Elevation of Cytotoxic Lymphocyte Gene Expression Is Predictive of Islet Allograft Rejection in Nonhuman Primates

The RNA purification reagent RNA Now-LW was purchased from Biogentex (Seabrook, TX). The first-strand cDNA synthesis kit SuperScript preamplification system was purchased from Gibco BRL (Grand Island, NY). Oligonucleotides for PCR were synthesized by Keystone Laboratory (Menlo Park, CA). LightCycler DNA Master SYBR Green I was purchased from Roche (Indianapolis, IN). TaqStart antibody was purchased from Clontech (Palo Alto, CA). All other chemicals were of the purest grade available and were obtained from commercial sources.

Four long-term rhesus monkey islet allograft recipients with stable graft function were used in the study. These monkeys were originally treated with humanized anti-CD154 specific monoclonal antibody (hu5c8, ANTOVA; Biogen, Boston, MA) (3). Three of the monkeys had stable partial graft function, and one was insulin-independent at the time this study was initiated. We have observed rejection 3–5 months after cessation of anti-CD154 therapy in rhesus monkey islet allograft recipients (4). To determine whether ECLG transcripts correlates and/or is predictive of islet rejection, maintenance therapy was discontinued on these four monkeys, and weekly peripheral blood (PB) samples were collected in EDTA-containing tubes to assess CL gene transcript expression. We froze 0.5 ml aliquots of whole blood at −80°C until processing for RNA. The experiments described in this study were conducted according to the principles set forth in the Guide for Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, Department of Health and Human Services [National Institutes of Health publ. no. 86-23]).

Total RNA was extracted from PB using the RNA Now-LW method (Biogentex, Seabrook, TX). RNA concentration was measured by a RiboGreen RNA Quantitation kit (Molecular Probe, Eugene, OR) and a fluorometer (Bio-Rad, Hercules, CA). First-strand cDNA synthesis was performed in a 21-μl reaction volume containing 250 ng of total RNA, 25 ng/μl oligo dT, 0.5 mmol/l of each dNTP, 2.5 mmol/l MgCl₂, 10 mmol/l dithiothreitol, and 200 units of SuperScript II RNase H⁻ reverse transcriptase (Gibco BRL, Grand Island, NY).

PCR primers were originally designed from the gene sequences of human perforin, GB, and FasL. Preliminary experiments to establish the methodologies were performed on RNA samples isolated from phorbol 12-myristate 13-acetate (PMA) and ionomycin-stimulated rhesus monkey PBL by using the human CL primers. The PCR-amplified fragments were cloned in a PCR 2.1 vector (Introgen, San Diego, CA) and confirmed by double-strand DNA sequencing in the DNA core laboratory (Department of Biochemistry, University of Miami, Miami, FL). Nonhuman primate specific primers of the three CL genes were then designed based on the sequence of each of the three nonhuman primate CL fragments as follows: perforin sense 5′-GCCGGCAACGTGCATGTGTCT-3′, antisense 5′-GTGTGTACCACATGGTAACTG-3′; GB sense 5′-GGGATCAGAAGTCTCTGAAGAG-3′, antisense 5′-CTTTCGATCTTCCTGCACTGTC-3′; FasL sense 5′-GTAGGATTGGGACTGGGGAT-3′, antisense 5′-AGTTGGGCTTGCCTGTTAAA-3′. β-Actin primer sequences were obtained from A.D. Kirk and colleagues (18). The sequence homologies of the 140-bp perforin, 395-bp GB, and 181-bp FasL fragments between monkey and human were 96.4, 93.4, and 95.0%, respectively.

PCR amplification was performed with the LightCycler DNA Master SYBR Green I kit in a PCR containing 0.5 μmol/l of each primer, 3 mmol/l MgCl₂, and 2 μl sample. TaqStart antibody was systematically added to the amplification reaction mixture to block TaqDNA polymerase activity during set-up of the PCR at ambient temperature (Clontech, Palo Alto, CA). The amplification and detection were carried out in a LightCycler instrument as follows. The reaction mixture was initially incubated at 96°C for 30 s to inactivate the TaqStart antibody and to denature the DNA. Amplification was performed for 45 cycles, with the following cycle parameters: denaturation (95°C for 1 s), annealing (62°C for 10 s), and extension (72°C for 15 s). The ramp rate was 20°C/s. Fluorescence was acquired at the end of each annealing phase, with the acquiring temperature set at 85°C for perforin, 88°C for GB, and 82°C for FasL.

The melting curves were obtained at the end of amplification by cooling the sample to 60°C at a rate of 20°C/s and then increasing the temperature to 95°C at a rate of 0.2°C/s. Fluorescence was acquired every 0.1°C. The conversion of the melting curves into melting peaks (plot of the negative derivative of florescence versus temperature) allowed identification of each specific gene.

Respective cloned CL plasmids, with serial dilutions from copy number 10,000,000 to 100, with 10-fold intervals, were used as standards to construct each standard curve in the LightCycler PCR quantification. A reaction mixture without the template and a reaction mixture without SuperScript II reverse transcriptase were used as negative controls. Quantification was carried out using LightCycler analysis software. Background fluorescence was removed by setting a noise band. The log-linear portion of the standard’s amplification curve was identified, and the crossing point was the intersection of the best-fit line through the log-linear region and the noise band (19). The standard curve was constructed by plotting the log of copy number versus the crossing points. β-Actin amplification was used to rule out failure in each RNA purification, reverse transcriptase reaction, and PCR amplification reaction and to control variation in cDNA quantity among samples. All results were expressed as the ratio of the copy number of the target gene to the copy number of β-actin.

We carried out a preliminary melting curve analysis to determine the proper temperature for the acquisition of the fluorescent data. The melting curve analyses for the GB, perforin, and FasL genes of the rhesus monkey are shown in Fig. 1A. The center of the melting peak is the melting temperature (T_m) of the DNA product. The target DNA product can be uniquely identified from other fragments based on these melting peaks. Nonspecific amplification products tend to melt at much lower temperatures and over a broader range. The melting temperatures for rhesus GB, perforin, and FasL were 89.8, 87.7, and 84.3°C, respectively. Therefore, the quantitation of GB, perforin, and FasL was performed, acquiring the fluorescence at, respectively, 88, 85, and 82°C, temperatures at which any nonspecific fluorescence is excluded. The size of each LightCycler PCR amplified fragment was confirmed by separating the products on an ethidium bromide-stained agarose gel (395, 140, and 181 bp for GB, perforin, and FasL, respectively) (Fig. 1B).

A representative amplification curve for the perforin PCR product is shown in Fig. 2A. After completion of PCR, the LightCycler software generates the amplification curve and sets a baseline x-axis. The baseline identifies the cycle in which the log-linear signal can be distinguished from the background for each sample. The plot of the x-axis “crossing point” for each standard against the logarithm of standard concentration generates the standard curve (Fig. 2B), from which the concentration of the target sequence in the sample can be extrapolated. The primers used for analysis of these genes in rhesus monkeys work equally well in a cynomolgus monkey model (data not shown).

Figure 3 shows the expression of mRNA levels of GB, perforin, and FasL in the PB of four rhesus monkey islet allograft recipients subsequent to discontinuation of anti-CD154 therapy, and the date relative to postoperative day (POD) is summarized in Table 1. ECLG expression preceded islet allograft rejection, defined as a loss of C-peptide production, by 83–197 days in the four monkeys studied (Table 1). ECLG occurred 165–176 days after discontinuation of 5c8 therapy in three monkeys and lasted 2–2.5 months. For the other monkey, ECLG occurred 33 days after cessation of the monoclonal antibody therapy and lasted 25 days (Table 1). Comparing the POD for ECLG duration with the POD for elevation of 2-h PB glucose (Table 1), it can be observed that ECLG expression correlated with the elevation of 2-h postprandial blood glucose in two of the four monkeys.

Hyperglycemia and increased insulin requirements are currently used as indicators of islet rejection; however, these parameters are not predictive and occur at the time when significant islet mass has been lost. Our study demonstrates that ECLG expression in PB precedes islet graft rejection, defined as a loss of C-peptide production, by 2.5–6.5 months, with a duration of 1–2.5 months. Antirejection therapy initiated within 3 days of elevated PPG enabled reversal of rejection and rescue of partial islet mass in anti-CD154-treated nonhuman primates (3,4); however, it would be difficult to define the level of PPG at which antirejection therapy should be undertaken in patients with varying degrees of graft function. It will be important to conduct further studies to demonstrate that detection of elevated CL gene expression enables reversal of rejection episodes in a clinically feasible time frame and results in reproducible maintenance of islet mass.

Competitive quantitative RT-PCR techniques have been widely used to detect gene expression in biological samples. The use of the LightCycler PCR instrument enables monitoring of the fluorescence of a PCR continuously and on-line. The subsequent ease of identification of the log linear cycles of a PCR allows for rapid, accurate, and sensitive quantification of nucleic acid.

In summary, analysis of CL gene expression in PB samples may allow for detection of islet allograft rejection several weeks before loss of C-peptide production. The maintenance of increased expression for a period of 1–2.5 months suggests that testing of these parameters may have practical applications in clinical islet cell transplantation.

This work was supported by National Institutes of Health Grant 1U19-AI43900 and 1R01-DK55178 and the Diabetes Research Institute Foundation, Hollywood, FL.

The authors gratefully acknowledge the expert assistance of Jenifer Leith, Waldo Diaz, and Jose Gomez.