Objectives: Regulation of immune responses in renal transplant recipients is complex, and the CTLA-4 gene plays a crucial role in maintaining immune tolerance. Methylation of CpG regions in the CTLA-4 promoter may affect its expression, potentially influencing transplant outcomes. This pilot study investigated the relationship between CTLA-4 promoter methylation and gene expression in CD8+ T cells of renal transplant recipients.
Materials and Methods: We analyzed the CTLA-4 promoter methylation profile in CD8+ T cells from 29 renal transplant recipients using bisulfite sequencing. The methylation status of specific CpG regions was correlated with CTLA-4 expression levels to assess any potential influence of methylation on gene regulation.
Results: Two patients with hemi-methylated CpG regions in the CTLA-4 promoter showed an increase in CTLA-4 expression. However, no overall correlation between methylation levels and gene expression was observed across the patient cohort.
Conclusions: The findings suggested that immune responses and treatment outcomes may differ considerably between individuals. In addition, other epigenetic mechanisms may play a role in modulating CTLA-4 expression. These results highlight the complexity of immune regulation in renal transplant recipients and the need for further investigation into the epigenetic factors influencing transplant outcomes.

Key words : Cytotoxic T-lymphocyte-associated antigen 4, Immune regulation, Kidney transplan

Introduction

During T-cell maturation, naive T cells need more than 1 stimulatory signal. Binding of T-cell receptor and major histocompatibility complex ensures specificity of T-cell activation.¹ B7-1 (CD80) or B7-2 (CD86) mole-cules on the surface of antigen-presenting cells bind with CD28 on the T-cell surface. T-cell proliferation begins with CD28-CD80/86 binding.² However, T-cell activation should be under control because overprolife-rated T cells can attack self-antigens and cause autoim-munity.³ At this point, immune checkpoint regulator molecules come into play and limit T-cell proliferation. The most studied of these regulatory molecules that limit T-cell activation are cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1) proteins. CTLA-4 stops autoreactive T cells at the initial stage of naive T-cell activation, whereas the PD-1 pathway regulates activated T cells in the later stages of the immune response.⁴
Studies aimed at understanding the role of CTLA-4 in organ transplant have been generally aimed at its role as the target of immunosuppressives.^5,6 Apart from this, animal experiments conducted to understand the role of CTLA-4 in peripheral tolerance have generally focused on CTLA-4. Increased CTLA-4 expression has been shown to affect tolerance after pancreatic islet cell transplant, with a study deter-mining that CTLA-4 upregulation had a direct effect on allograft survival.⁷ CTLA-4 expression in T-regulatory (Treg) cells is under the control of FoxP³; thus, expression of CTLA-4 is thought to have cell-specific variations. Transcriptional control of CTLA-4 may also be important in immune regulation. In patients with rheumatoid arthritis, downregulation of CTLA-4 expression in Treg cells is linked to methylation of a nuclear factor of activated T-cell binding site within the CTLA-4 gene promoter, leading to impaired suppressor function.⁸ Similarly, in head and neck squamous cell carcinoma, lower CTLA-4 promoter methylation correlated with positive res-ponse to immune checkpoint blockade therapy, indicating the predictive value of DNA methylation in CTLA-4 regulation.⁹ Furthermore, studies on drug addiction have highlighted the effects of various substances on CTLA-4 mRNA expression, suggesting a role for epigenetic factors in altering gene expression.¹⁰ In colorectal cancer, DNA hypomethy-lation and repressive histones have been implicated in the upregulation of CTLA-4, showing the complex epigenetic regulation of this immune checkpoint in cancer progression.¹¹ These finding led to the idea that regulation of gene expression through epigenetic mechanisms could be used in immunotherapy.¹²
In our previous study, we reported lower CTLA-4 mRNA expression levels and serum levels of the higher soluble form of CTLA-4 in CD8+ T cells of renal transplant recipients who had rejection episodes after transplant.¹² These changes in expression may be the reason for methylation; therefore, we aimed to compare the methylation status of CTLA-4 promoter regions of patients before and after transplant to analyze the interaction between rejection episodes. Moreover, to evaluate whether CTLA-4 protein levels are in parallel with CTLA-4 mRNA expression levels, we performed Western blot analyses.

Materials and Methods

Patient group and isolation of CD8+ T cells We included 29 renal transplant patients in this study. The Tepecik Training and Research Hospital Clinical Research Ethics Committee approved this study on October 9, 2019, under decision number 02. Written informed consent was obtained from patients.
Patient samples were collected before and 3 months after transplant. None of the patients had infections during the sample collection time, and all patients had negative crossmatch test results before transplant. Lymphocyte isolation was performed by using lymphocyte separation medium (Serox), and CD8+ T cells were isolated by using the magnetic bead system (Miltenyi Biotec). Details of the lymphocyte isolation and CD8+ T-cell separation were provided in our previous study.¹²

DNA isolation
We used the PureLink Genomic DNA Mini Kit (Invitrogen) to perform DNA isolation in accordance with the manufacturer’s manual. Briefly, 20 μL of proteinase K and 20 μL of ribonuclease A were added to cells (up to 5 × 106 cells) and incubated at room temperature for 2 minutes. We then added 200 μL of genomic lysis/binding buffer onto cell suspension and incubated cells for 10 minutes at 55 °C. Next, we added 200 μL of 100% ethanol, mixing well, and added lysate to the spin column. After lysate preparation, we performed 2 washing steps (wash buffer 1 and 2). We purified in 50 μL of genomic elution buffer.

Bisulfite conversion
We used the EZ DNA Methylation Gold Kit (Zymoresearch) for bisulfite modification. We added 130 μL of CT conversion reagent to 20 μL of DNA sample, which we incubated at 98 °C for 10 minutes and at 64 °C for 2.5 hours. Bisulfite-modified samples and 600 μL of M-binding buffer were added onto Spin IC column and centrifuged for 30 seconds at 10 000 g. Next, we added 100 μL of M-washing buffer and centrifuged the solution for 30 seconds at 10 000 g; we then incubated for 15 minutes at room temperature after adding 200 μL M-desulfonation buffer. We added 200 μL of M-washing buffer and centrifuged for 30 seconds at 10 000 g. Finally, we placed the column placed on a new centrifuge tube and centrifuged at 10 000 g for 30 seconds with 10 μL of M-elution buffer added. We stored samples at -20 °C until analyses.

Polymerase chain reaction and gel electrophoresis
We performed polymerase chain reaction (PCR) with ABI thermal cycle to analyze methylation status of the CpG islands in the promoter region of CTLA-4 gene. We purchased Taq polymerase from Amplicon. Primer sequences and CpG regions of the CTLA-4 promotor region are shown in Table 1. To visualize PCR products, we used 2% agarose (Invitrogen) in 1× Tris borate EDTA preparation (ABT Biotech).

Sanger sequencing
Sequencing of the samples was performed by Letgen Biotechnology (Turkey), and data were analyzed by using the Ugene tool.

Western blot
We used Western blotting to determine protein expression levels of CTLA-4. We used β-actin as the housekeeping protein. In our previous study, we analyzed CTLA-4 mRNA expression levels and soluble CTLA-4 levels in patients.¹² In this study, we analyzed CTLA-4 protein levels of the CD8+ T cells and evaluated the interactions between gene and protein expression levels. In brief, total protein was extracted from CD8+ T cells by using RIPA lysis buffer (Merck), protease inhibitory cocktail, 1 mM phenylmethylsulfonyl fluoride (PVDF), 1 mM sodium fluoride, and 1 mM sodium orthovanadate. We calculated total protein concentration by using the Bradford method (Applichem). Protein samples (30 μg) were stacked on 2% and separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrop-horesis. Protein bands were transferred onto PVDF membranes, and membrane blocking was performed by using 1% skimmed milk, washed with Tris-buffered saline with Tween 20(TBST) buffer, and incubated overnight at 4 °C with CD152/CTLA-4 rabbit polyclonal antibody (Elabscience) and β-actin rabbit (Cell Signaling). After we washed membranes with TBST, we allowed them to incubate with anti-rabbit immunoglobulin G horseradish peroxidase secondary antibody (Cell Signaling). We washed membranes again with TBST and used Luminata Forte HRP Solution (Millipore, Merck) for detection and radiography for visualization of bands. We used the Image J analysis program to analyze bands.

Results

Patient demographics and antibody detection test results
Our study group consisted of 21 male and 8 female patients with mean age of 44.9 and 44.5 years, respectively (Table 2). According to donor types, 12 patients had deceased donor transplant, 6 patients received living donor transplants from siblings, and 4 patients received living donor transplants from parents. When we evaluated the crossmatch results before transplant, none of the patients had positive crossmatch results (Table 2). After transplant, 1 patient (patient 28) had flow cytometry crossmatch and complement-dependent cytotoxicity crossmatch B-cell positivity; this patient had positive panel reactive antibody (PRA) results both before and after transplant. We noted that this patient had a transplant in 2001 that ended with rejection in 2019. Another patient (patient 29) had both T- and B-cell positive crossmatch results after transplant and had class I-positive PRA results before and after transplant. It was noteworthy to report that this patient’s donor had HLA-B*51 antigen and the patient had anti-HLA-B*51 positivity after transplant (mean fluore-scence intensity [MFI] = 1000).
Two patients (patient 8 and patient 11) had class I PRA-positive results after transplant. Donor-specific antibody test results showed that these antibodies were de novo (donor of patient 8 had HLA-B*07 antigen and patient 8 had anti-HLA-B*07 antibody with a 5000 MFI; donor of patient 11 had HLA-A*03 antigen and patient 11 had anti-HLA-A*01 and A*03 positive results with <2000 MFI). Another patient (patient 19) had class II positivity before and after transplant. However, according to the donor-specific antibody test results, the antibodies were not de novo. When we evaluated the alloimmunization history of the patient, she had 2 transplants, in 1997 from a sibling and in 2017 from her mother. In 2018, she had a rejection episode. Patient 24 had class I-positive PRA results before and after transplant. However, he only had blood transfusion history. Patient 27, who had a rejection episode, had class II-positive PRA results before transplant; however, after transplant, positivity returned to negative PRA results.

Polymerase chain reaction by gel electrophoresis before Sanger sequencing to determine CTLA-4 methylation levels We used patient DNA samples for CTLA-4 amplification, with PCR products checked on 1% agarose gel before Sanger sequencing (Figure 1). Size of the bands was controlled by using 100-basepair DNA ladder (Genesta).

Methylation status of patients
We analyzed 4 CpG regions of the CTLA-4 gene for methylation status. In results before versus after transplant, CpG region 3 and region 4 were evaluated as methylated in all patients both before and after transplant (Figure 2). Patients 1 and 2 had hemi-methylation in the CpG region 1 before and after transplant. No associations were shown among alloimmunization, anti-HLA antibody profiles, and methylation status of patients. In patients 7 and 11, CpG region 1 and region 2 were hemi-methylated after transplant; however, we observed no significant difference between CTLA-4 expression levels and methylation status. Patients 8, 27, and 29 had rejection episodes as reported previously.¹² However, methylation status of these patients did not change before and after transplant.

CTLA-4 protein levels of patients
After transplant, CTLA-4 protein levels were normalized to β-actin and calculated (Figure 3). Lysate of HEK293 cells were used as positive control for CTLA-4. All patients who showed decreased or increased levels of CTLA-4 mRNA levels were tested with Western blot analyses; results showed that protein levels and gene expression changes were correlated (Figure 3).

Discussion

The expression of immune checkpoint molecules in T cells is an important mechanism that regulates the response of immune cells to their own antigens. The relationship of these molecules, expressed on surfaces of immune cells, in many diseases has been identified. In cancer progression, the expression of these cells and their control through epigenetic mechanisms have been shown. However, the effects of these molecules in organ transplant and their roles in immune system regulation are not known.¹³ Therefore, in our study, we examined the methylation levels of 4 CpG sites in the promoter region of the CTLA-4 gene in patients undergoing kidney transplant.
CTLA-4 is a glycoprotein expressed on the surface of T lymphocytes. Although it acts as a regulatory molecule in the early stages of cell activation, its expression levels subsequently decrease. This molecule, which competes with CD28 for common ligands, also reduces interleukin 2 production. Minguela and colleagues reported that the ratio of CTLA-4 to CD28 on the surface of peripheral lymphocytes may be important in graft acceptance or rejection.¹⁴ Furthermore, the group also identified an increase in the expression of CD80/86 costimulatory molecules in patients who had acute rejection episodes. Rosik and colleagues determined that CTLA-4 gene polymorphism could provide information about posttransplant rejection in solid-organ and hemato-poietic stem cell transplant.¹⁵
Epigenetic mechanisms, including changes in CpG motif methylation, alterations in chromatin structure, and histone modifications, can regulate gene expression.^16,17 However, CpG methylation does not always indicate that a gene is inactive. Noncoding RNAs such as microRNAs can also inhibit mRNA translation. Abnormal microRNA expression has been found in many inflammatory diseases and cancer types, and it is noted that disease-specific microRNAs may be particularly effective.¹⁸ For DNA methylation to be an indicator in transplant, extensive patient follow-up is required. Many epigenetic changes can occur in both the recipient’s cells and the donor’s tissues and organs during transplant; therefore, all biological events during the posttransplant period can affect DNA methylation.^19,20 The ideas that DNA methylation patterns in kidney transplant recipients can be determined, especially in urine samples, and that determining the level of methylation due to ischemia-reperfusion may be an indicator of early acute kidney injury during kidney transplant, are gaining impor-tance.²¹ The use of epigenomic DNA methylation technologies to understand the effect of DNA methy-lation on pathological processes in graft damage may be beneficial in discovering new loci and patterns.²²
In this study, when the methylation rates of the CTLA-4 gene determined by the Sanger sequencing method were examined, although an increase in CTLA-4 expression was observed in 2 patients with hemi-methylated CpG regions, no correlation was observed between methylation level and gene expression level when evaluated overall for the patients. It can be concluded that individual immune responses and treatment strategies may differ, and different epigenetic mechanisms may also be effective. Considering the diversity in the clinical conditions of patients, mechanisms such as noncoding RNAs or histone modifications may play a role in each clinical parameter in this process.

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