*Purpose: While the negative impact of anti-HLA donor specific antibodies (DSA) on graft outcomes has been extensively investigated, little is known about early changes in the transcriptional profile and phenotype of circulating B and T cells before DSA development.

*Methods: We performed time-of-flight mass cytometry (CyTOF) and RNAseq on prospectively collected PBMC from pediatric kidney transplant recipients who developed (n=10) or did not develop (n=11) DSA within the first year after transplantation. PBMC were obtained at 2 months post-transplant, 3 months prior to DSA development, and at first DSA detection (PBMC from DSA^NEG controls were collected at the same time-points).

*Results: DSA^POS and DSA^NEG patients had similar baseline characteristics and comparable B and T cell frequencies across the different time-points. Within DSA^POS patients, only those that went on to develop antibody-mediated rejection (AMR; n=5) had increased B cells with an antibody-secreting phenotype (CD27⁺CD38⁺, Figure 1A, B p=0.0042) and a memory phenotype (CD27⁺CD95⁺IgD^–CD38^–, Figure 1A, C p=0.0005) compared to DSA^NEG and DSA^POSAMR^NEG recipients at the time of DSA detection. RNAseq analyses revealed differences in transcriptional programs involved in immunoglobulin formation and B cell activation (Gene Ontology enrichment) between DSA^NEG and DSA^POS patients at 3 months prior to DSA development, specifically in antibody-secreting B cells (CellCODE analysis). There was no difference in DSA titers between patients who developed AMR and those who did not (13,687±4,159 vs. 11,375±1,894 MFI, respectively; p=0.63).

*Conclusions: Our extensive CyTOF phenotypic and transcriptional analysis shows that circulating memory and antibody-secreting B cells expand and initiate a specific transcriptional profile months prior to the development of DSA and >1 year prior to biopsy-proven AMR.

Figure 1. Increased antibody-secreting and memory B cells detected in DSA^POS AMR^POS recipients by CyTOF. A) B cells from all patients were pooled together and 20 subclusters identified by Phenograph using 15 cellular markers (shown below heatmap). Heatmap of normalized marker expression across the 20 Phenograph B cell subclusters. B) Frequency of CD27⁺CD38⁺ antibody secreting cells and C) CD27⁺CD95⁺IgD^–CD38^– memory B cells at serial time points after transplant stratified by DSA and AMR status. Plots show mean ± SEM. *p < 0.05, **p < 0.01 and ***p<0.001.

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