The Association of Pre-Transplant Mixed Lymphocyte Reaction (MLR) with the Function of the Kidney Allograft and Antibody Response

Introduction

Organ transplantation is a life-saving procedure that replaces damaged or non-functional organs with healthy donor organs, significantly increasing life expectancy and enhancing the quality of life for patients suffering from organ failure.¹ Among transplanted organs, the kidney holds a particularly critical role. Kidney failure results in the accumulation of toxic substances in the body, creating a life-threatening condition.² While temporary treatments such as dialysis can provide short-term detoxification, they do not fully replicate the comprehensive functions of a healthy kidney. Kidney transplantation offers a definitive, long-term solution, enabling patients to regain normal physiological function and enhance their overall health.³

Donor characteristics and recipient variables—including age, sex, duration of dialysis, and comorbidities—along with the type of immunosuppressive drugs used, immunologic factors such as HLA compatibility, and the presence of donor-specific anti-HLA antibodies (DSA) are all associated with long-term graft survival.⁴ Crossmatch tests are assays designed to assess the immunological compatibility between allogeneic donor and recipient, significantly improving prognosis in various types of transplantation.⁵ Among the most important pre-transplant tests are serological and molecular HLA typing, panel reactive antibody (PRA) testing, recipient serum screening for antibodies against different HLA alleles, and the mixed lymphocyte reaction (MLR) assay.⁵ While DSA contributes to the immune response against the graft, cellular immunity plays a critical and distinct role in mediating immunologic rejection.^6,7 Although molecular and serological HLA typing facilitate donor-recipient matching, they do not correlate precisely with clinical outcomes. Immunologic reactions can still arise despite the avoidance of high-risk HLA alleles, while graft acceptance may occur even in the presence of potentially high-risk alleles such as A*11:01.⁸ This highlights an important gap in current practice: existing assays provide incomplete information about cellular allo-reactivity and cannot reliably predict transplant outcomes, underscoring the need for complementary approaches. One of the tests that simulates clinical conditions in this context, is the MLR assay. The MLR can be performed in either a one-way or two-way format. In the one-way MLR, donor cell proliferation is inhibited by irradiation or treatment with mitomycin C, allowing assessment of only the recipient’s response against the donor. In contrast, the two-way MLR evaluates both responses: the recipient’s reaction against the donor and the donor’s reaction against the recipient, the latter corresponding to graft-versus-host disease (GVHD).⁸ Early methods for MLR focused on detecting lymphocyte proliferation using radioactive techniques, such as the incorporation of H3-thymidine into the lymphocyte genome, which required specialized equipment.⁹ With advancements in flow cytometry-based proliferation assays, new techniques like CFSE-MLR have been developed.¹⁰ This method relies on the progressive dilution of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) within the cytoplasm of daughter cells after division. In this assay, recipient lymphocytes are initially labeled with CFSE and then co-cultured with mitotically inactivated donor lymphocytes. Finally, the cells are analyzed by flow cytometry to quantify the percentage of proliferating cells and determine the number of lymphocyte generations.¹⁰ Unlike other assays and given the novelty of this technique, standardization and determination of cut-off values for correlating the percentage of cell proliferation obtained from this test with clinical outcomes have not yet been established. Furthermore, owing to the assay’s inherent characteristics, standardized protocols must be independently developed and validated at each laboratory to ensure reproducibility and reliability of results.^8,11 Considering the significance of the MLR as a critical pre-transplant assay for evaluating immunologic compatibility between donor and recipient, and accepting the technical complexity and specialized infrastructure required for radioactive cell labeling techniques, the implementation of a CFSE-based MLR assay offers a viable and practical alternative. Accordingly, this study was designed to address this unmet need by evaluating and standardizing the CFSE-MLR method, thereby contributing exploratory evidence on its potential correlation with post-transplant DSA levels and allograft function as assessed by serum creatinine levels.

Materials and Methods

Study Population

This study enrolled 14 donor-recipient pairs who met the cross-match criteria necessary for kidney transplantation. The present study was rather an exploratory study to assess the rationale and clinical relevance of CFSE-MLR to post-transplant findings. The study population comprised patients deemed eligible and medically prepared for transplantation. Inclusion criteria required both donors and recipients to be considered suitable candidates for transplantation by the attending physician, including living donors or those from whom peripheral blood samples could be obtained, and the use of Corticosteroid, Mycophenolate Mofetil, and Calcineurin Inhibitor in therapeutic doses as a common immunosuppressive regimen. Exclusion criteria encompassed recipients with a history of inflammatory diseases or malignancies. All kidneys were donated voluntarily with written informed consent, and the donation and transplantation procedures were carried out in accordance with the principles of the Declaration of Istanbul. Peripheral blood samples were collected from all participants after obtaining written informed consent, which included a thorough explanation of the study objectives and potential outcomes. The study protocol was approved by the Ethics Committee of the Tabriz University of Medical Sciences (Ethical approval Code: IR.TBZMED.REC.1400.590) and was conducted in accordance with the principles outlined in the Declaration of Helsinki on research involving human subjects.

PBMC Isolation

Five milliliters of peripheral blood were collected from each kidney donor and recipient into heparinized tubes. The blood samples were diluted 1:1 with RPMI-1640 medium and gently layered onto an equal volume of Ficoll-Paque solution (Baharafshan Co., Tehran, Iran) (density 1.077 g/mL). Samples were centrifuged at 300 ×g for 20 minutes at room temperature to isolate the PBMC layer. The mononuclear cells were carefully collected, counted using a Neubauer hemocytometer, and cell viability was assessed by trypan blue assay.

Mixed Lymphocyte Reaction (MLR) Assay

Recipient PBMCs (responder cells) were labeled with the CFSE (Biolegend, San Diego, CA, USA) according to the following protocol: CFSE stock solution was prepared by dissolving 18 µL of dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) in a vial. Splenic cells at a concentration of 1×10⁶ cells/mL were suspended in PBS containing 0.1% bovine serum albumin (BSA) (Biosera, Nuaillé, Pays de la Loire–France). CFSE was added to the cell suspension at a final volume of 2 µL per 1×10⁶ cells, and cells were incubated at 37°C for 10 minutes in the dark. Subsequently, five volumes of ice-cold complete culture medium were added to quench the staining reaction. Cells were then placed on ice for 5 minutes, centrifuged at 300 ×g for 5 minutes, and washed three times with cold culture medium. A total of 2×10⁵ CFSE-labeled responder cells were co-cultured with an equal number of donor PBMCs, which had been pretreated with mitomycin C (10 µg/mL) (Accord, London, UK) and incubated for 3 hours to inhibit proliferation. Co-cultures were maintained in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA) at 37°C in a humidified 5% CO₂ incubator for 5 days. At the end of incubation, cells were harvested, washed with PBS, and 5000–7000 events were analyzed by a FACSCalibur flow cytometry instrument (Becton Dickinson Biosciences, CA, USA). The lymphocyte population was first gated based on an FSC-H and SSC plot. Then, the mean fluorescent intensity (MFI) of CFSE was evaluated in the gated lymphocytes within FL1 channel. The fluorescence peak was adjusted to be placed in right-side of the histogram and the low MFI region was gated as an indicator of the proliferated cells. The percentage of low MFI cells, which were the result of dye dilution during cell division, was quantified as indicator of the percentage of activated cells.

Panel Reactive Antibody (PRA) Evaluation

The PRA values were obtained using a LABScreen Flow-PRA kit (One Lambda, CA, USA) according to the manufacturer’s instructions. Briefly, 20 µL patient serum was added to 5 µL of class I PRA beads and class II PRA beads in separate microtubes. The samples were incubated at room temperature for 30 minutes in the dark with gentle shaking. Then, 150 µL of wash buffer was added to each sample. The samples were washed 3 times with wash buffer and centrifuging at 1300 × g for 5 minutes. Then, 100 µL of diluted anti-human FITC secondary antibody was added to each sample and were incubated at room temperature for 30 minutes in dark with gentle shaking. The samples were washed 3 times with wash buffer and re-suspended in PBS for flow cytometric analysis by a FACSCalibur instrument (Becton Dickinson Biosciences, CA, USA).

Donor-Specific Antibody (DSA) Detection

The DSA evaluation was carried out as previously described.¹² Briefly, donor PBMCs (2 × 10⁶ cells) were lysed using RIPA buffer supplemented with a proteinase K inhibitor to extract total protein. Protein concentration was determined by the Bradford assay. Protein samples were diluted to a final concentration of 5 µg/mL in PBS and coated onto high-binding ELISA plates (200 µL per well). Plates were incubated overnight at 4°C, followed by washing with PBS and blocking with 2% BSA for 1 hour at room temperature to minimize nonspecific binding. Subsequently, wells were washed three times with PBS containing 0.1% Tween-20. Recipient sera collected at 1 week, 1 month, 2 months, and 3 months post-transplant were diluted 1:10 and incubated in the coated wells for 1 hour at room temperature. After washing, horseradish peroxidase (HRP)-conjugated anti-human IgG antibody was added and incubated for 30 minutes at room temperature. Following additional washes, 100 µL of TMB substrate was added to each well and incubated for 15 minutes in the dark. The enzymatic reaction was terminated by adding 50 µL of stop solution, and optical density was measured at 450 nm with 650 nm as the reference wavelength. Absorbance values correlated directly with DSA titers.

Assessment of Graft Function and Correlative Analyses

Graft function was evaluated by measuring serum creatinine. The creatinine levels were correlated with cellular proliferation indices derived from the MLR assay. Furthermore, the association between pre-transplant MLR proliferation rates and DSA titers was analyzed. Correlations between MLR, DSA results, PRA percentages, and other pre-transplant immunological tests were also assessed.

Statistical Analysis

Comparison of mean values of dividing cells MLR assay was carried out by one-way analysis of variance followed by Tukey’s multiple comparison test. In the case of correlation analyses, for datasets exhibiting a Gaussian distribution, Pearson’s correlation coefficient was used and for those without a normal distribution, Spearman’s rank-order correlation was employed to determine the strength and significance of associations.

Results

Demographic Data of the Study Population

Peripheral blood samples were collected from 14 kidney transplant candidates and their corresponding donors. Recipients included in the study were non-sensitized patients with PRA indices below 5%. Table 1 summarizes the demographic characteristics of the study population.

Table 1 Demographic and Clinical Characteristics of Study Population

MLR Between Donor and Recipient

Lymphocyte proliferation in the MLR assay was quantified based on the dilution of the cytoplasmic fluorescent dye CFSE among daughter cells. Significant increase in the percentage of proliferating cells was observed at days 3 and 5 compared to baseline (day 0) (p < 0001) (Figure 1).

Figure 1 Proliferation of peripheral blood mononuclear cells in the MLR assay. Histograms demonstrating CFSE fluorescence shifts over time (a), bar chart of proliferation rates at different time points (b).

Correlation Between Cellular Proliferation and the Laboratory Findings

The potential correlation between the percentage of cell proliferation on day 5 and PRA class I and II levels was evaluated. A statistically significant positive correlation was observed between day 5 proliferation and PRA class II levels (Figure 2a). In contrast, no significant association was detected between pre-transplant cellular proliferation and DSA titers measured at six months post-transplant by ELISA using donor cell lysates (Figure 2b). The donor age did not show significant correlations with pre-transplant lymphocyte proliferation in MLR assay and post-transplant DSA formation (Figure 3c).

Figure 2 Correlation of the donor-related parameters and the laboratory findings. (a) Correlation of the percentage of cell proliferation in the MLR assay with PRA before transplantation. (b) Correlation of the MLR cell proliferation and the post-transplant DSA formation. (c) Correlation of donor age and cell proliferation in MLR assay (left) and DSA formation (right).

Figure 3 Correlation between the percentage of peripheral blood mononuclear cell proliferation before transplantation and serum creatinine levels at 1, 3, and 6 months post-transplantation.

Post-Transplant Serum Creatinine and Correlation with MLR Proliferation

Correlation analyses were conducted between pre-transplant MLR proliferation percentages on days 3, 5, and serum creatinine levels at 1, 3, and 6 months post-transplant. A significant positive correlation was observed between day 5 proliferation and serum creatinine level at 6 months post-transplant (Figure 3).

Discussion

In a study involving 19 kidney transplant recipients from deceased donors, a reduction in donor-specific response assessed by MLR at 3 and 6 months post-transplantation was associated with improved graft outcomes within the first year.¹³ However, conventional MLR exhibits poor reproducibility and limited predictive value in clinical transplantation.¹⁴ Therefore, CFSE-MLR, which more precisely measures T cell proliferation using a fluorescent dye, is preferred over the traditional 3H-thymidine-based MLR.¹⁵ This method enhances both accuracy and reproducibility, thereby providing a better evaluation of immune responses and the risk of graft rejection. Moreover, CFSE-MLR allows a more specific assessment of cellular responses to donor antigens compared to traditional MLR.¹⁵ Based on CFSE-MLR results, lower incidences of acute rejection and improved graft survival have been observed.¹⁶ The CFSE-MLR technique, a flow cytometry-based tool, enables high-precision tracking of cellular proliferation without the need for radioactive substances. This feature makes it a safer and more practical option for pre-transplant assessment.¹⁷ However, the lack of well-defined standards for this method has limited its widespread application. Therefore, the present study aims to establish standardized parameters and evaluate the predictive value of CFSE-MLR in donor-recipient matching, providing an effective approach to enhance pre-transplant diagnostic procedures.

In a study conducted in 2022 involving 461 patients with end-stage renal disease (ESRD) undergoing hemodialysis, the predictive value of various tests, including the MLR, was evaluated for prognostic assessment. The findings revealed that the mortality rate among patients with high MLR levels was approximately 32.35%. Elevated MLR values were also associated with prolonged hospital stays and a greater number of dialysis sessions.¹⁸ In a study by Tanaka et al, donor-specific alloreactive responses were investigated to accurately diagnose acute rejection following living-donor liver transplantation using MLR assay combined with CFSE. Despite moderate-to-severe liver dysfunction in 41% of patients within 6 months post-transplant, the incidence of acute rejection was low, reported at approximately 13.8%. These findings suggest that the CFSE-MLR method can serve as a reliable tool for precise monitoring of graft rejection.¹⁰ Consistent with these findings, in our study, CFSE-MLR demonstrated a preliminary potential to be harnessed as a tool to estimate the allograft’s fate. However, there have been attempts on other cellular assays such as ELISPOT and flowcytometric cytotoxic T-cell evaluations.^19,20 While all these approaches have been accompanied with accomplishments, they seem to have similar challenges such as complexity in development of standardized protocols and difficulty in determination of normal ranges.

In a 2024 study by Sica and coworkers, the impact of DSA on outcomes following allogeneic hematopoietic stem cell transplantation (alloSCT) was investigated. The study found that DSA positivity was directly associated with neutrophil and platelet engraftment failure within 30 days post-transplant. This underscores the importance of pre-transplant DSA screening to optimize donor selection and transplantation strategies. Presence of DSAs at transplantation is also linked to graft rejection and reduced survival in solid organ transplants.²¹ However, in our study, no significant correlation was observed between pre-transplant cellular proliferation percentage and serum DSA titer at six months post-transplant. Although the CFSE‑MLR assay may offer preliminary insights into cellular allo‑reactivity, several limitations should be noted. The limited sample size reduces the statistical power and generalizability of the results, the absence of adjustment for potential confounding factors may have affected the observed relationships, and the single‑center setting restricts external validity. Consequently, these findings should be interpreted with caution. Future investigations involving larger, multicenter cohorts and rigorous control of confounders will be essential to clarify whether CFSE‑MLR can be established as a dependable approach for evaluating transplant prognosis and graft survival.

Conclusion

This study investigated the correlation between the results of the MLR assay and the function of the transplanted kidney, as well as its association with immune indicators such as DSA titers and percentage PRA. Our findings may contribute to a better understanding of the relationship between pre-transplant immunologic responses and transplant outcomes. Importantly, while the CFSE‑MLR method may provide exploratory insights into cellular allo‑reactivity, the small sample size of this study limits the strength of its predictive utility. Validation in larger, multicenter cohorts will be necessary to determine whether CFSE‑MLR can serve as a reliable tool in the assessment of transplant prognosis and graft survival.