Introduction: Regulatory B cells (Breg) are postulated as major mediators of tolerance in the context of kidney transplantation. Several Breg signatures have been linked to kidney transplantation positive prognostic, however, a unique signature with potent discriminating and prognostic value is missing, setting back their application in the clinical setting
Methods: In this work we propose the use of Mesenchymal stromal cell (MSC)-B cell in vitro coculture and a T-cell mimicking cocktail to induce Bregs (iBregs), which have upregulated regulatory potential, may constitute an optimal platform for biomarker discovery. With this aim, we performed RNAseq and proteomic analysis.
On the other hand, we recruited 51 kidney transplant recipients and followed them up for a year monitoring their peripheral blood immunophenotype by flow cytometry pre- and at 7 days, 3-, 6- and 12-months post-transplantation and assessed the graft outcome. To monitor Bregs we used the immunophenotype CD19+ CD24hi CD38hi in absolute counts and in percentage among CD19+ in fresh blood and in frozen PBMCs.
Results: The iBregs were enriched in CD19+ CD24hi CD38hi phenotypes and showed immunomodulatory potential on activated autologous T cells which we proved was independent of IL-10 in vitro. In a RNAseq analysis, we identified over 1700 differentially expressed genes among which we validated a set of 9 genes of interest upregulated in iBreg compared to activated B cells.
In the patient immunophenotyping study we found statistically significant differences on Bregs % at 3m being higher in patients with stable kidney function (NKF) up to 12 months (3.9% ± 2.6) compared to patients who were biopsied for clinical indication (AKF) (1.8% ± 0.78) with 87.5% sensibility and 70% specificity. Patients with biopsy proven acute rejection (BPAR) had even lower Bregs % at 3m (1.4% ± 0.56) compared to biopsied patients with no acute rejection (1.9% ± 0.82).
Our data allowed us to establish a cut-off value of 2.73% of transitional B cells at 3 months above which 100% of the patients remained with stable kidney function up to 12 months and free from rejection events (p<0.006). Patients with transitional B cells at 3 months below 2.085% presented 25% chances to present rejection episodes in the 12months follow-up (p<0.01). 

The trends indicating the prognostic value of transitional B cells % for a stable kidney function were also seen in frozen PBMCs and the specific iBreg gene signature could be evaluated in the frozen material.
Conclusions: Higher % of Bregs at 3m has great potential as a biomarker of improved graft outcome. Bregs % at 3m may be useful to stratify rejection risk and identify low-risk patients who might benefit from immunosuppression minimization regimes. New Breg molecular / functional markers can be identified in vitro and used as biomarkers in patients’ material.