Humoral rejection is the most challenging immunological barrier to overcome in allotransplantation in humans. Currently, either in its acute or chronic forms of clinical presentation, it accounts for one of the most prevalent etiology of kidney allograft loss. Moreover, today up to almost 40 percent of patients in the waiting list for receiving a kidney transplant are considered as allosensitized, that is, with detectable presence of circulating antibodies against HLA antigens in peripheral blood. As a consequence, the accurate detection of donor-specific antibodies (DSA) has allowed transplant physicians an optimal prevention of early post-transplant antibody-mediated rejection in terms of establishing preventive therapeutic strategies and also even precluding patients undergoing transplantation due to the excessive rejection risk.
Two major B-cell populations that contribute to the maintenance of immunological memory are long-lived plasma cells and memory B cells. The long-lived plasma cells reside primarily in the bone marrow and continuously secrete antibodies that act rapidly on invading microbes. Memory B cells reside primarily in peripheral lymphoid tissues and can, upon re-encounter with the priming antigen, differentiate into antibody-secreting cells (ASC) and thus amplify the antibody response During a sensitization process such as transplantation, the body produces both long-lived plasma cells and memory B cells that provide an immunological memory. Conventionally, in the transplant setting B-cell responses are assessed by the serological measurement of specific antibodies against donor-antigens (DSA). In fact, there are many immune assays evaluating donor-specific circulating HLA antibodies such as complement dependent cytotoxicity (CDC), ELISA, flow cytometry or Luminex-based assays. However, although these assays are valuable for determination of DSA in the circulation, they are likely to underestimate the magnitude of the humoral immune response as it excludes the detection of the memory B-cell pool. In fact, memory B cells can exist in the absence of detectable serum antibody levels and their rapid differentiation and antibody production may be of high relevance for a protective humoral response.
In fact, there are different pathological situations in the kidney transplant setting in which circulating anti-donor HLA antibodies cannot be found using highly sensitive assays despite strong clinical evidence of humoral allosensitization, for instance in patients on the waiting list for a subsequent kidney transplant and not showing circulating antibodies against HLA antigens against previous graft alloantigens, or in kidney transplant recipients showing hystopathological features of antibody-mediated damage but without any evidence of DSA in peripheral blood. Indeed, these features may be apparent due to the eventual absorption of antigen-specific antibodies by the HLA mismatch graft or because of low antigen immunogenicity of donor antigens triggering transient low B-cell stimulation, thus leading to undetectable antibody release. Noteworthy, these biological conditions have relevant clinical implications since an individual considered non-sensitized by means of detectable circulating alloantibodies, could indeed display a very robust antigen-specific memory B-cell response, ready to generate a strong secondary immune response in case of a subsequent exposure to a previously recognized HLA-antigen. Therefore, detecting and quantifying memory B cells capable of producing donor-directed anti-HLA antibodies at different time points of the transplant setting (both before and after) would significantly refine current immune-monitoring tools of the effector alloimmune response.
In this regard relevant clinical situations in which such assay would provide outstanding information from the clinical perspective could be enumerated as follows: (a) before the transplant surgery to demonstrate lack of allosensitization in patients awaiting for a first or subsequent allograft with or without circulating anti-HLA antibodies, (b) before transplantation in well-known sensitized individuals in which specificity and degree of sensitization (MFI or mean fluorescence intensity) varies while being on the waiting list, (c) in sensitized patients undergoing desensitization programs in order to prove a significant inhibition not only of circulating DSA but also of the frequency of donor-specific ASC after treatment and furthermore, (d) after kidney transplantation in patients showing critical histological lesions highly suggestive of antibody-mediated damage but without any evidence of anti-HLA antibodies circulating in peripheral blood.
So far, reproducible assays aiming to quantify the number of cells contributing to alloantibody production are scarce. A HLA-tetramer staining of CD19+ B cells has been reported (Mulder et al., J. Immunol. 2003, 171: 6599-6603) and further developed by Zachary et al. (Zachary et al., Transplantation 2007, 83: 982-988; Zachary et al., Transplantation 2007, 83: 989-994) for the detection and enumeration of B cells which harbor a HLA-specific B-cell receptor, but per se this technique does not quantify B cells that are actually capable of antibody production. More recently, a previously described technique to estimate the precursor frequency of B cells with HLA specificity was based on a CD40L-driven B-cell culture stimulation method including standardized supplements and B-cell sorted responder cells, followed by a visualization phase in ELISPOT format with synthetic HLA molecules as the detection matrix (Mulder et al., Clin. Exp. Immunol. 2001, 124: 9-15; Heidt et al., A. Am. J. Transplant. 2012, 112(6): 1469-78). However, this approach renders it not feasible to standardization as would be required for a clinical test.
Therefore, there is still a need in the art for a reliable and reproducible method for determining and quantifying B cells that are actually capable of producing antibodies directed to HLA.