# US 2026/0094716 A1 · Biomarker for transplantation tolerance induced by apoptotic donor leukocytes

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- Number: US 2026/0094716 A1
- Document ID: US20260094716A1
- Status: Published application
- Kind: A1
- Application number: 19/342,280
- Filed: 2025-09-26
- Published: 2026-04-02
- Earliest claimed: 2024-09-27
- Assignees: Regents of the University of Minnesota
- Applicants: Regents of the University of Minnesota
- Inventors: Amar Singh; Bernhard J. Hering; Sabarinathan Ramachandran
- Web page: https://patentgrep.com/patent/US20260094716A1

## CPC

- G16H 50/20: for computer-aided diagnosis, e.g. based on medical expert systems
- G01N 33/505: {involving T-cells}
- G01N 33/56972: {White blood cells}
- G01N 2800/245: Transplantation related diseases, e.g. graft versus host disease
- G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
- G16H: HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
- 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)

## Abstract

In certain embodiments, the present invention provides methods of identifying and treating a transplant recipient patient having transplantation tolerance induced by apoptotic donor leukocytes infused under cover of transient immunotherapy.

## Claims

### Claim 1

A method of identifying a transplant recipient patient having transplantation tolerance induced by donor antigen administered under cover of transient immunotherapy, comprising:
(a) assaying a first blood sample from the patient to detect a baseline frequency of target cells, wherein the first blood sample is obtained pre-tolerization, pre-transplant, and pre-initiation of transient immunotherapy,
(b) assaying a second blood sample from the patient to detect a post-procedure frequency of target cells, wherein the second sample is obtained post-tolerization, post-transplant, and post-initiation of transient immunotherapy; and
(c) identifying the patient as having transplantation tolerance/immune acceptance induced by the donor antigen when the post-procedure frequency is at least 1.5-fold greater than the baseline frequency,
wherein the target cells are T regulatory (Treg) cells having one or more combination of markers HelioshiUSP22hi, CCR2hiTIGIThi, CCR2hiTIGIThiAreg+, and/or CD4+CD25+CD127−FOXP3+; and/or
wherein the target cells are T regulatory Type 1 (Tr1) cells having one or more combination of markers PD-1hiCD69hiHelioshi, TIGIThi CCR2hiST2hi GrzB+FOXP3−, Areghi TIGIThi CCR2hi GrnzB+ ST2hi FOXP3− and/or CD4+CD49b+LAG3+; and/or
wherein the target cells are exhaust CD4 T cells (Tex) having markers PD-1+EOMES+ CD127hi Helios+TOX+TCF-1−.

### Claim 2

A method, comprising:
(a) obtaining a first blood sample from a transplant recipient patient to detect a baseline frequency of target cells, wherein the first blood sample is obtained pre-tolerization, pre-transplant, and pre-initiation of transient immunotherapy,
(b) obtaining a second blood sample from the patient to detect a post-procedure frequency of target cells, wherein the second sample is obtained post-tolerization, post-transplant, and post-initiation of transient immunotherapy,
(c) assaying the first and second blood samples to detect levels of target cells before and after tolerization, and
(d) identifying the transplant recipient patient as having transplantation tolerance/immune acceptance induced by donor antigens infused under cover of transient immunotherapy when the post-procedure frequency is at least 1.5-fold greater than the baseline frequency,
wherein the target cells are T regulatory (Treg) cells having one or more combination of markers HelioshiUSP22hi, CCR2hiTIGIThi, CCR2hiTIGIThiAreg+, and/or CD4+CD25+CD127−FOXP3+; and/or
wherein the target cells are T regulatory Type 1 (Tr1) cells having one or more combination of markers PD-1hiCD69hiHelioshi, TIGIThi CCR2hiST2hiGrnzB+FOXP3−, Areghi TIGIThi CCR2hi GrnzB+ST2hi FOXP3− and/or CD4+CD49b+LAG3+; and/or
wherein the target cells are exhaust CD4 T cells (Tex) having one or more combination of markers PD-1+EOMES+ CD127hi Helios+TOX+TCF-1−,
wherein the identification is with a multiparameter single flowcytometry panel comprising 12 binding reagents that specifically recognize CD4, FOXP3, CD49b, LAG-3, PD-1, Helios/USP22, CCR2, ST2, TIGIT, TOX/EOMES, CD127, and Areg to determine these regulatory and exhaust subsets.

### Claim 3 (depends on 1)

The method of claim 1, wherein the donor antigens are apoptotic donor leukocytes (ADLs), donor-specific transfusion (DST) nanoparticles conjugated with donor peptides or encapsulating donor peptides, and/or apoptotic recipient leukocytes conjugated with donor peptides.

### Claim 4 (depends on 1)

A method of treating a transplant recipient patient, the method comprising:
(a) identifying the transplant recipient patient as using the method of claim 1, and
(b) treating the transplant recipient patient by ceasing to administer immunosuppressants.

### Claim 5 (depends on 4)

The method of claim 4, wherein the Tr1 cells exhibit indirect specificity for at least one mismatched donor MHC class I peptide.

### Claim 6 (depends on 1)

The method of claim 1, wherein the target cells are blood cells.

### Claim 7 (depends on 1)

The method of claim 1, wherein the patient has received two peritransplant, intravenous infusions of apoptotic donor leukocytes.

### Claim 8 (depends on 1)

The method of claim 1, wherein the transient immunotherapy comprises at least one immunosuppressant.

### Claim 9 (depends on 8)

The method of claim 8, wherein the immunosuppressant is an inhibitor of CD40:CD40L co-stimulation, an mTOR inhibitor, and concomitant anti-inflammatory therapy targeting proinflammatory cytokines.

### Claim 10 (depends on 1)

The method of claim 1, wherein the transient immunotherapy comprises an anti-inflammatory agent.

### Claim 11 (depends on 1)

The method of claim 1, wherein the transplant is an allotransplant.

### Claim 12

A kit comprising a panel of binding reagents, wherein the reagents individually specific for CD4, CD127, FOXP3 (i.c.), Areg, CD49b, LAG-3, ST2, CCR2, PD-1, TOX/EOMES (i.c.), TIGIT, and Helios and/or USP22.

### Claim 13 (depends on 12)

The kit of claim 12, wherein the binding reagents are antibodies.

### Claim 14 (depends on 12)

The kit of claim 12, wherein the kit is a single-tube format.

### Claim 15 (depends on 14)

The kit of claim 14, wherein the kit is compatible with BD/FACS and Cytek Aurora.

### Claim 16 (depends on 14)

The kit of claim 14, further comprising preloaded gating templates for Treg, Tr1, Tex Tol modules.

### Claim 17 (depends on 16)

The kit of claim 16, wherein the preloaded gating templates for the Treg module comprises % CD4+FoxP3+CD127low×USP22/Helios, CCR2 MFI.

### Claim 18 (depends on 16)

The kit of claim 16, wherein the preloaded gating templates for the Tr1, module comprises % CD4+FOXP3−CD49b+LAG-3+×AREG expression.

### Claim 19 (depends on 16)

The kit of claim 16, wherein the preloaded gating templates for the Tex Tol module % CD4+PD-1+CD127hi TIGIT+TOX+ (±EOMES) vs TIGIT-TOX-PD-1+/CD4 (activated T cell).