# US 10,371,696 B2 · Multiparametric method for assessing immune system status

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- Number: US 10,371,696 B2
- Document ID: US10371696B2
- Status: Granted
- Kind: B2
- Application number: 15/678,994
- Filed: 2017-08-16
- Published: 2017-11-30
- Granted: 2019-08-06
- Earliest claimed: 2005-06-03
- Assignees: University of Pittsburgh—Of the Commonwealth System of Higher Education
- Applicants: University of Pittsburgh—Of the Commonwealth System of Higher Education
- Inventors: Rakesh Sindhi
- Web page: https://patentgrep.com/patent/US10371696B2

## CPC

- G01N 33/5091: {for testing the pathological state of an organism}
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor
- G01N 33/56972: {White blood cells}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

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.

## Claims

### Claim 1

A method of assessing the reaction of a patient's immune system to an antigen stimulant, comprising:
contacting a first sample comprising T-cells obtained from the patient with a viable antigen under conditions sufficient to induce T cell proliferation by the viable antigen;
contacting a second sample comprising T-cells obtained from the patient with a viable third-party antigen under conditions sufficient to induce T- cell proliferation by the viable third-party antigen;
measuring T cell proliferation induced by the viable antigen in the first sample by detecting a plurality of markers in the first sample following contacting the first sample with the viable antigen, and at least one additional marker, respectively, wherein the plurality of makers comprises CD154, CD45RO and CD8;
measuring T cell proliferation induced by the viable third party antigen in the second sample by detecting the plurality of markers in the second sample following contacting the second sample with the third-party antigen, and the at least one additional marker, respectively; and
comparing T cell proliferation induced by the viable antigen in the first sample to T cell proliferation or an immune response induced by the viable third-party antigen in the second sample,
thereby determining that the patient's immune system responds to the viable third party antigen and that the viable third party antigen induces reaction of the patient's immune system.

### Claim 2 (depends on 1)

The method of claim 1, wherein the additional marker is PD-1, PD-L1, ICOS, or IFN-gamma.

### Claim 3 (depends on 2)

The method of claim 2, further comprising assessing viability of T-cells in the first sample and viability of T-cells in the second sample.

### Claim 4 (depends on 3)

The method of claim 3, wherein assessing viability of T-cells comprises use of a cell viability dye.

### Claim 5 (depends on 4)

The method of claim 4, wherein the cell viability dye is ethidium monoazide (EMA) or 7-amino-actinomycin D (7-AAD).

### Claim 6 (depends on 1)

The method of claim 1, wherein the first sample and the second sample are peripheral blood lymphocytes or peripheral blood leukocytes.

### Claim 7 (depends on 1)

The method of claim 1, wherein the patient is a human.

### Claim 8 (depends on 7)

The method of claim 7, wherein the patient is an adult.

### Claim 9 (depends on 7)

The method of claim 7, wherein the patient is a child.

### Claim 10 (depends on 1)

The method of claim 1, wherein the plurality of markers further comprises one or more of CD45RA, CD27, CD62, CD28, CCR4, CCR5, CD34, CD134, HLA-DR, CD11c, CD123, CD3, CD4, CD16/56, CD19, CD14, ILT3, CD25, 7-AAD, CD69, CD71, CD86, IL-2, TNF alpha, CFSE, TGF-beta, Annexin V, CFSE, a caspase, IL10, IL-12, IL-2, CCR7, CD56, and CD54.

### Claim 11 (depends on 1)

The method of claim 1, wherein the viable antigen or the viable third party antigen comprise an antigenic peptide, an antigenic peptide labeled with a flourochrome, or a combination thereof.

### Claim 12 (depends on 1)

The method of claim 1, wherein the T-cells are T-helper or T-cytotoxic lymphocytes.

### Claim 13 (depends on 1)

The method of claim 1, wherein the first sample and the second sample are further contacted with an immunomodulatory drug.

### Claim 14 (depends on 13)

The method of claim 13, wherein the immunomodulatory drug is an anti-rejection agent.

### Claim 15 (depends on 1)

The method of claim 1, wherein the viable antigen is an autoantigen, viral peptide, bacterial peptide, antigen attached to tetramer, or an antigenic peptide attached to a monomeric protein, polymeric protein or peptide.

### Claim 16 (depends on 1)

The method of claim 1, wherein the the at least one additional marker is-CTLA4, IFN-gamma.

### Claim 17 (depends on 1)

The method of claim 1, wherein the at least one additional marker is PD-1 or PD-L1.

### Claim 18 (depends on 1)

The method of claim 1, wherein the at least one additional marker is IFN-gamma.