Antibody-mediated rejection (ABMR) is the most challenging immunological barrier to overcome in human transplantation and one of the most prevalent causes of kidney allograft loss. Detection of circulating donor-specific antibodies (DSA) against donor HLA identifies sensitized patients at high risk of ABMR. In order to fully characterize the DSA repertoire, identify its targets and investigate the associated pathogenic mechanisms it is necessary to determine the full-length paired immunoglobulin heavy and light chains (IgH,L) expressed by individual donor-specific B cells from sensitized kidney transplant patients.

Unfortunately, this methodology has so far only been successfully applied to plasmablast populations, since antigen-specific memory B cells are present at very low frequencies in the blood and contain small amounts of mRNA. Here, we establish a methodology using combined polyclonal activation of circulating memory B cells with single cell IgHL paired sequencing analysis, as an approach for evaluating antigen-specific memory B cells in sensitized transplant patients.

PBMC from healthy donors were stimulated for 0h, 6h, 24h, 48h with a TLR7 and TLR8 agonist plus IL-2 to differentiate memory B cells (CD19+CD27+) to plasmablasts (CD19+CD20^int/lowCD27+CD38^bright) as determined by flow cytometry. We found plasmablasts increased in proportion over time, from 1.12%±0.37 (0h) to 1.78%±1.42 (6h), 6.05%±2.02 (24h) and 20.1%±3.39 (48h). CD19+CD20^int/lowCD27+CD38^bright plasmablasts were single cell sorted, bar coded, and underwent next generation sequencing to obtain paired IgH and IgL sequences. Forty-eight hour stimulation of memory B cells led to a 6-fold increase of detectable paired IgHL, relative to resting memory B cells, and reached a level comparable to freshly isolated blood plasmablasts. We observed significantly higher levels of CD38 (p<0,0001) on those cells that yielded productive paired IgHL sequences than in cells where sequences were not obtainable.

In summary, we developed a methodology using polyclonal activation of memory B cells to generate plasmablast-like cells for single cell high fidelity analysis of the full-length paired IgHL. This approach allows for antigen-specific analysis of the antibody repertoire of donor-specific B cells in transplant recipients.

CITATION INFORMATION: Lucia M, Gomez A, Luque S, Bestard O, Robinson W, Martinez O. Strategy for Analysis of Donor-Specific Antibody Repertoire: Paired Immunoglobulin Heavy and Light Chain Analysis of Individual Memory B Cells. Am J Transplant. 2017;17 (suppl 3).

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