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
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 . 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 . 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 . 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 . 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 . 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  (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 . 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 . More than 40% of patients whose biopsies persistently grew lymphocytes developed GCD. In contrast, only 6% with non-grower biopsies had subsequent GCD .
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 . Often these cultured lymphocytes exhibit restricted allospecificity patterns towards one or few of donor HLA class I and class II antigens . 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 .
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  (Table 2). Furthermore the propagation of cytolytic positive CD8+ T cells from histologically negative biopsies correlates with subsequent rejection .
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 .
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 . 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 . Sundaresan et al also reported the frequent detection of IL-2 and TNF-a in BAL during allograft rejection .
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 . We also analyzed the effectiveness of ACsA in a group of patients with refractory rejection prior, during and after 180 days of ACsA treatment . 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 . IL-10 mRNA expression of BAL cells did not change during the ACsA . 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 . 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 . 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 . Conversely, long term graft acceptance correlates with a decrease of donor-specific HTL . 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 . 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 . 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 . Patients who exhibited donor-specific hyporeactivity had fewer episodes of late acute rejection than the reactive patients and were free of chronic rejection . In one center, donor-specific hyporeactivity in mixed lymphocyte culture has been used as criterion to discontinue prednisone . 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 . 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 . 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 .
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].
As summarized in this chapter, the evaluation of recipient immune status may be extremely important in assisting the clinicians. Sequential evaluation of patient's immune status should be considered in any protocols that incorporate modification of the immunosuppression.
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