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.

Inclusion Criteria:

- Age 18-75 inclusive

- Patients on UWHC Kidney Transplant waitlist identified as sensitized or

- UWHC kidney transplant recipient patients diagnosed with antibody mediated rejection

Exclusion Criteria:

- Inability to provide informed consent to participate in study

- Diagnosed with an autoimmune disorder or kidney problems, currently on
immunosuppressive or immunomodulatory medication, or any current malignancies (healthy
controls only)