We recently reported that facilitating cell (FC) enriched hematopoietic progenitor stem cell (HSPC) allografts (FCRx) induced tolerance without graft-versus-host disease (GVHD) or engraftment syndrome in HLA-mismatched living donor renal transplant recipients. Recent studies suggest that dysfunction of B cells that maintain homeostasis may play a role in the pathogenesis of chronic GVHD after HSPC transplantation and alterations in B cells may be a biomarker for transplantation tolerance in kidney transplant recipients. We therefore analyzed B cell progenitors and interleukin 10 (IL-10)-producing B cell subsets in FC obtained from mobilized peripheral blood mononuclear cell (mPBMC) for clinical transplantation, control un-mobilized PBMC and G-CSF mPBMC from healthy volunteer donors. Clinical donors received 8 doses of 10 [micro]g/Kg G-CSF while volunteer donors received 4 doses of 10 [micro]g/Kg G-CSF. FC obtained from mPBMC for FCRx contained significantly more CD19⁺CD24^hiCD27⁺ activated B cells and memory B cells (p<0.01; n=7-15) and CD19⁺CD24^hiCD27⁺CD38^-/loIgM⁺ memory IL-10-producing B cell subsets (p<0.05; n=7-15), but contained a similar proportion of B cell progenitor subsets (Pro-B CD34⁺CD19⁺CD38⁺CD10⁺IgM^–, Pre-B CD34^–CD19⁺CD38⁺CD10⁺IgM^– and Immature B CD34^–CD19⁺CD38⁺CD10⁺IgM⁺) compared to the two control groups. The percentage of CD8⁺TCR^– FC in the lymphoid gate was 9 fold less than that of the Non-FC cell population (reverse gate of CD8⁺TCR^–) in mPBMC for FCRx. However, the FC population contained a significantly higher proportion of activated and memory B cells (p<0.05, n=7) and IgM⁺ memory IL-10-producing B cell subsets (p<0.01, n=7) compared to the Non-FC cell population. We then evaluated the impact of depleting CD19⁺ FC on in vivo engraftment of mPBMC in a non-obese diabetic / severe combined immune deficiency mutation and IL2 receptor gamma chain deficiency (NSG) mouse model. NSG mice were conditioned with 325 cGy TBI and transplanted with 50-70 x 10⁶ mPBMC for FCRx with or without CD19⁺ FC. Depletion of CD19⁺ FC resulted in a significant impairment in engraftment (6 % vs. 14 %; p = 0.03, n = 6) compared to mPBMC for FCRx containing CD19⁺ FC. In summary, our results suggest that CD8⁺TCR^– facilitating cells in mPBMC from clinical donors for FCRx are enriched with IL-10 producing B cell subsets, which may contribute to the prevention of GVHD and the induction of transplantation tolerance.

CITATION INFORMATION: Wen Y, Yolcu E, Huang Y, Kahn L, Ildstad S. CD8⁺TCR^– Facilitating Cells in Mobilized Peripheral Blood Mononuclear Cells Are Enriched with IL-10 Producing B Cells. Am J Transplant. 2016;16 (suppl 3).

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