2.5. Determination of FOXP3 mRNA splice variants as the exposure variables of interest

FOXP3 mRNA splice variants were measured in peripheral blood mononuclear cells using quantitative reverse transcription polymerase chain reactions. Peripheral blood mononuclear cells were isolated from blood samples by density gradient centrifugation using Histopaque (Sigma-Aldrich, St. Louis, MO, USA; density 1.077 g/mL), the cell interphase was washed by centrifugation in Hanks’ balanced salt solution (Thermo Fisher scientific, Waltham, MA, USA), and suspended in TRIzol (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Total RNA was isolated using RNeasy Mini kit including RNase-free DNase set (Qiagen, Hilden, Germany). Complementary DNA was synthesized from 300 ng of total RNA by using a QuantiTect Reverse Transcription kit (Qiagen). Genomic DNA was eliminated by incubation of each RNA sample with the genomic DNA elimination mix for 4 minutes at 42°C followed by incubation with the manufacturer mix of quantiTect RT primer, reverse transcriptase, and reverse transcription buffer for 60 minutes at 37°C, followed by heating to 95° for 5 minutes. Finally, complementary DNA was used in SYBR green quantitative reverse transcription polymerase chain reaction, LightCycler 96 (Roche Diagnostics, Basel, Switzerland). The FOXP3 gene, FOXP3 mRNA splice variants and primer sets are illustrated in Figure 2 .

Schematic presentation of Forkhead Box P3 (FOXP3) gene, mRNA splice variant mRNA and detailed description of primers used in quantitative polymerase chain reaction. (A) structure of FOXP3 gene. (B) primer locations for different FOXP3 splice variants, Total FOXP3 detects both FOXP3fl and FOXP3d2. (C) Detailed primer locations and primer pair forward and reverse sequences. NC, non-coding exon; Bp, base pair.

We measured three FOXP3 splice variants with the following primer sets: Total mature RNA FOXP3 (Total FOXP3) which accounts for the two major variants of FOXP3 in human T regulatory cells (9), namely FOXP3fl and FOXP3d2; Pre-mature mRNA FOXP3 (pre-mRNA FOXP3) which accounts for FOXP3 mRNA that has not undergone RNA splicing, and thus contains intron and exon RNA; FOXP3 that lack exon 2 (FOXP3d2) which accounts for spliced FOXP3 mRNA where exon 2 has been skipped (9); Full length FOXP3 (FOXP3fl) which accounts for spliced FOXP3 mRNA in which exon skipping has not occurred, and all coding exons are present (9).

The quantitative reverse transcription polymerase chain reaction method was adapted from an already published method by our group (26). Quantitative reverse transcription polymerase chain reaction was performed using a solution containing 10 µL Fast Start Essential DNA Green Master mix (Roche Diagnostics, Denmark), 4 µL H₂O and 2 µL of forward and reverse primer. For each sample, a mixture of solution and complementary DNA was added to a final volume of 20 µL. 15 µL of this solution was added with 5 µL of complementary DNA to measure Total FOXP3 and pre-mRNA FOXP3, while 18 µL was added to 2 µL of complementary DNA to measure FOXP3d2 and FOXP3fl. The reverse transcription settings were performed as follows: pre-incubation at 95°C for 10 minutes, 55 cycles with denaturation at 95°C for 10 seconds, annealing at 63°C for 10 seconds and extension at 72°C for 10 seconds. Quantification cycle values (Cq) for each reaction were determined using LightCycler 96 Software 1.1 (Roche Diagnostics, Denmark). We calculated a normalized target splice variant expression relative to β-actin with the following equation: Normalized ratio = ET^(CqR-CqT) with ET, efficiency of target amplification; CqT and CqR, quantification cycle at target/reference detection (26). The sizes of polymerase chain reaction products were 238 bp for Total FOXP3, 352 bp for pre-mRNA FOXP3, 128 bp for FOXP3d2 and 154 bp for FOXP3fl. Details of all primers, annealing temperature and efficiency are provided in Supplementary Table 1 .

Repeated measures of normalized values of the splice variants did not differ when using 5 µL or 2 µL cDNA ( Supplementary Table 2 ). We performed gel electrophoresis of the quantitative polymerase chain reaction product ( Supplementary Figure 1 ) to ensure that each primer pair produced only one product. We also ensured that each sample had one melting peak in quantitative polymerase chain reaction analysis ( Supplementary Figure 2 ). Finally, we observed low Inter- and intra-sample variability with repeated measures ( Supplementary Tables 3 , 4 ). The treating physicians were unaware of the FOXP3 results.

We developed several primer pairs to measure the FOXP3 mRNA variant that lacks exon 2 and 7 ( Supplementary Table 5 ). This FOXP3 splice variant accounts for less than 3% of FOXP3 transcripts in human T regulatory cells (9), and its expression may even be lower in kidney transplant recipients because immunosuppressive therapy may decrease T regulatory cell abundance and FOXP3 expression (6). The low abundance could be observed using several primer pairs, as these produced high Cq values, irregular melting peaks, and gel electrophoresis did not show a specific primer product in 55% of samples ( Supplementary Figure 3 ). Thus, the low abundance of the FOXP3 variant lacking exons 2 and 7 in peripheral blood mononuclear cells hinders its accurate measurement with quantitative polymerase chain reaction (27). Furthermore, the FOXP3 variant lacking both exon 2 and exon 7 is not involved in T regulatory cell differentiation, lineage stability or establishment of suppressive function (9). Therefore, we measured the two most abundant FOXP3 splice variants, FOXP3d2 and FOXP3fl, which is in agreement with the literature (27, 28).