Identification of Donor Specific B Cells and Antibody Mediated Rejection

NCT ID: NCT02133248

Last Updated: 2022-01-05

Basic Information

• Recruitment Status: TERMINATED
• Total Enrollment: 86 participants
• Study Classification: OBSERVATIONAL
• Study Start Date: 2014-06-05
• Study Completion Date: 2021-11-17

Brief Summary

Many people who are on the wait list for a kidney transplant have harmful antibodies, called donor specific antibodies (DSA), which will attack foreign tissue such as the transplanted organ. These people are considered to be"sensitized". Prior to receiving a kidney, these patients undergo desensitization treatments to remove these harmful antibodies. Levels of DSA are measured after desensitization, but the cells that produce the DSA, donor specific B cells (DSB), have not generally been measured. Additionally, if a person experiences chronic rejection due to antibodies they are also desensitized, but only the DSA are measured. This study will measure the DSA and, using new techniques, the DSB in two study groups: those who are receiving an organ and those experiencing chronic antibody mediated rejection after receiving an organ. The hypothesis is people with higher levels of DSB after desensitization are more likely to develop antibody mediated rejection.

Detailed Description

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.

Conditions

Sensitized Kidney Transplant Recipients

Study Design

• Observational Model Type: OTHER
• Study Time Perspective: PROSPECTIVE

Study Groups

kidney recipient waitlist

Subjects on waitlist for Kidney transplant who are sensitized

No interventions assigned to this group

chronic antibody mediated rejection

Kidney transplant recipient who are diagnosed with chronic antibody mediated rejection

No interventions assigned to this group

control

Normal subjects-no kidney transplant or chronic rejection

No interventions assigned to this group

Eligibility Criteria

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)

• Minimum Eligible Age: 18 Years
• Maximum Eligible Age: 75 Years
• Eligible Sex: ALL
• Accepts Healthy Volunteers: Yes

Sponsors

University of Wisconsin, Madison

OTHER

Sponsor Role: lead

Responsible Party

Responsibility Role: SPONSOR

Principal Investigators

Arjang Djamali, MD

Role: PRINCIPAL_INVESTIGATOR

University of Wisconsin Madison School of Medicine and Public Health

Locations

University of Wisconsin Hospital and Clinics

Madison, Wisconsin, United States

Countries

United States

References

Niederhaus SV, Muth B, Lorentzen DF, Wai P, Pirsch JD, Samaniego-Picota M, Leverson GE, D'alessandro AM, Sollinger HW, Djamali A. Luminex-based desensitization protocols: the University of Wisconsin initial experience. Transplantation. 2011 Jul 15;92(1):12-7. doi: 10.1097/TP.0b013e31821c93bb.

Reference Type: BACKGROUND

PMID: 21512428 (View on PubMed)

Lobashevsky AL, Higgins NG, Rosner KM, Mujtaba MA, Goggins WC, Taber TE. Analysis of anti-HLA antibodies in sensitized kidney transplant candidates subjected to desensitization with intravenous immunoglobulin and rituximab. Transplantation. 2013 Jul 27;96(2):182-90. doi: 10.1097/TP.0b013e3182962c84.

Reference Type: BACKGROUND

PMID: 23778648 (View on PubMed)

Djamali A, Muth BL, Ellis TM, Mohamed M, Fernandez LA, Miller KM, Bellingham JM, Odorico JS, Mezrich JD, Pirsch JD, D'Alessandro TM, Vidyasagar V, Hofmann RM, Torrealba JR, Kaufman DB, Foley DP. Increased C4d in post-reperfusion biopsies and increased donor specific antibodies at one-week post transplant are risk factors for acute rejection in mild to moderately sensitized kidney transplant recipients. Kidney Int. 2013 Jun;83(6):1185-92. doi: 10.1038/ki.2013.44. Epub 2013 Feb 27.

Reference Type: BACKGROUND

PMID: 23447068 (View on PubMed)

Tait BD, Susal C, Gebel HM, Nickerson PW, Zachary AA, Claas FH, Reed EF, Bray RA, Campbell P, Chapman JR, Coates PT, Colvin RB, Cozzi E, Doxiadis II, Fuggle SV, Gill J, Glotz D, Lachmann N, Mohanakumar T, Suciu-Foca N, Sumitran-Holgersson S, Tanabe K, Taylor CJ, Tyan DB, Webster A, Zeevi A, Opelz G. Consensus guidelines on the testing and clinical management issues associated with HLA and non-HLA antibodies in transplantation. Transplantation. 2013 Jan 15;95(1):19-47. doi: 10.1097/TP.0b013e31827a19cc.

Reference Type: BACKGROUND

PMID: 23238534 (View on PubMed)

Other Identifiers

A534280

Identifier Type: OTHER

Identifier Source: secondary_id

SMPH\MEDICINE\NEPHROLOGY

Identifier Type: OTHER

Identifier Source: secondary_id

2014-0076

Identifier Type: -

Identifier Source: org_study_id