WO2014020345A1 - Themis protein - Google Patents

Abstract

The present invention relates to isolated recombinant proteins, in particular Themis proteins and also to methods of providing isolated recombinant themis protein. Themis (thymocyte-expressed molecule involved in selection),is a member of a family of proteins whose functions are not all known. It is highly conserved among vertebrates. Themis has been found to have high expression in thymocytes between the pre-T cell antigen receptor (pre-TCR) and positive-selection checkpoints and low expression in mature T cells. Themis protein is not easy to produce and purify in large quantities. The present invention provides an isolated, soluble protein comprising an amino acid sequence selected from: a) the sequence set out in SEQ ID NO: 1; b) the sequence of amino acids 1 to 261 of SEQ ID NO: 1; c) a sequence having at least 70% identity to a) or b); d) an active fragment or variant of a), b) or c); e) a fusion protein comprising the sequence of a), b), c) or d) and a fusion partner; f) a sequence comprising the sequence of a), b), c), d) or e) and a purification tag.

Description

THEMIS PROTEIN

The present invention relates to isolated recombinant proteins, in particular Themis proteins and also to methods of providing isolated recombinant themis protein.

Themis (thymocyte-expressed molecule involved in selection), is a member of a family of proteins with unknown functions. It is highly conserved among vertebrates. Themis has been found to have high expression in thymocytes between the pre-T cell antigen receptor (pre-TCR) and positive-selection checkpoints and low expression in mature T cells. Themis-deficient thymocytes showed defective positive selection, which results in fewer mature thymocytes. Negative selection is also impaired in Themis-deficient mice. A greater percentage of Themis-deficient T cells have CD4(+)CD25(+)Foxp3(+) regulatory and CD62L(lo)CD44(hi) memory phenotypes than do wild-type T cells. In support of the idea that Themis is involved in TCR signalling, this protein is phosphorylated quickly after TCR stimulation and is needed for optimal TCR-driven calcium mobilization and activation of the kinase Erk.

THEMIS and the adapter molecule growth factor receptor-bound protein 2 (GRB2) associate constitutively through binding of a conserved PxRPxK motif within the proline-rich region 1 of THEMIS to the C-terminal SH3-domain of GRB2. This association is indispensable for THEMIS recruitment to the immunological synapse via the transmembrane adapter linker for activation of T cells (LAT) and for THEMIS phosphorylation by Lck and ZAP-70. Two major sites of tyrosine phosphorylation were mapped to a YYmotif close to proline-rich region 1. The YY-motif was crucial for GRB2 binding, suggesting that this region of THEMIS might control local phosphorylation-dependent conformational changes important for THEMIS function. Finally, THEMIS binding to GRB2 was required for thymocyte development. Data firmly assigns THEMIS to the TCR-proximal signalling cascade as a participant in the LAT signalosome and suggest that the THEMIS-GRB2 complex might be involved in shaping the nature of Ras signalling, thereby governing thymic selection.

Themis protein is not easy to produce and purify in large quantities. Full-length

Themis has not successfully been produced in bacteria or Eukaryotic cells. Previously only short peptides from the C-terminal end of Themis has been produced. It would be advantageous to provide a full-length and fragments of Themis protein that is stable, soluble and can be produced in large quantities.

Accordingly the present invention provides an isolated, soluble protein comprising an amino acid sequence selected from:

a) the sequence set out in SEQ ID NO: 1 ;

b) the sequence of amino acids 1 to 261 of SEQ ID NO: 1 ;

c) a sequence having at least 70% identity to a) or b);

d) an active fragment or variant of a), b) or c);

e) a fusion protein comprising the sequence of a), b), c) or d) and a fusion partner;

f) a sequence comprising the sequence of a), b), c), d) or e) and a purification tag. The protein may comprise or consist of the sequence set out in SEQ ID NO: 1 which is the full-length sequence of Themis protein. The protein may be encoded by a polynucleotide having the sequence set out in SEQ ID NO: 2 which is the cDNA sequence that encodes full-length Themis protein. The protein may have the sequence set out in SEQ ID NO: 3 which is construct F from Figure 5 which includes a polyhistidine tag, a linker sequence, amino acids 1 to 261 of Themis protein and a Maltose Binding Protein. The protein may have the sequence set out in SEQ ID NO: 4 which is the amino acid sequence of full-length Themis protein with a Strep-tag.

The protein may comprise a sequence having 70%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 1. The protein may comprise a sequence having 70%, 80%, 90%, 95%, 98% or 99% sequence identity to amino acids 1 to 261 of SEQ ID NO: 1. The protein may have one or amino acids added, deleted or substituted from the sequence set out in SEQ ID NO 1 , SEQ ID NO 3 or SEQ ID NO: 4. The protein may comprise a sequence according to SEQ ID NO: 1 with one or more amino acid additions, substitutions or deletions. The protein may comprise a sequence according to amino acids 1 to 261 of SEQ ID NO: 1 with one or more amino acid additions, substitutions or deletions. The substitutions may be conservative amino acid substitutions. The protein may comprise a homologue or variant of Themis protein having 70%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 1 or having 70%, 80%, 90%, 95%, 98% or 99% sequence identity to amino acids 1 to 261 of SEQ ID NO: 1.

The protein may be an isolated protein that is not inside or part of a cell. The isolated protein may be purified to remove other proteins or cell-debris. The protein may be pure if it accounts for 70%, 80%, 90%, 95%, 98% or 99% or 99.9% of the proteins in a composition. The protein may be pure if there is only a trace amount of other proteins in a composition. Purity of the protein may be analysed by standard laboratory methods for example SDS PAGE analysis, chromatography, column chromatography or mass spectrometry.

The protein may be soluble so that it does not precipitate in suitable buffers. The protein may be correctly folded. The Themis protein may be a fragment of the Themis protein which forms a correctly folded domain of the Themis protein. When the protein is produced in cells the majority of the protein may not form inclusion bodies. The protein may be an active Themis protein or a fragment of the Themis protein having Themis protein activity. Themis protein activity may be to bind, via the universal adaptor protein GRB2, to the scaffold protein LAT. In cells, LAT hangs from the cytosolic site of the plasma membrane. The recombinant Themis protein may be a fragment comprising part of the sequence of SEQ ID NO: 1 and having the activity of binding to the scaffold protein LAT via the universal adaptor protein GRB2. The Themis protein may form a signalosome complex with the scaffold protein LAT. The signalosome complex may comprise Themis, GRB2 and LAT. The protein may be a fusion protein comprising a fusion partner fused to a sequence having 70%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 1 or 70%, 80%, 90%, 95%, 98% or 99% sequence identity to amino acids 1 to 261 of SEQ ID NO : 1. The fusion partner may be fused via a linker peptide . The fusion partner may be fused to the N-terminus or the C-terminus of the Themis protein. The fusion partner may be any suitable fusion protein. The fusion partner may be Maltose Binding Protein (MBP). The protein may be a fusion protein comprising a polypeptide having 70%, 80%, 90%, 95%, 98% or 99% sequence identity to amino acids 1 to 261 of SEQ ID NO: 1 and Maltose binding protein. The protein may be produced in E. coli. A soluble Themis protein or fragment thereof may be made by expressing a fusion protein comprising the sequence set out in SEQ ID NO : 1 or a sequence having %, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 1 and a fusion partner and then cleaving of the fusion partner, for example by enzymatic cleavage. A soluble Themis protein or fragment thereof may be made by expressing a fusion protein comprising the sequence set out in amino acids 1 to 261 of SEQ ID NO: 1 or a sequence having %, 80%, 90%, 95%, 98% or 99% sequence identity to amino acids 1 to 261 of SEQ ID NO: 1 and a fusion partner and then cleaving of the fusion partner, for example by enzymatic cleavage.

The protein may be a fusion protein comprising Maltose Binding Protein and a sequence having 70%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 1. The protein may be a fusion protein having the sequence set out in SEQ ID NO 3.

The protein may be a fusion protein comprising Maltose Binding Protein and a sequence having 70%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 1 for expression in bacteria, preferably expression in E. coli. The protein may be a fusion protein having the sequence set out in SEQ ID NO 3 for expression in bacteria, preferably expression in E. coli.

The protein may comprise any suitable purification tag, for example a polyhistidine tag (his tag), an HA -tag or a Strep-tag. The purification tag may be fused to the N- terminus or to the C-terminus of the Themis protein.

The tag may be a Strep-tag and the Themis protein may be a full-length Themis protein having a sequence set out in SEQ ID NO: 1. The protein may have the sequence set out in SEQ ID NO: 4. The Strep-tag system is a method which allows the purification and detection of proteins by affinity chromatography. The Strep -tag may be a synthetic peptide consisting of eight amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). This peptide sequence exhibits intrinsic affinity towards Strep-Tactin, a specifically engineered streptavidin and can be N- or C- terminally fused to recombinant proteins. By exploiting the highly specific interaction, Strep-tagged proteins can be isolated in one step from crude cell lysates. Because the Strep-tag elutes under gentle, physiological conditions it is especially suited for generation of functional proteins.

The Themis protein or fusion protein may be expressed in any suitable bacterial cells, for example E. coli cells. The Themis protein or fusion protein may be expressed in any suitable eukaryotic cells, for example a mammalian cells, for example Chinese Hamster Ovary cells (CHO cells) or in other eukaryotic cells, for example yeast cells, for example Pichia Pastoris cells. In a further aspect the present invention may provide an immunogenic composition comprising a protein of the invention. The immunogenic composition may further comprise any suitable diluents, buffers, excipients or carriers. The immunogenic composition may further comprise one or more suitable adjuvants. The immunogenic composition may be useful in medical treatment. The immunogenic composition may be used to generate antibodies that bind to Themis protein.

In a further aspect the present invention provides an antibody that binds to a protein of the invention. The antibody may bind to an epitope within the sequence of SEQ ID NO: 1. the antibody may bind to an epitope within the sequence of amino acids 1 to 261 of SEQ ID NO: 1.

The antibody may bind within the CABIT 1 or CABIT 2 region of Themis protein. The antibody may not bind to the N-terminal section of Themis protein. The antibody may not bind to the section of Themis protein C-terminal of CABIT 2. The antibody may be a polyclonal antibody or a monoclonal antibody. Preferably the antibody may be a monoclonal antibody.

The antibody may bind to Themis protein with high affinity. An antibody that binds to Themis protein with high affinity may bind to Themis protein with a dissociation constant of about 1 nanomolar to about 5 nanomolar.

The antibody may have high affinity and high signal to nose ratio. The antibody may be labelled. The antibody may be an antibody reagent for cell staining. The antibody may be an IgGl or IgB2 antibody. The antibody may be coupled to a fluorophore. The antibody may be suitable for Elisa or FACS analysis.

The protein of the present invention is suitable for raising high quality antibodies because it is a recombinant protein that can be produced with high purity and in high quantities. The protein of the present invention has several epitopes intact and can therefore induce a variety of different antibodies.

The antibody of the present invention may bind within the CABIT l or the CABIT 2 region of Themis.

The antibody may be used in in vitro testing. The antibody may be used in a diagnostic test. The antibody may be used in a diagnostic test to assess the level of Themis in a sample from a subject. The antibody may be used in a diagnostic test to identify autoimmune diseases and/or immunodeficiency. The antibody may be used in FACS analysis for intracellular staining of human Themis. The antibody may be a monoclonal antibody for use as a reagent to identify Themis in T cells and may be used to precisely assess levels of Themis expression as a correlative parameter in autoimmunity and/or immunodeficiency. The antibody may be used to assess the lack of Themis as a negative marker in T regulatory cells to distinguish them from conventional activated T cells more precisely than currently performed.

The antibody may be used in an in vitro test to obtain an indication useful in the detection or diagnosis of an autoimmune disease, an immune deficiency, an allergy, an intolerance, rejection of grafted tissue. The antibody may be used in an in vitro test to obtain an indication useful in the detection or diagnosis of Celiac Disease, Multiple sclerosis, Type I Diabetes or in Inflammatory Bowel Disorder (IBD) .

In a further aspect the present invention provides a method of providing an antibody that binds to Themis protein wherein the antibody is obtained or obtainable by inoculating an animal with a protein of the present invention.

The method of providing an antibody may comprise the step of inoculating an animal with a protein of the present invention. The antibody may bind to an epitope of Themis protein that is within the sequence set out in SEQ ID NO: 1. The antibody may not bind to the fusion partner. The antibody may not bind to MBP.

In a further aspect the present invention provides a pharmaceutical composition comprising a protein of the present invention. The pharmaceutical composition may comprise one or more physiologically acceptable carriers or excipients.

In a further aspect the present invention provides a protein according to the present invention for use in medical treatment. The medical treatment may be treatment or prevention of an immunological or inflammatory disease . The medical treatment may be treatment of an autoimmune disease. The medical treatment may be treatment of an immune deficiency, an allergy, an intolerance, rejection of grafted tissue. The medical treatment may be treatment of Celiac Disease, Multiple sclerosis, Type I Diabetes or in Inflammatory Bowel Disorder (IBD).

Themis protein may bind specifically to T-cells.

The present invention provides the use of a protein of the invention in the manufacture of a medicament for the treatment or prevention of Celiac Disease, Multiple sclerosis, Type I Diabetes or in Inflammatory Bowel Disorder (IBD) .

In a further aspect the present invention provides an isolated polynucleotide encoding a Themis protein having an amino acid sequence selected from:

a) the sequence set out in SEQ ID NO: 1 ;

b) the sequence of amino acids 1 to 261 of SEQ ID NO: 1 ;

c) a sequence having at least 70% identity to a) or b);

d) an active fragment or variant of a), b) or c);

e) a fusion protein comprising the sequence of a), b), c) or d) and a fusion partner.

In a further aspect the present invention provides an expression vector suitable for expression of a Themis protein having a sequence selected from:

a) the sequence set out in SEQ ID NO: 1 ;

b) the sequence of amino acids 1 to 261 of SEQ ID NO: 1 ;

c) a sequence having at least 70% identity to a) or b); d) an active fragment or variant of a), b) or c);

e) a fusion protein comprising the sequence of a), b), c) or d) and a fusion partner. The expression vector may be any expression vector that is suitable for expression of Themis protein. The expression vector may be any vector that is suitable for expression in bacteria or in eukaryotic cells. The expression vector may be pETM-44.

In a further aspect the present invention provides a method of obtaining an indication useful in the diagnosis of an autoimmune disorder or an immune deficiency disorder comprising the steps of allowing an antibody of the invention to bind to Themis protein in a sample and assessing the expression level of Themis protein in the sample. The skilled man will appreciate that preferred features of any one embodiment and/or aspect of the invention may be applied to all other embodiments and/or aspects of the invention.

The present invention will be further described in more detail, by way of example only, with reference to the following figures in which:

Figure 1 - shows SEQ ID No 1 , the sequence of themis protein from humans. The underlined section is amino acids 1 to 261 ;

Figure 2 - shows the predicted schematic structure of the Themis protein which is predicted to have the following schematic organisation. CABIT = 'cysteine- containing, all-in Themis'. P = Proline-rich region;

Figure 3 - shows the cDNA sequence corresponding to Homo sapiens thymocyte selection associated (THEMIS), transcript variant 1 , mRNA (http://www.ncbi.nlm.nih.gOv/nuccore/NM_001 164685. l) used for the generation of the constructs (coding sequence of the entire Themis is in bold letters with start codon atg and stop codon taa. Themis nucleotide sequence included in MBP-Themis 1 -261 (encoding amino acids 1 - 261 of Themis) is underlined); Figure 4 - shows preliminary structure prediction studies to identify subdomains of Themis feasible for expression in E.coli;

Figure 5 - shows expression screening of MBP-Themis constructs in E.coli. (amino acid numbering is indicated in column 2 and corresponds to the protein sequence numbering indicated in figure 1);

Figure 6 - shows Purification of MBP- 1 -261 Themis fusion protein by NiNTA affinity chromatography. Elution profile of MBP- 1 -261 Themis fusion protein on NiNTA affinity chromatography. Absorption at A280 shows peaks where the proteins elute; percentage Buffer B ( 1 M imidazole in PBS) is shown as steps; fractions collected marked on the x axis;

Figure 7 - shows coomassie Blue-stained SDS-PAGE gel showing fractions eluted from NiNTA affinity column. The molecular species at ~ 75 kD is the expected size of MBP- 1 -261 Themis fusion protein. The second major species at ~ 43kD is MBP. Because of these contaminations pooled fractions containing Themis MBP- 1 -261 were further purified on a Superdex 200 16/600 gel filtration column (Figure 8);

Figure 8 - shows purification of MBP- 1 -261 Themis by gel filtration on Superdex 200. Elution profile of MBP- 1 -261 Themis fusion protein on Superdex 200 gel filtration. Peaks of absorbance at A280 are shown; fractions collected and analysed by SDS-PAGE are marked on the x axis (the SDS-PAGE is shown in Figure 9). The main peaks from left to right are indicated as peaks I, II, III and IV;

Figure 9 - shows Coomassie Blue-stained SDS-PAGE gel showing fractions eluted by Superdex 200 gel filtration;

Figure 10 - shows Coomassie Blue stained SDS-PAGE gel showing purification of full-length Themis (~ 75 kD species) by Strep-tactin affinity chromatography. L, cell lysate; FT, column flow through; Wl , high salt, detergent wash step; W2, PBS wash step; 1 to 4, elution fractions. Fractions containing Themis protein were pooled and further analysed on a Superdex 200 (Figure 1 1 , below). The profile after Superdex 200 chromatography shows that recombinant full length themis elutes as a single, soluble mono-dispersed species;

Figure 11 - shows Elution profile of full length Themis on Superdex 200 10/300 column;

Figure 12 - shows - MBP-His tag- Themis 1 -261 (F construct in Figure 5) His-tag (underlined); MBP (normal font); Themis 1 -261 (bold); Figure 13 - shows Full length Themis amino acid and nucleotide (below) sequence with linker and Streptag (OST) (Streptag is underlined);

Figure 14 - shows the results of an experiment showing that GRB2-SH3C binds an RxxK motif on THEMIS . (A) A peptide scanning array covering full-length human THEMIS (Uniprot Q8N 1K5 isoform 1) was spot synthesized as 29 aa peptides, sliding 3 aa with each step. (Top panel) Membrane probed with 0. 1 mM GST-GRB2 (full length); (bottom panel) 0.1 mM GST-GRB2 SH3C domain. Peptides surrounding the single binding region are displayed to the right with the probable binding region emphasized in bold type . (B) Alanine scanning peptide array through the identified binding region, wt residues were sequentially mutated to alanine (or glycine, when alanine present). Peptides were spotted in duplicates and the array was probed as in (A). Key GRB2 binding residues are indicated in the box. (C) Stepwise N- and C-terminal truncations of the THEMIS peptide used for alanine-scanning substitution as seen in (B). (D) HEK293 cells were cotransfected with a human THEMIS construct carrying a C-terminal OneStrepTag (THEMIS-Strep) and either empty vector, GRB2-wt-Myc, GRB2-49L-Myc (SH3N mutant), GRB2-203R-Myc (SH3C mutant), or GRB2-49L-203R-Myc (SH3C and N mutant). THEMIS was precipitated by Streptactin pull down and analyzed for bound GRB2 by immunoblotting. Data are representative of three independent experiments;

Figure 15 - shows the results of an experiment showing that GRB2 binding is required for THEMIS recruitment to the LAT signalosome. (A) Themis-Strep mutants of PRR1 and 2 (P555/558A and P582/585A, respectively) expressed in Jurkat cells were pulled down with Streptactin-Sepharose and probed for GRB2 association by immunoblotting. (B) As in (A), but from resting and CD3 mAb stimulated cells and analyzed for tyrosine phosphorylation, GRB2, LAT, and PLCg l association. (C) LAT-deficient Jurkat J.CaM2.5 were reconstituted with LATStrep wt or 3YF (Y 171/191/226F) mutant. LAT pull downs of CD3 Ab or NaPV-treated cells were probed for GRB2 and THEMIS binding. Relative amounts of THEMIS pulled down and normalized to LAT are shown. (D) LAT- deficient Jurkat J.CaM2.5 were transduced with THEMIS-Strep and either LAT wt or 3YF (Y 171F, Y 191F, and Y226F) mutant. THEMIS was pulled down from resting and stimulated cells, and assessed for tyrosine phosphorylation by immunoblotting. Data are representative of three independent experiments;

Figure 16 - shows results of an experiment showing thatrecruitment of THEMIS to the IS is mediated via GRB2. (A) GRB2 association to murine THEMIS wt and PRR1 mutant (P557/560A) GFP constructs in HEK293 cells. Data are representative of three independent experiments. (B and C) Live-cell imaging of conjugates between 5C.C7 T cells (MCC82- 103 in context of I-Ek) transduced with muTHEMIS-GFP (B) or muTHEMI S -PRR 1 -GFP (C) with MCC-pulsed ( 10 mM) CH27 B cells . Time point zero marks the formation of a tight cell contact. Differential interference contrast images are shown in the top rows, with top-down, maximum projections of three-dimensional fluorescence data in the bottom rows (intensities in rainbow-like false-colour scale) . The graphs display the accumulation of GFP sensors with the indicated patterns expressed as percentages of the total number of tight cell couples formed over time (see Fig. 21 for description of the individual patterns). Forty-three and 54 couples were analyzed for THEMIS-wt-GFP and THEMIS-PRR1 -GFP, respectively;

Figure 17 - shows results of an experiment showing that THEMIS is a target of Lck and ZAP70-mediated phosphorylation. (A) THEMIS-Strep was pulled down from Jurkat cells treated as indicated and tyrosine phosphorylation was probed by immunoblotting. (B) Jurkat cells were left untreated or pretreated as indicated with either BMS-509744 ( 10 mM), PP2 (50 mM), or vehicle control (DMSO) for 30 min before CD3 mAb stimulation and THEMIS immunoprecipitation. THEMIS tyrosine phosphorylation was assessed by immunoblotting. Compiled data of two independent experiments can be found in Fig. 22. (C) HEK293 cells were transfected with THEMIS-Strep alone or in combination with Lck and/or ZAP70. Tyrosine phosphorylation of pulled-down THEMIS was assessed by immunoblotting. (D) Quantification of THEMIS tyrosine phosphorylation by ZAP70 alone or in combination with Lck across multiple experiments expressed as fold increase over Lck-mediated phosphorylation (n = 4; *p , 0.05, Student t test) . (E) As in (C), but probed for

ZAP70-pY493. Data are representative of at least three independent experiments, except for (B), which has been repeated twice;

Figure 18 - shows results of an experiment showing that interdependence of Y540 and Y541 phosphorylation and PRRl function. (A) HEK293 cells were cotransfected with Lck and either THEMIS-Strep wt, an all tyrosine to Phe mutant ( 19YF) and swap mutants between wt and 19YF (residue 220 as swapping boundary). Tyrosine phosphorylation of THEMIS pull-downs was assessed by immunoblotting. (B) THEMIS-Strep wt or Y540 and Y541 to Phe mutant were isolated from Jurkat cells treated as indicated and probed for tyrosine phosphorylation, GRB2, and LAT association. (C) Peptide scanning array as in Fig. 14A with phosphorylation and substitutions at Y540 and Y541. (D) HEK293 cells were cotransfected with THEMIS-Strep wt or PRRl mutant and increasing amounts of Lck. Precipitated THEMIS was probed for tyrosine phosphorylation and GRB2 association. (E) A total of 500 ng recombinant

THEMIS-Strep was phosphorylated in vitro with 25 ng recombinant Lck. Where indicated, THEMIS-Strep was preincubated with recombinant GST-GRB2 ( 10 μΜ) for lh. THEMIS tyrosine phosphorylation was quantified by immunoblotting. (F) HEK293 cells were transduced with C-terminally Strep- tagged lentiviral constructs of either full-length wt and Y540/541F mutant

THEMIS or truncation constructs consisting of CABIT domain 1 (aa 1-260) and CABIT domain 2 including the tail sequence (aa 261-641). After Streptactin pull downs, THEMIS constructs were assessed for GRB2 binding by immunoblotting. Data are representative of three independent experiments;

Figure 19 - shows results of an experiment showing that critical role of the THEMIS-GRB2 complex for thymocyte development. (A) Development of wt or PRRl -mutant THEMIS expressing thymocytes in bone marrow chimeric mice. Irradiated B6.SJL recipient mice (CD45. 1+) were reconstituted with Themis2/2 bone marrow cells (CD45.2+) transduced with a bicistronic GFP-based retroviral vector harbouring either no insert, muTHEMIS-wt, muTHEMIS-PRRl (P557/560A), or muTHEMIS-Y542/43F. Four mice were injected per experiment. Eight weeks postreconstitution, only recipient mice bearing .5% CD45.2+GFP+ thymocytes were included and analyzed by flow cytometry. In the far left scheme, each symbol represents an individual mouse . Shown are CD4 versus CD8 stains after gating on CD45.2+GFP+ (transgene expressing) thymocytes. (B) Reconstitution of the peripheral T cell compartment in the bone marrow chimeric mice described in (A). Shown are B220 versus CD3 FACS stains after gating on CD45.2+GFP+ splenocytes. Data shown are representative of two independent experiments. Statistical analysis of THEMIS mutants versus THEMIS-wt as control is shown. *p # 0.05, * *p # 0.01, one-way ANOVA (Dunnett's post hoc test, THEMIS-wt as control) . Vector control reconstitutions frequently fail because of low cellularity (failed thymus reconstitution) and only serve to illustrate the background signal of the analysis;

Figure 20 - shows astructural overview of human THEMIS

(A) Multiple sequence alignment of the C-terminal region of THEMIS surrounding PRR1 and PRR2 between human (Q8N 1K5), mouse (Q8BGW0), fowl (E 1C820), platypus (F7CHG4) and zebrafish (A5PF62). (B) Graphical overview of THEMIS with putative structural features as proposed by Johnson and colleagues ( 1). The two Pro-rich regions (PRRs) and the key Tyr residues 540 and 541 are indicated;

Figure 21 - shows subclasses of spatiotemporal patterns of fluorescent protein tagged imaging sensors. Definition of patterns, schematic representations and sample images for each of the subclasses used for analysis of live-cell imaging data in Figure 16. The en face view shows the T cell from an APC's point of view whereas the top down view looks at the T cell-APC couple from the side with the APC at the top. For more details see (2);

Figure 22 - shows compiled data of two independent experiments of Figure 17B. Jurkat cells were left untreated or pretreated as indicated with either BMS- 509744 ( 10μΜ), PP2 (50 μΜ) or vehicle control (DMSO) for 30 min prior to CD3 mAb stimulation and THEMIS immunoprecipitation. THEMIS Tyr- phosphorylation was assessed by immunoblotting. Tyrphosphoyrlation as normalized to total THEMIS signal is shown for two independent experiments.

Thymocyte-expressed molecule involved in selection (THEMIS) has recently been identified as a new T cell lineage-specific gene. Themis -/- mice show impaired positive selection during thymocyte development and severe reduction of mature thymocytes and peripheral T cells. THEMIS expression is high in double-positive (DP) thymocytes, with a marked down regulation after positive selection and in peripheral T cells. Whereas knockout mice do not show hallmarks of autoimmunity, the recently described BNm rat strain has linked a frameshift mutation in the Themis gene to inflammatory bowel disease caused by defective regulatory T cell function. THEMIS is a highly conserved, 73-kDa cytoplasmic protein without any obvious catalytic or protein-protein interaction domains. Bioinformatics analysis predicts a tandem repeat of a novel, cysteine-containing globular domain (cysteine-containing all β in THEMIS [CABIT]) found in a number of metazoan proteins. A putative bipartite nuclear localization sequence is present within the CABIT-2 domain of THEMIS, but no nuclear translocation was detected upon TCR ligation. The C-terminal end of THEMIS, predicted to contain little or no secondary structure, harbours two proline- rich regions (PRRs). In previous work, we identified THEMIS as an early target of tyrosine phosphorylation downstream of TCR. TCR-induced tyrosine phosphorylation of THEMIS depended on the adapters linker for activation of T cells (LAT) and Src homology (SH)2 domain-containing leukocyte protein of 76 kDa (SLP76), and THEMIS appeared to associate to LAT upon TCR stimulation. THEMIS is constitutively associated to the adapter protein growth factor receptor-bound protein 2 (GRB2) ( 1 , 2, 5, 7), suggesting a potential mechanism of THEMIS recruitment onto LAT to regulate the TCR signalling cascade.

Initial studies reported only subtle alterations in TCR-proximal signalling in Themis - /- DP thymocytes ( 1), and THEMIS implication in the TCR signalling machinery has been disputed. Thus, precise delineation of when, where, and how THEMIS relocates and undergoes posttranslational modifications during TCR triggering can help clarify its molecular function and role in T cell development.

THEMIS PRRl , an atypical binding motif for the C-terminal SH3 domain (SH3C) of GRB2, mediates the constitutive association of THEMIS to GRB2 and THEMIS recruitment via LAT to the immunological synapse (IS) after Ag stimulation. The Lck and ZAP70 kinases control phosphorylation of two tyrosines located shortly upstream of PRR1 , and these tyrosines are required for GRB2 binding, which, in turn, boosts THEMIS phosphorylation, revealing an unusual proximal interplay between these two events. Finally, THEMIS mutants defective in GRB2 association do not rescue positive selection in Themis -/- mice . These data definitively support a model of THEMIS regulating key TCR signalling events, and suggest that in DP thymocytes, THEMIS-GRB2 may compete with son of sevenless (SOS)-GRB2 for LAT binding, thus favouring positive selection.

Examples

The structure of Themis was investigated to find the boundaries of the domains. Themis was found to have two CABIT domains, CABIT 1 and CABIT 2. CABIT 1 is amino acids 1 to 261 of SEQ ID NO: 1. CABIT 1 provides a stable polypeptide and may be fused to a fusion partner such as Maltose Binding Protein and also a tag for purification. A fusion protein of amino acids 1 to 261 fused to Maltose binding protein and with a polyhistidine tag (SEQ ID NO 3) was expressed in E. Coli and purified. A fusion protein of the full length Themis protein with a Strep-tag (SEQ ID NO: 4) was expressed in Chinese Hamster Ovary cells and purified using the Strep-tag binding to Strep-Tactin.

Two recombinant Themis proteins have been generated. One is a truncated Themis molecule fused to Maltose binding protein (MBP) to help stabilise the recombinant Themis moiety and increase its solubility. The fusion protein comprises residues 1 -261 (underlined in Figure 1) of the Themis protein with MBP fused to the N-terminus via a linker peptide (MBP- 1 -261) (as shown in Figure 12). The soluble MBP- 1 -261 protein was produced in E.coli. Themis MBP- 1 -261 can be obtained routinely with > 95% purity at 2-5 milligrams/litre of bacteria culture. Full-length Themis was expressed in CHO cells.

Intact full length Themis is obtained at > 95% purity from CHO cell lysates at ~ 200 micrograms/109 cells.

These themis proteins are not post-translationally modified. Both products are readily available and could be used for immunization to obtain mAbs to Themis.

Preliminary structure prediction studies to identify subdomains of Themis feasible for expression in E.coli.

Initial studies were carried out to identify boundaries of presumed Themis protein domains using first a IUPred- Prediction of intrinsically unstructured proteins software (http://iupred.enzim.hu/) to search for potential secondary structures in Themis protein. This initial survey (not shown) indicated that there are at least two main structured regions in human Themis, largely corresponding to the regions called CABIT 1 and CABIT 2, followed by C-terminal segment of ~ 100 aa residues. Three distinct tertiary structure prediction software were used (HHPred (PBD), Homology detection & structure prediction by HMM-HMM comparison (from MPI); FFAS03 (PBD) Fold & Function Assignment System and Phyre - Protein Homology/analogY Recognition Engine. The results for these searches are illustrated in Figure 4. This study allowed the boundaries to be set of the Themis segments to be fused to MBP

Expression screening of MBP-Themis constructs in E.coli.

The screening results are shown in Figure 5 (amino acid numbering is indicated in column 2 and corresponds to the protein sequence numbering indicated in figure 1) .

Several DNA constructs of Themis were made, corresponding to the boundaries indicated in figure 5. These were cloned into the indicated prokaryotic expression vectors fused to the maltose binding protein (MBP) and expressed in E.coli. Cultures were induced by IPTG to express recombinant fusion proteins that were screened for expression, solubility after centrifugation and degradation using SDS-PAGE analysis. Based on the positivity to all three of these criteria (Y = Yes, N = NO), only the MBP fusion protein corresponding to aa residues 1 - 161 (fusion protein F) was found to provide suitable expression to obtain large quantities of recombinant protein for further studies.

Detailed protocol for the construction, production and purification of fusion protein F (MBP-1-261 Themis)

Sequence encoding the first 261 amino acids of Themis spanning the first CABIT domain was amplified by PCR and cloned in-frame with N-terminal hexahistidine tag fused to Maltose binding protein (MBP) nucleotide sequence in the expression vector pETM-44. The plasmid vector was transformed into E.coli strain Tuner(DE3) and transformants selected on Kanamycin. Large-scale cultures were inoculated from overnight pre-cultures and grown in TB medium at 21 °C to an optical density of 0.6. (measured at 600 nm wavelength). Cultures were induced by the addition of 1 mM IPTG and incubated for 16 h at 21 °C to allow for protein production. Cells were harvested by centrifugation, re-suspended in lysis buffer containing in PBS, 30 mM imidazone, 1 mM EDTA, ImM 2-mercaptoethanol, 5% glycerol, l OOmg/mL Lysozyme, l OOmg/mL DNase I and protease inhibitors. Cells were lysed in an EmulsiFlex C5 homogenizer operated at 15000 to 20000 psi and cleared lysates applied to a NiNTA column. After extensive washings, bound material was eluted with 500 mM imidazole (see Figure 6 for protein elution profile) . Positive fractions as analysed by SDA-PAGE (Figure 7) loaded on a Superdex 200 16/600 gel filtration column and resolved at a flow rate of 1 ml/min (see Figure 8 for protein elution profile).

Figure 9 demonstrates that essentially all the fractions from Superdex 200 column containing protein show the presence of MBP- 1 -261 Themis. This experiment indicates formation of aggregates (peak I and II) in addition to monomers and dimers (peaks IV and III, respectively) that are all soluble. The fraction of MBP- 1 -261 Themis eluting very late indicates non-specific absorbtion, likely due to protein malfolding. More recent work that re-analysed peaks III (dimers) and IV (monomers) separately on Superdex 200 show that each one displays monomers and dimers. This suggests that they form reversible monomers and dimers in equilibrium.

For the purpose of immunisation all fractions can be pooled. LC-MS/MS analysis of the pooled protein detected only MBP and Themis tryptic peptides (not shown). All the procedures used for purification used standard reagents and procedures. Production of recombinant full length Themis in the CHO-K1 mammalian cell system

An expression cassette containing full length Themis isoform 1 fused to a C-terminal One-STrEP-tag, was cloned into pEE 14.1 (Lonza Biologies) and transfected into CHO-K1 cells. Highly expressing clones were expanded to 109 cells, harvested, re-suspended in 5 ml PBS and lysed by adding 5 ml 1 % lauryl maltoside in PBS in the presence of protease inhibitors. Cleared lysates were passed over a Strep-tactin column (IB A GmbH), washed extensively and eluted with 2.5 mM desthiobiotin in PBS . Eluted fractions were collected and analysed by SDS-PAGE, together with total lysate and flow through (Figure 10). Further purification was achieved by separation on a Superdex 200 gel filtration column and resolved at a flow rate of 1 ml/min (Figure 1 1).

All the procedures used for purification used standard reagents and procedures. Materials and Methods

Plasmids and Abs

Full-length cDNA encoding human THEMIS was obtained from Open Biosystems (NM_001010923.2; giving rise to a 641 -aa protein: Uniprot Q8N 1K5- 1) and used as the PCR template to generate THEMIS-Strep, carrying a C-terminal One-STrEP-Tag (IBA BioTAGnology, Gottingen, Germany). THEMIS-Strep was cloned into the lentiviral expression vector pHR-SIN-BX-IRES-Emerald (kindly provided by Dr. V. Cerundolo, Weatherall Institute of Molecular Medicine, Oxford, U.K.) to give rise to pHR-THEMIS-Strep. All mutants described were based on pHR-THEMIS Strep and derived by site-directed mutagenesis (QuickChange II Kit; Agilent Technologies). The lentiviral helper plasmids psPAX2 (Addgene 10703) and pMD2.G (Addgene 12259) were provided by Dr. Didier Trono (Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland) via Addgene. Myc-tagged human GRB2-49L, GRB2-203R, and GRB2-49L/203R constructs (kind gift of Dr. R.A. Weinberg, Massachusetts Institute of Technology Ludwig Center for Cancer Research, Cambridge, MA) were cloned into pHR-SIN-BX-IRES-Emerald by PCR. Plasmids pCEFL-LAT-wild-type (wt)-Myc and pCEFL-LAT-3YF-Myc were kindly provided by Dr. Lawrence E. Samelson (National Institutes of Health, Bethesda, MD) and used as PCR templates to generate LAT-Strep and LAT-3YF-Strep in pHR-SIN-BX- IRESEmerald. For BM reconstitution experiments, murine THEMIS was cloned into the retroviral expression vector pCMV-IRES-GFP to give rise to pCMVmuTHEMIS- IRES-GFP. Mutants of murine Themis described in this study were derived by site- directed mutagenesis. For expression in 5C.C7 T cells, GFP-tagged versions of the above constructs were expressed from the retroviral pGC vector. All constructs were verified by sequencing. Human wt Lck and ZAP70 were cloned into pEF-BOS expression vector. Mouse mAbs used included anti-phosphotyrosine (clone 4G10; Millipore); anti-LCK (clone 3A5 ; Santa Cruz); anti-ZAP70 (2F3.2), anti-LAT (2E9); anti-One-STrEP-Tag mAb (StrepMAB Classic; IBA Bio-TAGnology); anti-human CD3e (clone UCHT- 1 ; BioLegend), anti-SLP76 (clone SLP76/03 ; AbD Serotec), and anti-Myc (clone 4A6; Millipore). Rabbit polyclonal Abs used were anti- phospholipase C g 1 (anti-PLCg l ; 1249; Santa Cruz); anti-GRB2 (C-23 ; Santa Cruz); anti-phospho-ZAP70 Y493 (Cell Signaling Technology); anti-actin (Sigma), and anti- THEMIS (Sigma). Goat anti-murine Themis is from Abeam PLC.

Cells, transfections, and lentiviral transductions

CD4+ Jurkat subclone 20 and LAT-deficient Jurkat cells (J.CaM2.5) were maintained in RPMI 1640 (PAA Laboratories) medium supplemented with 10% FBS (Perbio). Human embryonic kidney epithelial cells (HEK293) were maintained in DMEM, 10% FBS . HEK293 cells were transfected by standard calcium phosphate precipitation. Lentiviral particles were produced in HEK293 cells by cotransfection of the pHR-SIN- BX-IRES-Emerald vector with the packaging plasmids psPAX2 and pMD2.G. Forty- eight hours after transfection, viral supernatants were harvested, filtered, and used for the transduction of cells in the presence of 5 mg/ml Polybrene . Puromycin selection was applied 48 h after transduction where appropriate at 1 mg/ml. Immunoprecipitations and pull-down assays

Resting or anti-CD3-activated wt Jurkat cells or Jurkat cells expressing THEMIS-OST were lysed in ice-cold lysis buffer (20 mM Tris pH 7.5, 150mM NaCl, 0.5% dodecyl- b-D-maltoside [Calbiochem], 1 mM Na3V04, protease inhibitor mixture [Roche]). Lysates were cleared by centrifugation at 14,000 3 g for 10 min. THEMIS-Strep pull- downs were carried out on cleared lysates for 15-20 min at 4°C with Streptactin- Sepharose beads (IBA BioTAGnology). After pull down, beads were washed three times with lysis buffer, and bound proteins were eluted with 5 mM biotin. For GFP immunoprecipitations, anti-GFP-agarose (rat monoclonal, clone RQ2; MBL International) was used.

Production of recombinant Themis

HEK293 cells were transduced with the lentiviral expression construct pHR-THEMIS- Strep. GFP expression from the IRES-Emerald cassette was used to sort for highly expressing cells by FACS . Cells were harvested with PBS/EDTA and lysed in standard lysis buffer (see above). Lysates were cleared by centrifugation at 14,000 3 g for 10 min and loaded onto a gravity-flow Streptactin-Sepharose column (IBA BioTAGnology, Go ^'ttingen, Germany). After washing with 10-column volumes of lysis buffer, bound protein was eluted with elution buffer ( 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 2.5 mM desthiobiotin).

In vitro phosphorylation assay for mass spectrometry

A total of 500 ng recombinant Themis was incubated with 50 ng recombinant Lck (Millipore, Billerica, MA) in kinase buffer (20 mM Tris-HCl pH 7.5, 10 mM MgC12, 10 mM MnC12, 1 mM ATP) for 30 min at 30°C. Reactions were stopped by adding reducing SDS NuPAGE sample buffer (Invitrogen) and incubation for 5 min at 95°C, followed by alkylation with 55 mM iodoactamide (Sigma) . Proteins were separated on 4- 12% gradient Bis-Tris NuPAGE gels (Invitrogen), gels were washed in distilled water, lightly stained with Colloidal Blue (Invitrogen), and subjected to GeLC-mass spectrometry (MS)/MS as described previously.

MS data analysis

Samples were analyzed on a Q Exactive (Thermo Scientific) coupled to an Ultimate 3000RSLCnano system (Dionex). Samples were resolved on a 25-cm-long by 75 -mm internal diameter home-packed Picotip emitter (New Objective) at a flow rate of 300 nl min21 using a 120-min gradient. The mass spectrometer was operated in a "top 10" data-dependent mode in which the 10 strongest precursors were selected for fragmentation by HCD. 1+ charged ions were excluded from isolation. Data were converted to .mzXML format using MSconvert (Proteowizard) and uploaded into the central proteomics facility pipeline (CPFP) (Trudgian, D. C, B . Thomas, S . J. McGowan, B . M. Kessler, M. Salek, and O. Acuto. 2010. CPFP: a central proteomics facilities pipeline. Bioinformatics 26: 113 1- 1 132) for analysis. Enzyme was set to trypsin allowing for up to two missed cleavages. Carbamidomethyl cysteine was set as a fixed modification and oxidation (methionine), deamidation (NQ), acetylation (Protein-N), and phosphotyrosine as variable modifications. Mass tolerances for MS and MS/MS peak identifications were 20 ppm and 0.1 Da, respectively. Assignment of a tyrosine phosphorylation site required identification by searches in the CPFP at 1 % false discovery rate. InterProphet probability is derived by the combination of results from multiple search engines within CPFP, and improves coverage and confidence over use of a single search engine . Modification localization scoring was performed using the ModLS algorithm (Trudgian, D. C, R. Singleton, M. E. Cockman, P. J. Ratcliffe, and B. M. Kessler. 2012. ModLS : Post-translational modification localization scoring with automatic specificity expansion. J. Proteomics Bioinform. 5 : 283-289), a method similar to the AScore algorithm (Beausoleil, S. A., J. Ville 'n, S. A. Gerber, J. Rush, and S. P. Gygi. 2006. A probability-based approach for high- throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 24: 1285- 1292). ModLS expands the AScore method to incorporate automatic specificity expansion. For each variable modification chosen for the database search, all amino acid specificities defined in the Unimod database are considered during localization. Image acquisition and image analysis

Image acquisition and analysis were performed as described in detail elsewhere (Singleton, K. L., K. T. Roybal, Y. Sun, G. Fu, N. R. Gascoigne, N. S. van Oers, and C. Wu lfing. 2009). In brief, T cell-APC interactions were imaged at 37°C. Every 20 s, 1 differential interference contrast and 21 fluorescence images that spanned 20 mm in the z-plane at 1 -mm intervals were acquired. The acquisition and analysis software was MetaMorph (Molecular Devices) . The formation of a tight cell couple, time 0 in our analysis, was defined as either the first time point with a fully spread T cell-APC interface or 40 s after first membrane contact, whichever occurred first. A region of sensor accumulation was defined by an average fluorescence intensity of 135% of the background cellular fluorescence. To classify spatial accumulation features, we used six mutually exclusive interface patterns: central, invagination, diffuse, lamellal, asymmetric, and peripheral, as defined by strict geometrical constraints (for details see Fig. 21). Distal accumulation was scored independently. A T cell was scored to have a uropod as long as an inversion of curvature of the plasma membrane could be detected at the distal pole in the differential interference contrast images. Data were routinely analyzed by two investigators independently to ensure the reliability of this analysis.

Bone marrow reconstitution experiments

Bone marrow cells were isolated from donor Themis 12/2 mice (B6.129SThemis tmlGasc; Jackson Laboratory Stock no. 010919) ( 1), which were pretreated with 5 - fluorouracil 5 d before isolation. A total of 2 3 106 bone marrow cells were cultured in 1 ml DMEM supplemented with 10% FBS, Pen/Strep/Glut, 2-ME, and nonessential amino acids in one well of a 24- well plate . Cytokines (PeproTech) were also added to the media as IL-3 (20 ng/ml), IL-6 (25 ng/ml), and stem cell factor ( 100 ng/ml) . For virus generation, retroviral vectors (i.e ., empty vector, WT- Themis vector, or mutated Themis vector) were transfected into Plat-E packaging cells that were preseeded 1 d before (as 5 3 106 cells per 10-cm petri dish). Twentyfour hours post-transfection, spent media were aspirated and replaced with 5 ml fresh media. At 48 and 72 h post- transfection, the derived viral supernatants were collected and concentrated by centrifugation with Amicon Ultra4 Centrifugal Filter Unit (UFC810024). Concentrated viral supernatants were used to spin-infect cultured bone marrow cells in the presence of 8 mg/ml Polybrene . The spin infection was carried out at 2500 rpm, 32°C for 2 h. The infection efficiency of bone marrow cells was determined by analyzing the percentage of GFP+ cells with flow cytometry. A typical infection efficiency falls between 30 and 70%. Infected bone marrow cells were i.v. injected into lethally irradiated (i.e ., 1 100 rads in two equally split doses) B6. SJL recipient mice (CD45. 1 + versus CD45.2+ in donor cells) . Eight weeks postreconstitution, mice bearing .5% CD45.2+GFP+ thymocytes were included and phenotyped for thymocyte and mature T cell development.

Protein expression and purification

GST and GST-Grb2 fusion proteins (both full-length and SH3C) were expressed in Escherichia coli growing in terrific broth medium at 18°C after induction with 50 mM IPTG Cells were lysed by sonication in TPE lysis buffer ( 1 % Triton X- 100, PBS, and 100 mM EDTA) with a protease inhibitor mixture added. The mixture was centrifuged at 48,000 3 g for 1 h, and the soluble fraction was incubated overnight with glutathione Sepharose beads. The next day, beads were washed extensively with cold wash buffer (50 mM Tris pH 7.5, 100 mM EDTA, and 0.1 % Tween 20) before overnight incubation with elution buffer ( 100 mM reduced glutathione brought to pH 8.0 with a concentrated Tris buffer stock, pH 8.8) . The eluted GST fusion proteins were further purified by gel filtration chromatography on a Superdex 75 column (GE Healthcare) in running buffer (20 mM Tris pH 7.5, 150 mM NaCl). Finally, purified proteins were dialyzed extensively against 5 mM Tris pH 7.5 and concentrated to 10 mg/ml before storage at 280°C.

Peptide arrays

The full amino acid sequence of human THEMIS (Uniprot code : Q8N 1K5 , isoform 1) was chemically synthesized as an array of spots of overlapping peptides (Multipep synthesizer; Intavis) with a peptide length of 29 amino acids, sliding 3 residues along the sequence with each consecutive peptide spot. Membranes were incubated for 4 h in blocking buffer (3% OVA, 20 mMTris-HCl pH 7.5, l OOmMNaCl, 0. 1 % Tween 20, 2mMDTT, ImM sodium molybdate, 1 mM sodium orthovanadate) and probed first with 0.1 mM GST overnight, followed by incubation with anti-GST primary Ab, and then HRP-coupled secondary Ab, and finally visualized by ECL detection to confirm no background binding. The same membranes were then incubated again in blocking buffer before reprobing overnight with 0. 1 mM purified GST-Grb2 (full-length) or GST-Grb2 SH3C followed by detection, as described earlier, of Grb2-interacting epitopes. The Grb2 SH3C binding motif in THEMIS harbouring the RxxK motif was incorporated into additional membrane spot-synthesized peptides. These feature an alanine scan and sequence truncations for the mapping of key binding determinants; multiple mutants of the proximal double tyrosine motif (Y540 and Y541) and peptides both without tyrosine phosphorylation and singly tyrosine phosphorylated. Membrane probing and detection was as described earlier for the scanning array.

Results

GRB2 binding is required for THEMIS recruitment via LAT to the IS

The highly conserved proline-rich sequence PPPRPPKHP (residues 553-561 , Fig. 20A, 20B) of THEMIS (here designated PRR1) resembles consensus binding sites for the SH3C of GRB2 (GRB2-SH3C) on Gab proteins and SLP76 lacking the typical PxxP core motif. To directly verify this prediction, we assessed binding of recombinant GST-fusion proteins of GRB2-SH3C and full-length GRB2 to an array of 29-mer peptides scanning the entire THEMIS sequence (Fig. 14A). Binding of full length GRB2 and GRB2-SH3C was observed only to 29-mers containing the intact PRR1 sequence . A core-binding motif of PxRPxK was defined by alanine-scanning substitution and successive truncations (Fig. 14B, 14C). Pull-down experiments of Strep tagged THEMIS from transfected HEK293 cells confirmed that binding of GRB2 to THEMIS was mainly mediated via GRB2-SH3C, as mutation of the N-terminal SH3 domain (SH3N) had only a minimal effect (Fig. 14D). This agrees with RxxK motifs having considerably higher affinity for GRB2-SH3C than for SH3N ( 12, 15) . In agreement with a single GRB2-binding site, THEMIS carrying alanine substitutions at P555 and P558 of PRR1 (THEMIS-PRR1) expressed in Jurkat cells showed defective binding to GRB2, whereas mutating PRR2 had no effect (Fig. 15A). THEMIS-PRR1 showed a loss of TCR-induced association with LAT, was not tyrosine phosphorylated, and exhibited decreased interaction with PLCg l (Fig. 15B), a key component of the LAT-SLP76 complex. The N-terminal moiety of LAT contains three (two YVNV and one YENL) binding motifs for the SH2 domain of GRB2 and may thus recruit the THEMIS-GRB2 complex. When a LAT construct with all three tyrosines at these sites mutated to phenylalanine (Y 171F, Y 191F, and Y226F, i.e., LAT-3YF) was expressed in the LAT-deficient Jurkat line J.CaM2.5, the TCR-induced LAT association to GRB2 and THEMIS was lost (Fig. 15C), as was THEMIS phosphorylation on tyrosine (Fig. 15D). Taken together, these data strongly suggested that constitutive binding of GRB2-SH3C to THEMIS serves to allow THEMIS recruitment onto the LAT signalosome after TCR stimulation.

An earlier study failed to detect changes in the distribution of THEMIS upon TCR triggering. In light of the data presented earlier, we re-examined this aspect by live- cell imaging using GFP reporters in primary T cells. Primed T cells were used from 5C.C7 TCR transgenic mice recognizing a peptide from moth cytochrome c (MCC; aa 82- 103) in the context of I-Ek. Activated 5C.C7 T cells were transduced with retroviral vectors expressing GFP fusion constructs for murine THEMIS-wt or THEMIS-PRR1 (P557A/P560A), the latter showing no detectable binding to GRB2 (Fig. 16A). Upon conjugate formation with MCC peptidepulsed murine CH27 B cell lymphoma cells, THEMIS-wt-GFP exhibited a rapid recruitment to the T cell-APC interface, mostly in a diffuse and lamellal pattern (Fig. 16B, Fig. 15 for description of the individual patterns.). By contrast, interface recruitment of THEMIS-PRR1 -GFP was significantly reduced (Fig. 16C). The distribution of THEMIS at the T-APC interface was less central than LAT, suggesting THEMIS localization with peripheral signalling TCRs. These data demonstrated that upon TCR stimulation, THEMIS is rapidly recruited from an intracellular pool to the T cell-APC interface in a GRB2 dependent manner, likely via LAT, supporting the idea that THEMIS has a role in regulating TCR signalling.

THEMIS is a target of Lck and ZAP70-mediated phosphorylation

Tyrosine phosphorylation of THEMIS may generate binding sites for interacting partners and/or help induce conformational changes required for protein activation. Three major protein tyrosine kinases, Lck, ZAP70, and IL-2-inducible T cell kinase (Itk), control tyrosine phosphorylation during the earliest phases of TCR signaling. In Jurkat T cells, THEMIS was rapidly tyrosine phosphorylated after CD3 stimulation or sodium pervanadate (NaPV) addition (Fig. 17A). An involvement of Itk could be ruled out by using the specific inhibitor BMS-509744 ( 17), which, in contrast with the Src family kinase inhibitor PP2, had no effect on THEMIS phosphorylation (Fig. 17B). To investigate whether Lck and/or ZAP70 phosphorylated THEMIS, we performed coexpression experiments in HEK293 cells. The data showed that Lck and ZAP70 together induced stronger phosphorylation of THEMIS than either kinase alone ( 17. 4C, 17D), an effect likely caused by Lck-mediated activation of ZAP70 ( 18), as evidenced by a strong ZAP70-pY493 signal only when Lck was coexpressed (Fig. 17E).

THEMIS can be a substrate of both Lck and ZAP70. Y34, Y95, Y 174, Y540, and Y541 were predicted as possible target sites by Netphos 2.0 (score . 0.8). Recombinant THEMIS was phosphorylated in vitro by recombinant Lck or ZAP70, and phospho- sites Y95, Y 174, Y220, Y353, Y429, Y540, and Y541 were detected with high confidence by MS (Supplemental Table I). In agreement with this, Y540 and Y541 were predicted as potential Src phosphorylation sites by the NetphosK l .O tool and as potential Lck and ZAP70 target sites by the group-based phosphorylation scoring system. These data were corroborated by chimeric swap constructs between wt and an all-YF mutant (all 19 tyrosines of THEMIS mutated to phenylalanine) cotransfected with Lck in HEK293 cells (Fig. 18A). An all-YF mutant except for Y540/541/ 546 was still efficiently tyrosine phosphorylated. In addition, considering conservation of tyrosines both on the interspecies and intraspecies level (i.e ., between THEMIS and THEMIS2 [21] ; Supplemental Table I) led us to conclude that Y540 and Y541 are major TCR-induced phosphorylation sites. Attempts to detect pY540/pY541 - containing tryptic peptide(s) by MS from THEMIS isolated from TCR-stimulated Jurkat cells failed maybe because of inefficient trypsin digestion as previously noted at other phosphorylation sites. TCR-induced tyrosine phosphorylation of a double Y540/541F THEMIS mutant expressed in Jurkat cells was profoundly affected (Fig. 18B). However, surprisingly, THEMISY540/ 54 IF lost constitutive association with GRB2 and could no longer bind to LAT. Substitutions, deletions, or phosphorylation of Y540 or Y541 in peptide-array analysis did not affect GRB2 binding (Fig. 14B, 14C, 18C), nor could we detect any obvious changes in GRB2 associated to increasingly phosphorylated THEMIS in coexpression studies with Lck in HEK293 cells (Fig. 18D). It seems therefore unlikely that tyrosine phosphorylation directly modulates THEMIS-GRB2 complex formation. Nevertheless, our data suggested that a secondary /tertiary structure connecting Y540/Y541 and PRRl makes them mutually sensitive to mutations. Indeed, tyrosine phosphorylation of THEMIS-PRR1 by Lck in HEK293 was substantially reduced (Fig. 18D). Moreover, recombinant THEMIS purified as a monomer without GRB2 was poorly phosphorylated by Lck in vitro, whereas addition of recombinant GRB2 reconstituted THEMIS tyrosine phosphorylation (Fig. 18E). In this context, it is of interest that a soluble fragment of THEMIS lacking the CABIT- 1 domain did not bind GRB2 (Fig. 18F). This suggests that the PRRl motif and/or its surrounding region are somehow connected to a distal region of THEMIS, hinting at a global compact structure of the entire protein. Thus, GRB2 association might keep THEMIS accessible to protein tyrosine kinases.

PRRl is crucial for THEMIS function in vivo

Finally, we determined whether the constitutive THEMIS-GRB2 complex was relevant in thymocyte development. PRRl in murine THEMIS was disrupted by point mutations (muTHEMIS-PRRl ; see Fig. 16A). Lethally irradiated B6.SJL (CD45.1+) recipient mice were reconstituted with Themis2/2 bone marrow cells (CD45.2+) retrovirally transduced either with empty vector, muTHEMIS-wt, or muTHEMIS- PRR1. GFP expression driven by an IRES-GFP cassette on the retroviral vector allowed gating on "truly" transduced cells. THEMIS-wt-transduced donor bone marrow cells reconstituted SP thymocyte development (Fig. 19A) and the peripheral T cell compartment (Fig. 19B). By contrast, perturbations of THEMIS-PRR1 led to a severe reduction in CD4 SP thymocytes and peripheral T cells. In line with the critical role of Y540/41 (Y542 and Y543 in mouse) in THEMIS phosphorylation and GRB2 binding (Fig. 19B), muTHEMIS-Y542/43F failed to reconstitute normal T cell development. Taken together, our findings demonstrate that TCR-proximal positioning of THEMIS via GRB2-LAT is physiologically important for thymocyte development.

Discussion

Protein components that channel and tune TCR-proximal signaling show stereotypical molecular signatures. Thus, soon after TCR engagement, they are recruited directly to the TCR or LAT and are phosphorylated on tyrosine residues, inevitably localizing at the IS. These three properties are satisfied by THEMIS, conclusively defining it as an element of the TCR-proximal signaling machinery. THEMIS requires constitutive association with the adaptor GRB2, which permits recruitment onto LAT, followed by THEMIS tyrosine phosphorylation by active Lck and ZAP70. Consistently, mutations affecting GRB2 binding strongly affected THEMIS accumulation at the IS and T cell development in THEMIS deficient mice. GRB2-SH3C binding to THEMIS was defined at the conserved PRRl site, mediated by the core-binding motif of PxRPxK. These data contradict recent reports suggesting that GRB2-SH3N mediates binding to THEMIS . Although the reason for this discrepancy is unclear, our data do agree with published reports indicating that GRB2- SH3N preferentially binds to motifs conforming to the consensus PxxPxR, such as the type II polyproline helix in SOS, and displays only negligible affinities toward RxxK motifs. This leaves open the possibility that GRB2, via SH3N, helps in bridging LAT and THEMIS to an unknown partner. Live-cell imaging of THEMIS-GFP showed GRB2-dependent dynamic recruitment not exclusively at the center of the IS but also into lamellar structures that transiently cover the entire interface . Such structures appear to be active sites of membrane signaling as defined by the presence of LAT and active signaling proteins.

When bound to LAT, THEMIS becomes a substrate for Lck and ZAP70, which prominently phosphorylate Y540 and Y541. Database searches found no apparent consensus binding motifs surrounding pY540 and/or Y541 , suggesting that these pTyr may serve another function. Enhanced tyrosine phosphorylation of THEMIS when in complex with GRB2 was somewhat surprising, as was the dependency of GRB2 binding on Y540 and Y541. These data evoke similarity to GAREM, a protein regulating EGFR-proximal signaling that contains a single CABIT domain and constitutively associates to GRB2. Mutation of a tyrosine proximal to the GRB2 binding site also abolished GAREMGRB2 association. These perturbations at the GRB2 binding site (a short, supposedly poorly structured sequence), induced by medium- and long-range distal alterations in THEMIS structure, indirectly suggest the existence of a complex network of intramolecular interactions. Thus, Y540/541 may be gatekeepers at a CABIT domain-proximal region undergoing conformational changes upon their phosphorylation. This may be indicative of a "closed" structure that could be unleashed for functional activation, similar to tyrosine phosphorylation- dependent mechanism that releases autoinhibition in Vav proteins. Notably, the THEMIS PRR1 mutant construct tested in this study gave an intermediate phenotype between wt and empty vector in thymocytes. This is in apparent contradiction to the biochemical data where we observed a complete loss of GRB2 binding. However, using a more sensitive imaging approach (Fig. 16B, 16C), we saw severely reduced and delayed, but still detectable, IS recruitment of THEMIS-PRR1 when compared with THEMIS-wt. A residual binding affinity for GRB2 of the disturbed, but not deleted, RXXK region might yield the intermediate phenotype of THEMIS-PRR1, perhaps because of the innate flexibility of this poorly structured region. Of note, the incomplete loss of function was only evident in the thymus, but not in peripheral T cells.

It is of particular interest that both THEMIS-GRB2 and SOS-GRB2 complexes can bind to the same sites on LAT, and that in immature CD4+8+ thymocytes, THEMIS and SOS 1 expression substantially increases or decreases, respectively, relative to their expression in earlier stage CD4282 thymocytes. During positive selection, association of the THEMIS-GRB2 complex to LAT may prevail over SOS 1-GRB2, the former favouring a LAT-SLP76 signalosome configuration triggering the weak but sustained ERK and calcium signaling required for positive selection. Agonist-driven negative selection (that elicits strong and transient ERK activation) might instead favour SOS l -GRB2/LAT-type signalosomes during the late phases of (or post) positive selection, when THEMIS levels decrease, thus favouring cell death. The observation that positive section requires THEMIS, but not SOS proteins, and recent data that SOS 1 is instead required for negative selection agree with this model. A functional role of constitutive THEMIS-GRB2 association in thymocyte positive selection agrees with data on conditional ablation of GRB2 in thymocytes, which showed that GRB2-SH2 and -SH3C (required for association to THEMIS) were found to be indispensable for thymocyte development.

Su plemental Table I

A) hek ph $hmyMimi sates

ΧΑΨ79 |sij«spli«i~yfcfiiss sites

Supplemental table 1 shows THEMIS Tyr-phosphorylation sites identified by mass spectrometry. A gel band corresponding to 500ng rTHEMIS-OST phosphorylated in vitro by 50ng rLck or 50ng rZAP70 was subjected to in-gel tryptic digest and MS analysis. Highest ranking peptide fo each phosphosite identified is shown.

IP Prob = Interprophet Probability; ModL Score = Modifiication Localization Score (see Material and methods). 1 = human, mouse, dog, fowl, platypus and zebrafish.

The binding site on the Themis protein of the adaptor grt>2 was precisely mapped and also where Themis binds to the scaffold protein Lat.

Themis has been shown to endow the TCR proximal signalling machinery with a critical negative feedback that prevents apoptosis during T cell development in the thymus and T cell activation. In another aspect the present invention provides use of an isolated Themis protein according to the first aspect of the present invention to provide negative feedback in the TCR proximal signalling machinery and reduce or prevent apoptosis during T cell development in the thymus and/or reduce or prevent T cell activation.

In another aspect the present invention provides an isolated Themis protein according to the present invention for use in reducing or preventing apoptosis during T cell development in the thymus and/or T cell activation in vivo. The Themis protein may be formulated in a pharmaceutical composition.

In another aspect the present invention provides an isolated Themis protein according to the present invention for use in reducing or preventing apoptosis during T cell development and/or T cell activation in vitro. In another aspect, the present invention provides a method of treating a subject comprising administering to said subject an isolated Themis protein according to the present invention to reduce T cell development in the thymus and/or T cell activation. The Themis protein may be formulated in a pharmaceutical composition. In another aspect the present invention provides a peptide, antibody or small molecule that binds to a sequence consisting of PPPRPPKHP and modulates binding of Themis to GRB2. The peptide, antibody or small molecule may bind to the sequence consisting of PPPRPPKHP on a Themis protein. The peptide, antibody or small molecule may be an agonist of Themis binding to GRB2. The peptide, antibody or small molecule may be an antagonist of Themis binding to GRB2.

In conclusion, our data firmly establish that thymocyte development crucially depends on THEMIS as a component of the TCRproximal signalling machinery.

Claims

1. An isolated, soluble Themis protein comprising an amino acid sequence selected from:

a) the sequence set out in SEQ ID NO: 1 ;

b) the sequence of amino acids 1 to 261 of SEQ ID NO: 1 ;

c) a sequence having at least 70% identity to a) or b);

d) an active fragment or variant of a), b) or c);

e) a fusion protein comprising the sequence of a), b), c) or d) and a fusion partner;

f) a sequence comprising the sequence of a), b), c), d) or e) and a purification tag.

2. An isolated protein according to claim 1 , wherein the Themis protein is active.

3. An isolated protein according to claim 1 or claim 2, wherein the fusion partner is Maltose Binding Protein (MBP).

4. An isolated protein according to claim 3, wherein the fusion partner is MBP.

5. An isolated protein according to any one of the preceding claims, wherein the protein comprises a tag for use in purification.

6. An isolated protein according to claim 1 wherein the protein has the amino acid sequence shown in SEQ ID NO: 3.

7. An isolated protein according to claim 1 wherein the protein has the amino acid sequence set out in SEQ ID NO: 4.

8. An immunogenic composition comprising an isolated protein described in any one of claims 1 to 7.

9. An antibody raised against an isolated protein described in any one of claims 1 to 7.

10. A pharmaceutical composition comprising an isolated protein described in any one of claims 1 to 7.

1 1. A pharmaceutical composition according to claim 10 further comprising a physiologically acceptable carrier or excipient.

12. A protein according described in any one of claims 1 to 7 for use in medical treatment.

13. A protein as described in any one of claims 1 to 7 for use in the treatment or prevention of Celiac Disease, Multiple sclerosis, Type I Diabetes or in Inflammatory Bowel Disorder (IBD).

14. Use of a protein as described in any one of claims 1 to 7 in the manufacture of a medicament for the treatment or prevention of Celiac Disease, Multiple sclerosis,

Type I Diabetes or in Inflammatory Bowel Disorder (IBD) .

15. An isolated polynucleotide encoding a Themis protein having an amino acid sequence selected from:

a) the sequence set out in SEQ ID NO: 1 ;

b) the sequence of amino acids 1 to 261 of SEQ ID NO: 1 ;

c) a sequence having at least 70% identity to a) or b);

d) an active fragment or variant of a), b) or c);

e) a fusion protein comprising the sequence of a), b), c) or d) and a fusion partner.

16. An expression vector suitable for expression of a Themis protein having a sequence selected from:

a) the sequence set out in SEQ ID NO: 1 ;

b) the sequence of amino acids 1 to 261 of SEQ ID NO: 1 ;

c) a sequence having at least 70% identity to a) or b);

d) an active fragment or variant of a), b) or c);

e) a fusion protein comprising the sequence of a), b), c) or d) and a fusion partner.

17. A method of obtaining an antibody that binds to Themis protein comprising the step of inoculating an animal with a protein according to any one of claims 1 to 7.

18. Use of an isolated protein according to any one of claims 1 to 7 to provide negative feedback in the TCR proximal signalling machinery and reduce or prevent apoptosis during T cell development in the thymus and/or T cell activation.

19. An isolated protein according to any one of claims 1 to 7 for use in reducing or preventing apoptosis during T cell development in the thymus and/or T cell activation in vivo.

20. An isolated protein according to any one of claims 1 to 7 for use in reducing or preventing apoptosis during T cell development and/or T cell activation in vitro.

21. A method of treating a subject comprising administering to said subject an isolated protein according to any one of claims 1 to 7 to reduce T cell development in the thymus and/or T cell activation.

22. A peptide, antibody or small molecule that binds to a sequence consisting of PPPRPPKHP and modulates binding of Themis to GRB2.