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WO2007042778A2 - Lymphocytes t reactifs a la hsp60 - Google Patents

Lymphocytes t reactifs a la hsp60 Download PDF

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Publication number
WO2007042778A2
WO2007042778A2 PCT/GB2006/003740 GB2006003740W WO2007042778A2 WO 2007042778 A2 WO2007042778 A2 WO 2007042778A2 GB 2006003740 W GB2006003740 W GB 2006003740W WO 2007042778 A2 WO2007042778 A2 WO 2007042778A2
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Prior art keywords
cells
kir2ds2
hhsp60
cell
absence
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WO2007042778A3 (fr
Inventor
Christina Baboonian
Juan Carlos Kaskim
Behnam Zal
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St Georges Enterprises Ltd
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St Georges Enterprises Ltd
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Priority claimed from GB0520655A external-priority patent/GB0520655D0/en
Priority claimed from GB0608057A external-priority patent/GB0608057D0/en
Application filed by St Georges Enterprises Ltd filed Critical St Georges Enterprises Ltd
Publication of WO2007042778A2 publication Critical patent/WO2007042778A2/fr
Publication of WO2007042778A3 publication Critical patent/WO2007042778A3/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70514—CD4
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70521—CD28, CD152
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

Definitions

  • the present invention relates to methods for diagnosing and monitoring acute coronary syndrome and other cardiovascular disorders.
  • the invention also relates to methods for treating or preventing acute coronary syndrome and methods for identifying agents useful in treating or preventing acute coronary syndrome.
  • Acute coronary syndrome is a collection of disorders characterised by myocardial ischemic discomfort at rest, including unstable angina, Q-wave and non- Q-wave myocardial infarction.
  • the inflammatory infiltrate in atherosclerotic plaques is composed mainly of CD4 + T cells and macrophages.
  • Patients with acute coronary syndrome (ACS) experience both circulatory and intraplaque expansion of CD4 + CD28 nu " T cells. These T cells produce high levels of IFN- ⁇ and perform, and alongside other proinflammatory mechanisms, are considered to play an important role in the events leading to coronary plaque destabilisation.
  • the killing function of CD4 + CD28 nu11 cells has been attributed to both antigen recognition by TCR and expression of killer immunoglobulin-like receptors (KIRs) encoded by 12 independent loci on chromosome 19ql3.4.
  • KIRs killer immunoglobulin-like receptors
  • CD4 + CD28 nu11 cells from patients with ACS and rheumatoid vasculitis exhibit a predominant expression of the activating KIR2DS2 receptor usually in the absence of the opposing inhibitory homologues KIR2DL2 and KIR2DL3.
  • the present inventors have, for the first time, defined the molecular components and pathways leading to CD4 + CD28 nu " cell activation. Accordingly, the present invention provides novel approaches to monitoring and managing progression of acute coronary syndrome.
  • the present inventors have investigated whether the KIR receptors on CD4 + CD28 nuI1 cells interact with hHSP60 presented by MHC class I.
  • the inventors have surprisingly found that KIR2DS2 receptors of CD4 + CD28 nu " cells from patients with acute coronary syndrome (ACS) specifically interact with MHC class I molecules bearing hHSP60 peptides and that the presence of inhibitory KIR receptors on CD4 + CD28 nu11 cells from ACS patients does not serve to inhibit activation of these cells by MHC class I bearing hHSP60 peptides.
  • ACS acute coronary syndrome
  • KIR2DS2 CD4 + CD28 nu11 cells from ACS patients are not activated by hHSP70/MHC class I. Accordingly, the present invention provides a method of treating or preventing ACS by blocking the activation of KTR2DS2 expressing CD4 + CD28 nu " cells by MHC type I bearing hHSP60 peptide.
  • the blocking of KIR2DS2 CD4 + CD28 nuI1 cells may be achieved using an antibody or antibody fragment.
  • the antibody may bind to KIR2DS2 or to hHSP60/MHCl complexes. Further agents that modulate the interaction between KJR2DS2 and hHSP60 may be identified by monitoring the interaction between KIR2DS2 and hHSP60/MHCl complexes.
  • the present inventors have also investigated the role that KTR inhibitory receptors play in the activation of CD4 + CD28 nu11 cells.
  • the inventors have shown that, although the expression of the corresponding inhibitory receptors, KTR2DL2 and KIR2DL3 was largely down-regulated on CD4 + CD28 null KIR2DS2 + cells, the inhibitory receptors, when present, were unable to oppose KTR2DS2-directed hHSP60-specific killing.
  • the inventors have found that all CD4 + CD28 null KTR2DS2 + clones exhibit a pro-inflammatory cytokine response and that some additionally exhibit cytotoxicity, as determined by monitoring the perforin response.
  • the clones that exhibited cytotoxicity all expressed DAP- 12 whilst the non-cytotoxic clones did not.
  • the DAP- 12 molecule was thus found to be an essential component of antigen- specific CD4 + CD28 null KIR2DS2 + cell cytotoxicity.
  • the inventors have concluded that the pro-inflammatory response precedes cytotoxicity. Accordingly, prevalence of the autoreactive KIR2DS2 + DAP-12 + clones correlated with disease status. Monitoring KIR2DS2 and DAP- 12 expression may thus be used to give an indication of ACS disease status.
  • the inventors have additionally shown that systemic lupus erythematosus
  • SLE SLE patients, who are susceptible to atherosclerosis, have increased levels of CD4 + CD28 nu " cells similar to those of ACS.
  • the CD4 + CD28 nu " cells of SLE patients also have a similar expression pattern of KIR2DS2, KIR2DL2 and KIR2DL3 to CD4 + CD28 nu " cells of ACS patients. Therefore, monitoring of KIR2DS2 and DAP- 12 may also be used to monitor onset of cardiovascular disease in SLE patients.
  • the present invention provides: a method for monitoring cardiovascular disease in a subject, the method comprising determining the presence or absence of DAP- 12 expression in CD4 + CD28 nu11 cells from said subject, wherein the presence of DAP-12 expression in CD4 + CD28 nu11 cells correlates with disease progression; a method for monitoring cardiovascular disease in a subject, the method comprising determining the presence or absence of functional KIR2DL2 and/or KIR2DL3 receptor expression in CD4 + CD28 null KIR2DS2 + cells from said subject, wherein the presence or absence of functional KIR2DL2 and/or KIR2DL3 receptor expression indicates the status of the syndrome; a method of identifying an agent which modulates the interaction between KIR2DS2 and hHSP60, the method comprising:
  • KIR2DS2 binds the hHSP60/MHC class I complex; and (ii) monitoring the interaction between KIR2DS2 and the hHSP60/MHC class I complex thereby determining whether or not said test agent modulates the interaction between KIR2DS2 and I1HSP6O; - use of an antibody, or antibody fragment, which binds to KIR2DS2 in the manufacture of a medicament for treating or preventing acute coronary syndrome; and a method of treating or preventing acute coronary syndrome, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antibody fragment that binds to KIR2DS2.
  • FIG 1 shows the profile of KIRs on CD4 + CD28 nu " T cell clones from patients with acute coronary syndrome (ACS), chronic stable angina (CSA), systemic lupus erythematosus (SLE) and healthy individuals (Normals).
  • ACS acute coronary syndrome
  • CSA chronic stable angina
  • SLE systemic lupus erythematosus
  • Normal healthy individuals
  • Figure 2 illustrates the KIR2DS2, KIR2DL2 and KIR2DL3 phenotyping of CD4 + CD28 nu " cells.
  • TriZol reagent total RNA was extracted from CD4 + CD28 nu11 T cell clones established from patient and control groups, followed by cDNA synthesis.
  • Group I ACS The patients had HSP60 reactive CD28 nu11 cells when the total CD4 + CD28 nu11 cells were tested and reactivity was also present when the CD4 + CD28 nu!1 cells were cloned.
  • Group II ACS The patients did not have HSP60 reactive CD28 nu11 cells when the total CD4 + CD28 nu " cells were tested, but reactive cells were present when the CD4 + CD28 nu11 cells were cloned.
  • Clones were established from patients with ACS and exposed to hHSP60- or PHA-pulsed target cells prior to the assessment of perforin transcription levels.
  • Panels 1, 5 and 9 show the blocking effect of MHC class I antibodies on hHSP ⁇ O activation of clones expressing 2DS2 2DL2 2DL3 (panel 1), 2DS2 alone (panel 5) or total CD4 + CD28 nu11 cell fraction (panel 9).
  • Panels 2 (2DS2 2DL2 2DL3), 6 (2DS2) and 10 (CD4 + CD28" uU cell fraction) show perforin production by replica clones stimulated by hHSP60- pulsed target cells in the absence of MHC class I blocking antibodies.
  • Panels 3, 7 and 11 show the blocking effect of MHC class I antibodies on T cells exposed to PHA-pulsed target cells (panel 3: 2DS2 2DL2 2DL3; panel 7: 2DS2 alone; and Panel 11: total CD4 + CD28 nu11 cell fraction).
  • Panels 4 (2DS2 2DL2 2DL3), 8 (2DS2) and 12 (total CD4 + CD28 nu " cell fraction) show perfornin production in response to PHA-pulsed target cells in the absence of blocking.
  • FIG. 1 is a picture of gels representing the reactivity to different antigens of the total CD4 + cell fraction and of CD4 + CD28 nu11 cells expressing different isoforms of the activating KIRs from an ACS patient. Assessment of perforin levels was used as marker of activation. Panels 1-5 represent responses to cells pulsed with hHSP ⁇ O, hHSP70, HCMV, no antigen and PHA, respectively.
  • CD4 + CD28 nu11 cells and CD4 + CD28 nu11 cell clones expressing KIR2DS2 were reactive to target cells pulsed with hHSP ⁇ O, but not to other antigens tested. None of the clones expressing the other receptors (2DSl, 2DS3, 2DS4 and 3DS 1) reacted with any of the antigens. The total CD4 + CD28 + fraction was also analysed as control and showed previous exposure of the patient to all three antigens.
  • Figure 5 shows an assessment of the cytotoxicity of CD4 + CD28 nu " clones expressing the activating 2DS2 alone or in the presence of the inhibitory 2DL2 and 2DL3 analogues using CytoTox96 kit (Promega).
  • Target cells pulsed with different antigens were exposed to T cell clones and the release of LDH was measured by detecting the change in supernatant colour in order to assess the number of lysed cells.
  • the results are shown as mean percentage cytotoxicity (mpc) for each clone.
  • Clones expressing 2DS2 alone showed a mpc of 95% against cells pulsed with hHSP60 in the absence of MHC class I blocking. This response was reduced to 35% in the presence of anti-MHC class I antibodies.
  • FIG. 6 illustrates the prevalence of hHSP60-reactive CD4 + CD28 null 2DS2 + T cells in different populations. The prevalence of CD4 + CD28 null 2DS2 + T cell clones responding to hHSP60 by perforin and/or IFN- ⁇ in patient and control groups was determined.
  • Figure 7 shows the 2DS2-mediated effector function of CD4 + CD28 nuII 2DS2 + clones against hHSP60-pulsed cells was reduced by blocking with anti-human KDR2DS2 antibody (clone DX27).
  • the blocking effect was demonstrated by treating the clones with different concentrations of the antibody for 1 hour at room temperature prior to target cell exposure (effector to target cell ratio- 30:1).
  • Ten clones were analyzed and sufficient replicas of each were prepared for the experiment.
  • Panel 1 represents perforin response of an untreated clone.
  • Panels 2-4 respectively represent the effect of 2,10 and 20 ⁇ g/ml of the antibody on the perforin response by the replica clones.
  • Panel 5 represents the effect of blocking (20 ⁇ g/ml) on a clone exposed to PHA-pulsed cells.
  • Figure 8 shows that SLE patients have increased frequencies of CD4 + CD28 nu " cells similar to those observed in patients with unstable angina.
  • the Figure is taken from Liuzzo et al. (1999) Circulation 100:2135-2139 and the SLE data have been transposed on to the graph.
  • SEQ ID NO: 1 shows the nucleic acid and amino acid sequence of hHSP60.
  • SEQ ID NO: 2 shows the amino acid sequence of hHSP60.
  • SEQ ID NO: 3 shows the nucleic acid and amino acid sequences of human KIR2DS2.
  • SEQ ID NO: 4 shows the amino acid sequence of human KTR2DS2.
  • SEQ ID NOS: 5 to 16 show the amino acid sequences of the peptide fragments of hHSP60 which have been tested and shown to activate KTR2DS2.
  • the invention provides methods for monitoring the status of cardiovascular disease, such as acute coronary syndrome (ACS) in a subject.
  • the methods for monitoring cardiovascular disease of the invention are useful for determining the status of disease in a subject.
  • the methods may, therefore, be used to determine disease progression.
  • the methods may be used to predict the risk of coronary plaque destabilisation in a subject.
  • the method may also be used to monitor the effectiveness of therapy and/or to evaluate the efficacy of new treatments of cardiovascular disease, such as new ACS treatments.
  • the cardiovascular disease is typically ACS but subjects having other cardiovascular diseases or subjects at risk of cardiovascular disease may be monitored.
  • SLE patients have an increased risk of cardiovascular disease and the onset and progression of cardiovascular disease in SLE patients may be monitored by a method of the invention.
  • a subject may be tested on a regular basis, for example monthly, six monthly or yearly, to monitor disease progression within the subject.
  • the invention provides a method for monitoring cardiovascular disease in a subject, the method comprising determining the presence or absence of functional KIR2DL2 and/or KIR2DL3 receptor expression in CD4 + CD28 null KTR2DS2 + cells from said subject, wherein the presence or absence of functional KIR2DL2 and/or KIR2DL3 receptor expression indicates the status of the syndrome.
  • the absence of functional KIR2DL2 and/or KIR2DL3 receptor expression may be due to an absence of the receptor proteins.
  • the receptor proteins may be present in the cell, but the function of the proteins may be compromised, hi particular, the receptor proteins may have a reduced capacity to protect hHSP60-presenting cells. The proteins may, however, retain their survelliance function.
  • the presence or absence of functional KIR2DL2 and/or KIR2DL3 receptor expression may be determined by any suitable method.
  • the presence or absence of receptor protein may be determined by assessing the presence or absence of mRNA transcripts encoding the KIR2DL2 and/or KIR2DL3 receptors, for example by RT-PCR.
  • the presence or absence of the proteins may be determined directly, for example by antibody binding.
  • the function of the receptors may be determined, for example, by monitoring cell activation in the presence and absence of MHC class I blocking antibodies. Cell activation in response to an autoantigen, such as hHSP60, is typically determined.
  • the KTR2DL2 and/or KIR2DL3 receptor proteins may be considered to have a reduced capacity to protect autoantigen- presenting cells. As such, the receptor proteins may be said to have a compromised function.
  • Cell activation may be determined by any suitable method such as by monitoring perforin release or by assessing cytolytic capability of the cells. Cytolytic capability may be monitored, for example, by measuring lactate dehydrogenase (LDH) released upon cell lysis such as by using the CytoTox96 R assay (Promega).
  • LDH lactate dehydrogenase
  • the invention provides a method for monitoring cardiovascular disease in a subject, the method comprising determining the presence or absence of DAP- 12 expression in CD4 + CD28 nu11 cells from said subject, wherein the presence of DAP- 12 expression in said cells correlates with disease progression.
  • the method may further comprise determining the presence or absence of KIR2DS2 expression in the CD4 + CD28 nu " cells. The frequency of cells expressing DAP- 12 and/or KIR2DS2 may be determined, wherein the frequency of such cells correlates positively with disease progression.
  • DAP- 12 expression may be monitored by any suitable method.
  • the level of mRNA transcripts in the cell may be determined (for example, by RT- PCR).
  • DAP- 12 protein may be detected using anti-DAP-12 antibodies.
  • the level of DAP- 12 protein may be determined, for example, by using quantitative RT-PCR.
  • KIR2DS2 expression may be monitored by any suitable method.
  • the level of mRNA transcripts in the cell may be determined (for example, by RT-PCR).
  • KIR2DS2 protein may be detected using anti-KIR2DS2 antibodies.
  • the level of KIR2DS2 protein may be determined, for example, by using quantitative RT-PCR.
  • the present invention provides a method for monitoring cardiovascular disease in a subject, the method comprising determining the presence or absence of DAP- 12 expression in CD4 + CD28 null KIR2DS2 + cells from the subject. The presence of DAP-12 expression in the
  • CD4 + CD28 null KIR2DS2 + cells correlates with disease progression.
  • DAP-12 expression marks a significant phase or functional change in CD4 + CD28 nu11 cells in cardiovascular disease, such as in ACS patients.
  • DAP- 12 functions as an adaptor protein which mediates cell activation via KIR2DS2 leading to cytotoxicity.
  • DAP- 12 expression may begin during the chronic phase of ACS and to upregulated with changes in vascular environment and re-modeling.
  • the level of DAP- 12 expression on the CD4 + CD28 nu11 cells may, therefore, indicate the likelihood of coronary plaque destabilisation in a patient, with levels of D AP- 12 being positively correlated with the risk of plaque destabilisation. High levels of DAP- 12 may indicate the presence of unstable plaques.
  • Levels of DAP- 12 may be compared to standards to determine disease status.
  • the methods of the invention for monitoring acute coronary syndrome are typically carried out in vitro on a sample of cells from the subject.
  • the subject is typically a mammal, preferably a human.
  • the human has typically been diagnosed as having ACS.
  • the CD4 + CD28 nu " cells which are analysed in the method are taken from the subject in a blood sample, although other types of samples which contain T- cells/NK-cells can be used.
  • the sample may be used directly in the assay or may be processed further.
  • the processing may comprise separation of components of the sample.
  • processing may involve separating CD4 + CD28 nu11 cells from the other cells in the sample.
  • mononuclear cells (PBMCs) are separated from the sample.
  • CD4 + CD28 nu11 fractions may then be isolated, for example, by magnetic separation on MACS columns (Miltenyi Biotec) or by FACS.
  • the CD4 + CD28 nu11 cells may have undergone clonal expression in culture.
  • the invention also provides kits for carrying out the monitoring methods of the invention.
  • the kit may comprise means for detecting KIR2DL2 and/or KIR2DL3 expression and means for monitoring function of the KIR2DL2 and/or KIR2DL3 receptors.
  • Means for detecting receptor expression may comprise probes or primers for detecting mRNA or antibodies for detecting expressed protein.
  • the means for determining function of the KIR2DL2 and/or KIR2DL3 receptor may comprise MHC Class I antibodies and/or means for monitoring cytolytic capability and/or perforin release, for example the means described in the Examples herein.
  • Instructions for using the kit to monitor ACS status may also be included.
  • the instructions may comprise a reference chart to assist in interpreting the results of the assay.
  • the kit may comprise means for detecting DAP- 12 and optionally means for determining the level of DAP- 12 expression.
  • the kits may additionally comprise means for detecting KIR2DS2 receptor expression.
  • kits to monitor ACS status may also be included.
  • the instructions may comprise a reference chart to assist in interpreting the results of the assay.
  • a kit of the invention may be suitable for detecting one, two, three or all of DAP-12, KIR2DS2, KIR2DL2 and KIR2DL3.
  • the invention also provides a method for identifying an agent which modulates the interaction between KIR2DS2 and hHSP60.
  • the agent preferably inhibits the interaction between KXR2DS2 and hHSP60.
  • Such inhibitory agents may be used to block the activation of CD4 + CD28 nu " cells expressing KIR2DS2 upon binding of these cells to MHC class I/hHSP60 complexes and so may be used in the treatment or prevention of acute coronary syndrome (ACS).
  • ACS acute coronary syndrome
  • a method of identifying an agent which modulates the interaction between KIR2DS2 and hHSP60 typically comprises, or consists essentially of:
  • KIR2DS2 binds the hHSP60/MHC class I complex
  • the KIR2DS2 may be recombinantly expressed.
  • the amino acid sequence of KIR2DS2 is shown in SEQ ID NO: 4.
  • Recombinant expression of KIR2DS2 may be achieved using methods well known in the art.
  • a recombinant expression vector comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4 under the control of suitable control sequences is introduced into a host cell and the host cell is cultured under conditions suitable for transcription and translation of the coding sequences in the expression vector.
  • KIR2DS2 may have the sequence shown in SEQ ID NO: 3.
  • KIR2DS2 used in a method of the invention may be a variant or homologue of the protein having the amino acid sequence shown in SEQ E ) NO: 4.
  • the variant or homologue typically has at least 80%, such as at least 85%, 90%, 95% or 99% sequence identity to SEQ E) NO: 4.
  • Sequence identity may be calculated using methods well known in the art.
  • the UWGCG Package provides the BESTFIT program which can be used to determine identity (for example, used on its default settings) (Devereux et al. (1984) Nucleic Acids Research 12:387-395).
  • the PE,EUP and BLAST algorithms can also be used to calculate identity or line up sequences (typically on their default settings), for example as described in Altschul (1993) J. MoI. Evol. 36:290-300;
  • Suitable host cells include eukaryotic cell lines, such as mammalian, insect or yeast cell lines. Particular examples of cell lines include HEK293, CHO, HeLa and COS cells.
  • the KIR2DS2 may be expressed in a
  • CD4 + CD28 nu " cell is preferably derived from a human subject having acute coronary syndrome (ACS).
  • CD4 + CD28 nu11 cells may be obtained from a blood sample which has been taken from a patient having ACS.
  • peripheral blood mononuclear cells PBMC
  • CD4 + CD28 nu11 cell fractions are then isolated, for example, by magnetic separation on MACS column (Miltenyi Biotec).
  • Clones of CD4 + CD28 nuil cells maybe generated from the separated CD4 + CD28 nu11 cell cultures. This may be achieved, for example, by plating cells at a dilution of from 0.25 to 1 per well, for example, at a dilution of 0.5 cells per well.
  • the wells may contain allogeneic feeder cells, phytohemagglutinin (PHA) and E,-2.
  • PHA phytohemagglutinin
  • CD4 + CD28 nu11 cell clones expressing KE12DS2 may be identified by any suitable method, such as RT-PCR which is a technique well known in the art.
  • hHSP60/MHC class I complexes for use in a method of the invention may be obtained by any suitable method.
  • the hHSP60/MHC class I complexes are expressed on the surface of a target cell.
  • the target cell may be a professional antigen presenting cell, such as a dendritic cell or monocyte.
  • the monocyte may be a macrophage-derived monocyte.
  • the target cell is typically obtained by exposing the target cell to hHSP60.
  • the hHSP60 is internalised into the target cell by receptor-mediated endocytosis.
  • the internalised hHSP60 is processed and loaded on to MHC class I molecules which are transported to the surface of the target cell.
  • the hHSP60 which is used may be derived from any suitable source.
  • the I1HSP6O may be recombinant hHSP60. Recombinant hHSP60 may be obtained by methods well known in the art.
  • the amino acid sequence of hHSP60 is shown in SEQ ID NO: 2.
  • a recombinant expression vector comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 under the control of suitable control sequences is introduced into a host cell and the host cell is cultured under conditions suitable for transcription and translation of the coding sequences in the expression vector.
  • the nucleic acid encoding hHSP60 may have the sequence shown in SEQ ID NO: 1. Full length hHSP60 may be used to contact the cells. Alternatively, one or more hHSP60 fragments may be used. AU tested hHSP60 fragments interact with the MHCI molecule such that cells comprising the peptide are killed by CD4 + CD28 nu " cells expressing KIR2DS2.
  • the hHSP60 fragment may be from 9 amino acids in length to about 540 amino acids in length.
  • the fragment may be from 10 to 100, 15 to 150, 20 to 200, 30 to 300, 40 to 400 or 50 to 500 in length. Examples of suitable fragments are shown in SEQ ID NOS: 5 to 16.
  • the interaction between KIR2DS2 and the hHSP60/MHC class I complex may be monitored by any suitable method. For example, binding of KIR2DS2 to the hHSP60/MHC class I complex may be monitored directly. Any suitable binding assay format may be used to monitor binding.
  • Suitable assays for monitoring inhibition of the binding of KIR2DS2 to a hHSP60/MHC class I complex include label displacement, surface plasmon resonance, fluorescence resonance energy transfer, fluorescence quenching, fluorescence polarisation and radioligand binding assays.
  • KIR2DS2 is expressed in a CD4 + CD28 nu " cell
  • the interaction between KIR2DS2 and hHSP60/MHC class I complex may be determined by monitoring activation of the CD4 + CD28 nuI1 cell.
  • Activation may be monitored by any appropriate method, for example, activation of the KIR2DS2 maybe determined by monitoring perforin release from the CD4 + CD28 nu " cell.
  • Activation of the KIRDS2 may also be monitored by monitoring killing of the target cell expressing the hHSP60/MHC class I complex.
  • Target cell killing may, for example, be monitored using CytoTox96 R cytotoxicity assay kit from Promega. This assay kit qualitatively measures lactate dehydrogenase (LDH) released upon cell lysis.
  • LDH lactate dehydrogenase
  • the test agent may be any natural or synthetic substance, including, for example, small molecules, compounds contained in extracts of animal, plant, bacterial or fungal cells, as well as condition medium from such cells.
  • Preferred test agents include antibodies and antibody fragments.
  • Especially preferred test agents are antibodies or antibody fragments that bind to KIR2DS2.
  • the invention provides the use of an antibody, or antibody fragment, that binds to KIR2DS2 in the treatment of ACS.
  • the antibody, or antibody fragment, that binds to KIR2DS2 typically binds specifically to KIR2DS2 or may bind specifically to KIR2DS2 and to other KIRs such as the inhibitory KIRs, KIR2DL2 and KIR2DL3.
  • the KTR2DS2 antibodies typically bind to KIR2DS2 on CD4 + CD28 nu11 cells resulting in killing of the KIR2DS2 expressing CD4 + CD28 nu " cells.
  • the cell killing may be localised to the site of a atherosclerotic plaque by administering the KIR2DS2 antibody directly to the site of the plaque, for example by injection.
  • KIR2DS2 expressing CD4 + CD28 nu11 cells may also be removed from a patient by killing the cells in blood ex vivo and returning the blood to the body. Accordingly, the invention provides a method of killing KIR2DS2 expressing CD4 + CD28 nu11 cells, the method comprising contacting the cells with an antibody that binds KIR2DS2, which may be an in vivo or ex vivo method.
  • An antibody, or antibody fragment "specifically binds" to a protein, or proteins, when it binds with preferential affinity to the protein(s) for which it is specific compared to other proteins, such as other cell surface receptors.
  • a variety of protocols for competitive binding or immunoradiometric assays to determine the specific binding capability of an antibody are well known in the art (see, for example, Maddox et al. (1993) J. Exp. Med. 158:1211-1266).
  • Such immunoassays typically involve the formation of complexes between the specific protein and its antibody and the measurement of complex formation.
  • the antibody may be a monoclonal or polyclonal antibody.
  • the antibody may be a chimeric antibody, a CDR-grafted antibody or a humanised antibody.
  • Antibodies can be produced by any suitable method. Means for preparing and characterising antibodies are well known in the art (see, for example, Harlow & Lane (1988) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). An antibody may, for example, be produced by raising an antibody in a host animal against the whole polypeptide or one or more fragments thereof, for example, an antigenic epitope thereof, herein after the "immunogen". The fragment typically has at least 10 or at least 15 amino acids.
  • a method for producing a polyclonal antibody comprises immunising a suitable host animal, for example, an experimental animal, with the immunogen and isolating immunoglobulins from the animal serum.
  • a method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody.
  • Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler & Milestin (1975) Nature 256:495-497).
  • An immortalised cell producing the desired antibody may be selected by a conventional procedure.
  • the hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the bloodstream of an allogenic host or immunocompromised host.
  • Human antibodies may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.
  • the experimental animal is typically a goat, rabbit, rat, mouse, guinea pig, chicken, sheep or horse.
  • the immunogen may be administered as a conjugate in which the immunogen is coupled, for example, via a side chain of one of the amino acid residues, to a suitable carrier.
  • the carrier molecule is typically a physiologically acceptable carrier.
  • the antibody obtained may be isolated and, if desired, purified.
  • Antibodies that bind to KIR2DS2 are known in the art and are commercially available from, for example, Beckman Coulter, Biolegend, Serotec and United States Biological.
  • the purified anti-KIR2DS2 antibody available from Beckman Coulter is GLl 83 and that available from Biolegend is Clone DX27.
  • KIR antibodies are also described in WO2005/003172, which is incorporated herein by reference.
  • the KIR2DS2 antibody, or fragment thereof typically binds to an extracellular epitope of KIR2DS2.
  • Computer modelling of the extracellular domains of KIR proteins based on their published crystal structures (Fan et al. (2001) Nature Immunology 2:452-460; Boyington et al. (2000) Nature 405:537-543) may be used to predict epitopes for antibodies that bind to the extracellular domain (WO2005/003172). It can also be determined whether an antibody binds to the extracellular domain by monitoring binding of the antibody to unpermeabilised cells expressing KIR2DS2 (i.e. in the absence of detergent).
  • the antibody fragment is typically an antigen-binding fragment.
  • antigen-binding fragment refers to one or more fragments of a full-length antibody that are capable of binding to KIR2DS2.
  • suitable fragments include (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CHl domains); (ii) a F(ab') 2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulphide bridge at the hinge region); (iii) a Fd fragment (consisting of the VH and CHl domains); (iv) a Fv fragment (consisting of the VH and VL domains of a single arm of an antibody); (v) a dAb fragment (consisting of the VH domain); (vi) an isolated CDR; (vii) a single chain Fv (scFv) (consisting of the VH and VL domains of a single arm of an antibody joined by a synthetic linker using recombinant
  • a therapeutically effective amount of the antibody or antibody fragment is administered to an individual in need thereof.
  • a therapeutically effective amount is an amount effective to alleviate or attenuate one or more symptom of ACS, when administered to treat ACS.
  • a therapeutically effective amount is an amount effective to prevent the onset of one or more symptom of ACS.
  • a prophylactically effective amount is an amount that delays or inhibits the formation of atherosclerotic plaques.
  • the agent may be administered or delivered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), parenterally, subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion or inhalation techniques.
  • the agent may also be administered or delivered as suppositories.
  • a physician will be able to determine the required route of administration or delivery for each particular patient.
  • the antibody, or antibody fragment may be administered directly to the site of an atherosclerotic plaque, typically by injection at the affected site.
  • the agent is formulated for use with a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical carrier or diluent may be, for example, an isotonic solution.
  • solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g.
  • diluents e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch
  • lubricants e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols
  • binding agents e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrroli
  • Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.
  • Liquid dispersions for oral administration may be syrups, emulsions and suspensions.
  • the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • the suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
  • Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • the dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.
  • a typical daily dose is from about 0.1 to 50 mg per kg, preferably from about 0.1mg/kg to 10mg/kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.
  • daily dosage levels are from 5 mg to 2 g.
  • Example 1 Methods Study Population: Nine patients with non-ST elevation (NSTE) acute coronary syndrome (ACS) (unstable angina [UA] or NSTE myocardial infarction [MI]) were studied. Six of these nine ACS patients had hHSP60 reactive CD4 + CD28 nu " cells and the other three ACS patients had hHSP60 non-reactive CD4 + CD28 nu " cells. Unstable angina included patients belonging to Braunwald class IIIB who had diagnostic ST segment or T wave changes.
  • NSTE non-ST elevation
  • ACS acute coronary syndrome
  • MI myocardial infarction
  • ST segment myocardial infarct was be defined according to established criteria (Myocardial Infarction Redefined: A Consensus Document of the Joint European Society of Cardiology/American College of Cardiology Committee for the Redefinition of Myocardial Infarction, J. Am. Coll. Cardiol. 2000, 36:959-969).
  • CSA chronic stable angina
  • CAD coronary artery disease
  • BMI body mass index
  • venous blood (10ml of clotted and 30ml of heparinized) was obtained from patients with CSA at study entry. Samples from patients with ACS were drawn at admission to Coronary Care Unit. The healthy volunteers were recruited from staff members working at St George's Hospital Medical School.
  • PBMC Peripheral blood mononuclear cells
  • CD4 + CD28 nu " and CD4 + CD28 + fractions were isolated by magnetic separation on magnetic cells sorting and separation of biomarkers (MACS) columns (Miltenyi Biotec). CD4 + CD28 + fraction was first separated from total PBMC using MACS CD4 + T cell isolation kit on MACS LS type column according to the manufacture's instructions. CD4 + CD28 + cells were then stained with phycoerythrin (PE)- conjugated anti-CD28 antibody (Pharmingen) for 10 minutes followed by treating with 20 ⁇ l of MACS anti-PE microbeads.
  • PE phycoerythrin
  • CD4 + CD28 nu11 cells were then separated from total CD4 + cells on MACS column. Activation status of separated T cells was analysed using perform mRNA transcription as previously described (ZaI et al. (2004) Circulation 109:1230-1235). Antigen reactivity of the CD4 + CD28 + cells in individual volunteers was used as experimental control, confirming previous exposure.
  • CD4 + CD28 nu T cell cloning: Cloning of CD4 + CD28 nu " cells from patients and control groups were carried out according to previously published protocols (Markovic-Plese at al (2001) J. Clin. Invest. 108:1185-1194). Cells were dispensed into 96 well plates at a dilution of 0.5 cell per well. The wells contained 1x10 5 of irradiated allogeneic feeder cells, 2.5 ⁇ g/ml PHA, and 40 U/ml rIL-2 (Sigma).
  • Iscove's modified Dulbecco's medium (Life Technologies, UK) supplemented with 5% human AB serum (Sigma, UK), 2mM L-Glutamine (Sigma, UK), 5000 U/ml Penicillin/streptomycin (Sigma, UK) was used. The cultures were incubated at 37 0 C and 5% CO 2 for 4 days before fresh medium was added. Wells containing single T cell clones were identified on days 12-14 and used for expansion.
  • each T cell clone was expanded in the absence of PHA. This was carried out by sub-culturing each T cell clone in the presence of allogeneic feeders and 40 U/ml rIL-2 and in the absence of PHA.
  • the Iscove's modified Dulbecco's medium contained 5% human AB serum (Sigma, UK), 2mM L-Glutamine (Sigma, UK), 5000 U/ml Penicillin/streptomycin (Sigma). The medium was replaced on day 3, and each clone was further sub-cultured on day 6. Expansion of clones was continued until sufficient replica of each clone was available for experiments.
  • KIR screening on CD4 + CD28 nu " cell clones Screening of both activating and inhibitory KIR transcripts was carried out by RT-PCR. Briefly, RNA was extracted from each T cell clone and cDNA synthesis was performed as previously described (ZaI et al. (2004)). Prior to screening for KIR transcripts, the CD4 + CD28 nu11 phenotype of each sample was further confirmed by RT-PCR analysis of mRNA for the CD4 and CD28 markers using previously published primers and conditions (ZaI et al. (2004)).
  • Adherent cells were pulsed with 5 ⁇ g/ml of I1HSP6O in the presence or absence of lO ⁇ g/ml anti-human MHC class I antibodies (HLA A, B, C - clone W6/32) (DAKO) and kept at 37 0 C overnight.
  • Target cells kept under the same conditions but without addition of hHSP60 were used as controls.
  • target cells pulsed with human cytomegalovirus (HCMV) proteins (Abingdon Oxon), hHSP70 (Bioquote UK) and PHA were also included as controls. Li some experiments, the cells were pulsed with peptide fragments of hHSP60 instead of with full length hHSP60.
  • HCMV human cytomegalovirus
  • Antigen-specific KIR-mediated cytotoxicity of CD4 + CD28 nu " clones measured by CytoTox96 R Assay The functional killing of CD4 + CD28 nu " clones expressing different KIRs in response to different antigens in the presence and absence of MHC class I blocking was assessed using the CytoTox96 R Cytotoxicity Assay kit (Promega).
  • LDH release lactate dehydrogenase (LDH) release, which reflects the target cell lysis levels as a measure of T cell cytotoxicity. LDH release was measured according to the manufacturer's protocol following the exposure of target cells to the effector T cells for 5 hours at 37 0 C. This was done by taking 50 ⁇ l aliquots from all wells and transferring into fresh wells containing equal volume of the reconstituted substrate (provided by the manufacturer).
  • the procedures for the preparation of target and effector cells, antigen pulsing and MHC class I blocking are carried out as described earlier for the assessment of perforin transcription.
  • the CytoTox96 R cytotoxicity assay is then used as described above to measure lactate dehydrogenase (LDH) release, which reflects the target cell lysis levels as a measure of T cell cytotoxicity. Percentage of antigen specific cytotoxicity is calculated for each sample.
  • LDH lactate dehydrogenase
  • Autologous monocyte-derived deiitritic cells are prepared as described above.
  • Adherent cells are pulsed with 5 ⁇ g/ml of hHSP60, or a hHSP60 peptide, in (a) the presence of 2 ⁇ g/ml, lO ⁇ g/ml or 20 ⁇ g/ml anti-human KTR2DS2 antibody (Clone DX27 " (Biolegend)) for 1 hour at room temperature; (b) the presence of lO ⁇ g/ml anti-human MHC class I antibodies (HLA A, B, C - Clone W6/32-DAKO) for 40 minutes at room temperature; (c) in the presence of lO ⁇ g/ml anti-human class I antibodies (HLA DR, DP, DQ-DAKO) for 40 minutes at room temperature; (d) the absence of any blocking antibodies.
  • Target cells kept under the same conditions but without addition of hHSP60 are used as controls.
  • hHSP60-pulsed and control target cells are re-seeded into 96-well microtitre plates at a density of 3x10 4 cells per well.
  • Effector CD4 + CD28 nu11 cell clones expressing activating KJRs in the presence or absence of the opposing inhibitory receptors are added to the target cells at an effector to target ratio of 30: 1 in lOO ⁇ l final volume. After 5 hours of incubation at 37 0 C, measurement of perforin transcription is carried out for each well by RT-PCR as described previously (ZaI et al. (2004)).
  • Example 2 Characterisation of the repertoire of KIR receptors on CD4 + CD28 nu " cell clones established from patients and control subjects
  • KIR2DS2 was expressed by 79% of the T cell clones. 26% of the clones expressed the KIR2DS2 receptor in the presence of at least one inhibitory homologue while 10% expressed this activating receptor in the presence of both inhibitory receptors. In patients with ACS who had non-reactive total CD4 + CD28 nu " cells to hHSP60, KIR2DS2 was expressed by only 55% of the clones and its presence coincided with a much higher occurrence of the inhibitory receptors (50%).
  • Example 3 Investigation of correlation between prevalence of KIR2DS2 and hHSP60-specif ⁇ c response
  • the activating KJR receptors have truncated cytoplasmic domains lacking tyrosine-based inhibitory motifs (ITIMs) but possess extracellular domains with a high degree of homology to the inhibitory receptors and are known to bind ligands encoded by HLA-C locus.
  • ITIMs tyrosine-based inhibitory motifs
  • Macrophage-derived monocytes were used as target cells because presentation of exogenous antigens on MHC class I molecules by these cells is well documented (Suto & Srivastava (1995) Science 269:1585-1588; Brossard et al. (1997) Blood 90:1594-1599 and Lipsker et al. (2002) Eur. J. Immunol. 32:322- 332).
  • hHSP60 and hHSP70 in particular are reported to share common receptors, which allow their internalisation via receptor-mediated endocytosis.
  • the internalised proteins then reach endosomal compartments, including HLA-DR-enriched compartments.
  • the internalisation pathway provides a mechanism which directs hHSP60 and 70 to compartments where they are processed and loaded onto HLA class I and class II molecules (Lipsker et al. (2002)).
  • Perforin transcription levels following exposure of T cell clones to antigen- pulsed target cells were measured to determine CD4 + CD28 nuU cell activation.
  • 15 hHSP60-reactive T cell clones from 9 unstable patients, which expressed the activating KDR2DS2 alone, showed elevated levels of perforin upon exposure with hHSP60-pulsed target cells.
  • AU replica clones examined under the same conditions but in the presence of MHC class I blocking exhibited a significant decline in perforin transcription (Fig. 3).
  • inhibitory receptors and class-I are therefore essential for generation of inhibitory signals and blocking of this pathway would result in effector responses.
  • Example 5 Selectivity of hHSP60-induced cell activation for KIR2DS2 expressing cells
  • T cell clones expressing the activating KIRs 2DSl, 2DS3, 2DS4 and 3DSl were established from ACS patients and were allowed to react with target cells pulsed with hHSP60 or PHA as positive control. None of the clones expressing these activating receptors gave a detectable response, although the replica clones were activated by PHA stimulation (Fig. 4). Our data so far demonstrate that activation of CD4 + CD28 nu " cells by hHSP60 is receptor specific and is preferentially skewed towards KTR2DS2 receptor.
  • Example 6 Assessment of the functional killing and levels of cytotoxic response by CD4 + CD28 nu " clones expressing different KIRs
  • CytoTox96 R Cytotoxicity Assay kit from Promega was employed to monitor activation of CD4 + CD28 nu11 cells.
  • CytoTox96 kit is colorimetric alternative to Cr 51 assay that quantitatively measures lactate dehydrogenase (LDH) released upon cell lysis.
  • LDH lactate dehydrogenase
  • LDH is a stable cytosolic enzyme, which when released into culture supernatants can be measured with a 30-minute coupled enzymatic assay that results in the conversion a Tetrazolium salt into a red formazan product.
  • the amount of colour change is proportional to the number of lysed cells.
  • the assay can therefore be used to measure membrane integrity for cell- mediated cytotoxcicity in which target cells are lysed by effector cells.
  • CD4 + CD28 nuI1 cells Prior to using CD4 + CD28 nuI1 cells in cytotoxicity experiment, the assay was first optimised for target cell number. This was because various cell types contain different amounts of LDH, and preliminary experiments using the target cells were therefore carried out to determine the optimum number of target cells for use in the CytoTox96 assay.
  • CD4 + CD28 nu " cell clones expressing different KIR receptors were examined for their cytolytic function against hHSP60 pulsed target cells.
  • Example 7 The prevalence of hHSP60-reactive CD4 + CD28 null 2DS2 + cells in ACS and other patient and control groups
  • CD4 + CD28 null 2DS2 clones were responsive to hHSP60 (Fig. 6). The remaining clones were non-reactive.
  • CD4 + CD28 nu11 cells from two patients with CSA were screened and found to be non-reactive to hHSP60. When twenty three clones from these patients were individually analysed, 8% reacted to hHSP60 (Fig. 6).
  • Total CD4 + CD28 nuU cells from healthy volunteers were non-reactive on initial screening. Cloned CD4 + CD28 nu11 cells from the healthy individuals did not react with hHSP ⁇ O.
  • Example 8 The proinflammatory response by CD4 + CD28 nu11 cells may precede cytotoxicity
  • TCR T cell receptor-
  • NK cell activation via KIR2DS2 is believed to be mediated by DAP- 12 adaptor dimers, which activate the ZAP70/Syk pathway, leading to phospholipase C- y phosphorylation and cytotoxicity (Nakajima et al. (2003)).
  • DAP- 12 adaptor dimers which activate the ZAP70/Syk pathway, leading to phospholipase C- y phosphorylation and cytotoxicity.
  • DAP-12 expression marks a significant phase of functional change in CD4 + CD28 nu11 cells, which may begin during the chronic phase and be up-regulated with progressive changes in vascular environment and re-modeling. Evaluation of circulating levels of autoreactive CD4 + CD28 null 2DS2 + DAP-12 + may prove to be a useful marker for monitoring disease status.
  • Example 10 Activation of CD4 + CD28 nu " cells by hHSP60 is blocked by antibodies Anti-human MHC class-II antibodies significantly reduced IFN- ⁇ response of
  • Example 11 Accelerated atherosclerosis in patients with systemic lupus erythematosus: the role of CD4 + CD28 nu " cells

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Abstract

L'invention concerne une méthode de surveillance de maladie cardiovasculaire chez un sujet. Cette méthode consiste à déterminer la présence ou l'absence de l'expression de DAP-12 et/ou de KIR2DS2 dans des cellules CD4+CD28nuIl du sujet, la présence de l'expression de DAP-12 et/ou de KIR2DS2 dans lesdites cellules corrélant avec la progression de la maladie.
PCT/GB2006/003740 2005-10-11 2006-10-06 Lymphocytes t reactifs a la hsp60 Ceased WO2007042778A2 (fr)

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