Simultaneous stimulation and concentration of cells

• US 20020058019A1
• Filed: 02/26/2001
• Published: 05/16/2002
• Est. Priority Date: 02/24/2000
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Xcyte Therapies, Inc. (Cyclacel Pharmaceuticals Incorporated)
• Inventors: Bonyhadi, Mark, Kalamasz, Dale, Craig, Stewart, Hardwick, Alan, Saund, Narinder, Law, Che, Berenson, Ronald, McMillen, David
• Granted Patent: US 6,905,874 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 2039/57 characterised by the type of response, e.g. Th1, Th2
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/464499 Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
• CPC Class Codes: A61L 27/3804 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• CPC Class Codes: A61L 27/3895 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2806 against CD2
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2812 against CD4
• CPC Class Codes: C07K 16/2815 against CD8
• CPC Class Codes: C07K 16/2818 against CD28 or CD152
• CPC Class Codes: C07K 16/2821 against ICAM molecules, e.g. CD50, CD54, CD102
• CPC Class Codes: C07K 16/2827 against B7 molecules, e.g. CD80, CD86
• CPC Class Codes: C07K 16/2833 against MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C07K 16/2845 against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
• CPC Class Codes: C07K 16/2854 against selectins, e.g. CD62
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 16/2875 against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154 against NGF C07K16/22, against TNF C07K16/241
• CPC Class Codes: C07K 16/2878 : against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 16/289 : against CD45
• CPC Class Codes: C07K 16/2896 : against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C12N 2500/32 : Amino acids
• CPC Class Codes: C12N 2501/23 : Interleukins [IL]
• CPC Class Codes: C12N 2501/51 : B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
• CPC Class Codes: C12N 2501/515 : CD3, T-cell receptor complex
• CPC Class Codes: C12N 2501/53 : CD2
• CPC Class Codes: C12N 2533/12 : Glass
• CPC Class Codes: C12N 2533/14 : Ceramic
• CPC Class Codes: C12N 2533/18 : Calcium salts, e.g. apatite, Mineral components from bones, teeth, shells
• CPC Class Codes: C12N 2533/30 : Synthetic polymers
• CPC Class Codes: C12N 2533/40 : Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
• CPC Class Codes: C12N 2533/54 : Collagen; Gelatin
• CPC Class Codes: C12N 2533/70 : Polysaccharides
• CPC Class Codes: C12N 2533/72 : Chitin, chitosan
• CPC Class Codes: C12N 2533/90 : Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0635 : B lymphocytes
• CPC Class Codes: C12N 5/0636 : T lymphocytes
• CPC Class Codes: C12N 5/0647 : Haematopoietic stem cells; Uncommitted or multipotent progenitors

Abstract

The present invention relates generally to methods for stimulating cells, and more particularly, to a novel method to concentrate and stimulate cells that maximizes stimulation and/or proliferation of such cells. In the various embodiments, cells are stimulated and concentrated with a surface yielding enhanced proliferation, cell signal transduction, and/or cell surface moiety aggregation. In certain aspects methods for stimulating a population of cells such as T-cells, by simultaneous concentration and cell surface moiety ligation are provided by contacting the population of cells with a surface, that has attached thereto one or more agents that ligate a cell surface moiety and applying a force that predominantly drives cell concentration and cell surface moiety ligation, thereby inducing cell stimulation, cell surface moiety aggregation, and/or receptor signaling enhancement. Also provided are methods for producing phenotypically tailored cells, including T-cells for the use in diagnostics, drug discovery, and the treatment of a variety of indications, including cancer, viral infection, and immune related disorders. Compositions of cells having specific phenotypic properties produced by these processes are further provided.

Claims

1. A method for stimulating a population of T-cells by simultaneous T-cell concentration and cell surface moiety ligation, comprising:

(a) providing a population of cells wherein at least a portion thereof comprises T-cells;

(b) contacting said population of cells with a surface, wherein said surface has attached thereto one or more agents that ligate a cell surface moiety of at least a portion of said T-cells and stimulates at least said portion of T-cells.

(c) applying a force that predominantly drives T-cell concentration and T-cell surface moiety ligation, thereby inducing T-cell stimulation.

2. The method of claim 1, wherein said surface has attached thereto a first agent that ligates a first cell surface moiety of a T-cell;

and the same or a second surface has attached thereto a second agent that ligates a second moiety of said T-cell, wherein said ligation by the first and second agent induces proliferation of said T-cell.

3. The method of claim 1, wherein said surface is biocompatible.

4. The method of claim 3, wherein said surface is natural or synthetic.

5. The method of claim 4, wherein said surface comprises a polymer.

6. The method of claim 5, wherein said surface is selected from the group consisting of collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, and extracellular matrix compositions.

7. The method of claim 6, wherein the polysaccharides are selected from the group consisting of chitosan, alginate, dextran, hyaluronic acid, and cellulose.

8. The method of claim 5, wherein the polymer is selected from the group consisting of polystyrene, polyesters, polyethers, polyanhydrides, polyalkylcyanoacrylates, polyacrylamides, polyorthoesters, polyphosphazenes, polyvinylacetates, block copolymers, polypropylene, polytetrafluoroethylene (PTFE), and polyurethanes.

9. The method of claim 8, wherein the polymer comprises lactic acid.

10. The method of claim 5, wherein the polymer is a copolymer.

11. The method of claim 10, wherein the copolymer comprises lactic acid and glycolic acid (PLGA).

12. The method of claim 3, wherein the biocompatible surface is biodegradable.

13. The method of claim 3, wherein the biocompatible surface is non-biodegradable.

14. The method of claim 13, wherein the non-biodegradable substance comprises a polymer selected from the group consisting of poly(dimethysiloxane) and poly(ethylene-vinyl acetate).

15. The method of claim 3, wherein the biocompatible surface is selected from the group consisting of collagen, metal, hydroxyapatite, glass, aluminate, bioceramic materials, hyaluronic acid polymers, alginate, acrylic ester polymer, lactic acid polymer, glycolic acid polymer, lactic acid/glycolic acid polymer, purified proteins, purified peptides, and extracellular matrix compositions.

16. The method of claim 3, wherein the biocompatible surface is associated with an implantable device.

17. The method of claim 16, wherein the device is selected from the group consisting of:

a stent, a catheter, a fiber, a hollow fiber, a patch, and a suture.

18. The method of claim 4, wherein said surface is selected from the group consisting of glass, silica, silicon, collagen, hydroxyapatite, hydrogels, PTFE, polypropylene, polystyrene, nylon, and polyacrylamide.

19. The method of claim 4, wherein said surface is selected from the group consisting of a lipid, a plate, a dish, a bag, a rod, a pellet, a fiber, and a mesh.

20. The method of claim 4, wherein said surface is a particle.

21. The method of claim 20, wherein the particle is selected from the group consisting of a bead, a microsphere, a nanoparticle, and a colloidal particle.

22. The method of claim 21, wherein said bead is about 5 nanometers to about 500 microns in diameter.

23. The method of claim 1, wherein said agents are independently selected from the group consisting of a protein ligand, a natural ligand, and a synthetic ligand.

24. The method of claim 23, wherein said agents are independently selected from the group consisting of an antibody, an antibody fragment, a peptide, a polypeptide, a glycopeptide, a receptor, a steroid, a hormone, a mitogen, an antigen, a superantigen, a growth factor, a cytokine, a lectin, a viral protein, an adhesion molecule, and a chemokine.

25. The method of claim 24, wherein at least one agent is an antibody or an antibody fragment.

26. The method of claim 24, wherein a first agent is an antibody and a fragment thereof, and a second agent is an antibody or a fragment thereof.

27. The method of claim 26, wherein said first and said second agents are different antibodies.

28. The method of claim 24, wherein said first agent is an anti-CD3 antibody, an anti-CD2 antibody, or an antibody fragment of an anti-CD3 or anti-CD2 antibody.

29. The method of either claim 24 or 27, wherein said second agent is an anti-CD28 antibody or antibody fragment thereof.

30. The method of either claim 24 or 27, wherein said second agent is a natural ligand for CD28.

31. The method of claim 30, wherein said natural ligand comprises B7-1 or B7-2.

32. The method of claim 1, wherein said force is selected from the group consisting of a force greater than gravitational force, a hydraulic force, a filtration force generated by transmembrane pressure, a centrifugal force, and a magnetic force.

33. The method of claim 32, wherein the magnetic force is generated by a magnet having a magnetic field strength ranging from between about 200 gauss to about 12,000 gauss at the surface of the magnet.

34. The method of claim 1, wherein said surface is a surface of a paramagnetic particle.

35. The method of claim 1, wherein said agents'"'"' attachment to the surface is covalent, noncovalent, electrostatic, or hydrophobic.

36. The method of claim 1, wherein the T-cells that are ligated are separated from the T-cells that are not ligated.

37. The method of claim 1, wherein said T-cells ameliorate immune response dysfunction.

39. The method of claim 38, wherein said time sufficient to achieve desired stimulation is from 1 minute to 8 days.

40. The method of claim 39, wherein said time sufficient to achieve desired stimulation is from 1 day to 5 days.

41. The method of claim 38, wherein said surface is biocompatible.

42. The method of claim 41, wherein said surface is natural or synthetic.

43. The method of claim 42, wherein the surface is selected from the group consisting of glass, silica, silicon, collagen, hydroxyapatite, hydrogels, PTFE, polypropylene, polystyrene, nylon, dextran, and polyacrylamide.

44. The method of claim 38, wherein said surface is selected from the group consisting of a plate, a dish, a bag, a rod, a pellet, a fiber, and a mesh.

45. The method of claim 38, wherein said surface is a particle.

46. The method of claim 45, wherein the particle is selected from the group consisting of a bead, a microsphere, a nanoparticle, and a colloidal particle.

47. The method of claim 46, wherein said bead is about 5 nanometers to about 500 microns in diameter.

48. The method of claim 38, wherein said ligand is selected from the group consisting of a protein, a natural ligand, and a synthetic ligand.

49. The method of claim 38, wherein said ligand is selected from the group consisting of an antibody, an antibody fragment, a peptide, a polypeptide, a glycopeptide, a soluble receptor, a steroid, a hormone, a mitogen, an antigen, a ligand, a superantigen, a growth factor, a cytokine, a lectin, and a chemokine.

50. The method of claim 49, wherein at least one ligand is an antibody or a fragment thereof.

51. The method of claim 49, wherein at least two ligands are an antibody or a fragment thereof.

52. The method of claim 49, wherein at least two ligands are present and are different antibodies or fragments thereof.

53. The method of claim 49, wherein at least one ligand is an anti-CD3 antibody, an anti-CD2 antibody, or an antibody fragment of an anti-CD3 or anti-CD2 antibody.

54. The method of either claim 49 or 53, wherein at least one ligand is an anti-CD28 antibody or antibody fragment thereof.

55. The method of either claim 49 or 53, wherein at least one ligand is a natural ligand for CD28.

56. The method of claim 55, wherein said natural ligand comprises B7-1 or B7-2.

57. The method of claim 38, wherein said force is selected from the group consisting of a force greater than gravitational force, a hydraulic force, a filtration force generated by transmembrane pressure, a centrifugal force, and a magnetic force.

58. The method of claim 57, wherein the magnetic force is generated by a magnet having a magnetic field strength ranging from between about 200 gauss to about 12,000 gauss at the surface of the magnet.

59. The method of claim 38, wherein said surface is a surface of a paramagnetic particle.

60. The method of claim 38, wherein said ligand attachment to the surface is covalent, noncovalent, electrostatic, or hydrophobic.

61. The method of claim 38, further comprising prior to or concurrently with step (d), separating T-cells concentrated with surface from non-concentrated cells.

91. The method of claim 32, wherein the magnetic force is generated by a magnet having a magnetic field strength ranging from between about 200 gauss to about 12,000 gauss at the magnet surface.

38. A method for stimulation of T-cells by simultaneous cell surface moiety ligation and T-cell aggregation, comprising:

(a) providing a cell population comprising T-cells;

(b) contacting said cell population with a surface, wherein said surface has attached thereto one or more ligands specific for a cell surface moiety;

(c) applying a force that drives concentration of T-cells and surface; and

(d) incubating said cells for a period of time sufficient to achieve desired stimulation.

62. A method adapted for inducing T-cell activation in vivo, comprising providing paramagnetic particles to an animal, said particles having attached thereto, ligands specific for a T-cell surface moiety that induces T-cell activation;

applying a magnetic field to a discrete region of the animal; and

thereby inducing localization and activation of T-cells bound to said particles at said discrete region.

63. A method for stimulating a population of target cells by simultaneous target cell concentration and target cell surface moiety ligation, comprising:

(a) providing a population of cells wherein at least a portion thereof comprises target cells;

(b) contacting said population of cells with a surface, wherein said surface has attached thereto one or more agents that ligate a cell surface moiety of at least a portion of said target cells and stimulates at least said portion of target cells.

(c) applying a force that predominantly drives target cell concentration and target cell surface moiety ligation, thereby inducing target cell stimulation.

64. The method of claim 63, wherein said surface has attached thereto a first agent that ligates a first cell surface moiety of a target cell;

and the same or a second surface has attached thereto a second agent that ligates a second moiety of said target cell, wherein said ligation by the first and second agent induces signal transduction in said target cell.

65. The method of claim 63, wherein said surface is biocompatible.

66. The method of claim 65, wherein said surface is natural or synthetic.

67. The method of claim 66, wherein said surface comprises a polymer.

68. The method of claim 67, wherein said surface is selected from the group consisting of collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, and extracellular matrix compositions.

69. The method of claim 68, wherein the polysaccharides are selected from the group consisting of chitosan, alginate, dextran, hyaluronic acid, and cellulose.

70. The method of claim 67, wherein the polymer is selected from the group consisting of polyesters, polyethers, polyanhydrides, polyalkylcyanoacrylates, polyacrylamides, polyorthoesters, polyphosphazenes, polyvinylacetates, block copolymers, polypropylene, polytetrafluoroethylene (PTFE), and polyurethanes.

71. The method of claim 70, wherein the polymer comprises lactic acid.

72. The method of claim 67, wherein the polymer is a copolymer.

73. The method of claim 72, wherein the copolymer comprises lactic acid and glycolic acid (PLGA).

74. The method of claim 65, wherein the biocompatible surface is biodegradable.

75. The method of claim 65, wherein the biocompatible surface is non-biodegradable.

76. The method of claim 75, wherein the non-biodegradable substance comprises a polymer selected from the group consisting of poly(dimethysiloxane) and poly(ethylene-vinyl acetate).

77. The method of claim 65, wherein the biocompatible surface is selected from the group consisting of collagen, metal, hydroxyapatite, bioglass, aluminate, bioceramic materials, hyaluronic acid polymers, alginate, acrylic ester polymer, lactic acid polymer, glycolic acid polymer, lactic acid/glycolic acid polymer, purified proteins, purified peptides, and extracellular matrix compositions.

78. The method of claim 65, wherein the biocompatible surface is associated with an implantable device.

79. The method of claim 78, wherein the device is selected from the group consisting of:

a stent, a catheter, a fiber, a hollow fiber, a patch, and a suture.

80. The method of claim 66, wherein said surface is selected from the group consisting of glass, silica, silicon, collagen, hydroxyapatite, hydrogels, PTFE, polypropylene, polystyrene, nylon, and polyacrylamide

81. The method of claim 66, wherein said surface is selected from the group consisting of a lipid, a plate, a dish, a bag, a rod, a pellet, a fiber, and a mesh.

82. The method of claim 66, wherein said surface is a particle.

83. The method of claim 82, wherein the particle is selected from the group consisting of a bead, a microsphere, a nanoparticle, and a colloidal particle.

84. The method of claim 83, wherein said bead is about 5 nanometers to about 500 microns in diameter.

85. The method of claim 63, wherein said agents are independently selected from the group consisting of a protein ligand, a natural ligand, and a synthetic ligand.

86. The method of claim 85, wherein said agents are independently selected from the group consisting of an antibody, an antibody fragment, a peptide, a polypeptide, a glycopeptide, a receptor, a steroid, a hormone, a mitogen, an antigen, a superantigen, a growth factor, a cytokine, a lectin, a viral protein, an adhesion molecule, and a chemokine.

87. The method of claim 86, wherein at least one agent is an antibody or an antibody fragment.

88. The method of claim 86, wherein a first agent is an antibody or a fragment thereof, and a second agent is an antibody or a fragment thereof.

89. The method of claim 86, wherein said first and said second agents are different antibodies or fragments thereof.

90. The method of claim 63, wherein said force is selected from the group consisting of a force greater than gravitational force, a hydraulic force, a centrifugal force, a filtration force generated by transmembrane pressure, and a magnetic force.

92. The method of claim 63, wherein said surface is a surface of a paramagnetic particle.

93. The method of claim 63, wherein said agents'"'"' attachment to the surface is covalent, noncovalent, electrostatic, or hydrophobic.

109. The method of either claim 63 or 94, wherein said target cells are selected from the group consisting of T-cells, B-cells, or stem cells.

94. A method for stimulation of target cells by cell surface moiety ligation and target cell concentration, comprising:

(a) providing a cell population comprising target cells;

(b) contacting said cell population with a surface, wherein said surface has attached thereto one or more ligands specific for a cell surface moiety;

(c) applying a force that drives concentration of target cells and concentration of said cells on said surface; and

(d) incubating said cells for a period of time sufficient to achieve desired stimulation.

95. The method of claim 94, wherein said time sufficient to achieve desired stimulation is from about 1 minute to about 30 days.

96. The method of claim 95, wherein said time sufficient to achieve desired stimulation is from about 1 day to about 5 days.

97. The method of claim 94, wherein said surface is biocompatible.

98. The method of claim 97, wherein said surface is natural or synthetic.

99. The method of claim 98, wherein the surface is selected from the group consisting of glass, silica, silicon, collagen, hydroxyapatite, hydrogels, PTFE, polypropylene, polystyrene, nylon, dextran, and polyacrylamide.

100. The method of claim 94, wherein said surface is s elected from the group consisting of a plate, a bag, a dish, a rod, a pellet, a fiber, and a mesh.

101. The method of claim 94, wherein said surface is a particle.

102. The method of claim 101, wherein the particle is selected from the group consisting of a bead, a microsphere, a nanoparticle, and a colloidal particle.

103. The method of claim 102, wherein said bead is about 5 nanometers to about 500 microns in diameter.

104. The method of claim 94, wherein said ligand is selected from the group consisting of a protein, a natural ligand, and a synthetic ligand.

105. The method of claim 94, wherein said ligand is selected from the group consisting of an antibody, an antibody fragment, a peptide, a polypeptide, a glycopeptide, a receptor, a steroid, a hormone, a mitogen, an antigen, a ligand, a superantigen, a growth factor, a cytokine, a lectin, and a chemokine.

106. The method of claim 105, wherein at least one ligand is an antibody or a fragment thereof.

107. The method of claim 105, wherein at least two ligands are an antibody or a fragment thereof.

108. The method of claim 105, wherein at least two ligands are present and are different antibodies or fragments thereof.

110. The method of claim 94, wherein said force is selected from the group consisting of a force greater than gravitational force, a hydraulic force, a filtration force generated by transmembrane pressure, a centrifugal force, and a magnetic force.

111. The method of claim 110, wherein the magnetic force is generated by a magnet having a magnetic field strength ranging from between about 200 gauss to about 12,000 gauss at the surface of the magnet.

112. The method of claim 94, wherein said surface is a surface of a paramagnetic particle.

113. The method of claim 94, wherein said ligand attachment to the surface is covalent, noncovalent, electrostatic, or hydrophobic.

114. The method of claim 94, further comprising prior to or concurrently with step (d), separating the concentrated target cells from the non-concentrated cells.

118. The method of claim 117, wherein said cell population comprises lymphocytes.

119. The method of either claim 116 or 117, wherein said solid surface is selected from the group consisting of a plate, a bag, a dish, a rod, a pellet, a fiber, a microsphere, and a bead.

120. The method of either claim 116 or 117, wherein said ligand is selected from the group consisting of an antibody, a natural ligand, and a synthetic ligand.

121. The method of claim 120, wherein said ligand comprises an antibody, a peptide, a polypeptide, a growth factor, a cytokine, or a chemokine.

122. The method of either claim 116 or 117, wherein said force is selected from the group consisting of a force greater than gravitational force, a hydraulic force, a force generated by transmembrane pressure, a centrifugal force, and a magnetic force.

123. The method of claim 122, wherein the magnetic force is generated by a magnet having a magnetic field strength ranging from between about 200 gauss to about 12,000 gauss at the surface of the magnet.

124. The method of either claim 116 or 117, wherein said solid surface is paramagnetic.

125. The method of either claim 116 or 117, wherein said receptor binding leads to downregulation or suppression of a cellular event.

126. The method of either claim 116 or 117, wherein said receptor binding leads to upregulation or activation of a cellular event.

127. The method of claim 116, wherein said cellular event is receptor mediated signal transduction.

128. The method of claim 94, wherein said force drives concentration or orientation of cell surface moieties.

130. The method of claim 129 wherein said period of time is between about two hours and about forty-eight hours.

131. The method of claim 130 wherein said period of time is between about two hours and about twelve hours.

132. The method of claim 129 wherein said period of time is between about two and about eight days.

133. The method of claim 129 wherein said period of time is between about three and about six days.

134. The method of claim 129 wherein said first and second agent are immobilized on the same solid surface.

135. The method of claim 134 wherein said solid surface is selected from the group consisting of a flat surface, an irregular surface, a spherical surface.

136. The method of claim 129 wherein said solid surface is a bead.

137. The method of claim 135 wherein said irregular surface is a plastic surface.

138. The method of claim 129 wherein said first agent comprises an antibody or fragment thereof that binds CD3 and said second agent comprises an antibody or fragment thereof that binds CD28.

140. A composition comprising the population of T-cells according to claim 139 and a pharmaceutically acceptable excipient.

142. The method of claim 141, wherein said bead has a diameter of between about 80 microns and about 500 microns.

143. The method of claim 142, wherein said bead has a diameter of between about 100 microns and about 400 microns.

144. The method of claim 143, wherein said beads have a diameter of between about 250 microns and 300 microns.

146. The method of claim 141, wherein the first agent comprises an anti-CD3 antibody and the second agent comprises an anti-CD28 antibody.

147. The method of claim 146, wherein the population of T-cells comprises helper T-cells.

149. The method of claim 148, wherein the first agent comprises an anti-CD3 antibody and the second agent comprises an anti-CD28 antibody.

150. The method of claim 149, wherein the population of T-cells comprises helper T-cells.

155. The population of claim 154 wherein said period of contact is from about four hours to about eight days.

156. The population of claim 154 wherein said period of contact is from about four hours to about eight days.

158. The population of claim 157 wherein said T-cells produce peak interleukin-4 levels between about two and about seven days post-primary and secondary signal activation.

159. The population of claim 157 wherein said T-cells produce peak interleukin-2 levels between about two and about seven days post-primary and secondary signal activation.

160. The population of claim 157 wherein said T-cells produce peak tumor necrosis factor-alpha or interferon-gamma levels between about two and about seven days post-primary and secondary signal activation.

163. The method of claim 162, wherein the suspension is a whole blood cell suspension.

165. The method of claim 164, wherein the antibody is a non-specific antibody.

166. A method of treating tumors in a patient, comprising administering to the patient the pharmaceutical composition of claim 161.

167. The method of claim 166, wherein the patient is depleted of endogenous lymphocytes prior to administration.

168. The method of claim 166, wherein the T-cells are derived from the patient prior to activation.

169. The method of claim 166, wherein the T-cell population has been depleted of monocytes.

115. A method of inducing target cell stimulation in vivo, comprising providing paramagnetic particles to an animal, said particles having attached thereto, ligands specific for a target cell surface moiety that induces target cell stimulation;

applying a magnetic field to a discrete region of the animal; and

thereby inducing localization and stimulation of the target cells bound to said particles at said discrete region.

116. A method for inducing receptor polarization in receptor bearing cells, comprising:

a) providing a cell population;

b) contacting said cell population with a solid surface, wherein said solid surface has attached thereto one or more ligands specific for a cell surface receptor present on at least a portion of said cell population; and

c) applying a force that drives cell concentration and cell surface receptor ligation.

117. A method for inducing aggregation of cell surface molecules, comprising:

a) providing a population of cells having a target cell surface molecule;

b) contacting said population of cells with a solid surface, wherein said solid surface has attached thereto a ligand for at least one target cell surface molecule

c) applying a force that drives aggregation of targeted cell surface molecules.

129. A method for inducing a population of T-cells to proliferate, comprising contacting the T-cells with a solid surface for a period of time of between about two hours and about nine days, said solid surface having immobilized thereon a first agent and second agent, and wherein said first agent provides an activation signal and said second agent provides a co-stimulatory signal to said T-cells.

139. A population of T-cells produced by any one of methods 129-138.

141. A method for inducing a population of T-cells to proliferate, comprising:

a. activating a population of T-cells by contacting said T-cells with a first agent immobilized on a bead, wherein said bead has a diameter of between about 20 microns to about 1 millimeter; and

b. stimulating an accessory molecule on the surface of said T-cells with a second agent that binds the accessory molecule, wherein said second agent is immobilized on the bead, and thereby inducing proliferation of the T cells.

145. The method of any one of claims 141-144, where said bead is a paramagnetic bead.

148. The method of any one of claims 141-144, further comprising separating the bead from the T-cells by filtration.

151. A population of activated T-cells, wherein at least a subset of the cells have a phenotype in which CD154 levels peak between about one and about four days post-stimulation.

161. A composition comprising the T-cells of any one of claims 151-160 and a pharmaceutically acceptable excipient.

152. A population of activated T-cells, wherein said population comprises greater than about 60% CD4⁺ T-cells.

153. A population of activated T-cells, wherein said population comprises at least about 70% CD4⁺ T-cells.

154. A population of activated T-cells previously stimulated to proliferate by contact with anti-CD3 and anti-CD28 antibodies or fragments thereof for a period of about two hours to about nine days.

157. A population of activated T-cells wherein said T-cells have previously been induced to proliferate by contact with an immobilized first agent and second agent for a period of between about two hours and about nine days, wherein said first agent provides an activation signal and said second agent provides a co-stimulatory signal to said T-cells.

162. A method for depleting monocytes from a suspension comprising T-cells and monocytes, comprising contacting the suspension with para-magnetic beads, wherein said beads have a diameter of between about 2.8 μ

m and about 10 μ

m, and subsequently separating the monocytes from the suspension by magnetic attraction.

164. The method of any one of claims 162 and 163, wherein the bead has at least one antibody attached thereto.

170. A population of stimulated T-cells having CD154 expression levels at least 10% higher than T-cells that have been stimulated with anti-CD3 and anti-CD28 antibodies in the absence of simultaneous concentration and stimulation, wherein said levels are determined by flow cytometry between days 1 to 4 following T-cell stimulation.

171. A population of stimulated T-cells having CD25 expression levels at least 10% higher than T-cells that have been stimulated with anti-CD3 and anti-CD28 antibodies in the absence of simultaneous concentration and stimulation, wherein said levels are determined by flow cytometry between days 1 to 4 following T-cell stimulation.