Methods of identifying compounds that upmodulate T cell activation in the presence of a PD-1 mediated signal
The invention provides isolated nucleic acids molecules, designated PD-L2 nucleic acid molecules, which encode novel B7-related molecules which are ligands for PD-1. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing PD-L2 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a PD-L2 gene has been introduced or disrupted. The invention further provides isolated PD-L2 polypeptides, fusion proteins, antigenic peptides and anti-PD-L2 antibodies. The invention still further provides methods for promoting or inhibiting the interaction between PD-L2 and PD-1. The invention further provides methods of identifying compounds that upmodulate T cell activation in the presence of a PD-1-mediated signal. Diagnostic and treatment methods utilizing compositions of the invention are also provided.
1. A method of identifying a compound which upmodulates T cell activation at both a low and high antigen concentration comprising:
a) contacting a T cell expressing PD-1 with a test compound at a low concentration of antigen to which the T cell is specific in the presence of a costimulatory signal and a PD-1 ligand having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5, wherein the low concentration of antigen is minimally sufficient to induce T cell proliferation to levels above background in the absence of a PD-1-mediated signal and wherein the low concentration of antigen is 0.001 μg/ml−0.01 μg/ml;
b) determining the ability of the test compound to upmodulate T cell proliferation or cytokine production at the low antigen concentration;
c) contacting a T cell expressing PD-1 with the test compound at a high antigen concentration in the presence of a costimulatory signal and a PD-1 ligand having the amino acid sequence selected from the group consisting of SEQ ID NO:2 or SEQ ID NO:5, wherein the high antigen concentration is 0.1 μg/ml−1.0 μg/ml; and
d) determining the ability of the test compound to upmodulate T cell proliferation or cytokine production at the high antigen concentration, wherein the ability of the compound to upmodulate T cell proliferation or cytokine production at both the low and high antigen concentration identifies the compound as one that upmodulates T cell activation at a both a low and high antigen concentration.
2. A method of identifying a compound which upmodulates T cell activation at both a low and high antigen concentration comprising:
a) contacting a T cell expressing PD-1 with a test compound at a low concentration of antigen to which the T cell is specific in the presence of a costimulatory signal and a PD-1 ligand having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5, wherein the low concentration of antigen is minimally sufficient to induce T cell proliferation to levels above background in the absence of a PD-1-mediated signal and wherein the low concentration of antigen is 0.001 μg/ml−0.01 μg/ml;
b) determining the ability of the test compound to upmodulate T cell proliferation or cytokine production at the low antigen concentration;
c) contacting a T cell expressing PD-1 with the test compound at a high antigen concentration in the presence of a costimulatory signal and a PD-1 ligand having the amino acid sequence selected from the group consisting of SEQ ID NO:2 or SEQ ID NO:5, wherein the high antigen concentration is 0.01 μg/ml−1.0 μg/ml and wherein the high antigen concentration is always greater than the low antigen concentration; and
d) determining the ability of the test compound to upmodulate T cell proliferation or cytokine production at the high antigen concentration, wherein the ability of the compound to upmodulate T cell proliferation or cytokine production at both the low and high antigen concentration identifies the compound as one that upmodulates T cell activation at a both a low and high antigen concentration.
3. The method of claim 1 or 2 , wherein T cell proliferation or cytokine production is measured at the low antigen concentration.
4. The method of claim 1 or 2 , wherein cytokine production is measured at the high antigen concentration.
5. A method of identifying a compound which overcomes a PD-1-mediated negative signal, comprising:
a) contacting a T cell expressing PD-1 with a test compound at low concentration of antigen to which the T cell is specific in the presence of a costimulatory signal and a PD-1 ligand having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5, wherein the low antigen concentration is minimally sufficient to induce T cell proliferation to levels above background in the absence of a PD-1-mediated signal;
b) contacting a T cell expressing PD-1 with a test compound at low concentration of antigen to which the T cell is specific in the presence of a costimulatory signal and in the absence of a PD-1 ligand having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5;
c) determining the ability of the test compound to upmodulate T cell proliferation or cytokine production under each condition;
d) comparing the ability of the compound to upmodulate T cell proliferation or cytokine production in the presence and the absence of the PD-1 ligand, wherein the ability of the compound to upmodulate T cell proliferation or cytokine production in the presence of the PD-1 ligand and not in the absence of the PD-1 ligand identifies the compound as one that overcomes a PD-1-mediated negative signal.
6. A method of identifying a compound which overcomes a PD-1-mediated negative signal, comprising:
a) contacting a T cell expressing PD-1 with a test compound and a weak TCR:CD28-mediated signal and a PD-1 ligand having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5, wherein the weak TCR:CD28-mediated signal is minimally sufficient to induce T cell proliferation to levels above background in the absence of a PD-1-mediated signal;
b) contacting a T cell expressing PD-1 with a test compound and a weak TCR:CD28-mediated signal in the absence of a PD-1 ligand having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5;
c) determining the ability of the test compound to upmodulate T cell proliferation or cytokine production under each condition;
d) comparing the ability of the compound to upmodulate T cell proliferation or cytokine production in the presence and the absence of the PD-1 ligand, wherein the ability of the compound to upmodulate T cell proliferation or cytokine production in the presence of the PD-1 ligand and not in the absence of the PD-1 ligand identifies the compound as one that overcomes a PD-1-mediated negative signal.
7. The method of claim 5 or 6 , wherein the cells express high levels of PD-1 and low levels of CD28.
8. The method of claim 5 or 6 , wherein the ability of the compound to upmodulate T cell proliferation is measured.
9. The method of claim 5 or 6 , wherein the ability of the compound to upmodulate cytokine production is measured.
10. The method of claim 9 , wherein upmodulation of cytokine production is indicated by an increase in cytokine mRNA expression.
11. The method of claim 9 , wherein the cytokine is a Th1 cytokine.
12. The method of claim 9 , wherein the cytokine is a Th2 cytokine.
13. The method of claim 9 , wherein the cytokine is selected from the group consisting of IL-2, IL-10 and IFN-y.