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CELLULAR AND
MOLECULAR METHODS FOR IMMUNOLOGIC
MONITORING OF SOLID ORGAN TRANSPLANT RECIPIENTS Adriana
Zeevi Ph.D., Professor of Pathology and Surgery Rene Duquesnoy Ph.D., Professor of Pathology
and Surgery Division
of Transplant Pathology Thomas
E. Starzl Transplantation Institute University
of Pittsburgh Medical Center INTRODUCTION Solid
organ transplantation has become an increasingly important therapeutic modality
for patients with various end-stage diseases. Despite improved immunosuppression
protocols, most transplant recipients face a variety of complications. Early
post transplant, infection and rejection are the major causes of morbidity and
mortality. Drug toxicity, chronic rejection and malignancies are long-term
complications. Many attempts have been made to
develop in vitro procedures that can asses the immunologic status of the
allograft reliably and accurately. Immunological monitoring could, in theory,
differentiate rejection from other forms of dysfunction, such as infection or
primary non-function. Furthermore, the ideal tool would be able to gauge
accurately a patient's response to anti-rejection therapy and might help
prevent over-immunosuppression. Immune monitoring could also be important in
predicting which recipients could have their immunosuppression markedly reduced
without increasing the risk of acute or chronic rejection. Although there is no one ideal
immunologic test which can accomplish all of the above, there are a number of
tests that can be used together to assess the immnunologic status of the
transplant recipient. ASSESSMENT
OF IMMUNOCOMPETENCE
Increased immunosuppression, which
is associated with the development of opportunistic infections, may also affect
immune reactivity. Strober et al have reported suppression of in vitro
proliferative responses and an increase in viral infections in kidney
transplant recipients one year following total lymphoid irradiation [1]. In lung transplant recipients we reported a systemic
depression in immunocompetence as evidenced by decreased mitogen responses of
bronchoalveolar lavage (BAL) lymphocytes and peripheral blood lymphocytes (PBL)
during opportunistic infection [2]. Proliferative responses to non-specific mitogens
(Phytohemagglutinin-PHA, Concanavalin A-ConA), and recall antigens (Tetanus
Toxoid, CMV) may provide an assessment of the immunocompetence of transplant
recipient's circulating lymphocytes.
PROPAGATION OF LYMPHOCYTE
CULTURES FROM ALLOGRAFT BIOPSIES - CLINICAL SIGNIFICANCE In solid organ transplantation the
histological analysis of allograft biopsies remains the "gold standard" for
diagnosing rejection [3]. Although
many cell types have been identified in rejecting allografts, including B
cells, macrophages, neutrophils and eosinophilis, it is generally accepted that
CD3+ T lymphocyte initiate allograft rejection [4-10]. Both CD4+ (helper) and CD8+ (cytotoxic) T cells are
present in the graft and express interleukin 2 (IL-2) receptor and HLA-DR
antigens, suggesting the presence of activated lymphocytes [7-10]. The phenotype studies provide little information
about the functional characteristics, and their specificity of allorecognition.
Another approach is to learn about the functional characteristics of graft
infiltrating cells through in vitro propagation in the presence of IL-2 [11-14]. Activation of T cells occurs as a result of
recipient allorecognition of donor antigens, which leads to the expression of
IL-2 receptors on the activated T cells. The lymphocyte growth assay is
straightforward. Biopsy fragments are incubated in tissue culture medium
supplemented with 5% human serum and 10-30 IU of recombinant IL-2. Lymphocyte
growth in cardiac transplant recipients correlates with the histological grade
of rejection, and positive growth from histologically negative biopsies was
associated with subsequent biopsy-proven rejection [15-20] (Table1).
Table 1.
Propagation of Lymphocyte Cultures from Heart and Lung Allograft
Biopsies with Different Histological Diagnosis
A
persistent lack of biopsy growth is also associated with increased
rejection-free cardiac allograft survival [21]. These studies indicate that in vitro culturing of
lymphocytes from endomyocardial biopsies is clinically useful for the early
diagnosis of acute cellular rejection. This conclusion may also apply to other
organs. In lung transplant recipients
transbronchial biopsies have been cultured to propagate infiltrating
alloreactive T cells [22]. Lymphocyte growth is virtually 100% for biopsies
with histological diagnosis of acute rejection or active bronchiolitis
obliterans (BO), a form of chronic lung rejection, while less than 30% biopsies
with no major abnormalities show biopsy growth [22] (Table 1). Lymphocyte growth from renal transplant
biopsies also correlated with acute cellular rejection, and the production of
IL-2 by the infiltrating cells was associated with irreversible rejection [23]. In liver transplant recipients more lymphocyte
growth was associated with rejection and less growth was seen for patients on
OKT3 treatment for rejection [24,
25]. A serious complication of heart
transplantation is the development of accelerated graft coronary disease (GCD) [26, 27]. Persistent biopsy growth during the first three
months post-transplant is associated with a higher risk of GCD [28]. More than 40% of patients whose biopsies
persistently grew lymphocytes developed GCD. In contrast, only 6% with
non-grower biopsies had subsequent GCD [28].
FUNCTIONAL CHARACTERIZATION OF GRAFT INFILTRATING CELLS
The
alloreactivity of biopsy grown cells toward donor cells presents an additional
risk factor for rejection. Two techniques are generally used to determine the
presence of donor-specific alloreactive T cells: the primed lymphocyte test
(PLT) and the cell mediated lympholysis
(CML) assay. The PLT assay is a short-term (2-3
days) proliferation assay of alloreactive T cells stimulated by irradiated
lymphoid cells from the donor. Most endomyocardial derived lymphocyte cultures
respond in PLT and the frequency of PLT reactivity shows some correlation with
the histological rejection grade [29,
30]. The majority of lymphocyte cultures propagated from
liver biopsies also express donor-specific alloreactivity [24]. Often these cultured lymphocytes exhibit restricted
allospecificity patterns towards one or few of donor HLA class I and class II
antigens [14]. We observed a correlation between liver biopsies
yielding class II specific lymphocytes and bile duct loss suggesting that
infiltrating anti-donor class II specific lymphocytes may play a role in bile
duct damage [25]. The CML assay determines the
presence of cytotoxic T cells that cause lysis of chromium labeled target cells
from the donor. Two types of target can be used: 3-day PHA activated T cells
that primarily express class I HLA antigens, and EBV transformed lymphoblastoid
cell lines that express both class I and class II HLA antigens. Many
endomyocardial biopsy cultures that contain CD8+ T cells exhibit cytolytic
activity against donor class I HLA antigens [16,
31-34]. This cytolytic activity correlates with the
histological grade of rejection [21] (Table 2). Furthermore the propagation of cytolytic
positive CD8+ T cells from histologically negative biopsies correlates with
subsequent rejection [21]. Table 2. Correlation between CML Activity of Cultured Lymphocytes and
Biopsy Histology
The
BAL has provided an accurate and sensitive method for diagnosing opportunistic
infections of the lung [35-37]. The procedure is non-invasive, and provides easy
access to cells and alveolar fluid from lung allografts. Serial BAL also offers
unique opportunities to study in situ intragraft immunity because of the direct
access to graft infiltrating cells. Functional studies on BAL cells recovered
from lung transplant recipients yield significant clinical information. The
incidence of donor-specific PLT activity in BAL lymphocytes during clinically
evident acute cellular rejection was 90% compared with an incidence of 11% in
patients whose clinical status was normal (p<0.001, Table 3) [38, 39]. Table 3.
Correlation of Clinical Status with Primed Lymphocyte Test Activity of
BAL Lymphocytes
* P < 0.001 acute rejection vs. normal, infection Most
PLT responses in the clinically normal group were associated with subclinical
acute rejection as verified by histological evaluation of lung biopsy. Although
33% of the BAL samples exhibited a PLT response during infection, this was
significantly less than during rejection. About 45% of PBLs had donor-specific
PLT reactivity and this alloreactivity did not correlate with clinical status.
These findings indicate that PLT activity of intragraft cells recovered in BAL
and not of the cells in PBL are clinically informative. Reinsmoen et al has
confirmed the presence of donor specific alloreactive T cells in BAL during
lung allograft rejection [40]. Obliterative bronchiolitis is the most significant long-term
complication following pulmonary transplantation [41-43]. This airway disease develops in more than 40% of
lung transplant recipients. Functional testing of BAL cells has been useful in
monitoring long-term lung transplant recipients for OB [39]. The PLT
activity of BAL cells precedes the decline in forced expiration volume (FEV1)
that is associated with progressive loss of pulmonary function. Furthermore
those patients that exhibit persisting donor specific PLT activity eventually
develop airway obstructive disease while the pulmonary function of the
long-term survivors who show little or no PLT activity in their BAL continues
to be normal [39, 44]. QUANTITATION
OF INTRAGRAFT CYTOKINE GENE EXPRESSION
Accurate diagnosis of the rejection
process in the solid organ allograft is critical for clinical management. Much
work has been done in the assessment of intragraft cytokine gene expression in
solid organ transplantation as an attempt to improve the diagnostic methods.
The development of sensitive molecular techniques permitted the detection of
cytokine mRNA in tissue biopsies and limited number of cells. In particular in
situ hybridization and polymerase chain reaction (PCR) amplification have been
successfully applied to learn more about the role of cytokines in allograft
rejection [45, 46]. Many cytokines are found during rejection of kidney,
liver, heart and lung allografts[47-49]. We and others have examined in human lung transplant
recipients the cytokine gene expression by using the reverse transcriptase PCR
(RT-PCR) technique [50, 51]. The up-regulation of IL-2, IL-4, IL-6, IFN-g and serine protease mRNA in BAL cells, but not in
peripheral blood (PBL) correlated with histological evidence of acute cellular
rejection. In contrast IL-8 and IL-10 mRNA can be detected in almost all BAL
and PBL samples from patients with rejecting and stable allografts [50]. Sundaresan et al also reported the frequent
detection of IL-2 and TNF-a in BAL during
allograft rejection [52]. Refractory rejection is a major cause
of morbidity and death in lung transplant recipients. Rescue therapies with
augmented immunosuppression have proved only modestly successful. At our
institution we introduced the aerosol cyclosporine as a new modality to treat
refractory lung rejection. Initially the efficacy of aerosol cyclosporine (ACsA) was evaluated in 12 patients and 9
exhibited histologic resolution within 3 months of treatment [53, 54]. All of the nine histologic responders exhibited 4 to
150 fold decreases (p<0.05) in IL-6 and IFN-g mRNA in BAL [53]. We also analyzed the effectiveness of ACsA in a
group of patients with refractory rejection prior, during and after 180 days of
ACsA treatment [55]. As shown in figure 2 control patients with
reversible rejection and those with refractory rejection expressed more BAL
cell IFN-g mRNA then
those controls without rejection (* p=0.003). IFN-g gene expression in recipients with refractory rejection
decreased after 50 and 180 days in treatment. Similar pattern was seen for IL-6
mRNA [55]. IL-10 mRNA
expression of BAL cells did not change during the ACsA [55]. Our results suggest that expression of IL-6 and IFN-g mRNA correlated with the histologic persistence of acute
rejection and these genes may be useful markers to assess the efficacy of
immunosuppressive drug regimens. The competitive RT-PCR assay that
permits a more quantitative determination of mRNA was applied for molecular
diagnosis of renal allograft rejection [56]. Quantification by the usage of competitor DNA
constructs in PCR revealed that intensity of rejection correlates with the
concentration of mRNA encoding the cytotoxic attack molecules Granzyme B (GB)
and perforin [56, 57]. We have also applied the quantitative RT-PCR method
for GB in lung transplant recipients during various clinical conditions. The
amount of intragraft mediator gene transcripts are calculated in picograms (pg)
and compared with the amount of GAPDH in pg [58]. As
illustrated in the example enclosed during CMV infection there was an increase
in GB over the baseline level detected during minimal rejection, while during
moderate to severe rejection the level of GB increased significantly (figure
3). Although the levels of GB dropped following treatment of rejection they
were still above the baseline. Histological examination of the lung biopsy
showed moderate rejection (3A) and the patient was treated again. After the second
bolus with steroids the patient's GB mRNA in BAL return to the baseline level.
These results suggest that the competitor RT-PCR can be useful to predict
effective response to augmented immunosuppression. DIRECT
AND INDIRECT ALLOPRESENTATION
T cell recognition of alloantigen is the central event that initiates graft rejection. There are at least two distinct pathways of allorecognition: in the "direct"� pathway, T cells recognize intact allo-MHC molecules on the surface of donor antigen presenting cells (APC) and in the "indirect"� pathway, T cells recognize processed antigen presented as allopeptides by self APCs [59-61]. The contribution of each pathway in the acute and chronic processes of allograft rejection is not completely understood. Several investigators have shown that early acute rejection is initiated by donor APC (direct pathway) while chronic rejection may occur without the persistence of donor APC (indirect pathway). Recent studies in cardiac and liver transplant recipients have shown that both T cell mediated pathways occurred early post transplantation [62-64]. New in vitro assays for quantitation of T helper cells recognizing donor antigen have been developed [64-68]. These assays permit the evaluation of immune reactivity toward donor graft using peripheral blood T cells. Although the allograft biopsy is considered the gold standard for monitoring graft rejection, it is invasive and may be associated with complications. The development of noninvasive immunologic methods for differential diagnosis of rejection is an important step in the management of transplant recipients. 1.
Quantitation of donor
antigen-reactive T helper cells The rise in frequency of IL-2 producing helper T cells (HTL)
is associated with cardiac allograft rejection [69]. Conversely, long term graft acceptance correlates
with a decrease of donor-specific HTL [70].
Donor-specific HTL can be quantitated by limiting dilution analysis [69, 70]. In brief, we stimulated various dilutions of
patients' PBL with a constant numbers of donor irradiated cells. After 48 hours
of incubation, an IL-2 dependent T cell line was added for an additional 24
hours. We measure proliferation by
adding 3H-thymidine to cultures for the last 6 hours. Cultures were considered positive for IL-2 release if cpm. exceeded the mean of 3H-Thymidine
incorporation in background control wells (20 wells that lack the responder
cells) by 3 fold. An example of T helper frequency for IL- 2 in a liver
transplant recipient is shown in figure 3. The donor-specific HTL was
significantly lower than the third party HTL at 5 and 9 months post
transplantation. This low donor-specific HTL frequency was associated with low
donor-specific mixed lymphocyte reaction (MLR) and stable allograft function.
In contrast, during a rejection episode at 14 months post transplantation the
liver transplant recipient had an increase in donor-specific HTL and exhibited
donor-specific MLR (figure 4).
2. Monitoring the donor-specific
reactivity via the direct T cell allorecognition
Recent observations in cardiac allografts have demonstrated
the utility of monitoring the expression of CD69, an early activation marker,
on memory peripheral blood lymphocytes [71]. PBL from
recipients depleted of monocytes (CD14+ cells) were stimulated for four hours
with donor derived B cells or with controls mismatched for both recipient and
donor. Cultures were stained with CD3,
CD69 and LDA1, a late differentiation antigen expressed on T helper cells. A ratio between the percent CD69+ T cells
stimulated by donor cells and control cells was calculated. Histopathologic
diagnosis of cardiac rejection was significantly associated (p<0.001) with a
ratio greater than 1, indicating that the determination of CD69 expression on
memory lymphocytes may represent a reliable method for differential diagnosis
of rejection. 3.
Monitoring the
donor-specific reactivity via the indirect T cell allorecognition
Indirect recognition of alloantigen
takes place in the peripheral lymphoid tissue where T cells recognize peptides
derived from HLA antigens of the graft processed by host antigen presenting
cells (APC). Prior to and during acute cellular rejection, allopeptide-reactive
T cells were detected both in peripheral blood and within the graft (heart,
liver) [62-64, 72]. The frequency of allopeptide-reactive T cells
estimated by limiting dilution assay was significantly elevated in patients
that were undergoing acute cellular rejection. Thus, monitoring both the direct
and indirect T cell alloactivation may provide an excellent tool for the
diagnosis of allograft rejection. The method used for determining the
frequency of allopeptide-reactive T cells was previously published [62-64]. Briefly, PBMCs are incubated in 96-well round-bottom
trays at various concentrations in 24 replicate reactions. The cells are
expanded in IL-2 for 7 days, washed and tested for donor-specific peptide
reactivity in a 3-day proliferation assay. The minimum number of alloreactive T
cells detected by this assay is 1.5 cells/million PBMC. ASSESSMENT
OF DONOR-SPECIFIC HYPOREACTIVITY
The ultimate goal after clinical whole-organ transplantation
is to achieve acceptance without chronic graft rejection and without the need
for continuous immunosuppression. The possibility of drug weaning has been
demonstrated in 25% of liver transplant recipients who were at least 5 to 10
years post transplantation with stable graft function [73, 74]. However, the risk of weaning liver patients too
early, too abruptly and without frequent testing of allograft function has also
been reported [75]. Although a
complete weaning may not be achieved in all allografts, the gradual reduction
of immunosuppressive drugs is beneficial. Improved long-term renal transplant
outcome has been associated with the development of in vitro donor-specific
hyporeactivity [76]. Patients who
exhibited donor-specific hyporeactivity had fewer episodes of late acute
rejection than the reactive patients and were free of chronic rejection [76]. In one
center, donor-specific hyporeactivity in mixed lymphocyte culture has been used
as criterion to discontinue prednisone [77]. Thus, stable
donor-specific hyporeactivity might be a useful parameter to identify the
recipients who may be eventually weaned from immunosuppression. Recent advancements in understanding
the dual role of donor-derived cells in promoting immunogenecity and tolerance
of solid organ allografts did provide the basis for designing new protocols for
organ allo-transplantation [78-81]. In an attempt to augment spontaneous donor cell
chimerism, a new therapeutic strategy was initiated which involved infusion of
donor bone marrow (BM) cells under conventional immunosuppressive treatment
with tacrolimus and steroids [80]. Single and/or multiple infusions of donor BM in
solid organ transplant recipients induced a significant augmentation of
donor-derived chimerism [80, 81] Sequential immunologic evaluations
of BM augmented and control solid organ transplant recipients were carried out
on peripheral blood mononuclear cells (PBMC) collected pre-transplant and at
one month, 3, 6, 9, and 12 months post-transplant [82]. The in vitro testing consisted of the following:
proliferative responses to mitogens (PHA and ConA) and recall antigens (TT and
CMV) to assess immunocompetence, proliferation of patients PBMC to allogeneic
stimulation (donor and third party unrelated panel cells). Donor-specific
hyporeactivity is defined as a significantly decreased (70%) post-transplant
versus pre-transplant mixed lymphocyte response to donor cells while
maintaining reactivity to third party HLA unrelated cells [82]. We have observed in
bone-marrow augmented solid organ transplant recipients that the development of
stable donor-specific hyporeactivity is achieved only 12 to 18 months
post-transplantation [82, 83]. Thus, any attempt to withdraw immunosuppression
should be done gradually even in clinically well patients at least three to
five years post-transplantation. BM-augmented liver transplant recipients who exhibited
stable donor-specific hyporeactivity had fewer episodes of late acute cellular
rejection than did those patients who remained donor MLR reactive [82, 83]. The development of chronic rejection in BM augmented
lung transplant recipients was significantly lower than in the control group
and those lung transplant recipients required less steroids [82, 83]. CONCLUSION As summarized in this chapter, the evaluation of recipient
immune status may be extremely important in assisting the clinicians.
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