD4 (glows blue) by confocal microscopy. Two representative panels are shown for both Ctla-4^+/+ and Ctla-4^'A conditions. Data are representative of 3 independent experiments. Scale bars = 10 μΜ.

Figure 5

(A) Gating strategy for analysis of CD86-GFP transfer to CTLA-4 expressing cells. CTLA-4- expressing cells and Far Red labelled CD86-GFP-expressing CHO cells (donor cells) were co-cultured for 3h. Following FACS acquisition of the total mixed population (i) cells were gated on low pulse width (ii) to exclude cell clusters. Far Red negative cells were then gated to exclude CD86-GFP donor cells from the analysis (iii). The resultant singlet CTLA-4 expressing cells were then analysed for GFP content (iv). (B) Depletion of CD86-GFP from donor cells is associated with concomitant GFP acquisition by CTLA-4 expressing recipient cells. CD86-GFP expressing CHO cells (donor cells) were co-cultured for 3h with control CHO or CTLA-4-expressing CHO cells. Compared with control CHO cells (upper panel), cells expressing CTLA-4 (lower panel, Far Red negative) acquire GFP. This is associated with a concomitant decrease in GFP signal on the CD86 donor cells (Far Red positive cells).

Figure 6

Staining for cell surface CD86 on CTLA-4 recipients post-transfer

Control CHO cells or CTLA-4 expressing CHO cells were incubated with CD86-GFP expressing CHO cells (Far Red labelled) for 3 hours. CD86 was then surface labelled by anti- CD86 PE at 4°C and gated using the strategy in Fig. 5. Whilst 36% of CTLA-4 positive cells capture CD86-GFP only 0.5% label for surface CD86. The CD86 staining of the donor cells following incubation with CTLA-4⁺ cells is shown in the lower panel, revealing a marked reduction in CD86 surface expression. Representative of 2 independent experiments.

Figure 7

Degradation of transferred CD86 in the absence of CTLA-4 degradation

(A). CTLA-4 expressing CHO cells were incubated alone (left panels) or with Far Red labelled CD86-GFP expressing CHO cells for 3 hours (right panels) in the presence or absence of the lysosomal inhibitor Bafilomycin A. Cells were then fixed and stained with anti-CTLA-4. Plots are gated on CHO-CTLA-4 (Far Red negative) cells. CD86 transfer does not reduce CTLA-4 expression in GFP+ cells. Additionally, in the presence of Bafilomycin A the CD86-GFP signal increases (due to blockade of lysosomal degradation) however the expression of CTLA-4 is not affected (lower right panel). This suggests CTLA-4 is not degraded along with ligand. (B) Histogram overlays of the data in A showing CTLA-4 expression in the presence of absence of bafilomycin. Data are representative of 2 experiments.

Figure 8

Live imaging of CD86-GFP transfer

(A) Time-lapse (0-80 minutes) confocal micrographs of GFP-tagged CD86 (green) transfer into CTLA-4 expressing cells (stained red by pulsing anti-CTLA-4 APC). White images show CD86 alone. (B) Quantitation of CD86 GFP mean fluorescence intensity of the cells shown in A. Fluorescence changes were analysed in the cells shown using Image J with each time point indicated by a circle.

Figure 9

Both CD80 and CD86 are acquired by CTLA-4 expressing cells.

CTLA-4 expressing CHO cells were incubated with Far Red-labelled donor CHO cells expressing either CD80-GFP or CD86-GFP. Cells were co-cultured for the times indicated in the presence or absence of ammonium chloride and analysed by FACS as shown in figure 5. Data are representative of at least 3 independent experiments.

Figure 10

Estimation of the number of CTLA-4 and CD86 molecules expressed in transfer experiments.

To quantify the number of CD86 molecules and CTLA-4 molecules expressed by cells, CD86 APC or CTLA-4 APC antibodies (BD Biosciences) were titrated on to beads that have known antibody binding capacity (ABC) (Quantum Simply Cellular anti-mouse IgG). The geometric mean fluorescence was determined for both CD86 and CTLA-4 antibodies generating a standard curve of MFI vs number of antibody molecules bound. This allows an estimation of the number of molecules expressed per cell for CD86 (A) or CTLA-4 (B). Arrows intersecting standard curve show fluorescence values of CD86 on CHO-CD86 transfectants or mature DC (A) and the values for CTLA-4 expressing Jurkat or CHO-CTLA-4 cells (B). CTLA-4 expressing Jurkat cells were incubated with CD86-expressing CHO cells overnight at various ratios and reduction in CD86 expression measured (C). At a ratio of 1 Jurkat cell to 1 CHO cell which corresponds to a ratio of approximately 8 CD86 molecules to 1 CTLA-4 molecule we observe a 35% reduction in CD86 expression. This reduction was sufficient to observe a functional inhibition in T cell proliferation when these cells were used to stimulate CFSE-labelled responder T cells (D). Data are representative of 2 experiments.

Figure 11

Transfer between cells is unidirectional.

(A) CD86 expression pattern is punctate in cells cultured with CTLA-4. Confocal micrograph of adherent CHO-CD86 cells cultured overnight either alone or with CHO-CTLA-4 cells. Cells were fixed, permeabilised and stained with goat anti-human CD86 (CI 9) green. Dtata are representative of more than 20 independent experiments (B) Quantification of the confocal images. The cells shown in Fig. 1C were quantified by outlining cells (n=35) using Image J software and measuring fluorescence intensity of CTLA-4 and CD86. This shows a decrease in CD86 expression in the donor cell and an increase of CD86 in the CTLA-4 recipient. The CD86 donor cell does not acquire CTLA-4. (C) CHO-CD86 expressing cells were labelled with the lipophilic dye PKH26 to label the plasma membrane and then incubated with CHO-CTLA-4 cells. Transfer of membrane dye was monitored by flow cytometry to assess the level of plasma membrane exchange between CTLA-4 and CD86 cells. CTLA-4⁺ recipents were gated on those that acquired CD86-GFP(red box) and those which did not (black box). The PKH transfer associated with these populations was then analysed. Data are representative of 2 experiments.

Figure 12

Removal of the CTLA-4 C-terminus prevents endocytosis.

(A) CHO cells stably expressing either wild type CTLA-4 or CTLA-4 del36 (lacking the cytoplasmic domain) were initially incubated with an unlabelled anti-CTLA-4 antibody at 37°C for 30 minutes to allow labelling of internalising CTLA-4. Surface receptors were then stained red (anti-mouse Ig Alexa 594) at 4°C. Cells were then fixed and permeabilized and stained with alexa 488 conjugated anti-mouse Ig. Cells that internalise the unlabelled CTLA- 4 antibody at 37°C antibody stain green. Bar chart shows quantification of plasma membrane (PM) to internalized (I) protein ratios of WT and mutant CTLA-4 plus SEM. PM/I ratios were determined by outlining cells using Image J(more 32 cells were analysed for each celltype). (B) Levels of surface expression of control (left peak) wild type (middle peak) and del36 (right peak) CTLA-4 receptors found on cells used in Figure 1 E and F analysed by flow cytometry. Data are representative of 3 experiments.

Figure 13

CTLA-4 specifically downregulates CD80 and CD86 but not CD40 on dendritic cells

(A) Dendritic cells were incubated with human CD4⁺CD25^" T cells and anti-CD3 (0^g/ml) for 4 days to allow T cell expression of CTLA-4. Cultures were carried out in the presence or absence of anti-CTLA-4 (20 μg/ml). Dendritic cells were then gated using anti-CD 11c and the levels of CD80, CD86 and CD40 analysed by flow cytometry. CD80 and CD86 levels were specifically increased by anti-CTLA-4 treatment whereas there was no change in CD40 levels.

Figure 14

Jurkat cell line expressing CTLA-4.

(A) WT control or CTLA-4-transduced (right peak) Jurkat cells were stained for total CTLA- 4 expression and analysed by flow cytometry. Isotype control staining is shown in the filled histogram and compared with wild-type untransduced (dotted line) and transduced CTLA-4 staining (red line) demonstating that WT Jurkat cells do not express CTLA-4. (B) Acquisition of CD86-GFP by CTLA-4 transduced Jurkat cells as measured by FACS compared to control Jurkat. Lower panels show the effect of Bafilomycin (BafA) (C) Electron micrograph showing CD86-HA transfer to a CTLA-4 expressing Jurkat cell. 70nm thick cryosections were stained using anti-HA antibody and detected using rabbbit anti-mouse antibody followed by protein A gold. Data are representative of 3 experiments.

Figure 15

Blocking the interaction of CTLA-4 with CD86 prevents transfer.

(A) CTLA-4 expressing Jurkat cells were incubated for 3 hours with CD86-GFP expressing CHO cells in the presence of NH₄C1. Cells were analysed as outlined in figure SI. Anti- CTLA-4 (ΙΟμ^πύ), CTLA-4 Ig (ΙΟμ^πύ) and CD80-Ig (ΙΟμ^πύ) inhibited the transfer of CD86. (B) Pooled analysis (n=4) of GFP acquisition by CTLA-4 transduced Jurkat T cells with and without anti-CTLA-4 blockade. Error bars show SEM. Figure 16

Anti-CD28 does not block CD86 transfer.

CTLA-4 expressing Jurkat cells were incubated for 3 hours with CD86-GFP expressing CHO cells in the presence of NH₄C1. Cells were analysed as outlined in figure SI. The effect of anti-CTLA-4 (ΙΟμ^πύ), anti-CD28 (ΙΟμ^πύ) or anti-CD45RO (ΙΟμ^πύ) on transfer of CD86-GFP was assessed. Data are representative of at least 3 experiments.

Figure 17

TCR stimulation drives CTLA-4 trafficking in T cells and Treg.

(A) Human CD4+ T cells were activated using anti-CD3 / anti-CD28 beads for 3 days. Cycling CTLA-4 was stained with anti-CTLA-4 labelling at 37°C for 30 minutes using a mouse anti-CTLA-4 antibody. Cells were then fixed and stained for total CTLA-4 using goat anti-CTLA-4 C-terminal antibody. Cells were then stained with anti-mouse Alexa 555 and anti-goat Alexa 488. These data demonstrate that CTLA-4 remains in a recycling pool in activated T cells. (B) T cell blasts were re-stimulated with anti-CD3/CD28 beads for lh and cycling and total CTLA-4 was detected by flow cytometry using anti-CTLA-4-PE. Left to right on cycling graph: isotype, blasts, blasts + anti CD3. Whilst the total CTLA-4 level does not change, anti-CD3/CD28 stimulation increases the CTLA-4 cycling pool. (C) Total expression of CTLA-4 in resting human peripheral blood T cells gated on CD25 expression. Levels of CTLA-4 were determined by intracellular staining of CD4⁺ T cells along with FoxP3. (D) Resting or activated (anti-CD3 and anti-CD28 beads) Tregs were stained for surface or cycling CTLA-4. Surface staining was carried out at 4 °C and cycling of CTLA-4 was detected by anti-CTLA-4-PE labelling at 37°C. "Activated" is right peak. Data are representative of at least 3 experiments.

Figure 18

CTLA-4 downregulation of costimulatory molecules is functionally relevant.

(A) Proliferation of CFSE-labelled CD4⁺CD25 T cells 4d after stimulation by DC re-isolated from culture with Tregs in the presence or absence of anti-CTLA-4 antibody (anti-CTLA-4 upper trace). (B) CFSE-labelled CD25^" T cells were stimulated with DC previously incubated with Treg. Cultures were supplemented with either control CHO cells or those transfected with CD86 CD86 CHO left peak. (C) T cell blasts expressing CTLA-4 were used as suppressor cells. Dendritic cells were incubated with T cell blasts with or without anti-CTLA- 4 and their capacity to stimulate proliferation of CD25^" CFSE labelled responder CD4 cells was assessed. T cell responses to DC in the absence of blasts (left panel) and presence of blasts (right panel) are shown. Data are representative of 3 experiments. CTLA-4 upper trace of blasts traces.

Figure 19

Mouse CD4+ CD25+ T cells can effectively capture CD86 in vitro.

(A) T cells from either WT or CTLA-4 -deficient mice (Ctla4^"/_) were incubated overnight with anti-CD3 (O^g/ml) and murine CD86-GFP expressing CHO cells. Transfer of CD86 was observed into wildtype T cells but not CTLA-4 knockout T cells. Wildtype cells did not acquire control GFP protein (B) DO 11 T cells were incubated with CHO cells expressing I- A^d and CD86-GFP either with or without OVA peptide and analysed by confocal microscopy. Cells were analysed for CD4 (blue) CD25 (red) and CD86 (green) expression. Data are representative of 2 experiments.

Figure 20

Workflow of in vivo trans-endocytosis experiment

Figure illustrates the workflow for in vivo analysis of ligand trans-endocytosis. Figure 21

Analysis of T cells from in vivo experiments

(A) FACS analysis showing Foxp3 staining of T cell populations shown in figure 4B and 4C. Plots are gated on CD4+CD25+ cells. (B) Quantification of confocal images from Fig. 4 shows that the mean GFP fluorescence of the CD86-GFP transferred to T cells approaches that of cells that were originally transduced with CD86-GFP indicating a high level of transfer. Fluorescence values were generated by measuring CD86-GFP content of recipient T cells and donor CD86-GFP+ cells using Image J software. Each circle represents an individual cell. Left column - recipient T cells, right column donor cells. (C) Quantification of in vivo experiments shown in Figure 4. Pooled data (n=2) from multiple confocal fields were scored for CD4+CD25+GFP+, CD4+CD25+GFP- and CD4+CD25- T cells. Approximately 100 cells were scored in each condition. Data show the requirement for CTLA-4 expression and peptide stimulation for CD86-GFP acquisition by T cells in vivo. (D) Representative images from in vivo experiments using control GFP transduced bone marrow. No endosomal GFP was observed in T cells in these experiments (n=2).

Figure 22

Shows points for intervention of CTLA-4 function. CTLA-4 internalises and directs its ligands for lysosomal degradation. Large arrows indicate targets for inhibition including internalisation of ligand (1) sorting to MVB (2) inhibition of lysosomal degradation (3), delivery of CTLA-4 to the cell surface (4) and re-cycling of CTLA-4 following ligand capture (5).

Figure 23

Cytoplasmic sequences determine the rate of CTLA-4 internalisation (Step 1 of Figure 22). Human CTLA-4 rate of internalisation. Human CTLA-4 transfected CHO cells were incubated at 4°C for 30 minutes with CTLA-4-PE and washed. Cells were either incubated at 4°C or raised to 37°C for times shown. Remaining surface CTLA-4 was then detected using an Alexa 647 coupled secondary Ab at 4°C. The use of different CTLA-4 species variants reveals that the cytoplasmic domain sequence controls the rate of internalisation.

Figure 24

Mutation of cytoplasmic lysines prevent CTLA-4 degradation (Steps 2 and 3 of Figure 22). CHO-CTLA-4 (wild type or mutant) were exposed to drug treatment (cycloheximide, NH₄C1, or both) for 3 h CTLA-4 protein was then measured by microscopy (A and C) or by FACS (B and D). Data shows that removal of cytoplasmic lysine residues stabilised the CTLA-4 protein (C and D). Data also show that inhibiting lysosomes with ammonium chloride prevents CTLA-4 degradation (A and B).

Figure 25

Ammonium chloride and bafilomycin prevent CTLA-4 degradation (Step 3 of Figure 22). CTLA-4 expressing CHO cells were treated with either NH₄C1 or bafilomycin to inhibit lysosomal degradation. Data shows a significant rescue by both compounds which inhibit different targets involved in degradation. Figure 26

Shows that KLESS re-cycles faster than WT CTLA in CHO cells. Figure 27

WT CTLA-4 re-cycles more quickly in the presence of ubiquitin inhibitor UBEI. Figure 28

Shows that MG132 prevented WT CTLA-4 degradation and increased CTLA-4 expression in WT but not KLESS CTLA-4 (MG132 is the right hand peak of the CTLA-4 graphs).

Materials and Methods

Cell purification and Culture

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coats (provided by the National Blood Transfusion Service, Birmingham, U.K) using Ficoll-paque density centrifugation. CD4⁺ T cells were isolated by incubating PBMC with human CD4⁺T cell- enrichment cocktail and magnetic colloid according to the manufacture's instruction (Stem Cell Technologies). To purify CD25⁺ T cells CD4⁺ cells were incubated with anti-CD25- microbeads (Miltenyi Biotech, Auburn, CA) at 4°C for 30 minutes. CD4⁺CD25^~ T cells which did not bind to the column were collected from the flow-through and washed before use. CD4⁺CD25⁺ T cells were subsequently retrieved from the column. Human monocytes were purified from PBMC by negative selection using human monocyte enrichment mixture and magnetic colloid according to the instructions of the manufacturer (StemCell, Meylan, France). To generate dendritic cells (DCs), monocytes (2 x 10⁶ cells/ml) were cultured in RPMI 1640 medium containing 10% FCS and antibiotics with GM-CSF (PeproTech, Rocky Hill, NJ; 800 U/ml) and IL-4 (PeproTech; 500 U/ml). Additional medium containing GM- CSF and IL-4 was added every 2 days. To generate SEB-specific T cell blasts PBMCs were incubated with ^g/ml SEB for 6 days. Live CD4⁺ cells were then negatively purified as above. Where stated DCs were re-isolated from T cell cultures using anti-CD2 microbeads (Miltenyi) to deplete T cells. DNA constructs and Transfectants

Full-length CTLA-4 cDNA was cloned into a CMV expression vector pCDNA3.1 as previously described (25). A CTLA-4 construct with the C-terminal 36 amino acids deleted was generated by PCR and cloned into the same vector to generate CTLA-4 del36. GFP- tagged CD80 and CD86 were generated by PCR to remove the stop codon and cloned into the EGFP-N1 vector (Clontech). Mouse CD86-GFP fusion was cloned into modified (IRES-GFP deleted) MIGR1 vector (a gift from Dr. W.S. Pear, University of Pennsylvania, Philadelphia, PA).

CHO Transfectants

CHO cell lines expressing either CD80, CD86 or CTLA-4 were generated by electroporation of human cDNAs cloned into a CMV expression vector. Cells were grown in DMEM containing 10% FBS as previously described (25). Cells expressing the plasmid were selected using G418 (500μg/ml) treatment and FACS. Cultures were maintained at 37°C in a humidified incubator containing 5% C0₂ and were passaged by trypsinization. CHO cells expressing IAd were obtained from Prof. Gordon Freeman (Dana Faber Cancer Institute) and transfected with mouse CD80-GFP using Amaxa nucleofection.

CTLA-4 expressing Jurkat Cells

CTLA4 was cloned into the MP71 retrovirus vector. Retroviral supernatants were obtained by transfecton of Phoenix-A packaging cells with retroviral vectors containing CTLA4 in combination with pCLampho, using the FUGENE6 transfection reagent (Roche Molecular Biochemical). Retroviral supernatants were harvested 48h after transfection and used for transduction of Jurkat cells. For transduction of Jurkat cells, non-tissue culture treated 6- well plates were coated with RetroNectin (TaKaRa) at 30 μg/ml and blocked by 2%BSA, 1 ml of retroviral supernatants were added to RetroNectin pre-coated wells and centrifuged at 1500 rpm at room temperature for 15 minutes. 2xl0⁶ Jurkat cell were added and centrifuged at 2000 rpm at 30°C for 60 minutes. 24 hours post- infection, media was changed to fresh RPMI 1640 with 10% FCS. Jurkat cells were maintained in RPMI containing 10% FBS. 72 hours post-infection, cells were sorted by staining with CTLA4-PE (BD) using MoFlow.

Human primary T cell Transfectants Purified human CD4⁺CD25^" T cells were transfected with wild-type CTLA-4 using the AMAXA nucleofector kit according to the manufacturers' instructions.

Generation of retroviral supernatants and bone marrow transduction

CD86-GFP fusion sequence was cloned into modified (IRES-GFP deleted) MIGR1 vector (a gift from Dr. W.S. Pear, University of Pennsylvania, Philadelphia, PA). Retroviral superntants were prepared by transfecting retroviral vector into the packaging cell line Plat-E (32) using Lipofectamine 2000 (Invitrogen). To prepare bone marrow (BM) cells, adult Rag- /- mice were treated with 150mg/kg 5-fluorouracil by intraperitoneal injection and total BM cells were flushed from femurs and tibias and used for retrovirus infection.

Retrovirus infection of cells was performed as described previously (33). Fresh BM cells were cultured in IMDM supplemented with 20% FCS, L-glutamine, sodium pyruvate, nonessential amino acids, penicillin, streptomycin, 50ng/ml stem cell factor, 50ng/ml IL-6, and lOng/ml IL-3. After 48, 72, and 96 hr, cells were spin-infected with retrovirus by centrifuging the culture plate in the presence of lOug/ml polybrene for 90 min.

Generation of CD86GFP Rag k/o mice and CD4+ T cell transfer

Retrovirally transduced bone marrow was intravenously injected into irradiated (450 Rad) Rag -/- mice. After 3 weeks, CD4+ cells were isolated from DOl l mice or from CTLA-4+/+ or CTLA-4-/- DOl lxRip-mOV A/Rag-/- mice and adoptively transferred I.V into Balb/c Rag- /- mice at l-5xl0⁶ cells per recipient. After 24 hours, mice received 100μg of OVA/alum i.p. At day 8, mice were given 100μg of OVA peptide I.V followed by 600μg of chloroquine I.P, 3 hours later. After a further 3 hours spleens were harvested and digested with 5mg/ml collagenase dispase (Roche) prior to staining for confocal.

Flow cytometry assays

Flow cytometry was carried out using a Dako Cyan flow cytometer and acquired using Summit software. Compensation and analysis was performed using FlowJo software (Treestar inc.) CD86-transfer

CD86 transfer into CTLA-4 expressing cells was carried out by labeling CD86 GFP expressing CHO cells with CellTrace Far Red DDAO-SE (Invitrogen). Cells were then incubated together with CTLA-4 expressing CHO cells for 3 hours in the presence or absence of 10 nM Bafilomycin A or 10 μΜ NH₄C1. To identify GFP transfer to single, CTLA-4 expressing cells and exclude clusters containing CD86-GFP, cells were gated for low pulse width and negative for Far Red staining as shown in Figure 5. CD80 GFP transfer was assayed in a similar way. Where used, PKH 26 (Sigma) labelling was carried out by incubating CHO-CD86 cells according to maufacturers instructions.

CFSE dilution

For assessing T cell proliferation, responder CD4⁺CD25^~ T cells were labeled using 5-(and- 6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE, Molecular Probes). CD4+ CD25- T cells were washed twice in PBS and resuspended at 2 x 10 cells/ml and an equal volume of CFSE in PBS added such that the final concentration was 2.5 μΜ. Cells were then incubated at RT for 10 minutes with gentle agitation and washed twice in normal growth medium. When stimulating with CD86 transfectants, CHO cells were fixed using 0.025% glutaraldehyde in PBS for 2-3 min and washed extensively. Fixed CHO cells and CFSE labeled responder cells with then incubated (1 CHO cell : 5 T cells unless otherwise specified) with anti-CD3 (0^g/ml) for 5 days before analysis by flow cytometry. When using Jurkat cells as suppressors, cells were incubated overnight with CHO cells and Jurkat cells removed using anti-CD2 microbeads (Miltenyi) prior to fixation. When using dendritic cells as stimulators, cells were cultured with CD4⁺CD25⁺ purified Tregs for 5 days with 0.5 μg/ml anti-CD3 (+/- anti-CTLA-4 20 μ§/πι1). DC was re-isolated by depleting CD2 positive cells with anti-CD2 microbeads (Miltenyi Biotech). Where indicated T cell blasts were used as CTLA4+ suppressor cells. Blasts were generated by CD3/CD28 bead stimulation for 5 days and added to along with CFSE-labelled responder T cells. Cells were stimulated with DC and anti-CD3 (0^g/ml) for 5 days in the presence or absence of anti-CTLA-4 (20 μg/ml) before analysis by flow cytometry.

CD86 surface downregulation For surface analysis of DC phenotypes, cells were collected into cold PBS and surface labeled at 4°C with antibodies against CD86, CD80, CD40 or CD 11c directly conjugated to FITC, PE or APC (BD PharMingen, San Diego, CA). DCs were gated based on forward and side scatter, and CD1 lc labeling. DCs were incubated with T cells at the ratios indicated.

CTLA-4 Antibodies

Surface staining of CTLA-4 was carried out on ice for 30 min. Staining of cycling CTLA-4 was carried out by incubation at 37°C for 30min. Total CTLA-4 was carried out by fixing cells with 3.0% formaldehyde solution in PBS and permeabilizing with 0.1% saponin and then stained with anti-CTLA-4 PE (BNI3-Pharmingen) or anti-CTLA-4 C-19 antibody (Santa Cruz) followed by an appropriate secondary antibody. Where stated T cells were stimulated using anti-CD3 anti-CD28 beads (Invitrogen) for 2h prior to staining. To study CTLA-4 cycling in Treg, cells were incubated overnight with anti-CD3 anti-CD28 beads and CTLA-4 staining carried out at 37°C. Where blocking anti-CTLA-4 was used unconjugated anti- CTLA-4 was used at 20μg/ml.

FOXP3 staining was performed using a FOXP3 staining kit (Ebioscience) according to the manufacturer's instructions.

Estimation of molecules of CD86 and CTLA-4

We used Quantum Simply Cellular anti-mouse IgG beads (Bangs Laboratories Inc, IN) according to the manufacturers instructions. Briefly, calibration curves were produced by titrating antibodies with beads of known antigen binding capacity. Flow cytometric staining of cells was then carried out using saturating titrations of antibody and used to estimate number of molecules from the calibration curves.

Confocal microscopy

Imaging was carried out using a Zeiss LSM 510 or a Zeiss LSM 780 inverted laser scanning confocal Microscope using a lOOx oil immersion objective with excitation at 488nm, 543nm and 633nm. Constant laser powers and acquisition parameters were maintained throughout individual experiments for analysis. Digital images were prepared using ImageJ (Wayne Rasband, NIH). For quantitation, cells were outlined and mean fluorescence intensity measured using ImageJ. Movies were prepared using ImageJ. All confocal images shown are representative of at least thirty micrographs taken from at least three independent experiments.

Live Cell Imaging

Cells were imaged using glass bottom culture dishes (MatTek). In single time point experiments CTLA-4 expressing cells were labelled with DDAO-SE and incubated with CD86-GFP CHO cells for 3h. For time course experiments CTLA-4 expressing CHO cells were identified by brief labelling with anti-CTLA-4 APC (BD) and incubated with CHO cells stably expressing CD86-GFP. Z-stacks were acquired as a time-series approximately every 3 minutes for 90 minutes or every 1 minute. Projections were then generated using Image J and converted into movies or single images.

Immunofluorescence staining

For co-localisation of CD86 and CTLA-4, CHO cells, were incubated together for 16 hours on a poly-L-lysine coated coverslip in a 24 well plate. Cells were then fixed with methanol at -20°C for 20 minutes. Non-specific binding sites were blocked by incubation in blocking solution (BS) which consisted of 5% donkey serum (Sigma) in PBS at room temperature (RT). Cells were incubated with anti-CD86 primary antibody (B7-2, C19 antibody, Santa Cruz, CA) and anti-CTLA-4 (BNI3 Pharmingen), washed and then labeled with donkey anti- goat Alexa 546 and anti-mouse Alexa 488 secondary antibodies (Molecular Probes).

Dendritic cells and T cells were added at a 1:3 ratio to poly-L-lysine coated coverslips in a 24 well plate. Cells were activated by the addition of anti-CD3 (Clone OKT3) at a concentration of 1 μ^πύ and were incubated for 72h (CD4⁺CD25^~) or overnight (CD4+CD25+) at 37 °C and 5 % C0₂. Where SEB stimulation was used, DC were pulsed with ^g/ml SEB for 2h and washed prior to use. Cells were centrifuged in a swing out rotor at 200 g for 10 minutes and fixed with methanol at -20°C for 20 minutes. Non-specific binding sites were blocked by incubation in blocking solution (BS). Cells were incubated with anti-CD86 primary antibody (B7-2, C19 antibody, Santa Cruz, CA), washed and then labeled with donkey anti-goat IgG Alexa 546. CTLA-4 was detected with anti-CTLA-4 APC. HLA-DR was labeled using anti- HLA-DR-FITC. For Treg experiments anti-CD3 was used to identify T cells and detected using anti-mouse IgG Alexa 633 secondary antibody.

Confocal staining of cycling CTLA-4 was carried out by incubating cells at 37°C with unconjugated anti-CTLA-4 for 30 minutes. Cells were then fixed, permeabilized and labeled with an anti-mouse Alexa 546. Total CTLA-4 was detected using a C-terminal anti-CTLA-4 antibody (Santa Cruz) detected with anti goat Alexa 488.

Following staining, coverslips were dried and mounted with Vectashield (Vector Laboratories, UK) prior to visualization by confocal microscopy.

Animals

DO 11.10 TCR transgenic mice and BALB/C mice were purchased from The Jackson Laboratory. RAG-2-/- mice were purchased from Taconic Farms. RIP-mOVA mice on a BALB/c background that express a membrane-bound form of OVA under the control of the rat insulin promoter (from line 296- IB) were a gift from W. Heath (Walter and Eliza Hall Institute, Melbourne, Australia). DO11.10 mice, RIP-mOVA mice and RAG-/- mice were crossed as previously described. CTLA-4-/- mice on a BALB/c background were a generous gift from A. Sharpe (Brigham and Women's Hospital, Boston, USA). All mice were housed in the University of Birmingham Biomedical Services Unit and used according to Home Office and institutional regulations.

For in vivo transfer experiments, splenocytes were labelled in glass-bottom dishes (MatTek) at 4°C with anti-CD25 PE and CD4 APC for 15 minutes before imaging by confocal microscopy. Quantitation of GFP fluorescence was carried out by outlining cells and measuring mean fluorescence in ImageJ. Electron Microscopy

Cryo-immuno-EM was performed on cells fixed with 4% paraformaldehyde in 0.1M phosphate buffer pH 7.4, supported in 10% gelatin, and infused with 2.3M sucrose. Sections (70 nm) were cut at - 120°C and picked up in 1 : 1 2.3M sucrose:2% methylcellulose. Mouse anti-HA ( HA.11, Covance) was followed by rabbit anti-mouse intermediate antibody (Dako) and lOnm protein A gold and then contrast stained/dried in 1 :9 4% uranyl acetate:2% methyl cellulose. Samples were viewed on a JEOL 1010 TEM, and images gathered with a Gatan OriusSClOOB CCD camera.

Results and Discussion

The T cell protein CTLA-4 is essential to the prevention of autoimmune disease (1-3). Although the molecular basis for CTLA-4 action has been suggested to be a cell-intrinsic inhibitory signal (4) possibly mediated by the cytoplasmic domain (5), a cell-extrinsic function for CTLA-4 is clearly evident from in vivo models (6-13). Therefore a molecular explanation of CTLA-4 function compatible with such a cell-extrinsic mechanism is needed. The intercellular transfer of a ligand from one cell to its receptor on a different cell is observed in both immune settings and elsewhere (14-19). Because of its highly endocytic behaviour, we tested whether CTLA-4 could potentially act in such a manner in order to deplete its ligands and thereby extrinsically inhibit T cell activation via CD28. We cultured CTLA-4-expressing (CTLA-4⁺) CHO cells with donor CHO cells expressing a C-terminally tagged CD86 protein (CD86-GFP). Using a flow cytometric assay we observed substantial transfer of CD86 into CTLA-4⁺ cells (Fig. 1A and fig. 5). This finding was confirmed by confocal microscopy where acquisition of ligand by CTLA-4 caused the appearance of CD86 containing vesicles within the CTLA-4⁺ cell (Fig. IB). Treatment with the lysosomal inhibitor bafilomycin A caused an increase in detectable CD86-GFP after transfer (Fig 1A), which indicated degradation of the transferred ligand inside the CTLA-4⁺ cell. Accordingly, CD86 did not appear on the cell surface of the recipient CTLA-4⁺ cell (fig 6). Interestingly, bafilomycin A treatment did not result in an increase in CTLA-4 expression, which suggested that although CTLA-4 could capture and deliver ligand for degradation, CTLA-4 itself was not degraded (fig. 7). Overall, this process appeared different to the generalized intercellular exchange or 'trogocytosis' reported for other receptors (20-22), where transferred proteins are detected at the cell surface. To investigate the time course of CD86 acquisition we performed live-cell imaging of CTLA- 4⁺ CHO cells interacting with CHO cells expressing CD86-GFP (fig. 8A). Within minutes of cell contact we observed a marked concentration of CD86-GFP at the site of cell-cell contact, from which CD86 positive vesicles emanated into the CTLA-4 expressing cell. Quantitation of this process revealed a significant depletion of GFP fluorescence from the plasma membrane of the CD86 donor cell and a corresponding increase in GFP inside the CTLA4⁺ recipient cell (fig. 8B). Kinetic analysis by flow cytometry also revealed that over 50% of CTLA-4⁺ cells acquired ligand within 3h (fig. 9). Furthermore, we estimated the number of CTLA-4 molecules and CD86 molecules expressed by our cell lines to determine the stoichiometry of CD86 depletion (fig. 10). This showed that a ratio of approximately 1 :8 (CTLA-4:CD86) molecules was sufficient for functionally relevant depletion.

To confirm that trans-endocytosis of ligand was not an artifact of using GFP-fusion proteins we also performed experiments with wild type CD86 expressed in CHO cells. CHO-CD86 cells were cultured alone or with CHO-CTLA-4 cells then stained for CD86 and CTLA-4 using antibodies (Fig. 1C). In the absence of CTLA-4, CD86-expressing cells display a characteristic plasma membrane staining pattern (fig. 11 A), however in the presence of CTLA-4, CD86 containing vesicles were observed inside CTLA-4 recipient cells (Fig. 1C, fig. 11 A). Moreover, because CD86 was stained using an antibody against the cytoplasmic domain this indicated that the whole ligand had been transferred into the CTLA-4⁺ cell. Analysis of immunofluorescence images showed no significant acquisition of CTLA-4 by the CD86-expressing cells (fig. 11B). In addition, studies using the membrane dye PKH26 revealed that trans-endocytosis was associated with transfer of small amounts of membrane lipids (fig. 11C). Taken together these data indicated that protein transfer was unidirectional and appeared to involve transfer of membrane lipid. Because the C-terminus of CTLA-4 is required for endocytosis (fig. 12) (23, 24), and shows a remarkable degree of evolutionary conservation, we tested the contribution of this region to CD86 trans-endocytosis, by deleting the C-terminus of CTLA-4 (CTLA-4 del36). Although CD86 still localized to regions of cell- cell contact (Fig. ID), wild-type-CTLA-4 was much more effective at depleting CD86 than CTLA-4 del36 (Fig. IE). As expected, when the CD86-depleted cells from these experiments were used to stimulate T cells, those exposed to wild-type CTLA-4 had impaired stimulatory capacity compared to those exposed to non-endocytic CTLA-4 (Fig. IF). Together, these results indicate that by a process of trans-endocytosis CTLA-4 removes CD86 from neighboring cells resulting in impaired T cell responses. To test these observations in a more physiological setting we activated human CD4 CD25 T cells in the presence of monocyte-derived dendritic cells to allow the induction of CTLA-4 and looked for evidence of trans-endocytosis. In the absence of T cells, CD86 was robustly expressed on the surface of dendritic cells (Fig. 2A). In the presence of activated T cells, however, CD86 on the plasma membrane of DCs was reduced and instead found in a punctate pattern that co-localized with CTLA-4 (Fig. 2B). Importantly, incubation with a blocking anti-CTLA-4 antibody prevented the down-regulation of CD86 on the DC as well as

+

the appearance of CD86 in CTLA-4 vesicles in the T cell (Fig. 2, B and C and fig. 13). Although CD86 was downregulated in a CTLA-4-dependent manner, expression of other molecules on the DC such as CD40 was unaffected (fig. 13). These results indicated that CTLA-4-mediated trans-endocytosis was specific to CD80 and CD86. To establish whether CTLA-4 was sufficient to confer this function to T cells we also generated a Jurkat cell line expressing CTLA-4 (fig. 14A). Jurkat cells transduced with CTLA-4 acquired the ability to capture CD86-GFP (fig. 14B). Analysis by electron microscopy also showed acquired CD86

+

in distinct intracellular vesicles within CTLA-4 Jurkat cells (fig. 14C). The use of a number of blocking reagents, including CTLA-4-Ig and anti-CD28, confirmed the specificity of CD86-GFP transfer to Jurkat cells (fig. 15 and fig. 16) and demonstrated that CD28 was not capable of trans-endocytosis. We have previously shown that transfection of resting human T cells with CTLA-4 confers suppressive activity (8). We therefore tested whether this approach also conferred the ability to capture CD86 from the APC. CTLA-4-transfected (but not mock transfected) resting T cells exhibited specific sequestration and internalisation of CD86, from the DC (Fig. 2, D and E), but had no effect on HLA-DR expression. Taken together, these data demonstrated CTLA-4 expression by T cells was sufficient to confer the ability to remove CD 86 from DCs.

Because TCR-engagement leads to enhanced trafficking of CTLA-4 to and from the plasma membrane (fig. 17, A and B) (25-27) we predicted that TCR stimulation should enhance

+

CD86 acquisition. To test this, human CTLA-4 T cell blasts were incubated in the presence of CD86-GFP-expressing CHO cells with or without anti-CD3. TCR stimulation increased the acquisition of CD86-GFP in a manner that was blocked by anti-CTLA-4 and enhanced by bafilomycin (Fig. 3, A). Similarly, staphylococcal enterotoxin B (SEB)-reactive T cell blasts incubated with dendritic cells also showed enhanced acquisition of CD86 (Fig. 3, B and C). We next determined whether CD4⁺ CD25⁺ regulatory T cells (Treg) could acquire CD86 from APC because CTLA-4 is constitutively expressed on Treg cells and is re-cycled upon

+ stimulation (fig. 17C and D). DC were incubated overnight with purified human CD4 CD25

T cells or CD4 CD25⁺ Treg cells and anti-CD3. In the presence of CTLA-4⁺ Treg, CD86 was downregulated from the APC surface and observed in intracellular puncta inside the Treg. In

+ contrast, CD86 remained on the plasma membrane of DC in the presence of CD4 CD25 T cells that lacked CTLA-4 (Fig. 3, D and E). To test whether downregulation of CD86 by Treg cells affected T cell stimulation, we stimulated Treg with DCs in the presence or absence of a blocking anti-CTLA-4 antibody. We then re-purified these DCs and used them to stimulate CFSE-labelled responder T cells. This revealed that blocking CTLA-4 on regulatory T cells maintained the stimulatory capacity of the DC compared to where CTLA-4 was available to deplete ligands (fig. 18 A). Moreover, suppression could be overcome by restoring co- stimulation using CD86-expressing transfectants (fig. 18B). We also observed that T cell blasts could act as suppressor cells in a CTLA-4-dependent manner (fig. 18C) again indicating that depletion of costimulatory molecules by CTLA-4 has functional consequences.

Our data suggest a model where antigen stimulation of either T cells or Treg promotes the removal and degradation of CD80 and CD86 from antigen presenting cells by CTLA-4. To test whether this process could also take place in vivo we generated a system to study trans-

+

endocytosis in mice. We first established that mouse CD4 T cells stimulated in vitro could acquire CD86-GFP from CHO cell targets (fig. 19A and B). We next developed an in vivo protocol (fig. 20) in which DOl l. lO TCR transgenic T cells (specific for a peptide fragment of chicken ovalbumin (OVA) presented by I-A^d) were transferred into Balbc Rag2^' mice, which 3 weeks prior, had been reconstituted with CD86-GFP-transduced Rag2^~/~ bone marrow. Recipient mice therefore lacked lymphocytes, except the adoptively transferred DOl l.lO T cells, and expressed CD86-GFP on their antigen presenting cells. Seven days after OVA immunization, mice were re-challenged with OVA peptide in the presence of the lysosomal inhibitor, chloroquine. T cells were then harvested and immediately analyzed by confocal imaging. This revealed CD86-GFP in endosomal compartments of antigen- stimulated, but not unstimulated, T cells (Fig. 4A). Moreover, internalized CD86-GFP was + +

restricted to CD4 CD25 T cells (Fig.4B), consistent with the expression of CTLA-4 by these cells. These cells did not express Foxp3 (fig. 21, left panel). The amount of CD86-GFP transfer was extensive because the mean GFP fluorescence inside T cells approached the amounts on donor cells themselves (fig. 21B). To establish the requirement for CTLA-4, we

+/+ -/- tested CD4⁺ cells from Ctla4 or Ctla4 , Rag2^"ADO11.10 mice bred to mice that express OVA under the control of the rat insulin promo tor (Rip-mOVA). These mice were useful because they develop OVA-specific Treg cells and we have shown that those deficient in

CTLA-4 fail to regulate diabetes (13). After in vivo re-challenge with OVA peptide, CD86-

+ +

GFP acquisition was only observed in CD4 CD25 T cells capable of CTLA-4 expression but not those from Ctla4 ^_/~ mice (Fig. 4C). Moreover, CD4⁺ CD25⁺ T cells from these mice were almost entirely Foxp3⁺ (fig. 21A right panel). Overall, analysis indicated that approximately

+ +

25-40% of wildtype CD4 CD25 T cells acquired ligand (fig. 21C). No transfer of control (unfused) GFP molecules was observed (fig. 21D). Together these data indicate that both Foxp3⁺ and Foxp3^" T cells are capable of CD86 acquisition in vivo.

The CTLA-4 molecule plays a critical role in suppressing autoimmunity and maintaining immune homeostasis; however, its precise mechanism of action has been a subject of debate. Recent data have provided evidence that CTLA-4 can perform a non-redundant effector function for Treg, requiring a cell extrinsic mechanism of action (9, 13). Here we demonstrate a cell-extrinsic model of CTLA-4 function which operates by the removal of co- stimulatory ligands from APCs via trans-endocytosis. Importantly, using both human and mouse T cells we establish that trans-endocytosis of ligand occurs in precisely the same settings where we have demonstrated CTLA-4-dependent regulation (8, 13). Moreover, this mechanism is specific for CTLA-4 /CD28 ligands and operates in an antigen-dependent manner in vivo. Our data are also compatible with several studies demonstrating reduced levels of costimulatory ligand expression in the presence of CTLA-4-expressing Treg cells (9, 28, 29) and consistent with a role for CTLA-4 on both activated and regulatory T cells (9, 30, 31). Taken together, these results not only provide a widely applicable basis for CTLA-4 function but they also offer cogent explanations for long-standing paradoxes in the field, namely, how CTLA-4 can function in a cell-extrinsic manner, why CTLA-4 shares ligands with the stimulatory receptor CD28 and why CTLA-4 exhibits endocytic behaviour. Whilst not excluding other mechanisms of CTLA-4 action, we suggest that CTLA-4 carries out the same molecular functions whether expressed by T cells or by Treg; a concept which has significant implications for our understanding of immune homeostasis. Together these data provide a new framework for studies of CTLA-4 and should facilitate our understanding of its immunoregulatory role in the key settings of cancer, HIV infection and autoimmune disease.

Possible targets for intervention of CTLA-4 function

Figure 22 shows possible targets including internalisation of ligand (1). Sorting to MVB (2), inhibition of lysosomal degradation (3), delivery of CTLA-4 to the cell surface (4) and recycling of CTLA-4 following ligand capture (5).

To investigate possible ways of targeting these targets, the inventors carried out a number of tests.

Figure 23 shows the effect of cytoplasmic domain sequences on CTLA-4 internalisation ("Step 2"). CTLA-4 was transfected into CHO (Chinese Hamster Ovary) cells and incubated at 4°C or 37°C for the times shown. Remaining surface CTLA-4 was detected using labeled antibody (labeled with Alexa 647) at 4°C. The amount of internalisation increased at 37°C. The graph shows different CTLA-4 species showing that the cytoplasmic domain sequence controls the rate of internalisation.

Figure 24 shows that mutant CTLA-4 without lysines does not degrade as much as wild type CTLA-4 (steps 2 and 3).

Wild Type CTLA-4 has the following lysine residues:

SLS KM L RSPLTTGVYVK ¹⁶⁵MPPTEPECEK ¹⁷⁵QFQPYFIPIN

A lysine deficient CTLA-4 known as "KLESS" was identified having the following mutations:

SLS|MLi|RSPLTTGVYVi^1MMPPTEPECEi^{1 3}QFQPYFIPIN

The data obtained by the Applicant suggests that lysines 165 and 175 are the predominant ones in controlling degradation of CTLA-4, however, all lysines are potentially modification targets

In wild type CTLA-4, cyclohexamide (CHX) treated cells did not stain for CTLA-4 showing that the protein was degraded. KLESS was not degraded to the same extent. The addition of ammonium chloride inhibited CTLA-4 degradation.

Figure 25 shows that bafilomycin also inhibits lysosomal degradation of wild type CTLA-4 re-cycling (step 3) and can be used to modify CTLA-4 re-cycling.

The KLESS mutant was shown to be internalised from the plasma membrane equally compared to WT CTLA-4 in CHO cells (data not shown). However when cells were stained with antibodies to detect surface or intracellular pools, then qualified by confocal microscopy, KLESS was observed to have a higher steady state plasma membrane to internalised ratio than WT. Endocytosis rates were similar from the plasma membrane.

Figure 26 shows that KLESS re-cycles faster than WT CTLA-4 using antibody labelling. Inhibitory ubiquitination using UBEI-41, a ubiquitin εΐ inhibitor (Biogenova) caused WT CTLA-4 to re-cycle more quickly (Figure 27) when used at 20 micromolar.

MG132 is a specific, potent proteosome inhibitor - it also depletes the availability of free cellular ubiquitin and thereby reduces degradation of ubiquitin conjugated proteins in mammalian and yeast cells. Figure 28 shows that it reduced WT CTLA-4 degradation and increased CTLA-4 expression when used at 5 micromolar.

When lysosomal degradation was blocked using bafilomycin or ammonium chloride, much less change was observed with KLESS than WT CTLA-4 by western blot analysis (data not shown).

Immunoprecipitation (not shown) from WT or KLESS expressing cells Western blotted for CTLA-4 and ubiquitin showed that WT but not KLESS were modified by ubiquitination.

This shows that ubiquitination may be a target to modulate CTLA-4 and hence the immune response. SEQUENCE LISTING

<110> The University of Birmingham

<120> Immune Assay

<130> p336523PCT

<150> GB1104950.9

<151> 2011-03-24

<160> 2

<170> Patentln version 3.5

<210> 1

<211> 39

<212> PRT

<213> Homo sapiens

<220>

<221> MI SC_FEATURE

<222> (1) .. (39)

<223> Partial wild-type CTLA-4 Sequence

<400> 1

Ser Leu Ser Lys Met Leu Lys Lys Arg Ser Pro Leu Thr Thr Gly Val 1 5 10 15

Tyr Val Lys Met Pro Pro Thr Glu Pro Glu Cys Glu Lys Gin Phe Gin

20 25 30

Pro Tyr Phe lie Pro lie Asn

35

<210> 2

<211> 39

<212> PRT

<213> Homo sapiens

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<221> MI SC_FEATURE

<222> (1) .. (39)

<223> Partial sequence of mutated lysine deficient CTLA-4

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Ser Leu Ser Arg Met Leu Arg Arg Arg Ser Pro Leu Thr Thr Gly Val 1 5 10 15

Tyr Val Arg Met Pro Pro Thr Glu Pro Glu Cys Glu Arg Gin Phe Gin

20 25 30 Pro Tyr Phe lie Pro lie Asn 35

Claims

1. A method of identifying a modulator of the immune response system, comprising assaying for the effect of a compound on the internalisation and recycling of CTLA-4 in a cell, wherein an increase in cycling of CTLA-4 compared to a control without the compound indicates that the compound suppresses the immune response and a decrease in the cycling of CTLA-4 compared to a control without the compound, indicates that the compound increases the immune response.

2. A method according to claim 1, wherein the cycling of CTLA-4 is determined by monitoring uptake of CTLA-4 dependent label into the cell.

3. A method according to claim 1 or 2, comprising detecting the CTLA-4 dependent uptake of CD80 or CD86, preferably CD80 or CD86 from a donor, cell.

4. A method according to claim 3, wherein the CD80 or CD86 is labelled.

5. A method according to claim 4, wherein the CD80 or CD86 is a fusion protein with a coloured label such as green-fluorescent protein (GFP).

6. A method according to claims 3 to 5, wherein CD80 or CD86 is provided by a donor cell.

7. A method according to any preceding claim, wherein the CTLA-4 is in a cell selected from a mammalian, insect or fish cell.

8. A method according to claim 5 or 7, wherein the cell is a T cell, especially regulatory T cell, a Chinese Hamster Ovary cell (CHO cell) or a Jurkat cell.

9. A method according to claims 7 or 8, wherein the cell is a part of the whole organism.

10. A method according to claim 8, wherein the donor cell is a dendritic cell or CHO cell.

11. A method according to any preceding claim, wherein the modulator is a lysosomal inhibitor, for example bafilomycin A, a modulator of CTLA-4 ubiquitylation or modulator of CTLA-4 delivery to endosomes, multivesicular bodies or lysosomes.

12. A method according to claim 11 wherein the moderator of ubiquitylation is a ubiquitin ligase inhibitor, ubiquitin ligase enhancer, an inhibitor of de ubiquitinating enzymes (DuBs) or a DuBs enhancer.

13. A method according to any preceding claim, wherein the modulator activates, enhances or inhibits binding of CD80 or CD86 to CTLA-4.

14. A method of modulating an immune response comprising the use of a CTLA-4 specific ubiquitin ligase enhancer or inhibitor; or an inhibitor of a CTLA-4 specific DuB inhibitor or enhancer.