This analysis presents data from a pilot initial validation group of subjects for the biomarker panel under consideration as a panel to measure both sides of the immunosuppression spectrum. We were able to demonstrate an absolute increase in the serum kyn/trp ratio with subsequent acute rejection events and a within–subject increase in urine kyn/trp ratio or drop in CD4-ATP level with subsequent major infection event.
Our results of serum kyn/trp ratios in relation to acute rejection are in accord with those of Brandacher et al. and Lahdou et al., who have previously demonstrated a rise in serum kyn/trp ratios in association with acute rejection events shortly thereafter, within 13 or 3 days, respectively (21
). However, it is pertinent to note that the apparent differences in the values presented in earlier studies relate to different ways of expressing individual ratios. For example, studies by Lahdou et al. (23
) and Holmes et al. (24
) utilized μmol units for both kyn and trp. In the study by Lahdou et al., kyn/trp ratios were represented without any transformation, such that kyn/trp ratios were presented as fractions, since kyn levels are much less than trp levels. In contrast, Brandacher et al. (21
) used μmol units for kyn and mmol units for trp in their kyn/trp ratio, a thousand fold difference in trp units. In the present study, we converted ng/ml to μmol units and multiplied by 100 to arrive at our kyn/trp ratio. If our ratios are multiplied by 10, our units become identical to the units used in the study by Brandacher et al and serum results then look almost identical to that study.
Apart from differential expression of ratios, prior studies utilized a high performance liquid chromatography platform while we have utilized a tandem mass spectrometry technique, which is considered a more robust detection system. The Brandacher et al. study also restricted to events within the first 3 weeks post-transplant, while the study by Lahdou et al. tested retrospectively collected serum samples out to 6 months post-transplant. In contrast to the study by Brandacher et al., we were unable to replicate a significant rise in urine kyn/trp ratios in subjects going on to experience an acute rejection event. The reasons for this contrasting result are not clear, but may relate to the longer time interval studied by us, the potentially greater duration between sample collection to event, or the relatively low frequency of acute rejection events in our study.
To our knowledge, ours is the first study to evaluate in detail the change in kyn/trp ratios from serum or urine, in relation to major post-transplant infection events, out to one-year post-transplant. Holmes et al. (24
) postulated that infections would be another major cause of increased IDO enzyme activity, since the enzyme is induced by interferon-γ, which is stimulated by viruses such as cytomegalovirus and Epstein-Barr virus. A growing body of literature has demonstrated increased IDO activity through interferon-γ mediated pathways in response to HIV infection (25
). The more recent era of transplantation is characterized by reduced frequency of acute rejection events and emergence of major infections such as BK virus. Consistent with this trend, our study group exhibited a greater number of major infection events than acute rejection episodes. Of note, the viral infection events detected by PCR monitoring did not lead to full blown disease in most cases, perhaps because of reductions in immunosuppression made in response to these standard of care tests. Holmes et al. found that serum kyn levels were markedly raised, on the day of infection, in 5 patients with Gram negative bacterial infection or viral infections, in a study that was restricted to the first 3 weeks post-transplant (24
). Kyn/trp ratios were not assessed in this study. The study by Brandacher et al. evaluated serum and urine kyn/trp ratios in a small group of 6 subjects with infection events (4 herpes simplex, 1 UTI, 1 sepsis). They were unable to detect any significant differences, probably due to a combination of small event number, time restriction to the first 21 days post-transplant only and 4 of the 6 infection events being relatively minor and localized.
Our study did not demonstrate lower mean levels of CD4-ATP in association with subsequent infection, as has been previously shown in a multi-center analysis (7
). However, within subjects, we were able to detect a significant drop in CD4-ATP levels with subsequent infection, suggesting considerable intra-subject variability. We did not see a spike in CD4-ATP levels with active viremia as has been reported with EB viremia (29
). Notably, only one subject in our study had EB viremia.
In studies of other biomarkers, fibrosis within the allograft could have represented a confounder variable. Two of the three prior studies of IDO enzyme activity post-transplantation were restricted to a very early time point within the first 3 weeks. Since we were looking to assess IDO enzyme activity out to 1 year, we prospectively measured serum TGF-β and CTGF levels as a surrogate marker of potential intragraft fibrosis. Prior studies have shown that, in serum, both TGF-β and CTGF levels rise in human kidney transplant recipients with documented chronic allograft nephropathy (30
).We do not perform protocol biopsies at our center, which would be the ideal measure of intragraft fibrosis. TGF-β may be affected by doses and levels of calcineurin inhibitors, so its absence may also reflect lower target tacrolimus levels. Nevertheless, our results of virtually undetectable serum TGF-β and CTGF levels suggest that fibrosis events were not significant in our subjects and would not represent any significant confounding.
Limitations of our pilot study include the relatively small sample size, the need for a separate validation group in future, and the absence of pre-transplant sample measures. However, prior studies by Lahdou et al. and Brandacher et al. have already demonstrated via pre-transplant samples that patients with end stage renal disease have higher serum kyn/trp ratios than normal controls, and that post-transplant recipients with early acute rejection have levels higher than those seen in end stage renal disease (21
). Neither we nor others have assessed changes in ratio with timing of meals, which may provide a trp load.
In summary, our pilot results suggest that less invasive immune monitoring may be able to detect a net state of immune suppression at either end of the spectrum. However, the results of this study do not possess sufficient discrimination ability for routine use in clinical practice. Further validation studies are needed to best define the combination of markers and time points that will provide the best prediction.