Lymphnodes with and without Alemtuzumab to Measure Broad Alloreactivity against Donor Antigens

Allograft rejection after kidney transplantation is a major determinant of
allograft survival [1-3]. Different cells of the adaptive and innate immune
response infiltrating the graft contribute to the allo-antigen specific organ
injury. Traditionally T-cells have been the main focus of transplantation
immunology research, while in recent years B-cells have been recognized to be
important not only in antibody production by plasma cells, but also as
regulators of immune responses and in providing T-cell help [4]. New detection
techniques for donor-specific antibodies for example contribute to the
understanding of antibody-mediated allograft injury [5]. Despite these new
laboratory techniques, the results of sampling the peripheral blood to study
specific cell populations and functions that can predict the risk for allograft
rejection are disappointing. The composition and function of lymphocyte subsets
in the peripheral blood poorly correlate with clinical outcomes like BPAR
(biopsy-proven acute rejection) [6].
Lymph nodes differ in lymphocyte composition and contain for example more
follicular T-helper cells and less cytotoxic CD4+ T cells than peripheral blood
[7]. It is known that the migration of antigen presenting cells from the
allograft to the draining lymph nodes is essential for the initiation of the
alloreactive T-cell response and subsequent rejection [8]. Therefore, the lymph
nodes may be a better site than the peripheral blood compartment to study cells
involved in allograft rejection.
Different phenotypic and functional characteristics of lymph node derived
versus peripheral blood derived lymphocytes in kidney transplantation have
mainly been studied following the administration of lymphocyte-depleting
agents. After T-cell depleting induction therapy, profound T-cell depletion is
found in bone marrow, spleen and peripheral blood, but not in lymph nodes [9].
It has been demonstrated that while B cells were completely depleted in the
peripheral blood after treatment with the B-cell depleting agent rituximab, B
cells in the kidney allograft could still produce donor-specific antibodies and
B-cell survival factors [10]. After rituximab administration, lymph node
derived B-cells shifted from a naïve to a memory phenotype [11]. In a similar
fashion the T- and B-cell depleting antibody against CD52 (alemtuzumab) is
highly effective for total and long lasting depletion of peripheral blood
lymphocytes but not of the central lymphoid compartment [12]. This observation
most likely explains why after induction therapy with alemtuzumab, the risk for
AR is only reduced by approximately 50% [13, 14].
Given these data, we would like to investigate whether the phenotypical
features and functions of lymph node derived lymphocytes are associated with
BPAR. Differences in lymph node derived versus peripheral blood derived
lymphocytes have not been studied so far in patients with renal failure before
the start of immunosuppressive medication.
To study lymphocellular composition and risk of BPAR, a patient cohort with a
relative high risk of BPAR is warranted. Patients with a high percentage of
panel reactive antibodies (PRA) of more than six per cent [15], and/ or more
than 3 HLA mismatches with the donor kidney are both at increased risk for
acute rejection.
Two research lines in our transplantation laboratory can be further explored
when studying lymph node derived lymphocytes. First, in an ongoing study we are
investigating the relation between a prematurely aged immune system in patients
receiving a kidney allograft, and the risk for acute rejection [16, 17]. Recent
results indicate that the numbers of cytotoxic T cells which circulate through
the lymphoid compartments (called central-memory CD8+ T cells) are in
particular associated with acute allograft rejection [Meijer and Betjes,
manuscript in preparation]. Therefore we would like to extend the research on
the ageing immune system to the composition of lymph nodes in patients
undergoing kidney transplantation. Second, the T-helper CD4+ subset named
follicular T-helper (Tfh) cells are of importance for the differentiation of
B-cells into immunoglobulin-producing plasmablasts [18]. In addition, higher
frequencies of Tfh cells were measured in the peripheral blood of patients with
antibody-mediated allo-reactivity; a poorly characterized immune response often
refractory to treatment with conventional immunosuppression. Tfh-cells are
mainly active in secondary lymphoid organs where these CD4+ T cells support
activated B cells via IL-21 after binding to the IL-21Receptor expressed by
these B cells. By studying the phenotypical characteristics and functions of
lymph node T, Tfh and B cells we aim to better understand their interaction in
the setting of allo-immunity. We hope to provide novel insights into
recruitment, compartment and function of the T, Tfh - B cell network.
Another group of patients is at particular risk for acute rejection: blood
group ABO-incompatible (ABOi) kidney transplant recipients have a high risk of
BPAR of approximately 40 per cent based on our own data (antibody-mediated
component 20% [19]). Therefore lymphocyte-depleting induction therapy is given
preoperatively (alemtuzumab). In contrast to rituximab, only non-human data are
available on the effect of alemtuzumab administration on lymphocyte composition
[12]. In a substudy the composition of lymph nodes after alemtuzumab induction
therapy administered three weeks before transplantation and its effect on BPAR
will be studied.
References:
1. Naesens, M., et al., The Histology of Kidney Transplant Failure: A Long-Term
Follow-Up Study. Transplantation, 2014. 98(4): p. 427-435.
2. El-Zoghby, Z.M., et al., Identifying Specific Causes of Kidney Allograft
Loss. American Journal of Transplantation, 2009. 9(3): p. 527-535.
3. Matignon, M., et al., Concurrent Acute Cellular Rejection Is an Independent
Risk Factor for Renal Allograft Failure in Patients With C4d-Positive
Antibody-Mediated Rejection. Transplantation, 2012. 94(6): p. 603-611.
4. Coelho, V., et al., Rethinking the multiple roles of B cells in organ
transplantation. Current Opinion in Organ Transplantation, 2013. 18(1): p.
13-21.
5. Lawrence, C., et al., Preformed Complement-Activating Low-Level
Donor-Specific Antibody Predicts Early Antibody-Mediated Rejection in Renal
Allografts. Transplantation, 2013. 95(2): p. 341-346.
6. van de Berg, P.J.E.J., et al., Circulating lymphocyte subsets in different
clinical situations after renal transplantation. Immunology, 2012. 136(2): p.
198-207.
7. Havenith, S.H.C., et al., CXCR5CD4 follicular helper T cells accumulate in
resting human lymph nodes and have superior B cell helper activity.
International Immunology, 2014. 26(3): p. 183-192.
8. Herrera, O.B., et al., A novel pathway of alloantigen presentation by
dendritic cells. J Immunol, 2004. 173(8): p. 4828-37.
9. Page, E.K., et al., Enhanced De Novo Alloantibody and Antibody-Mediated
Injury in Rhesus Macaques. American Journal of Transplantation, 2012. 12(9): p.
2395-2405.
10. Thaunat, O., et al., B cell survival in intragraft tertiary lymphoid organs
after rituximab therapy. Transplantation, 2008. 85(11): p. 1648-1653.
11. Kamburova, E.G., et al., A Single Dose of Rituximab Does Not Deplete B
Cells in Secondary Lymphoid Organs but Alters Phenotype and Function. American
Journal of Transplantation, 2013. 13(6): p. 1503-1511.
12. Marco, M.R., et al., Post-transplant repopulation of naive and memory T
cells in blood and lymphoid tissue after alemtuzumab-mediated depletion in
heart-transplanted cynomolgus monkeys. Transpl Immunol, 2013. 29(1-4): p. 88-98.
13. Group, C.S.C., et al., Alemtuzumab-based induction treatment versus
basiliximab-based induction treatment in kidney transplantation (the 3C Study):
a randomised trial. Lancet, 2014. 384(9955): p. 1684-90.
14. Hanaway, M.J., et al., Alemtuzumab induction in renal transplantation. N
Engl J Med, 2011. 364(20): p. 1909-19.
15. van den Hoogen, M.W., et al., Rituximab as induction therapy after renal
transplantation: a randomized, double-blind, placebo-controlled study of
efficacy and safety. Am J Transplant, 2015. 15(2): p. 407-16.
16. Betjes, M.G., et al., Premature aging of circulating T cells in patients
with end-stage renal disease. Kidney Int, 2011. 80(2): p. 208-17.
17. Betjes, M.G., et al., Terminally differentiated CD8+ Temra cells are
associated with the risk for acute kidney allograft rejection. Transplantation,
2012. 94(1): p. 63-9.
18. de Graav, G.N., et al., Follicular T-helper cells and humoral reactivity in
kidney-transplant patients. Clin Exp Immunol, 2014.
19. van Agteren, M., et al., The First Fifty ABO Blood Group Incompatible
Kidney Transplantations: The Rotterdam Experience. Journal of Transplantation,
2014. 2014: p. 6.

Primary objective of the study:
- The primary objective for this study is to determine the prognostic
characteristics for BPAR in the first 3 months after transplantation, as
assessed in the lymphocyte composition of the lymph node in immunologically
high-risk kidney transplantation.
Secondary objectives of the study are:
- to capture global composition of lymph node and blood leukocyte subsets [20].
- to compare the immunological ageing profile of T cells in the peripheral
blood to the T cells derived from the lymph node.
- to assess whether pre-transplant frequencies of lymph node derived Tfh, T and
B cells predict BPAR.
- to assess differences in lymphocyte composition of lymph nodes in alemtuzumab
treated versus untreated patients both in single cell suspension and within the
tissue. (ABO-incompatible kidney transplant recipients receive alemtuzumab
induction therapy three weeks before transplantation).
This study will result in a set of defined markers that will identify patients
at risk to develop rejection.

This is an investigator-driven, prospective, observational study to explore
immunological processes. Kidney transplant recipients will be asked for their
consent during admission before kidney transplantation. This consent relates to
extra blood sampling at one time point when blood is already drawn, and to
harvest a local lymph node during transplantation. During surgery, the surgeon
will harvest a lymph node from the inguinal area before revascularization (open
procedure). This harvesting of a local lymphnode is often necessary to have
good access for anastomosis and these extirpated lymphnodes are discarded as
residual material. Peripheral blood will be sampled before transplantation (and
thus before immunosuppressive therapy). Kidney transplant recipients will be
treated by current standard immunosuppressive protocol. 'For cause' kidney
transplant biopsies will be performed according to local protocol: in case of
deteriorating kidney transplant function in the absence of an obvious
alternative diagnosis, the radiologist will perform an ultrasound-guided kidney
transplant biopsy.

The risk of the venapuncture is small, as extra blood tubes are taken at a time
point when blood is already drawn. The risk of a venapuncture is the occurrence
of a bruise after puncture and possible pain-symptoms at the site of puncture.
The risk of harvesting a regional lymph node during transplantation by the
surgeon is negligible, since it is an open procedure. During transplantation,
the surgeon needs to harvest lymph nodes to have good access to the artery and
vein for anastomosis in approximately half of all standard kidney
transplantation procedures. These lymph nodes are then discarded and can be
regarded as *residual material*. In previous trials performed in the Erasmus
Medical Center, lymph nodes were harvested during kidney transplantation: [27,
28, 29]. There are no benefits for individual patients when participating in
this study.

References:
27. Tanis, W., et al., Human hepatic lymph nodes contain normal numbers of
mature myeloid dendritic cells but few plasmacytoid dendritic cells. Clin
Immunol, 2004. 110(1): p. 81-8.
28. Peters, J.H., et al., Human secondary lymphoid organs typically contain
polyclonally-activated proliferating regulatory T cells. Blood, 2013. 122(13):
p. 2213-23.
29. Eissens, D.N., et al., Defining early human NK cell developmental stages in
primary and secondary lymphoid tissues. PLoS One, 2012. 7(2): p. e30930.