QUALITY ASSAYS FOR ANTIGEN PRESENTING CELLS
The present invention provides methods for evaluating the quality of a preparation of antigen presenting cells, such as dendritic cells. Assays for antigen-independent co-stimulation of T cells and for presentation of predetermined antigen by APCs are provided.
1 . A method for determining antigen-independent, co-stimulatory activity of antigen presenting cells (APCs), comprising:
providing T cells having a known functional activity and being substantially free of co-stimulatory activity:
providing a sample of APCs of unknown co-stimulatory activity;
contacting the T cells with a sub-optimal concentration of an antigen-mimetic agent;
contacting the T cells with the sample of APCs of unknown co-stimulatory activity;
determining the activation of the T cells contacted with the antigen-mimetic agent and the sample of APCs; and
comparing the determined activation of the T cells with a standard activation index for the T cells to determine the co-stimulatory activity of the APCs.
2 . The method of claim 1 , wherein the T cells and the APCs are syngeneic.
3 . The method of claim 1 , wherein the T cells and the APCs are allogenic.
4 . The method of claim 1 , wherein the antigen-mimetic agent is a CD3 binding agent, a plant lectin or a mitogen.
5 . The method of claim 4 , wherein the CD3 binding agent is anti-CD3 antibody.
6 . The method of claim 1 , wherein the APCs are dendritic cells.
7 . The method of claim 6 , wherein the dendritic cells are mature dendritic cells derived from immature dendritic cells by contacting ex vivo with a dendritic cell maturation agent.
8 . The method of claim 6 , wherein the dendritic cells are immature dendritic cells.
9 . The method of claim 1 , wherein the T cells have been substantially depleted of peripheral blood mononuclear cells expressing CD14, CD54, CD80, CD83 or CD86 molecules on their cell surface.
10 . The method of claim 1 , wherein the T cells have been substantially depleted of peripheral blood mononuclear cells expressing MHC class II molecules on their cell surface.
11 . The method of claim 1 , wherein the activation of the T cells is determined by 3 H-thymidine uptake assay.
12 . The method of claim 1 , wherein the activation of the T cells is determined by assaying T cell cytokine production.
13 . The method of claim 12 , wherein the assayed T cell cytokine production is IFNγ or Interleukin 2 production.
14 . The method of claim 12 , wherein the assayed T cell cytokine production is extracellular cytokine production.
15 . The method of claim 12 , wherein the assayed T cell cytokine production is intracellular cytokine production.
16 . The method of claim 1 , wherein the activation of T cells is determined by detecting the modulation of expression of a T cell activation marker.
17 . The method of claim 16 , wherein the T cell activation marker is CD25, CD69, CD44 or CD125.
18 . The method of claim 16 , wherein the T cell activation marker is detected using labeled antibody capable of binding to the T cell activation marker.
19 . The method of claim 1 , wherein comparing the determined activation with the standard activation index includes comparing the determined T cell activation with activation of the T cells contacted with the sample of dendritic cells alone to determine the quality of the dendritic cells.
20 . The method of claim 1 , wherein the standard activation index is a threshold value.
21 . The method of claim 1 , wherein the standard activation index is a range of values, each value associated with a predetermined quality of dendritic cells.
22 . The method of claim 1 , further comprising determining presentation of a predetermined antigen by the APCs.
23 . The method of claim 22 , wherein presentation of the predetermined antigen is determined by Western blotting, flow cytometry or activation of antigen-specific T cells.
24 . A method for determining antigen-independent co-stimulatory activity of a preparation of dendritic cells, comprising:
contacting a first quantity of T cells, which are substantially free of co-stimulatory activity and have a known functional activity, with a suboptimal quantity of an antigen-mimetic agent and with a first sample of a dendritic cell preparation of unknown co-stimulatory activity:
determining a first activation value for the first quantity off cells;
contacting a second quantity off cells with a second sample of the dendritic cell preparation or the suboptimal quantity of the antigen-mimetic agent;
determining a second activation value for the second quantity of T cells; and
comparing the first and second activation values to determine the co-stimulatory activity of the dendritic cell preparation.
25 . The method of claim 24 , wherein the T cells are allogenic with respect to the dendritic cell preparation.
26 . The method of claim 24 , wherein the T cells are syngeneic with respect to the dendritic cell preparation.
27 . The method of claim 24 , wherein the antigen-mimetic agent is anti-CD3 antibody, a plant lectin or a mitogen.
28 . The method of claim 24 , further comprising determining presentation of a predetermined antigen by the dendritic cells.
29 . The method of claim 28 , wherein presentation of the predetermined antigen is determined by Western blotting, flow cytometry or activation of antigen-specific T cells.
30 . A method for determining the quality of a preparation of dendritic cells, comprising:
(1) providing a dendritic cell preparation of unknown co-stimulatory activity and unknown antigen presenting ability for a predetermined antigen;
(2) determining the co-stimulatory activity of the dendritic cell preparation, said determination of co-stimulatory activity comprising
(a) providing T cells of known functional activity and substantially free of co-stimulatory activity;
(b) contacting the T cells with a suboptimal quantity of an antigen-mimetic agent and with a first sample of the dendritic cell preparation;
(c) determining the activation of the contacted T cells; and
(d) comparing the determined activity of the contacted T cells with the standard activation index for the T cells to the determined co-stimulatory activity of the dendritic cell preparation;
(3) determining presentation of the predetermined antigen by the preparation of dendritic cells, said determination of presentation comprising:
(a) contacting a second sample of the dendritic cell preparation with the predetermined antigen; and
(b) determining the amount of predetermined antigen presented by the dendritic cells; and
(4) determining the quality of the dendritic cell preparation based on the determined co-stimulatory activity and determined antigen-specific presentation of the predetermined antigen.
31 . The method of claim 30 , wherein the antigen-mimetic agent is a CD3 binding agent, a plant lectin or a mitogen.
32 . The method of claim 31 , wherein the CD3 binding agent is anti-CD3 antibody.
33 . The method of claim 30 , wherein the dendritic cells are mature dendritic cells derived from immature dendritic cells by contacting ex vivo with a maturation agent.
34 . The method of claim 30 , wherein the dendritic cells are immature dendritic cells.
35 . The method of claim 30 , wherein the T cells have been substantially depleted of peripheral blood mononuclear cells expressing MHC Class II, CD14, CD54, CD80, CD83 or CD86 molecules on their cell surface.
36 . The method of claim 30 , wherein the activation of the T cells is determined by 3 H-thymidine proliferation assay.
37 . The method of claim 30 , wherein the activation of the T cells is determined by assaying T cell cytokine production.
38 . The method of claim 37 , wherein the T cell cytokine production is IFNγ or Interleukin 2 production.
39 . The method of claim 37 , wherein the T cell cytokine production is extracellular cytokine production.
40 . The method of claim 37 , wherein the T cell cytokine production is intracellular cytokine production.
41 . The method of claim 30 , wherein the activation of T cells is determined by expression of at least one T cell activation marker.
42 . The method of claim 41 , wherein the T cell activation marker is CD25, CD69, CD44 or CD125.
43 . The method of claim 41 , wherein the T cell activation marker is detected using labeled antibody capable of binding to the T cell activation marker.
44 . The method of claim 30 , wherein determining the co-stimulatory activity includes comparison of the determined T cell activation with a standard activation index for the T cells.
45 . The method of claim 44 , wherein the standard activation index is a threshold value.
46 . The method of claim 44 , wherein the standard activation index is range of values, the values associated with different predetermined co-stimulatory activities.
47 . The method of claim 30 , wherein presentation of the predetermined antigen is determined by Western blotting, flow cytometry or activation of antigen-specific T cells.