| Abstract|| |
Allograft rejection remains a major barrier to successful organ transplantation. Cellular and humoral immune responses play a critical role in mediating graft rejection. During the last few years, monoclonal antibodies have been used as a new specific therapeutic approach in the prevention of allograft rejection. Recently, the technology of flow cytometry has become a useful tool for monitoring immunological responses in transplant recipients. The application of this valuable tool in clinical transplantation at the present time is aimed at, i) determining the extent of immunosuppressive therapy through T-cell receptor analysis of cellular components, ii) monitoring levels of alloreactive antibodies to identify high-risk recipients (sensitized patients) in the pre-operative period and iii) to predict rejection by monitoring their development postoperatively. In future, further development of this technology may demonstrate greater benefit to the field of organ transplantation.
Keywords: Flow cytometric crossmatch, Organ transplantation, Sensitized patients, Allo-antibodies, Allograft rejection.
|How to cite this article:|
Al-Mukhalafi Z, Pyle R, Al-Hussein K. Monitoring Immune Responses in Organ Recipients by Flow Cytometry. Saudi J Kidney Dis Transpl 2001;12:32-41
|How to cite this URL:|
Al-Mukhalafi Z, Pyle R, Al-Hussein K. Monitoring Immune Responses in Organ Recipients by Flow Cytometry. Saudi J Kidney Dis Transpl [serial online] 2001 [cited 2019 Nov 19];12:32-41. Available from: http://www.sjkdt.org/text.asp?2001/12/1/32/33883
| Introduction|| |
In recent years, the application of flow cytometric technology in the field of medicine has grown dramatically resulting in numerous benefits for diagnosis and management of diseases. A wide range of fluorescent probes are available for directly estimating or analyzing cellular and biochemical factors including nucleic acid content, enzyme activity, calcium flux, antibodies and cell surface molecules.
Flow cytometry (FC) has begun to play a role in the evaluation of allograft recipients, both before and after transplantation. The techniques are promising, since they are more sensitive and specific than traditional methods used to evaluate transplant recipients. The application of flow cytometry in this area of research is likely to provide new insight into our understanding of the immunology of transplantation.
In this article, we will discuss briefly the application of flow cytometric analysis of labeled T-subset and allo-antibodies in the evaluation of organ transplantation.
Basic Principles of Flow Cytometry
Flow cytometry is known as the automated analytical cytology. As the term implies, FC is a technology which allows the simultaneous measurement of multiple physical properties (metry) of cells or particles (cyto). These measurements are made for cells or particles when they pass at a rate of 500 to 4000 per second through a fluidic system (flow) intersected by a laser beam. In the measurement region, cells that are stained with one or more fluorochrome markers for specific cellular epitopes are detected. At present, there are three to four fluorescent dyes, which relate to the fluorochrome markers for cell surface epitopes. Any fluorescent stain on the cell surface is excited as the cell passes through the laser excitation beam. The scattered light is detected by a set of detectors that results in an electrical signal equal to the intensity of fluorescence, which is proportional to the amount of bound fluorochrome. The forward scatter (FSC) is related to the cell size, while the side scatter (SSC) is related to the internal cellular complexity or granularity. The electrical signals that result from the scattering of all the parameters are transferred to a computer module connected to the flow cytometer. Data are collected and analyzed using various integrated softwares. 
Reagents for Flow Cytometry
The discovery of monoclonal antibodies by Kohler and Milstein in 1975  that resulted in awarding the Nobel prize to these scientists, led to a significant advancement in the analysis of cell surface marker expression. The advent of laser cytometry and defined monoclonal antibodies, has permitted precise identification and selection of cells by the surface marker expression, which provides valuable information for both research and clinical applications. Monoclonal antibodies are very pure immunological reagents that can be produced from heterogeneous antigenic epitopes that assign these antibodies a major advantage to define the antigenic structure expressed on cells, in various stages of differentiation or disease. 
| Clinical Applications of Flow Cytometry for Organ Transplantation|| |
Immunobiology of Allograft Rejection
During the immune response to the allograft, which leads to graft rejection, the recipient's T lymphocytes (CD3 + cells) are activated by recognizing the donor antigens of the graft. These antigens are processed and presented by the antigen presenting cells (APC), either by the direct or indirect pathways.  This activation is a very complicated process and is controlled by a series of cell-cell surface interaction, which requires delivering various signals, soluble protein interactions and secretion of cytokines. , These factors trigger phagocytes, natural killer cells, T cells including T helper CD4 + and T cytotoxic CD8 + cells, B cells that produce cytotoxic or non-cytotoxic antibodies, and other inflammatory cells. All of these initiate the development of a variety of immune effector mechanisms, which result in the acute rejection of grafts. The most important mechanism of acute rejection is mediated by antibody-dependent complement cytotoxicity (ADCC). 
Monitoring the Immunosuppressive Therapy
It is established that the T cells and their subsets play an important role in the initiation and maintenance of the immune response against the graft. Therefore, when using an anti-CD3 agent as immunosuppressive therapy, it is important to ensure a significantly low number of these cells in order to prevent their immune response.
One purpose of evaluating the lymphocyte subsets in transplant recipients is to detect rejection in its early stages. Consensus does not exist on many issues related to the analysis of these subsets and their correlation with graft rejection. However, with the continuing work of many researchers for a more complete understanding and classification of lymphocyte markers, a greater consensus may be reached.
In patients with stable grafts, the most consistent change in T cell subsets after organ transplantation is the decrease of CD4 lymphocytes and an increase of CD8 lymphocytes. ,, A decrease in CD4 subset by immunosuppression is found mainly in those patients who had initially cells with helper/inducer function. 
In patients with graft rejection, some early studies demonstrated that acute rejection rarely occurs in patients who display a low CD4/CD8 ratio in the post-transplant period.  However, this observation was not confirmed by subsequent reports.  Another marker apparently associated with acute rejection is HLA-DR expression on CD8 lymphocytes.  DR expression on T lymphocytes is usually very low and it increases during rejection on CD8 but not on CD4 lymphocytes. HLA-DR was restored to normal levels after reversal of the episode of rejection. Another marker of T-cell activation, interleukin-12 (IL-12) receptor, did not increase during rejection. 
Anti-CD3 murine monoclonal antibodies are the most widely utilized antibodies in immunosuppressive therapy at the present time. A significant reduction of CD3-positive lymphocytes occurs during therapy. AntiCD3 monoclonal antibody (mAb) therapy has been shown to be highly effective in the treatment and prevention of organ allograft rejection. ,, However, significant problems are associated with this form of therapy including first-dose morbidity, the generation of a strong humoral response against the mAb, and the lack of specific tolerance.
The most obvious explanation for the immunosuppression observed with antiCD3 mAb is T cell depletion. This was initially assumed to be the major mechanism of action; however, as more therapeutic experience was gained, it seemed unlikely to be the only mechanism. Observations in humans suggested that Tcell receptor (TCR) modulation occurred in vivo, , since the first T cells to reappear in the peripheral blood after administration of anti-CD3 mAb, expressed CD4 or CD8, but not CD3. Because the majority of CD4 + and CD8 + cells were CD3 - for almost 10 days after treatment, the observed acute T cell dysfunction after administration of antiCD3 may be consequent to the inability of the TCR to function adequately due to modulation or blockade. ,
Further evidence that additional mechanisms beside T cell depletion, TCR blockade, and TCR modulation are involved in immunosuppression arose from studies using the mouse model. T cells in the lymphoid organs are dysfunctional after the administration of anti-CD3 mAb, despite the re-expression of unbound TCR to the mAb. This illustrates a state of T cell hypo-responsiveness postanti-CD3 therapy. Thus, a number of points regarding the immunosuppressive effects of anti-CD3 mAb therapy have arisen. First, the rapid suppression of T cells that is observed in the peripheral blood does not appear to be solely consequent to the T-cell lysis. Second, induction of the state of hypo-responsiveness is responsible for at least a portion of the observed effect. 
One of the complications of treatment with the anti-CD3 mAb is the induction of a humoral response that is observed both in humans ,,, and in mice,  and often has a large anti-idiotypic component. Several approaches can be envisioned for suppressing this humoral response to the anti-CD3 mAb. First, concomitant immunosuppressive therapy can be used to suppress B cell function. Second, attempts could be made to reduce the immunogenicity of this xeno-antibody. Third, the mAb could be molecularly altered to remove the xeno-geneic portion. Concomitant immunosuppressive therapy is routinely used in the clinical setting by the administration of cyclosporine or azathioprine during anti-CD3 mAb therapy. , Reduction of the immunogenicity of the xeno-antibody has been accomplished in mice by removing the constant region of an immunoglobulin molecule (Fc portion). In mice, the variable region of the immunoglobulin molecule Fab2 is much less immunogenic than the whole mAb. Molecular alteration of the mAb is being considered to engineer a version of the antibody that would reduce the humoral response while maintaining the immunosuppressive properties.
Furthermore, anti-thymocyte globulin (ATG) therapy has been effectively used for more than 20 years in the prophylaxis and treatment of rejection of renal allograft. It has proved itself as a powerful suppressor of T cell function both before and during the cyclosporine era. ,,,,
Gorrie et al  demonstrated that ATG was a successful anti-rejection therapy with satisfactory graft function at 6-9 months post-therapy. Lower than recommended doses of ATG proved its effectiveness in the prophylaxis and treatment of renal allograft rejection. The major immunosuppressive effect of ATG is the depletion and functional alteration of T lymphocytes. Flow cytometry has been used in monitoring T cells treated by ATG.  Although ATG is effective in reversing or delaying allograft rejection, such treatment is frequently associated with many undesirable side effects, including bone marrow suppression and high incidence of viral and bacterial infections. ,,,
Laboratory monitoring of CD3 + lymphocyte cell counts should be performed during therapy with mAb. Pre-treatment counts of the circulating CD3 + lymphocytes should be performed prior to the first dose of antiCD3 mAb to establish baseline values. Thereafter, the reduction in the circulating CD3 + lymphocytes can be maintained at <200 CD3 + lymphocytes/µl of blood.  A specific measurement of both CD2 and CD3 markers helps to observe the different phases of response during treatment. The CD2 marker is present on all non-B lymphocytes; it is not modulated on T lymphocyte cell membrane when exposed to anti-CD3 mAb, as the CD3 marker.  The presence of CD2 + CD3 - lymphocytes in the peripheral blood is an indicator of successful treatment.
Monitoring lymphocyte subsets easily identifies the dose of anti-CD3 mAb needed to achieve optimal treatment. When performing lymphocyte subset analysis, CD2-FITC (FITC, fluorescent isothiocyanate) and CD3-PE (PE, phycoerythrin) and conjugates are utilized allowing the dual staining of the cells of interest. This dual staining is critical to demonstrate the coexpression of the target markers. Appropriate isotype controls should be employed to eliminate the possibility of non-specific staining which may be incorrectly interpreted as a positive result. 
An appropriate amount of the antibody (determined by an antibody titer or manufacturer's recommendation) is added to 100 µl of whole blood and incubated for 30 minutes at 4 o C. Incubation in cold temperature helps to prevent the capping effect of the bound antibody on the cell surface. After incubation, the cells are washed twice and then the RBCs are lysed using one of the standard commercial lysing kits. The remaining WBCs are then washed and fixed by 2% paraformaldehyde. At this time, the cells are ready for analysis on the flow cytometer and can be stored at 4 o C for up to 72 hours prior to analysis. 
The percentage of positive cells expressing either CD2/CD3 is determined and then an absolute number of cells can be calculated using the WBC count, which has been performed on the test sample with the automated hematology cell counter. The desired target range during treatment period for CD3 + lymphocytes in the whole blood is < 200 cells/µL. 
Flow Cytometric Analysis of Allo-antibodies
In 1983, a flow cytometric crossmatch method (FACS) capable of detecting donorreactive antibodies independent of complement fixation was developed.  In this method, serum of the potential recipient is incubated with the donor lymphocytes, then fluorochrome conjugated antibody directed against human IgG or IgM is added. The antibody is identified and measured by the surface fluorescence of the lymphocytes. This method detects two fluorescent peaks, a dim T-cell and a bright B-cell peak. 
Later studies indicated that the natural killer (NK) cells were present in the second peak and these cells posed a problem in the interpretation of the results.  In an attempt to resolve this problem, other workers have used manual separation of T and B cells. , The dual color flow cytometric test was developed with the objective of identifying the different cell types that stained with the recipient's sera. ,,, A fluorescent isothiocyanate anti-human IgGFc specific reagent (FITC anti-IgG-Fc) and phycoerythrin (PE) conjugated monoclonal antibodies (anti-CD3 and anti-CD20) have been recommended for use to discriminate between antibodies directed against T and B cells. Serum samples from normal, healthy, un-transplanted males have been used as negative controls and serum pooled samples that stained 100% of donor lymphocytes were used as positive controls. Using antiFc-IgG fragments as the antibody contributed to a further improvement in the methodology. ,,
Since then, the flow cytometric crossmatch has proven to be the most sensitive crossmatch procedure that could predict more episodes of acute graft rejection,  primary non-function  and graft loss. ,
With calibration beads, the surface concentration of the donor directed antibodies can be determined.  Conversely, the results reported from other centers indicated that the FACS crossmatch is not always a contraindication to organ transplantation. Lazda et al  reported an increased incidence of rejection episodes in patients with a positive FACS crossmatch, but no differences were found in graft survival when compared to the patients with negative FACS crossmatches. The specificity of FACS crossmatch was also questioned by Wang et al,  who suggested that the positive crossmatch determined by the flow cytometric protocols was falsely positive. They used donor platelets as cell targets and found that 50% of the renal allograft recipients with positive T cell FACS crossmatches showed positive platelet antibodies. Fifty-eight percent of these patients had a non-functioning renal allograft at one month post-transplant. Therefore, Wang et al proposed that only those cross-matches which were both T cell and platelet positive should be considered as contra indication to transplantation.  Moller et al,  demon strated the significance and relevance of the new crossmatching techniques. They concluded that the specificity of antibody detected in sera of patients undergoing any type of organ transplantation using standard or new crossmatch method should be determined. The class of immunoglobulin also should be known, since cytotoxic antibodies are detrimental to graft function while most antibodies of the IgM class are often autoreactive antibodies. The target for these antibodies would need clarification. However, it became clear that there is a significant prognostic role of the use of FACS crossmatch as a method for detecting antibodies. 
In addition to the FACS, a method of flow cytometry crossmatching (FCXM) has proven to be superior to the standard complementdependent cytotoxicity crossmatch (CDCXM). FCXM has been demonstrated as a reliable and useful predictor of long-term clinical outcome in renal transplant recipients.  Cook et al  also found FCXM effective in identifying kidney transplants at risk of early graft rejection. In a recent report, a strong association was found between a positive FCXM and subsequent development of chronic rejection.  This finding raises the possibility that more aggressive treatment of the patients with positive FCXM might ultimately result in a lower incidence of chronic rejection and improve overall graft survival.
Post-Transplant Alloantibodies Monitoring in Renal Recipients
The use of the crossmatch as a predictor of post-transplant rejection is not yet fully elucidated. Coppola et al  performed their experiments on a canine model and found antibodies that heralded or, were associated with acute rejection. In human beings, antibodies against living related donor lymphocytes in the post-transplantation period before the clinical detection of rejection,  or against a panel of unrelated lymphocytes have been identified. , Cochrum et al  used donor renal antigens as a target, and Trickett et al  used the indirect immunoperoxidase technique on sections of donor renal tissue, and conventional cytotoxic assays with donor lymphocytes; both found that the presence of antibodies correlated with graft damage. All these studies used the conventional cytotoxic assay.
With the use of the binding fluorescence assay, McPhaul et al  showed that, of those antibodies developing post-operatively, some targeted HLA antigens while others targeted structural epitomes such as endothelial antigens. Huber et al  utilized a binding assay on cultured renal cell lines to identify antibodies during rejection. Antidonor antibodies were detected after transplantation using the dual color FACS method. The introduction of the flow cytometric crossmatch technique in the preoperative period has provided a more sensitive and precise method to detect the presence of anti-donor antibodies and the association with graft loss. ,, Detection of low levels of IgG antibodies directed against donor lymphocytes by flow cytometry was associated with an increased number of rejection episodes in the immediate post-transplantation period. , Furthermore, some reports have shown a correlation between the presence of antibodies in the pre- and posttransplantation periods to endothelial and epithelial antigens using different cell lines. , Using the flow cytometric method for detection of antibodies in the posttransplantation period, we found that in renal transplant recipients, the presence of high levels of T and B-cell IgG antibodies, specific to the donor lymphocytes, were associated with both an increased number of severe rejection episodes and delayed graft function. , Moreover, IgG antibodies were also detected before the clinical diagnosis of rejection. Accordingly, alterations of immunosuppressive therapy were possible. ,
| Conclusion|| |
Flow cytometry offers many advantages for the study of humoral and cellular components of the immune system in the transplant patients. The greater sensitivity and specificity of the flow cytometry technique to analyze allo-reactive antibodies in comparison to the standard methods may help in detecting some forms of the antibodymediated rejection. The ease with which flow cytometry characterizes the major and minor lymphoid populations makes it a useful tool for monitoring the cell-mediated events during the post-transplant period.
The flow cytometric monitoring seems to be a simple, rapid and sensitive procedure to evaluate serum anti-CD3 antibody levels and circulating CD3 + cells. Thus, flow cytometry could be utilized for transplantation in i) diagnosing cellular or humoral rejection early, which helps in the management of immunosuppressive therapy, ii) defining highly sensitized recipients who have high levels of panel reactive antibodies, and iii) monitoring alloreactive antibodies in the post-transplant period.
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Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Center, MBC-03, P.O. Box 3354, Riyadh 11211