A multicolour HLA-specific B-cell FluoroSpot assay to functionally track circulating HLA-specific memory B cells

Area of Science:

• Immunology
• Transplantation Biology
• Clinical Diagnostics

Background:

• Donor-reactive memory B cells (mBC) are implicated in antibody-mediated rejection (ABMR) post-transplant.
• mBC possess a broader antigen repertoire than plasma cells, potentially existing without detectable donor-specific antibodies.

Purpose of the Study:

• To develop and characterize a novel immune assay for quantifying circulating donor-reactive mBC.
• To assess the utility of this assay in understanding the pathogenesis of humoral rejection and identifying at-risk transplant recipients.

Main Methods:

• Development of a novel HLA-specific B-cell FluoroSpot assay for enumerating HLA-specific Antibody Secreting Cells (ASC) from mBC.
• Characterization of selective in-vitro mBC activation methods (TLR7,8 agonist R848 + IL-2 or anti-CD40 agonist mAb).
• Optimization of culture conditions, cell sources, and activation time; comparison of HLA-specific IgG-ASC measurement versus HLA-IgG antibodies in supernatants.

Main Results:

• The assay precisely quantifies multiple HLA-specific mBC specificities using fluorescently-labeled multimerized HLA molecules.
• Demonstrated the ability to characterize anti-HLA alloreactivity by measuring HLA-specific IgG-ASC.
• Established optimal conditions for mBC activation and enumeration.

Conclusions:

• Evaluating circulating donor-reactive mBC with this new technology offers novel insights into ABMR pathogenesis.
• This assay may help identify transplant recipients at high risk of allograft rejection.
• The developed FluoroSpot assay provides a valuable tool for assessing anti-HLA alloreactivity.