Screening immunomodulatory agents by CTLA-4 upregulation
Screens that directly or indirectly measure or qualitatively observe CTLA-4 expression or function by animals or cell cultures, or that indirectly measure dependence of an agent on CTLA-4 by comparison to animals or cell cultures that lack CTLA-4, identify agents that are useful in immunosuppression and the generation of immunologic tolerance for the prevention and treatment of transplant rejection, autoimmune and inflammatory diseases.
1. A method for screening for an agent useful in promoting immunosuppression or immunologic tolerance in a mammal comprising:
(a) administering a test agent to an experimental animal;
(b) obtaining a biological sample comprising T-cells from the animal and a sample from a control animal of the same species;
(c) examining CTLA-4 expression on both the T-cells obtained from the experimental animal to which the test agent has been administered and the T-cells obtained from the control animal, by measuring the levels of at least one of cell surface CTLA-4, cytoplasmic CTLA-4, or CTLA-4 mRNA in the T-cells, and comparing the levels of CTLA-4 on the T-cells in the control and experimental animals;
(d) examining T-cell activation by measuring T-cell activation in the T-cells obtained from the experimental animal to which the test agent has been administered, by measuring upregulation of T-cell markers, T-cell proliferation, and entry of T-cells into cell cycle; and
(e) identifying whether the administered test agent can be used to promote immunosuppression or immunologic tolerance in a mammal by determining if the administered test agent increases levels of CTLA-4 on T-cells in the sample from the experimental animal as compared to the levels of CTLA-4 on T-cells in the control animal sample;
wherein the increase of CTLA-4 on the T-cells in the experimental sample occurs without observing T-cell activation.
2. A method according to claim 1 wherein an immunoassay is employed to determine the levels of CTLA-4 in the samples.
3. A method according to claim 2 which employs polyclonal, monoclonal or fusion phage antibodies to CTLA-4.
4. A method according to claim 1 wherein protein analysis is used to determine the levels of CTLA-4 in the samples.
5. A method according to claim 1 wherein the biological sample comprises cells that are fixed, permeabilized, and incubated with an anti-CTLA-4 tagged antibody prior to comparison.
6. A method according to claim 1 wherein the animals are mice or non-human primates.
7. A method for screening for an agent useful in promoting immunosuppression or immunologic tolerance in a mammal comprising:
(a) adding a test agent to a culture of mammalian cells comprising T-cells;
(b) measuring the levels of at least one of cell surface CTLA-4, cytoplasmic CTLA-4, or CTLA-4 mRNA in the T-cells in the culture of mammalian cells to which the test agent has been added and in T-cells in an untreated culture of mammalian cells comprising T-cells used as a control, and comparing the two;
(c) examining T-cell activation by measuring T-cell activation in the culture of mammalian cells comprising T-cells to which the test agent has been added, by measuring upregulation of T-cell markers, T-cell proliferation, and entry of T-cells into cell cycle, and
(d) identifying whether the administered test agent can be used to promote immunosuppression or immunologic tolerance in a mammal, by determining if the administered test agent increases levels of CTLA-4 on T-cells in the culture of mammalian cells to which the test agent has been added as compared to the levels of CTLA-4 on T-cells in the untreated culture of mammalian cells;
wherein the increase of CTLA-4 on T-cells in the culture of mammalian cells to which the test agent has been added occurs without observing T-cell activation.
8. A method according to claim 7 wherein an immunoassay is employed to determine the levels of CTLA-4 in the samples.
9. A method according to claim 8 which employs polyclonal, monoclonal or fusion phage antibodies to CTLA-4.
10. A method according to claim 7 wherein protein analysis is used to determine the levels of CTLA-4 in the samples.
11. A method according to claim 7 where the cells are fixed, permeabilized, and incubated with anti-CTLA-4 tagged antibody prior to comparison.
12. A method according to claim 7 where the cells are derived from mice, non-human primates, or human subjects.