A Novel ELISPOT Assay to Quantify HLA-Specific B Cells in HLA-Immunized Individuals

Endothelial chimerism and vascular sequestration protect pancreatic islet grafts from antibody-mediated rejection

“…Consequently, the chance for a percutaneous needle biopsy to sample an islet is estimated below 5/1,000. Although several techniques have been developed to monitor anti-HLA reactivity of peripheral blood lymphocytes (37)(38)(39), these surrogate approaches all involve the realization of labor-intensive and complex in vitro assays, precluding their routine use. Hence, rather than the treatment, it is the diagnosis of cellular rejection that seems to be the main hurdle to the improvement of islet graft survival.…”

Polyclonal B cell activation for accurate analysis of pre-existing antigen-specific memory B cells

“…Indeed, Lynch et al recently showed the presence of donor-specific memory B cells in the absence of donor-specific serum IgG antibodies in kidney transplant recipients [12]. These, and other data [11], made clear that in-vitro polyclonal B cell activation may be used to provide a snapshot of the B cell memory pool at a given time-point. By focusing upon defined antigens, such as viral antigens or HLA molecules, the actual frequency of antigenspecific memory B cells can be determined.…”

“…Our main interest is on HLA-specific memory B cells in the context of (solid organ) transplantation. As HLA-specific memory B cells are present at extremely low frequencies [11], our protocol includes a B cell purification step prior to activation to increase assay sensitivity. For B cell activation, we have selected the CD40-CD40L pathway as surrogate for T cell help in combination with ODN2006 CpG and IL-21, which are potent drivers of B cells into ASC plasma cells [18,33].…”

“…However, only a few studies aimed at the detection and enumeration of the relatively low levels of HLA-specific memory B cells [10,11]. Our group has recently developed an HLA-specific B cell enzyme-linked immunospot (ELISPOT) assay which allows for the quantification of memory B cell frequencies directed towards defined HLA molecules [11]. This technique was recently adapted by Lynch and colleagues to detect B cell memory towards donor-specific HLA class I on cultured fibroblasts from donor origin [12].…”

Antibody-mediated rejection across solid organ transplants: manifestations, mechanisms, and therapies

“…On the other hand, patients with a known history of sensitization events may not exhibit detectable circulating anti-HLA antibodies at the time of transplant evaluation. This has prompted development of alternative methods to measure alloreactivity, including an HLA-specific B cell enzyme-linked immunospot (ELISPOT) (63,64), ex vivo stimulation of B cells (65,66), and staining of peripheral B cells with HLA tetramers (67). More work is needed to understand the clinical relevance of alloreactive memory B cells in the absence of circulating HLA DSAs.…”