Clinical relevance of HLA-specific memory B cells in kidney transplant recipients undergoing desensitization

Kidney transplantation is the preferred treatment option for patients with
end-stage kidney failure. However, HLA-sensitized patients have limited access
to transplantation, and experience prolonged waiting times with increased
mortality rates due to positive crossmatches with their candidate donors. The
crossmatch is an assay, in which patient serum is tested against donor
lymphocytes to detect the presence of DSA. Sensitization to HLA occurs through
blood transfusions, pregnancies or previous transplantations. Approximately 20%
of patients waiting for transplantation in the Netherlands are HLA-immunized
(1, 2). Despite enhanced transplantation of HLA-immunized patients in the
Eurotransplant Acceptable Mismatch program and Dutch National Living Donor
Kidney Exchange program, still 40% are not transplanted within 2 years of
listing (3).
Desensitization is a therapy that removes or inactivates circulating antibodies
and provides a window of opportunity for transplantation of HLA-sensitized
patients. Patients transplanted following removal of circulating antibodies by
plasmapheresis and low dose intravenous immunoglobulins (IVIg) have improved
survival in comparison to those who remained on dialysis (4, 5). However,
depending on their broadness of HLA-sensitization, 30-70% of patients
transplanted upon desensitization experience acute ABMR due to DSA rebound as
early as 1 month after transplantation, which may progress to chronic ABMR and
ultimately graft loss (6-8). Furthermore, in some patients, for unknown
reasons, removal of DSA cannot be achieved and therefore transplantation cannot
commence (9). Recently, Immunoglobulin degrading enzyme of Streptococcus
pyogenes (IdeS) or Imlifidase was shown to rapidly cleave total serum IgG,
allowing for successful transplantation of HLA-sensitized individuals. Upon
Imlifidase treatment, DSA remained undetectable until 2 weeks after
transplantation and rebound DSA-triggered ABMR rate was 36% within 6 months
after transplantation (10, 11).
Current risk assessment in patients undergoing desensitization includes 1)
crossmatching, and 2) luminex SAB assays which are used to determine serum HLA
antibody specificities. Using luminex, serum samples are screened against
single HLA-coated microspheres yielding a readout called mean fluorescence
intensity (MFI). Depending on the policy of the transplant center, conversion
from a positive to negative crossmatch and/or a significant decrease in serum
DSA MFI values are required upon desensitization treatment to proceed with
transplantation.
Currently, only circulating HLA-specific antibodies are considered for clinical
decision making. However, humoral alloimmune responses to foreign HLA can occur
as circulating HLA-specific antibodies and/or HLA-specific memory B cells (12).
Memory B-cells can rapidly differentiate into antibody producing cells upon
antigen re-encounter or bystander activation, and harbour a more diverse
specificity profile than serum (13). Importantly, preliminary data indicate
that the current immunological risk assessment based on serum HLA-specific
antibodies may be incomplete due to the lack of information on HLA-specific
B-cell memory. To overcome this, we developed an HLA-specific memory B-cell
assay in which HLA-specific memory B-cell-derived antibodies can be detected at
a sensitivity similar to serum HLA-specific antibody detection. Using this
method, we demonstrated that memory and serum HLA-specific antibody profiles
can be different in a patient in means of specificity as well as strength of
antibodies. Furthermore, in a cohort of patients transplanted across DSA, we
demonstrated a higher incidence of ABMR and more persisting DSA in patients
with concurrent memory and serum DSA in comparison to those with only serum DSA
(14,15). In line with this, in a recent study, significant expansion of
isotype-switched total memory B-cells were found in patients with DSA and ABMR
in comparison to patients with DSA without ABMR. Correlation of circulating
memory B-cell expansion with the emergence of DSA in patients with ABMR
suggests that systematic monitoring of circulating B cell subsets may
anticipate ABMR (16). While these studies indicate the clinical relevance of
switched (IgG) memory B-cells, recent data show the presence of IgM memory B
cells serving as precursors of IgG memory B cells (17). In transplantation, IgM
HLA-specific memory B cells may serve as a novel early biomarker of an ongoing
or newly initiated donor-specific immune response.
Desensitization by plasmapheresis/IVIg ± ATG is effective in removing serum
antibodies without affecting plasma cells (18). However, effective DSA
depletion may facilitate a recall response by allowing memory B-cells to more
easily access the antigen. In the case of Imlifidase this response may be
delayed due to the cleavage of membrane bound IgG, but not IgM B cell
receptors. Therefore, monitoring both IgM and IgG HLA-specific memory B cells
upon Imlifidase treatment is of importance (19).
Secondary lymphoid organs are an important reservoir of memory B cells. Current
evidence shows that B cell depleting treatments hardly affect memory B cells in
secondary lymphoid organs (20), suggesting that expansion of these cells upon
antigen re-encounter can contribute to rebound DSA responses. In this regard,
assessment HLA-specific B cell memory in secondary lymphoid organs may prove
useful for immunological risk assessment.
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Pretransplant Donor-Specific Anti-HLA Antibodies and the Risk for
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Overcoming a positive crossmatch in living-donor kidney transplantation. Am J
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Primary Objective:
To analyze whether HLA-specific B-cell memory underlies rebound DSA formation,
donor HLA-specific memory B-cell-derived antibodies (DSM) (IgG) will be
determined before desensitization and will be associated to serum DSA rebound
at month 3 after transplantation.

This is a single center investigator-driven prospective, non-medicinal product
intervention study. Peripheral blood samples of 20 ml heparinized and 5ml EDTA
will be collected before and directly after desensitization and post-transplant
month 3, 6 and 12. There are two different study cohorts: living-donor
transplant recipients and deceased-donor transplant recipients. Desensitization
for these cohorts differs clinically: Living-donor HLA-incompatible kidney
transplantation is elective surgery and desensitization is scheduled one or two
weeks before transplantation. Deceased-donor HLA-incompatible kidney
transplantation is non-scheduled surgery and desensitization is scheduled hours
before emergency transplantation.
Samples ''after desensitization'' will be collected either at one day before or
at the day of transplantation. In addition to blood sampling, an iliac lymph
node will be collected from patients during the anastomosis phase of the
transplant surgery, provided that the surgeon has good view of the anatomy and
considers this safe.
All samples will be collected during patients' routine transplantation care and
therefore recipients do not have extra visits to the hospital and do not have
extra blood sampling scheduled.
At each time point, plasma will be isolated from heparinized blood and stored
at -20°C for HLA-specific antibody detection. Peripheral blood mononuclear
cells (PBMC) will be isolated using Ficoll-Hypaque density gradient
centrifugation. Peripheral blood and lymph node mononuclear cells will be kept
frozen in liquid nitrogen until further use for HLA-specific memory B-cell
detection. Multicolour flow cytometry for B cell immunophenotyping will be
performed using frozen PBMC from EDTA blood.
All biological material of all study patients will be stored in a coded way at
the laboratory of Department of Nephrology and Transplantation. Samples will be
transferred to the HLA laboratory located in Leiden University Medical Center
for further testing for HLA antibody screening, HLA-specific memory B cell
assay and peripheral blood B cell immunophenotyping.
Kidney transplant patients will be asked for their consent at the outpatient
clinic after reading the **patient information leaflet**. Transplant biopsies
and assessment of kidney function by serum creatinine will be performed as part
of the routine clinical follow-up in the EMC.

The burden of a venous puncture for the kidney transplant recipient is small (a
possible bruise and pain). In the current study protocol, extra vials are
obtained at the moment when blood is drawn for routine clinical purposes and an
extra venous puncture is thus not necessary. For lymph node collection during
transplant surgery, there is a very small risk of developing a lymphocele.
Overall, included patients do not directly benefit from participation. If the
HLA-specific memory B cell assay proves to be relevant at the end of the study,
it can be implemented into the immunological risk assessment of (future) kidney
patients.