Multiparameteric method for assessing immune system status
View Patent ↗The invention provides a multiparametric method of assessing the reaction of a patient's immune system to a test subject. The invention compares a patient sample reacted with a test sample and a third party sample and combines the assessments of the multiple parameters to correlate the test reaction with a clinical event.
1. A method of assessing organ rejection, comprising:
contacting a first sample comprising T-cells obtained from a patient in need of or having received an organ transplant from a donor with a viable donor antigen from a donor under conditions sufficient to induce T-cytotoxic memory cell proliferation by the viable donor antigen;
contacting a second sample comprising T-cells obtained from the patient in need of or having received an organ transplant with a viable third-party antigen under conditions sufficient to induce T-cytotoxic memory cell proliferation by the viable third-party antigen;
measuring T-cytotoxic memory cell proliferation or an inflammatory response induced by the viable donor antigen by detecting CD8, CD154 and CD45RO levels on T-cells in the first sample following contacting the first sample with the viable donor antigen;
measuring T-cytotoxic memory cell proliferation or its inflammatory response induced by the viable third party antigen by detecting CD8, CD154 and CD45RO levels on T-cells in the second sample following contacting the second sample with the viable third-party antigen; and
determining the ratio of T-cytotoxic memory cell proliferation or an inflammatory response induced by viable donor antigen in the first sample to T-cytotoxic memory cell proliferation or an inflammatory response induced by viable third-party antigen in the second sample, wherein a ratio of greater than one indicates organ rejection.
2. The method of claim 1 , further comprising assessing viability of T-cells in the first sample and viability of T-cell in the second sample.
3. The method of claim 2 , wherein assessing viability of T-cells comprises use of a cell viability dye.
4. The method of claim 2 , wherein the cell viability dye is ethidium monoazaide (EMA) or 7-amino-actinomycin D (7-AAD).
5. The method of claim 1 , wherein the first sample and the second sample are peripheral blood lymphocytes or peripheral blood leukocytes.
6. The method of claim 1 , wherein the organ is selected from the group consisting of bone marrow and a solid organ.
7. The method of claim 6 , wherein the solid organ is selected from the group consisting of a liver, intestine, kidney, heart, lung, pancreas, skin, and sections and combinations thereof.
8. The method of claim 1 , wherein the patient is an organ recipient.
9. The method of claim 1 , wherein the patient is a human.
10. The method of claim 9 , wherein the patient is a child.
11. The method of claim 1 , wherein measuring T-cytotoxic memory cell proliferation or an inflammatory response induced by the viable donor antigen further comprises detecting expression of at least one of CD25, CD3, CD27, CD4, 7-AAD, CD69, CD71, CD86 IFNgamma, IL-2, TNF alpha, CD45RA, CCR7 and CD54 in the first sample following contacting the first sample with the viable donor antigen; and measuring T-cytotoxic memory cell proliferation or its inflammatory response induced by the viable third-party antigen further comprises detecting expression of at least one of CD25, CD3, CD27, CD4, 7-AAD, CD69, CD71, CD86 IFNgamma, IL-2, TNF alpha, CD45RA, CCR7 and CD54 in the second sample following contacting the second sample with the viable third-party antigen.
12. The method of claim 1 , wherein the viable donor antigen or the viable third-party antigen comprises donor cells, an antigenic peptide, an antigenic peptide labeled with a fluorochrome, or a combination thereof.