*Purpose: Prospective randomized study of IL-6R blockade with Tocilizumab for treatment of subclinical inflammation (SCI) in renal transplantation, reveals unique profiles of resting and stimulated PBMCs by multiplex single-cell RNASeq (scRNA-seq).

*Methods: Fifteen patients were sequentially enrolled in a prospective randomized clinical trial of Tocilizumab vs. standard of care (SOC; observation only) for SCI at the time of stable kidney allograft function, as assessed by the 6 month protocol biopsy. Serial PBMC samples at enrollment, and at sequential time points until 1 year later, were profiled by multiplexed droplet scRNA-seq (Mux-Seq) on the 10X Genomics platform, either as resting cells or stimulated with CD3/CD28 antibodies. Cell genotyping was simultaneously performed using SNP arrays and Demuxlet was used for identifying individual cell identities. Seurat/SingleR packages and UMAP were used for Mux-Seq analysis. Patients had a follow-up biopsy 1 month and 12 months later, which was scored by Banff, and Tregs were measured in the circulation over time.

*Results: Following cell clustering using UMAP, multiple distinct PBMC cell subsets inclusive of different T, B, NK, Treg, and monocytes were noted (Fig. 1). IL-6 gene expression of stimulated cells from patients receiving Tocilizumab resulted in loss of IL-6 expression (Fig. 2), when compared to SOC patients. Other inflammatory genes also show marked loss of expression in Tocilizumab-treated stimulated cells, including interferon-gamma (IFNG) (Figure 2). Tregs expanded over time, and biopsy histology showed mild reduction in SCI in a few patients on Tocilizumab.

*Conclusions: Tocilizumab treatment for SCI results in mild reduction of graft inflammation by histology, expansion of peripheral Tregs, changes in different PBMC subsets over time, and marked reduction of IL-6, IL-6R, and pro-inflammatory gene expression in stimulated rather than resting PBMCs. Mux-Seq reduces batch variation, sequencing costs, and sample input. These results support unique cell-subset compositions with IL-6R blockade and possibly greater value in priming the allograft with Tocilizumab, prior to the development of allograft injury.

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