Monitoring indirect allorecognition in renal transplant recipients;(INDIRECT)

1. INTRODUCTION AND RATIONALE

Kidney transplantation has improved survival and quality of life for patients
with end-stage renal failure. Despite dramatic improvements in short-term
survival, long-term survival of transplanted renal allografts has changed
little during the past decades. The improvements in short-term survival is
mainly due to superior immunosuppressive drugs that are directly aimed at
T-cells and very efficiently inhibit their activation. However, since these
drugs are non-specific, this is a double edged sword as it will compromise the
recipient in its defense against different pathogens or against developing
tumors.
In transplantation recognition by T cells can occur through two separate
pathways named the direct and indirect pathway of allorecognition. The direct
pathway involves the direct recognition of donor non-self HLA (class I or II)
by recipient T cells. The direct pathway of allorecognition has an important
role in the first months after transplantation, but decreases over time. The
indirect pathway of allorecognition involves the recognition of a non-self
HLA-derived peptide presented in the context of recipient (self) HLA-II on
recipient APC. This pathway has been shown to be correlated with chronic
rejection. Importantly, T cells with indirect specificity are required to
function as helper cells for B cells producing alloreactive antibodies. It is
therefore of great importance to monitor indirect presentation, which can be
used as a tool to monitor the immune response against the graft. This will be
instrumental in improving the current treatment regimes.
Indirect allorecognition has been proven to be difficult to measure and as of
yet there is no clinical applicable test that can be used. We have recently
developed an in vitro model system in which a mismatched HLA monomer is used
and given to recipient APCs which in turn process and present the non-self HLA
and present it to T-cells in the context of their own HLA. We propose to
investigate whether this method could potentially be applied in the monitoring
of T cells with indirect specificity in renal transplant recipients. For this
proof of concept study we want to collect blood of 40 renal transplants
recipients during a regular visit at least 6 months after transplantation.
time progresses, donor APCs will disappear and the contribution of the direct
pathway to the rejection processes diminishes.
The indirect pathway of allorecognition involves the recognition of a non-self
HLA derived peptide processed and presented in the context of recipient HLA-II
on recipient APC, resembling the normal route of antigen presentation. Although
this pathway starts with low precursor frequencies, it increases in time. This
pathway has been shown to be correlated with chronic rejection. Importantly, T
cells with indirect specificity are required to function as helper cells for B
cells producing alloreactive antibodies.

Currently used immunosuppressive agents are very efficient in inhibiting T cell
activation, however they do this in an antigen nonspecific manner (6). Even
more, part of the long term problems in the transplant population are
associated with over-immunosuppression, including infectious problems,
malignancies and nephrotoxicity. Therefore, there is a medical need for reduced
dosing of immunosuppressive drugs, however without increasing the risk of
rejection. This requires an accurate monitoring of the alloimmune response (7).
Methods to monitor alloreactive T-cells with direct specificity are available
and have been used extensively in the transplantation field. However, a
reliable method to monitor indirectly alloreactive T-cells is currently not
available (8).
Previous studies have exploited different strategies to provide donor
alloantigen and to monitor T cell reactivity, ranging from loading with
synthetic peptides (9) or with freeze-thawed donor cells (10;11), till a
trans-vivo DTH reaction in mice (12). Synthetic peptides offer the advantage
that the exact antigen is known resulting in a highly reproducible assay.
However by selecting synthetic peptides, peptides may be selected that do not
occur in vivo, because synthetic peptides do not use the normal route of
processing and may end up being recognized by T-cells as a new epitope. Another
limitation is the choice of peptides, by limiting the response to only one
peptide the other possible natural occurring epitopes are ignored. The
advantage of the use of cellular fragments derived from donor cells is that the
full repertoire of alloantigen is covered. However, donor cells are not always
available and also other antigens are introduced in the system, and it has
turned out to be difficult to reproduce this method at different laboratories.
Furthermore the semi-direct pathway (integrating transmembrane proteins at the
surface of recipient APC) may activate T cells with direct specificity thereby
leading to false positivity for indirect allorecognizing T-cells (13).
Recently, we have developed a model system making using of recombinant
HLA-molecules as a source of donor antigen. This seems to offer an alternative
to allow for the normal route of processing by the APC and the presentation of
all the possible natural peptides. This method has been developed making use of
a CD4+ T cell clone directed against a peptide of HLA-A2, presented in the
context of DR1. Using typed PBMC, we confirmed that presentation occurred in a
dose dependent manner, resulting in T cell proliferation and IFN-gamma
production, thereby theoretically providing an opportunity for monitoring
indirect presentation.


Renal transplantation is the preferred treatment for end-stage kidney failure
as it delivers superior patient survival and quality of life compared with
dialysis. There have been significant improvements in rates of renal transplant
rejection over the last two decades, giving 1-year graft survival rates of over
95%. However, this reduction in rejection has not translated into improvements
in long-term graft survival. The reasons for the lack of improvement remains
unclear and may be multi-factorial including immunological (chronic
inflammation, humoral rejection) and non-immunological events
(ischemia-reperfusion injury, nephrotoxicity of immuno-suppressive agents,
donor risk factors)(1;2).

Transplantation mostly takes place between non-HLA matched individuals which
leads to a vigorous immune response as the recipient*s immune-system is
confronted with non-self HLA molecules which it recognizes as foreign and leads
to rejection of the organ (3). Allograft rejection is an important risk factor
in graft survival. In transplantation recognition by T cells can occur through
two separate pathways named the direct and indirect pathway of allorecognition
(4;5). The direct pathway of allorecognition involves the recognition of donor
non-self HLA (class I or II) by recipient T cells, highly expressed on donor
APCs. This pathway is unique for the process of transplantation and involves a
high frequency of *cross-reactive* allo-specific T cells. The direct pathway
was shown to be correlated with acute rejection and is especially important the
first months after transplantation. As

2. OBJECTIVES

Primary Objective: To test the proof of concept whether recombinant HLA
molecules (A1, A2, B7, B8) can be used for the monitoring of the indirect
allorecognition pathway in renal transplant recipients.
Secondary Objective(s): To investigate the correlation between frequencies of T
cells with indirect reactivity T-cells and simultaneous presence alloreactive
antibodies.

3. STUDY DESIGN
The study is designed as an observational proof of concept study. Eligible
patients will be identified a few weeks before their routine follow-up
appointed and informed consent will be asked before the hospital visit.
Experimental blood samples of participating patients will be combined with the
routine blood drawl. Blood samples will be taken at one time point.
The research question of the current study is to obtain proof of concept that
recombinant HLA molecules can be used for the detection and quantification of T
cells with indirect reactivity. It has been proposed that the frequency of
these T cells increases in time. Therefore, we will concentrate on transplant
recipients who are at least 6 months after transplantation. We will determine
in the same blood samples presence of anti-HLA antibodies. Blood samples will
be obtained at regular visits of these patients.

Nature and extent of the burden and risks associated with participation,
benefit and group relatedness: 50 ml (5x 10ml) of heparinized blood will be
obtained during the standard visits for blood and urine samples, for the
measurement of indirectly reactive T-cells. When this study has a positive
outcome (ie this method is indeed applicable in the clinical transplant
setting), there will be several opportunities for follow up: further
characterization of T cells with indirect reactivity (cytokine responsiveness,
regulatory properties), to perform analysis in patients with signs of (chronic)
allograft rejection, and longitudinal samples to predict long term functioning
of the graft. These analyses might benefit transplant recipients in general.