# Single cell analysis of transplant rejection

> **NIH NIH R21** · CINCINNATI CHILDRENS HOSP MED CTR · 2020 · $199,026

## Abstract

The ultimate goal of organ transplantation is to achieve sustained allograft acceptance with
minimal broad-based immunosuppression and off-target toxicity. Unfortunately, current
approaches using corticosteroids (CCS) and calcineurin inhibitors (CNIs) have substantial
nephrotoxicity. Our Co-Investigator, Dr. Woodle has shown that when combined with T cell-
depleting induction, belatacept, a biologic that inhibits T cell co-stimulation, provides
immunosuppression that is similar to CNI/CCS-based regimens without toxicity. However, the
remaining rejection under belatacept is more difficult to control and requires further
understanding. To this end, we have developed the ability to analyze rejection using single cell
RNA sequencing of cells isolated from tissue biopsies. Further, our novel approach will also
specifically identify the allo-reactive cells in the rejecting allograft. This will be done by performing
a functional mixed lymphocyte reaction (MLR) assay using PBMCs and identify the T cell receptor
complementarity determining region (CDR) of those cells responding in the MLR assay. These
sequences will serve as a fingerprint to identify CDRs from T cells in the allograft of the same
patient using a novel kit from 10x genomics, which combines analysis of CDR3 regions and whole
cell transcriptomes. We hypothesize that (i) the phenotype of donor reactive CD8+ T cells will
differ in PBMCs from belatacept-refractory (CD28-CD38+) vs CNI/CCS-refractory
(CD28+CD38+) patients; (ii) MLRs from peripheral blood cells can identify donor-specific T
cell receptors and their unique CDR3 sequences can be used to identify donor-specific T
cells in the rejecting allograft; and (iii) the transcriptomes and underlying biology of donor-
specific T cells from patients rejecting under belatacept will be distinct from those
rejecting under CNI/CCS-based regimens. To test these hypotheses, we Aim to (1) determine
the transcriptomes of CD8+ T cells in kidney biopsies during rejection under belatacept versus
CNI/CCS-based regimens, and (2) identify the donor reactive CD8+ T cells in the PBMCs of
belatacept vs CNI/CCS-refractory patients, determine their TCR and  CDR3 sequences, and
match them with CDR3 sequences from the graft.

## Key facts

- **NIH application ID:** 9840441
- **Project number:** 5R21AI142264-02
- **Recipient organization:** CINCINNATI CHILDRENS HOSP MED CTR
- **Principal Investigator:** David A Hildeman
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $199,026
- **Award type:** 5
- **Project period:** 2018-12-15 → 2021-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9840441

## Citation

> US National Institutes of Health, RePORTER application 9840441, Single cell analysis of transplant rejection (5R21AI142264-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9840441. Licensed CC0.

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