Receptor binding ligands, their use in the detection of cells with biological interest

Abstract

A method for the identification and quantification of the expression of membrane receptors present on the surface of target cells using at least two soluble receptor binding ligands derived from the soluble part of the glycoprotein of an enveloped virus that interacts with a cellular cognate receptor, the receptor binding ligand containing a part or the totality of one of the receptor binding domains (RBD) of the glycoprotein, and the soluble receptor binding ligand being liable to interact with the at least one membrane receptor of a target cell, for the identification and quantification of the expression of membrane receptors present on the surface of target cells, the identification and quantification taking place at a given time or during a given time interval, and allowing the determination of a physiological state of the target cell.

Claims

1. A method for determining a physiological state of target cells taking place at a given time or during a given time interval, comprising: contacting the target cells with at least two soluble receptor binding ligands comprising the totality of the receptor binding domains (RBD) of at least two glycoproteins of at least two enveloped viruses that interacts with a cellular cognate receptor, each of said at least two receptor binding ligands having a tag, detectable molecule or label coupled thereto, each of said at least two soluble receptor binding ligands containing the totality of one of the RBD of said glycoprotein, wherein said RBD does not comprise a transmembrane domain; and, wherein complexes are formed between each of said at least two soluble receptor binding ligands and each of at least two distinct membrane receptors, and the complexes are detectable by the tag, the detectable molecule or the label coupled to each of said at least two receptor binding ligands, wherein all receptor binding ligands contacting the cells contain the totality of RBD of said glycoprotein, and said all receptor binding ligands do not comprise a transmembrane domain, and wherein said at least two glycoproteins belong to viruses selected from the group consisting of: Amphotropic MLV (ampho, SEQ ID NO:1), Gibbon Ape Leukemia virus (GALV, SEQ ID NO:2), Feline endogenous virus (RD114, SEQ ID NO:3), vesicular stomatitis virus (VSV, SEQ ID NO:4), Xenotropic Murine Leukaemia Virus (NZB, Xeno, SEQ ID NO: 10), Feline Leukaemia Virus C (FeLV, SEQ ID NO: 19), Env Koala Retrovirus (KoV, SEQ ID NO: 20), Env Porcine Endogeneous Retrovirus-B (Perv B, SEQ ID NO: 22), Human T Leukaemia Virus-1 (HTLV1, SEQ ID NO:27), Human T Leukaemia Virus-2 (HTLV2, SEQ ID NO:28), Human T Leukaemia Virus-4 (HTLV4, SEQ ID NO: 31), and Env Bovine Leukaemia Virus (BLV, SEQ ID NO: 30).

2. The method according to claim 1, wherein said membrane receptors are selected from the group consisting of CAT1, PiT2, XPR1, SMIT1, PiT1, ASCT1/ASCT2, FLVCR, PAR1, PAR2, and GLUT1.

3. The method according to claim 1, wherein said target cells are animal stem cells.

4. The method according to claim 3, wherein said animal stem cells are human stem cells or cancer stem cells.

5. The method according to claim 3, wherein said target cells are haematopoietic stem cells or B or T cells.

6. The method according to claim 1, wherein said at least two soluble receptor binding ligands is a set of three to twelve soluble receptor binding ligands isolated from glycoproteins belonging to viruses selected from the group consisting of: Amphotropic MLV (ampho, SEQ ID NO:1), Gibbon Ape Leukemia virus (GALV, SEQ ID NO:2), Feline endogenous virus (RD114, SEQ ID NO:3), vesicular stomatitis virus (VSV, SEQ ID NO:4), Xenotropic Murine Leukaemia Virus (NZB, Xeno, SEQ ID NO: 10), Feline Leukaemia Virus C (FeLV, SEQ ID NO: 19), Env Koala Retrovirus (KoV, SEQ ID NO: 20), Env Porcine Endogeneous Retrovirus-B (Perv B, SEQ ID NO: 22), Human T Leukaemia Virus-1 (HTLV1, SEQ ID NO:27), Human T Leukaemia Virus-2 (HTLV2, SEQ ID NO:28), Human T Leukaemia Virus-4 (HTLV4, SEQ ID NO: 31), and Env Bovine Leukaemia Virus (BLV, SEQ ID NO: 30).

7. The method according to claim 1, wherein said soluble receptor binding ligand is a set of ten receptor binding ligands wherein the RBD of glycoproteins are selected from glycoproteins belonging to viruses selected from the group consisting of: Amphotropic MLV (ampho), Feline endogenous virus (RD114), Gibbon Ape Leukemia virus (GALV), Xenotropic Murine Leukaemia Virus (NZB, Xeno), Env Koala Retrovirus (KoV), Env Porcine Endogeneous Retrovirus-B (Perv B), Human T Leukaemia Virus-2 (HTLV2), Human T Leukaemia Virus-4 (HTLV4), Env Bovine Leukaemia Virus (BLV), and Env Feline Leukaemia Virus C (FeLV).

8. The method according to claim 1, wherein said soluble receptor binding ligand is a set of eight receptor binding ligands wherein the RBD of glycoproteins are selected from glycoproteins belonging to viruses selected from the group consisting of: Amphotropic MLV (ampho, SEQ ID NO:1), Gibbon Ape Leukemia virus (GALV, SEQ ID NO:2), Feline endogenous virus (RD114, SEQ ID NO:3), vesicular stomatitis virus (VSV, SEQ ID NO:4), Xenotropic Murine Leukaemia Virus (NZB, Xeno, SEQ ID NO: 10), Feline Leukaemia Virus C (FeLV, SEQ ID NO: 19), Env Koala Retrovirus (KoV, SEQ ID NO: 20), Env Porcine Endogeneous Retrovirus-B (Perv B, SEQ ID NO: 22), Human T Leukaemia Virus-1 (HTLV1, SEQ ID NO:27), Human T Leukaemia Virus-2 (HTLV2, SEQ ID NO:28), Human T Leukaemia Virus-4 (HTLV4, SEQ ID NO: 31), and Env Bovine Leukaemia Virus (BLV, SEQ ID NO: 30).

9. The method according to claim 1, wherein said RBD of glycoproteins belong to viruses selected from the group consisting of: Amphotropic MLV (ampho), Feline endogenous virus (RD114), Xenotropic Murine Leukaemia Virus (NZB, Xeno), Env Koala Retrovirus (KoV), Env Porcine Endogeneous Retrovirus-B (Perv B), Human T Leukaemia Virus-2 (HTLV2), Human T Leukaemia Virus-4 (HTLV4), and Env Bovine Leukaemia Virus (BLV).

10. The method according to claim 1, wherein said at least two soluble receptor binding ligands is a set of four soluble receptor binding ligands isolated from glycoproteins belonging to viruses selected from the group consisting of: Amphotropic MLV (ampho, SEQ ID NO:1), Gibbon Ape Leukemia virus (GALV, SEQ ID NO:2), Feline endogenous virus (RD114, SEQ ID NO:3), vesicular stomatitis virus (VSV, SEQ ID NO:4), Xenotropic Murine Leukaemia Virus (NZB, Xeno, SEQ ID NO: 10), Feline Leukaemia Virus C (FeLV, SEQ ID NO: 19), Env Koala Retrovirus (KoV, SEQ ID NO: 20), Env Porcine Endogeneous Retrovirus-B (Perv B, SEQ ID NO: 22), Human T Leukaemia Virus-1 (HTLV1, SEQ ID NO:27), Human T Leukaemia Virus-2 (HTLV2, SEQ ID NO:28), Human T Leukaemia Virus-4 (HTLV4, SEQ ID NO: 31), and Env Bovine Leukaemia Virus (BLV, SEQ ID NO: 30).

11. The method according to claim 1, for the implementation of a selection process of target cells, having a defined physiological state.

12. The method according to claim 11, wherein said target cells are stem cells sub-population expressing said at least two membrane receptors.

13. A process of identification and quantification of the expression of membrane receptors to a glycoprotein RBD of target cells comprising the following steps: a. contacting at least two soluble receptor binding ligands, as defined in claim 1, optionally marked with a tag, with a target cell to form at least two complexes, each complex being constituted by one said receptor binding ligand and one said membrane receptor of said target cell, b. identifying each complex formed, c. quantifying the expression of each membrane receptor of said target cell able to form said complex, d. optionally, distinguishing receptors expressed to the surface of the membrane of the target cell from the total receptors expressed within the target cell.

14. The process according to claim 13, wherein said target cells are animal stem cells.

15. The process according to claim 14, wherein said animal stem cells are human stem cells.

16. The process according to claim 14, wherein said target cells are haematopoietic stem cells or B cells or T cells.

17. A process of selection of target cells expressing at least one particular membrane receptor to a glycoprotein RBD in a given amount of expression, comprising the following steps: a. contacting at least two soluble receptor binding ligands, as defined in claim 1, optionally marked with a tag, with a target cell to form at least two complexes, each complex being constituted by one said receptor binding ligand and one said membrane receptor of said target cell, b. detecting each complex formed and quantifying said each complex formed at an instant T1, c. detecting and quantifying said each complex formed at a second instant T2, T2 being higher than T1, d. selecting at T2 said target cells presenting a variation in the expression of at least one particular membrane receptor having formed said complex.

18. The process of selection according to claim 17, wherein said target cells are animal stem cells.

19. The process according to claim 18, wherein said animal stem cells are human stem cells.

20. The process of selection according to claim 18, wherein said target cells are haematopoietic stem cells or B cells or T cells.

21. A process of amplification of target cells expressing at least one particular membrane receptor to a glycoprotein RBD in a given amount of expression, comprising the following steps: a. contacting at least two soluble receptor binding ligands, as defined in claim 1, optionally marked with a tag, with a target cell to form at least two complexes, each complex being constituted by one said receptor binding ligand and one said membrane receptor of said target cell, b. detecting each complex formed and quantifying said each complex formed at an instant T1, c. detecting and quantifying said each complex formed at a second instant T2, T2 being higher than T1, d. selecting at T2 said target cells presenting a variation in the expression of at least one particular membrane receptor having formed said complex, e. sorting out and amplifying said selected target cells.

22. The process of amplification according to claim 21, wherein said target cells are animal stem cells.

23. The process according to claim 22, wherein said animal stem cells are human stem cells.

24. The process of amplification according to claim 21, wherein said target cells are haematopoietic stem cells or B cells or T cells.

Description

(1) The following figures and examples illustrate the invention.

(2) FIG. 1 corresponds to the schematic representation of the mature Env protein of HTLV-1 (as a prototypic deltaretrovirus Env) and common motifs in the SU with Friend-MLV (as a prototypic gammaretrovirus Env). TM corresponds to the transmembrane domain and SU corresponds to the surface domain. RBD corresponds to the domain of SU that interact with the membrane receptor of the target cell.

(3) FIGS. 2A to J correspond to the cell surface expression kinetics of Gammaretrovirus membrane receptors of CD34+ umbilical cord blood stem cells (human) from day 0 to day 1 of amplification according to different amplification protocols and as detected after contacting with Ampho and GALV receptor binding ligands at 37° C. for 30 minutes.

(4) FIG. 2A represents the FACS of CD34+ umbilical cord blood cells isolated after Ficoll and CD34+ Miltenyi selection kits.

(5) FIG. 2B represents the Fisher amplification protocols at Day=0 (Black: Mock, light grey (ampho), Dark grey GALV) of R2 cells of FIG. 2A (upper zone).

(6) FIG. 2C represents the Fisher amplification protocols at Day=1 (Black: Mock, light grey (ampho), Dark grey GALV) of R2 cells of FIG. 2A (upper zone).

(7) FIG. 2D represents the FACS obtained with R1 zone of FIG. 2A after selection of CD34+ hematopoietic stem cells using Miltenyi selection kits.

(8) FIGS. 2E and 2H represents the Fisher amplification protocols at Day=0 and Day=1 respectively (Black: Mock, light grey (ampho), Dark grey GALV) of R6 zone of FIG. 2D (upper left zone).

(9) FIGS. 2F and 2I represents the Fisher amplification protocols at Day=0 and Day=1 respectively (Black: Mock, light grey (ampho), Dark grey GALV) of R3 zone of FIG. 2D (upper right zone).

(10) FIGS. 2EG and 2J represents the Fisher amplification protocols at Day=0 and Day=1 respectively (Black: Mock, light grey (ampho), Dark grey GALV) of R4 zone of FIG. 2D (lower right zone).

(11) FIG. 3A to F correspond to the expression of various receptors during ex vivo differentiation of CD34+ umbilical cord blood stem cells (human) by Thrasher's protocol in the presence of Ampho, GALV, RD114 and VSV receptor binding ligands from Day=0 to Day=3, at 37° C. for 30 minutes

(12) FIGS. 3A, 3B and 3C represent the FACS obtained from a buffy coat after Ficoll and CD34+ Miltenyi selection kit (3A) and further Thrasher protocol (3B: Black: Mock, light grey: VSV, dark grey: Ampho; 3C: Black: Mock, light grey: GALV, dark grey: RD114) at Day=0)

(13) FIGS. 3D, 3E and 3F represent represents the FACS obtained from a buffy coat after Ficoll and CD34+ Miltenyi selection kit (3D) and further Thrasher protocol (3E: Black: Mock, light grey: VSV, dark grey: Ampho; 3F: Black: Mock, light grey: GALV, dark grey: RD114) at Day=3)

(14) FIGS. 3B,C and 3E,F shows the variation of the expression of membrane receptors of umbilical cord blood stem cells versus the time, in particular with the receptors binding ligands of Ampho (SEQ ID NO:1), GALV (SEQ ID NO:2), RD114 (SEQ ID NO:3) and VSV (SEQ ID NO:4).

(15) FIG. 4 to FIG. 9 represent the RBD binding profile on human B and B− chronic lymphocytic leukaemia (B-CLL) cells using the following RBD:

(16) HTLV 2 (SEQ ID NO: 28), HTLV 4 (SEQ ID NO: 31), Ampho (SEQ ID NO: 1), Perv B (SEQ ID NO: 22), BLV (SEQ ID NO: 30), RD 114 (SEQ ID NO: 3), KoV (SEQ ID NO: 20, and Xeno (SEQ ID NO: 10).

(17) FIG. 4A to 4E represent the RBD binding profile to CD19+ cells and the FACS of a healthy donor (healthy donor 1):

(18) FIG. 4A: curve filled with grey: mock Light grey unfilled curve: HTLV 2 (SEQ ID NO: 28), Dark grey unfilled curve: HTLV 4 (SEQ ID NO: 31).

(19) FIG. 4B: curve filled with grey: mock Light grey unfilled curve: Perv B (SEQ ID NO: 22), Dark grey unfilled curve: Ampho (SEQ ID NO: 1).

(20) FIG. 4C: curve filled with grey: mock Light grey unfilled curve: Xeno (SEQ ID NO: 10), Dark grey unfilled curve: RD 114 (SEQ ID NO: 3).

(21) FIG. 4D: curve filled with grey: mock Light grey unfilled curve: BLV (SEQ ID NO: 30), Dark grey unfilled curve: KoV (SEQ ID NO: 20).

(22) FIG. 4E: FACS of B cells FIG. 5A to 5E represent the RBD binding profile to CD19+ cells and the FACS of another healthy donor (healthy donor 2):

(23) FIG. 5A: curve filled with grey: mock Light grey unfilled curve: HTLV 2 (SEQ ID NO: 28), Dark grey unfilled curve: HTLV 4 (SEQ ID NO: 31).

(24) FIG. 5B: curve filled with grey: mock Light grey unfilled curve: Perv B (SEQ ID NO: 22), Dark grey unfilled curve: Ampho (SEQ ID NO: 1).

(25) FIG. 5C: curve filled with grey: mock Light grey unfilled curve: Xeno (SEQ ID NO: 10), Dark grey unfilled curve: RD 114 (SEQ ID NO: 3).

(26) FIG. 5D: curve filled with grey: mock Light grey unfilled curve: BLV (SEQ ID NO: 30), Dark grey unfilled curve: KoV (SEQ ID NO: 20).

(27) FIG. 5E:FACS of B cells

(28) FIG. 6A to 6E represent the RBD binding profile to CD19+ cells and the FACS of a patient with CLL (patient 1):

(29) FIG. 6A: curve filled with grey: mock Light grey unfilled curve: HTLV 2 (SEQ ID NO: 28), Dark grey unfilled curve: HTLV 4 (SEQ ID NO: 31).

(30) FIG. 6B: curve filled with grey: mock Light grey unfilled curve: Perv B (SEQ ID NO: 22), Dark grey unfilled curve: Ampho (SEQ ID NO: 1).

(31) FIG. 6C: curve filled with grey: mock Light grey unfilled curve: Xeno (SEQ ID NO: 10), Dark grey unfilled curve: RD 114 (SEQ ID NO: 3).

(32) FIG. 6D: curve filled with grey: mock Light grey unfilled curve: BLV (SEQ ID NO: 30), Dark grey unfilled curve: KoV (SEQ ID NO: 20).

(33) FIG. 6E: FACS of B cells

(34) FIG. 7A to 7E represent the RBD binding profile to CD19+ cells and the FACS of another patient with CLL (patient 2):

(35) FIG. 7A: curve filled with grey: mock Light grey unfilled curve: HTLV 2 (SEQ ID NO: 28), Dark grey unfilled curve: HTLV 4 (SEQ ID NO: 31).

(36) FIG. 7B: curve filled with grey: mock Light grey unfilled curve: Perv B (SEQ ID NO: 22), Dark grey unfilled curve: Ampho (SEQ ID NO: 1).

(37) FIG. 7C: curve filled with grey: mock Light grey unfilled curve: Xeno (SEQ ID NO: 10), Dark grey unfilled curve: RD 114 (SEQ ID NO: 3).

(38) FIG. 7D: curve filled with grey: mock Light grey unfilled curve: BLV (SEQ ID NO: 30), Dark grey unfilled curve: KoV (SEQ ID NO: 20).

(39) FIG. 7E represents the FACS of B cells.

(40) FIG. 8A to 8E represent the RBD binding profile to CD19+ cells and the FACS of another patient with CLL (patient 3):

(41) FIG. 8A: curve filled with grey: mock Light grey unfilled curve: HTLV 2 (SEQ ID NO: 28), Dark grey unfilled curve: HTLV 4 (SEQ ID NO: 31).

(42) FIG. 8B: curve filled with grey: mock Light grey unfilled curve: Perv B (SEQ ID NO: 22), Dark grey unfilled curve: Ampho (SEQ ID NO: 1).

(43) FIG. 8C: curve filled with grey: mock Light grey unfilled curve: Xeno (SEQ ID NO: 10), Dark grey unfilled curve: RD 114 (SEQ ID NO: 3).

(44) FIG. 8D: curve filled with grey: mock Light grey unfilled curve: BLV (SEQ ID NO: 30), Dark grey unfilled curve: KoV (SEQ ID NO: 20.

(45) FIG. 8E:FACS of B cells.

(46) FIGS. 9A to 9E represent the RBD binding profile to CD19+ cells and the FACS of another patient with CLL (patient 4):

(47) FIG. 9A: curve filled with grey: mock Light grey unfilled curve: HTLV 2 (SEQ ID NO: 28), Dark grey unfilled curve: HTLV 4 (SEQ ID NO: 31).

(48) FIG. 9B: curve filled with grey: mock Light grey unfilled curve: Perv B (SEQ ID NO: 22), Dark grey unfilled curve: Ampho (SEQ ID NO: 1).

(49) FIG. 9C: curve filled with grey: mock Light grey unfilled curve: Xeno (SEQ ID NO: 10), Dark grey unfilled curve: RD 114 (SEQ ID NO: 3).

(50) FIG. 9D: curve filled with grey: mock Light grey unfilled curve: BLV (SEQ ID NO: 30), Dark grey unfilled curve: KoV (SEQ ID NO: 20).

(51) FIG. 9E: FACS of B cells.

(52) FIG. 10A to 10K represent the RBD binding profile of various RBD on human red blood cell (RBC):

(53) FIG. 10A: HTLV2 (SEQ ID NO: 28) tagged with a green fluorescent protein (GFP),

(54) FIG. 10B: HTLV2 (SEQ ID NO: 28).

(55) FIG. 10C: HTLV4 (SEQ ID NO: 31).

(56) FIG. 10D: Ampho (SEQ ID NO: 1).

(57) FIG. 10E: GALV (SEQ ID NO: 4).

(58) FIG. 10F: RD114 (SEQ ID NO: 3).

(59) FIG. 10G: BLV (SEQ ID NO: 30).

(60) FIG. 10H: KoV (SEQ ID NO: 20.

(61) FIG. 10I: Xeno (SEQ ID NO: 10).

(62) FIG. 10J: FelV (SEQ ID NO: 19).

(63) FIG. 10K: Perv B (SEQ ID NO: 22).

(64) FIGS. 11A to 11L and 12A to 12J represent the RBD binding profile of various RBD on unstimulated or TCR-stimulated human T cells:

(65) FIGS. 11A to 11F: unstimulated human T cells:

(66) FIG. 11A: HTLV2 (SEQ ID NO: 28) tagged with a green fluorescent protein (GFP),

(67) FIG. 11B: HTLV2 (SEQ ID NO: 28).

(68) FIG. 11C: HTLV4 (SEQ ID NO: 31).

(69) FIG. 11D: Ampho (SEQ ID NO: 1).

(70) FIG. 11E: GALV (SEQ ID NO: 4).

(71) FIG. 11F: RD114 (SEQ ID NO: 3).

(72) FIGS. 11G to 11L: TCR-stimulated T cells:

(73) FIG. 11G: HTLV2 (SEQ ID NO: 28) tagged with a green fluorescent protein (GFP),

(74) FIG. 11H: HTLV2 (SEQ ID NO: 28).

(75) FIG. 11I: HTLV4 (SEQ ID NO: 31).

(76) FIG. 11J: Ampho (SEQ ID NO: 1).

(77) FIG. 11K: GALV (SEQ ID NO: 4).

(78) FIG. 11L: RD114 (SEQ ID NO: 3).

(79) FIGS. 12A to 12E: Unstimulated T cells:

(80) FIG. 12A: BLV (SEQ ID NO: 30),

(81) FIG. 12B: KoV (SEQ ID NO: 20,

(82) FIG. 12C: Xeno (SEQ ID NO: 10),

(83) FIG. 12D: FelV (SEQ ID NO: 19),

(84) FIG. 12E: Perv B (SEQ ID NO: 22).

(85) FIGS. 12F to 12J: TCR-stimulated T cells:

(86) FIG. 12F: BLV (SEQ ID NO: 30),

(87) FIG. 12G: KoV (SEQ ID NO: 20,

(88) FIG. 12H: Xeno (SEQ ID NO: 10),

(89) FIG. 12I: FelV (SEQ ID NO: 19),

(90) FIG. 12J: Perv B (SEQ ID NO: 22).

(91) FIGS. 13A to 13O represent the RBD binding profile of various RBDs on murine T cells differentiated on Th0, Th1 and TH2 cells.

(92) FIGS. 13A, 13B and 13C: Ampho (SEQ ID NO: 1).

(93) FIGS. 13D, 13E and 13F: BLV (SEQ ID NO: 30),

(94) FIGS. 13G, 13H, and 13I: HTLV1 (SEQ ID NO: 27).

(95) FIGS. 13J, 13K and 13L: GALV (SEQ ID NO: 4).

(96) FIGS. 13M, 13N and 13O: RD114 (SEQ ID NO: 3).

(97) For each figure representing the RBD binding, the curves unfilled, from left to right, represent the mock and the RBD respectively.

(98) FIGS. 14A to 14J represent the RBD binding profile of various RBDs on murine T cells differentiated on Th17 and iTreg cells.

(99) FIGS. 14A and 14B: Ampho (SEQ ID NO: 1).

(100) FIGS. 14C and 14D: BLV (SEQ ID NO: 30),

(101) FIGS. 14E and 14F: HTLV1 (SEQ ID NO: 27).

(102) FIGS. 14G and 14H: GALV (SEQ ID NO: 4).

(103) FIGS. 14I and 14J: RD114 (SEQ ID NO: 3).

(104) For each figure representing the RBD binding, the curves unfilled, from left to right, represent the mock and the RBD respectively.

(105) FIGS. 15A to 15O represent the RBD binding profile BLV (SEQ ID NO:30) and GALV (SEQ ID NO:4) RBDs on CD34 progenitor cells at day 0, day 2, day 4 and day 6.

(106) FIGS. 15A, 15D, 15H, 15L represent the FACS of CD34 cells.

(107) FIGS. 15A, 15B and 15C represent day 0.

(108) FIGS. 15D, 15E, 15F and 15G represent day 2.

(109) FIGS. 15H, 15I, 15J and 15K represent day 4.

(110) FIGS. 15L, 15M, 15N and 15O represent day 6.

(111) FIGS. 15B, 15E, 15I and 15M represent the RBD binding on CD34+/CD38− cells.

(112) FIGS. 15C, 15F, 15J and 15N represent the RBD binding on CD34+/CD38+ cells.

(113) FIGS. 15G, 15K, 15O represent the RBD binding on CD34−/CD38+ cells.

(114) For each figure representing the RBD binding, the curved filled with grey represents the mock and the curves unfilled, from left to right, represent GALV and BLV RBDs respectively.

(115) FIG. 16A to 16F represent the RBD binding profile (BLV (SEQ ID NO:30), GALV (SEQ ID NO:4) and Ampho (SEQ ID NO: 1) RBDs) on CD34 progenitor cells cultured in two media.

(116) FIGS. 16A to 16C: STEMPAN medium.

(117) FIG. 16D to 16F: XVIVO15 medium.

(118) FIG. 16A, 16D represent the FACS of CD 34 cells.

(119) FIGS. 16B and 16E: CD34+/CD38− cells.

(120) FIGS. 16C and 16E: CD34+/CD38+ cells.

(121) For each figure representing the RBD binding, the curved filled with grey represents the mock and the curves unfilled, from left to right, represent Ampho, GALV and BLV RBDs respectively.

(122) FIGS. 17A to 17C represent the morphological changes in the postnatal development of cerebral cortex from day 6 after birth to day 19 after birth.

(123) FIG. 17A: day 6 after birth.

(124) Dashed arrow: external granular layer (EGL)

(125) Full line arrow: internal granular layer (IGL)

(126) FIG. 17B: day 12 after birth

(127) Dashed arrow (small dots): external granular layer (EGL)

(128) Full line arrow: internal granular layer (IGL)

(129) Dashed arrow (large underscores): molecular layer (ML)

(130) FIG. 17C: day 19 after birth

(131) Dashed arrow (large underscores): molecular layer (ML)

(132) Full line arrow: granular layer (GL)

(133) FIG. 18A to 18F represent the expression of nutrient transporters in the different layers after day 6-7 (grey zones).

(134) FIG. 18A: Hoechst+Alexa488 (Ampho/PiT2)

(135) FIG. 18B: Hoechst+Alexa488 (GALV/PiT1)

(136) FIG. 18C Hoechst+Alexa488 (HTLV1/GLUT1)

(137) FIG. 18D: Hoechst+CellTrace BODIPY (Ampho/PiT2)

(138) FIG. 18E: Hoechst+CellTrace BODIPY (GALV/PiT1)

(139) FIG. 18F: Hoechst+CellTrace BODIPY (HTLV1/GLUT1)

(140) The meaning of EGL, IGL and ML is the same as in FIG. 17A to 17C.

(141) Frequencies at which the images showing these patterns of labeling with the corresponding probes are observed among all the images including both of the EGL and IGL are the following:

(142) FIGS. 18A and 18D: 17/25 (68%)

(143) FIGS. 18B and 18E: 19/24 (78.2%)

(144) FIGS. 18C and 18F: 18/22 (81.8%)

(145) FIGS. 19A and 19B represent the expression of PiT2 in the different layers (arrows) after day 6-7.

(146) FIG. 19A: Hoechst+Alexa488 (Ampho/PiT2)

(147) FIG. 19B: Hoechst+CellTrace BODIPY

(148) Frequencies at which the images with these patterns of labeling with Ampho are observed among all the images including both areas that face and do not face the forming fissures are the following: 11/19 (57.9%).

(149) FIGS. 20A to 20D represent the expression of PiT1 in the different layers (arrows) after days 6-7 or days 12-14.

(150) Day 6-7:

(151) FIG. 20A: Hoechst+Alexa488 (GALV/PiT1)

(152) FIG. 20B: Hoechst+CellTrace BODIPY

(153) Days 12-14

(154) FIG. 20C: Hoechst+Alexa488 (GALV/PiT1)

(155) FIG. 20D: Hoechst+CellTrace BODIPY

(156) Frequencies at which the images with these patterns of labeling with GRBD are observed among all the images including both areas that face and do not face the forming fissures are the following:

(157) FIGS. 20A and 20B: 3/13 (23.1%)

(158) FIGS. 20C and 20D: 4/15 (26.7%)

(159) FIGS. 21A to 21D represent the expression profiles of PiT1 in the cerebellar cortex of adult mice (16-22 days after birth).

(160) FIG. 21A: Hoechst+Alexa488 (GALV/PiT1)

(161) FIGS. 21B and 21D: Hoechst+CellTrace BODIPY

(162) FIG. 21C: FIGS. 21A, 21B and 21D merged

(163) FIGS. 22A to 22D represent the expression profiles of GLUT1 in the cerebellar cortex of adult mice (16-22 days after birth).

(164) FIG. 22A: Hoechst+Alexa488 (HTLV1/GLUT1)

(165) FIGS. 22B and 21D: Hoechst+CellTrace BODIPY

(166) FIG. 22 C: FIGS. 22A, 22B and 22D merged

EXAMPLES

Example 1: General Method for the Production of Receptor Binding Ligands with 293T Cells Transfection

(167) At D-1: 293T Cells Spreading

(168) TABLE-US-00001 Plate type 6 wells 60 mm 10 cm Cell numbers 3 × 10.sup.5 10.sup.6 2 × 10.sup.6

(169) At D0: Transfection by Calcium Phosphate Precipitation

(170) TABLE-US-00002 Plate type 6 wells 60 mm 10 cm Volume (ml) 3 ml 5 ml 10 ml

(171) 1) Prepare the HBS+DNA of a receptor binding protein in an eppendorf tube (under hood):

(172) TABLE-US-00003 Plate type 6 wells 60 mm 10 cm DNA total quantity (μg) 6 10 20 PCSI 6 10 20 Vol. HBS (μl) 150 250 500

(173) 2) Add CaCl2 2M (sterile) up to a final concentration=125 mM:

(174) TABLE-US-00004 Plate type 6 wells 60 mm 10 cm Vol. CaCl2 2M (μl) 10 17 33

(175) 3) “Gently” Vortex for 10 sec,

(176) 4) Incubate 5 min at RT, a white precipitate is formed,

(177) 5) Gently add the precipitate on cells and homogenise,

(178) 6) Put the cells inside the incubator (37° C., 5% CO2).

(179) At D1: Medium change:

(180) The sooner the possible in the morning and gently (293T cells detach easily) with 10 ml of optipro SFM Medium (Gibco) without FBS-16H MAX,

(181) Then incubate (32° C., 5% CO2).

(182) After 48 h, i.e. at D3: Supernatant recovering and concentration Recover the conditioned medium in 50 ml falcon tube Spin at 1500 tr/min, 3 min, 4° C. Filter the supernatant on 0.45 μm Conserve the supernatant on ice Add 20 ml of ultrapure water in the concentrators (Icon concentrator, 20 ml/9 k, PIERCE) Spin at 3600 tr/min, 10 mM, (Swinging-bucket), 4° C. Add 20 ml of filtered RBD sample Spin at 3600 tr/min, 20 min, 4° C. Add sample, centrifuge 20 min (100 ml max of RBD for each concentrator) Spin until desired concentration factor is achieved (100×) Recover concentrated sample, aliquot and stock at −80° C.

Example 2: General Method of FACS

(183) The FACS assay of HRBD-EGFP (non antibody Glut1-ligand) is representative of the method for the receptors binding ligands:

(184) Target cells: Any mammalian cell lines/human RBC/Human activated PBLs or any subpopulation/any primary or established cell type of interest.

(185) For the binding assay: Entire binding assay should be performed on ice except for the actual binding step performed at 37° C.

(186) RBD stored at −80° C.

(187) Thaw RBD-containing conditioned medium and mock transfected conditioned medium. Avoid re-freezing the RBD preparation.

(188) Single Assay in Eppendorf Tubes

(189) 1-2×10.sup.5 cells per assay in 1.5 ml eppendorf tube Centrifuge 3 min at 3200 RPM. Aspirate supernatant gently. Gently resuspend pellet (tapping). Dilute the concentrated HRBD-EGFP 1/20 (v/v) dilution in PBS or medium Add 100 μl to 200 μl/tube of the dilution and resuspend gently. Incubate 30 min at 37° C. (no agitation is required). Keep cold during all the following steps Centrifuge 3 min at 3200 RPM 4° C., gently aspirate supernatant and gently tap pellet. Add 1 ml of cold PBA (PBS+2% FBS and 0.01% sodium azide) and gently tap pellet. Repeat last two steps, resuspend pellet with 500 μl of PBA and transfer to FACS tubes. FACS analysis
Multiple assays in 96 well-microplates (V bottom) 1-2×10.sup.5 cells for each binding assay per well. Centrifuge 3 min at 1500 RPM. Discard the supernatant by quickly flipping the plate (over sink for instance). Place the plate upside down on absorbing paper to eliminate remaining droplets. Gently vortex the plate. Dilute the concentrated HRBD-EGFP preparation 1/20 (v/v) in PBS or medium Add 50 μl/well of the diluted preparation of HRBD-EGFP and resuspend gently. Incubate 30 min at 37° C. (no agitation is required). Transfer to 4° C. for all the following steps. Centrifuge 3 min at 1500 RPM at 4° C. and discard supernatant as previously. Wash pellet with 200 μl of cold PBA twice, with 3 min centrifuge at 1500 RPM. Resuspend pellet with 200 μl of PBA and transfer the mix to FACS tubes. FACS analysis

(190) FIGS. 3B,C and 3E,F present the results obtained with the receptors binding ligands of SEQ ID NO:1 to SEQ ID NO:4.

Example 3: Expression of Nutrient Transporters on B-CLL Cells

(191) As compared to healthy donors with CD19+/CD5− B cells, patients with B-CLL harbor blasts with a CD19+/CD5+ phenotype. Assessment of cell surface nutrient transporters, as assessed by binding to tagged retroviral envelope receptor domains (Env RBDs), shows increased expression of the receptors for bovine leukemia virus (BLV), Xeno and RD114 (ASCT2) Env in some patients.

(192) It is also notable that binding of HTLV RBD to the HTLV Env receptor, the ubiquitous glucose transporter Glut1, is significantly decreased in all tested B-CLL patients as compared to healthy controls (Cf. FIGS. 4 to 9).

(193) Thus, the panel of Env RBDs will allow us to determine the signature of nutrient transporters that is associated with good and poor prognostic B-CLL.

Example 4: Expression of Nutrient Transporters on Human RBCs and Human T Cells

(194) Extensive assessment of nutrient transporters on human RBC, using tagged retroviral Env RBDs, shows expression only of the HTLV and PervB Env receptors; the former identified as Glut1 whereas the latter has not yet been identified (FIG. 10).

(195) Prior to activation, quiescent human T cells express only low levels of nutrient transporters serving as receptors for the HTLV2, GaLV, RD114, BLV, Xeno and FeLV envelopes. Receptors for all these envelopes, with the exception of Xeno, are upregulated following TCR stimulation. Interestingly, expression of the receptors binding the amphotropic and Koala RBDs (phosphate transporters) are highly expressed in quiescent cells and their levels decrease following TCR engagement (observed in some but not all experiments that we performed). Expression of the PervB receptor is not altered by TCR stimulation (FIGS. 12E and 12J).

Example 5: Use of Nutrient Transporter Expression to Track T Cell Activation and Polarization

(196) METHOD: Ligand binding on murine CD4+T cells upon Th1, 2, 17 or iTreg differentiation. Th1 and Th17 cells are CD4+ T cell subsets characterized by the secretion of IFNg and IL-17 respectively and are involved in inflammatory processes: these subsets have been involved in many disorders such as autoimmune diseases (MS, arthritis . . . ), TB infection, skin lesions. Th2 cells secrete IL-4, IL-5, IL-9, IL-10 . . . . These cells play a role in parasitic infections for example but are also implicated in allergy or asthma. iTreg cells are a population of regulatory T cells, implicated in immune suppression. This experiment was performed on murine naive CD4+T cells activated by anti-CD3/anti-CD28 and upon differentiation Th0: no polarizing cytokine added Th1: IL-12 and anti-IL-4 Th2: IL-4 and anti-IFNg Th17: IL-6 and TGFb iTreg: TGFb Polarizing cytokines were added at day 0, after 2 days in culture, cells were diluted and IL-2 was added to the medium (except for Th17: IL-23 was used instead of IL-2) At day 4, binding assay was performed.

(197) Results:

(198) Upon stimulation of murine T cells under either non-polarizing (Th0) or polarizing conditions (towards Th1, Th2, Th17 or Treg fates), nutrient transporter expression was assessed. Of note, expression of Glut1, the receptor for the HTLV RBD, is significantly higher in Th1 and Th2 conditions than in either Th17 or Treg conditions. Moreover, the PiT1 phosphate transporter, as recognized by GaLV RBD, is expressed in T cells modulated towards a Th1 fate but is minimal under all other conditions (FIGS. 13 and 14).

Example 6: Use of Nutrient Transporter Expression to Follow CD34 Progenitor Cell Activation and Differentiation

(199) Receptors for both BLV and GaLV RBDs were found to be expressed at significantly lower levels on primitive CD34+/CD38− progenitors as compared to more differentiated CD34+/CD38+ cells. Of note, ex vivo expansion (media/cytokines as used in the clinic) of these progenitors resulted in a significant upregulation of BLV and GaLV Env receptors, as early as 48 h post-stimulation and this upregulation was detected in primitive (CD34+/CD38−) as well as more differentiated subsets (CD34+ and CD34−) (FIG. 15).

(200) Finally, while both Stemspan and XVivo15 media are used for clinical use, expression of the PiT2 phosphate transporter, as assessed using the tagged ampho RBD, is higher in CD34+ cells (and particularly CD34+/CD38−) cultured in the former conditions. The significance of this change remains to be determined (FIG. 16).

Example 7: Distinct Expression of the Glucose (GLUT1) and Inorganic Phosphate (PiT1, PiT2) Transporters in the Developing Mouse Cerebellar Cortex Unveiled by New Ligands Based on Retrovirus Envelope Proteins HTLV 2 (SEQ ID NO: 28) for GLUT1, GALV (SEQ ID NO: 2) for PiT1, Ampho (SEQ ID NO: 1) for PiT2

(201) All the mammalian cells uptake necessary nutrients such as glucose, amino acids, inorganic phosphate . . . via “Nutrient Transporters” on the cell surface leading to the survival of the cell (inhibition of apoptosis, inhibition of autophag, proliferation . . . ).

(202) Using the novel probes based on the envelope proteins of retroviruses GALV (SEQ ID NO: 2), HTLV 2 (SEQ ID NO:28) and Ampho (SEQ ID NO: 1), the changes of expression profiles of some nutrient transporters during the postnatal development of cerebellar cortex of mice were deciphered.

(203) It must be noted that HTLV 1 (SEQ ID NO: 27) or HTLV 4 (SEQ ID NO: 31) could have been used instead of HTLV 2 as said RBDs are also GLUT1 ligands.

(204) Methods: a) Unfixed cerebellum from 6-7, 12-14 and 16-22 day old mice, b) Cryosectioning at 20 μm thickness, c) Fixation with 100% ethanol at room temperature, d) Blocking with normal serum and an endogenous biotin blocking reagent, e) Incubation with either of the soluble RBD-rFc fusion protein, 30 min. at 37° C.: HTLV-RBD (HRBD, SEQ ID NO:27) or SEQ ID NO: 28), ligand for GLUT1; Gibbon ape Leukemia Virus-RBD (GRBD, (SEQ ID NO: 2), ligand for PiT1 Amphotropic MLV-SU (ASU, (SEQ ID NO: 1), ligand for PiT2, f) Incubation with biotinylated anti-rabbit IgG 1 hr at room temperature, g) Incubation with Streptavidin-Alexa488 (transporter ligand), h) Counterstaining with Hoechst (cell nucleus) and CellTrace BODIPY (intracellular membranes), i) Z-series image acquisition of the three emissions at 3 μm distance (7 slices), j) Image restoration using Huygens professional software (Montpellier RIO Imaging), k) Creating the final images by the Maximum Intensity Projection method using Imaris 5.7.0.
Results:
RESULTS 1: Characteristic expression profiles of GLUT1, PiT1 and PiT2 during the postnatal development of cerebellar cortex of mice

(205) The postnatal development of cerebellar cortex is accompanied by drastic morphological changes (FIGS. 17A, 17B and 17C.).

(206) At the 6-7 days after birth, all the three nutrient transporters were shown to be expressed in larger amount in the EGL than in the IGL nearby. This is not obvious at the 12-14 days after birth (FIGS. 18A to 18F).

(207) At the 6-7 days after birth, in the EGL that do not face the forming fissures, PiT2 was revealed to be expressed in larger amount in the deeper layer of the EGL. In this deeper area of the EGL, it is well known that postmitotic granule cells are migrating toward the ML and IGL, changing their morphologies at this stage of postnatal development (FIGS. 19A and 19B). This is observed less frequently in the cases of the other two transporters.

(208) At the 6-7 and 12-14 days after birth, PiT1 were revealed to be preferentially localized in the areas that do not face the forming fissures (FIGS. 20A to 20D). This is not the case with the expression of PiT2 and GLUT1.

(209) RESULTS 2: Characteristic expression profiles of GLUT1, PiT1 and PiT2 in the cerebellar cortex of adult mice (16-22 days after birth).

(210) All the three transporters were shown to be localized in the GL and the Purkinje Layer (PL). It is worth noting that the expression of the transporters in the Purkinje cells is irregular in intensity.