B-cell antigen presenting cell assay
View Patent ↗Disclosed herein are methods for diagnosing or predicting B-cell rejection in a subject. In one example, for assessing transplant rejection, the method includes determining an antigen presenting index by comparing uptake of a donor antigen to uptake of a reference antigen in a biological sample obtained from the subject. In another example, for assessing GVHD, the method includes determining an antigen presenting index by comparing uptake of a recipient antigen to uptake of a reference antigen in a biological sample obtained from the subject.
1. A method, comprising:
obtaining antigen presenting cells (APCs);
contacting a first sample of the obtained APCs with a donor antigen from a donor under conditions sufficient to induce uptake of the donor antigen;
contacting a second sample of the obtained APCs with a third-party antigen under conditions sufficient to induce uptake of the third-party antigen; and measuring uptake of the donor antigen in the first sample and uptake of the third-party antigen in the second sample by flow cytometry.
2. The method of claim 1 , wherein the first sample and the second sample are peripheral blood lymphocytes or peripheral blood leukocytes.
3. The method of claim 1 , further comprising expressing donor antigen uptake relative to third party antigen uptake following measuring uptake of the donor antigen in the first sample and uptake of the third-party antigen in the second sample by flow cytometry.
4. The method of claim 1 , wherein the method is used to titrate immunosuppression within a subject or evidence effectiveness of an immunosuppressive regimen.
5. The method of claim 4 , wherein the subject is an organ recipient.
6. A method, comprising:
obtaining a first sample containing antigen presenting cells (APCs) from a subject in need of or having received an organ transplant;
contacting the first sample comprising APCs obtained from the subject in need of or having received an organ transplant with a donor antigen from a donor under conditions sufficient to induce uptake of the donor antigen;
obtaining a second sample containing antigen presenting cells (APCs) from the subject in need of or having received an organ transplant;
contacting the second sample comprising APCs obtained from the subject in need of or having received an organ transplant with a third-party antigen under conditions sufficient to induce uptake of the third-party antigen;
detecting a plurality of biomarkers following treatment of the donor antigen with the first sample and the third-party antigen with the second sample, wherein the plurality of biomarkers includes at least CD27CD5 and CD 10; and comparing the plurality of biomarkers detected following treatment with donor antigen to those detected following treatment with the third-party antigen to determine an antigen presenting index.
7. The method of claim 1 , wherein the donor antigen or the third-party antigen comprises donor cells, cell lysate, an antigenic peptide, an antigenic peptide labeled with a detectable label, such as a fluorochrome, or a combination thereof.
8. The method of claim 1 , wherein the donor antigen and third party antigen are labeled with carboxyflouresciensuccinimyldiester.
9. The method of claim 1 , further comprising labeling sample donor antigen and third-party antigen with a fluorescent molecule prior to contacting the first sample with the donor antigen or the second sample with the third-party antigen.
10. The method of claim 6 , wherein the organ is bone marrow or a solid organ.
11. The method of claim 10 , wherein the solid organ is selected from the group consisting of liver, intestine, kidney, heart, lung, pancreas, skin, and combinations thereof.
12. The method of claim 6 , wherein the method is used to titrate immunosuppression within the subject or evidence the effectiveness of an immunosuppressive regimen.
13. The method of claim 6 , wherein the subject is an organ recipient.
14. The method of claim 6 , further comprising labeling the donor antigen and third-party antigen with a fluorescent molecule prior to contacting the first sample with the donor antigen or the second sample with the third-party antigen.
15. The method of claim 6 , wherein the donor antigen or the third-party antigen comprises donor cells, cell lysate, an antigenic peptide, an antigenic peptide labeled with a detectable label, such as a fluorochrome, or a combination thereof.
16. A method, comprising:
obtaining antigen presenting cells (APCs);
labeling a donor antigen and a third-party antigen with a fluorescent molecule;
contacting a first sample of the obtained APCs with fluorescently labeled donor antigen from a donor under conditions sufficient to induce uptake of the donor antigen;
contacting a second sample of the obtained APCs with fluorescently labeled third-party antigen under conditions sufficient to induce uptake of the third-party antigen; and
measuring uptake of the donor antigen in the first sample and uptake of the third-party antigen in the second sample by flow cytometry; and
expressing donor antigen uptake relative to third party antigen uptake.
17. The method of claim 16 , wherein the donor antigen or the third-party antigen comprises donor cells, cell lysate, an antigenic peptide, an antigenic peptide labeled with a detectable label, such as a fluorochrome, or a combination thereof.