Identification of Donor Specific B Cells and Antibody Mediated Rejection

Patients that are sensitized, who have panel reactive antibodies (PRAs) > 20%, comprise a disproportionate and increasing cluster on the wait list (33%), and have to wait longer to receive a transplant than non-sensitized patients. By 36 months on the wait list, 10% died before receiving a transplant. Those who do receive transplants require desensitization. The current desensitization protocols include anti-CD20 monoclonal antibody to remove B cells, a proteasome inhibitor to eliminate plasma cells and plasma exchange and/or intravenous immunoglobulin (IVIG) to remove pre-formed donor specific antibodies (DSA). The success of these protocols can be measured using single antigen bead (SAB) luminex technology. These desensitization protocols have been shown to significantly decrease mean fluorescence intensity (MFI). Desensitization protocols typically are more effective in removing antibodies that recognize HLA Class I molecules than those that recognize HLA Class II molecules. Unfortunately, even after pre-conditioning, sensitized patients are more likely to develop antibody mediated rejection (ABMR) than patients that do not have donor specific antibodies prior to transplant. Currently, serum levels of DSA are measured after desensitization, but not donor specific B cells (DSB). It is possible that B cells and/or plasma cells remain after treatment. Donor specific B cells, upon transplant and the accompanying exposure to antigen, can be re-stimulated to both produce antibody and also to develop into plasma cells which produce antibody. Therefore, potentially a better means of determining whether desensitization has been successful would be to look at both the DSA level using SAB technology as described above and to look at DSB to determine if these cells have been adequately cleared by the desensitization protocol.

Specific Aim 1 Hypothesis: Patients who have adequate clearance of serum DSA, but who still have significant populations of DSB are more likely to develop ABMR.

In Specific Aim 1, the investigators will utilize cutting edge technology to observe levels of anti-HLA antibodies, particularly donor specific antibodies, present in sensitized patients by staining DSB with HLA class I tetramers. This method allows enumeration and characterization of the source of DSA, the DSB. The investigators will determine the number of DSB and donor specific plasma cells (DSPC) prior to and after desensitization as well as later post transplant. In addition, the investigators will look at the phenotype and activation state of the DSB prior to transplant and after transplant. HLA class I tetramers are MHC class I molecules of a particular allele which are refolded with a peptide in the peptide binding groove, biotinylated on the C terminal tail and tetramerized using streptavidin conjugated to a fluorophore such as phycoerythrin (PE). Similarly, HLA class II tetramers are made of MHC class II alleles. B cells retain membrane bound antibody which has the exact specificity of the soluble antibodies that it also produces. The tetramers will bind only to B cells that have antibodies against that specific HLA class I allele on the surface, since the binding is determined only by the specificity of the antibody. At the same time, the cells will be stained with other markers to determine the activation state and phenotype of the DSB. This assay is done using flow cytometry. Additionally, the investigators will measure BAFF and APRIL cytokine levels by Elisa at all time points. These cytokines are closely related to B cell development. There is data to suggest that BAFF is elevated after some forms of desensitization, which could enhance new B cell development.

Specific Aim 1 Hypothesis: Patients who have adequate clearance of serum DSA, but who still have significant populations of DSB are more likely to develop ABMR.

In Specific Aim 1, the investigators will utilize cutting edge technology to observe levels of anti-HLA antibodies, particularly donor specific antibodies, present in sensitized patients by staining DSB with HLA class I tetramers. This method allows enumeration and characterization of the source of DSA, the DSB. The investigators will determine the number of DSB and donor specific plasma cells (DSPC) prior to and after desensitization as well as later post transplant. In addition, the investigators will look at the phenotype and activation state of the DSB prior to transplant and after transplant. HLA class I tetramers are MHC class I molecules of a particular allele which are refolded with a peptide in the peptide binding groove, biotinylated on the C terminal tail and tetramerized using streptavidin conjugated to a fluorophore such as phycoerythrin (PE). Similarly, HLA class II tetramers are made of MHC class II alleles. B cells retain membrane bound antibody which has the exact specificity of the soluble antibodies that it also produces. The tetramers will bind only to B cells that have antibodies against that specific HLA class I allele on the surface, since the binding is determined only by the specificity of the antibody. At the same time, the cells will be stained with other markers to determine the activation state and phenotype of the DSB. This assay is done using flow cytometry. Additionally, we will measure BAFF and APRIL cytokine levels by Elisa at all time points. These cytokines are closely related to B cell development. There is data to suggest that BAFF is elevated after some forms of desensitization, which could enhance new B cell development.

The investigators will obtain a blood draw from subjects once consent is obtained and utilize this sample to establish a baseline of B cells that produce antibodies that recognize HLA class I tetramers of the same type as those previously identified by SAS anti-HLA antibody analysis. Immediately prior to and after the patient receives a transplant, the SAB anti-HLA antibody analysis, tetramer analysis, and BAFF/APRIL Elisas will be repeated. Analyses will also be performed between 6 weeks and 2 months after transplant.

Specific Aim 2 Hypothesis: During chronic rejection, patients who respond well to desensitization and who are able to maintain tolerance after desensitization will have fewer residual DSB.

In Specific Aim 2, the investigators will utilize the same technology to quantify and characterize DSA and DSB when a patient experiences chronic rejection due to ABMR. In this case, the patient may not have had DSA when transplanted, but developed de novo DSA (dnDSA) after transplant, causing rejection. Or the patient may have had DSA, been desensitized successfully, maintained tolerance for a period of time, then either lost tolerance or developed dnDSA. The timelines will be similar to specific aim 1 - the investigators will take samples at the following timepoints: upon diagnosis of ABMR, 1 week after desensitization, then 2 to 3 months after desensitization to look for rebound.

In addition to enrollment of new subjects, the investigators will also enroll healthy normal individuals to serve as controls.