Interleukin 17 in renal biopsies as risk factor for progression Sandrine Florquin, Amsterdam, The Netherlands Chairs: Mohamed R. Daha, Leiden, The Netherlands Pierre Ronco, Paris, France Prof. Sandrine Florquin Department of Pathology Academic Medical Center University of Amsterdam Amsterdam, The Netherlands Slide 1 Slide 2
Good morning ladies and gentlemen, Mr Chairman. First of all, I would like to thank the organizing committee of this meeting for giving me the opportunity to discuss with you some issues about the role of IL-17 in renal allografts as a risk factor for progression. As far as the literature I know goes there is no published data about the role of these cytokines in primary renal disease, I will focus my talk on allograft biopsies. Slide 3 So as you all will know, acute rejection is a major risk for graft failure and most importantly late episodes of acute rejection are associated with an increased risk of graft loss. Also the severity and the type of acute rejection is associated with an increase of risk of graft loss and also rejection episodes of rejections resistant to conventional therapy is also associated with an increased graft loss. Slide 4
Beyond the classical direct and indirect pathway of alloreactivity leading to acute rejection, it's not recognising that other alternative pathways might also be playing a role in an episode of acute rejection. Among which the TH-2 type cells have been involved in these memory CD8+ T cells, TH-17 cells, B cells, natural Killer cells, eosinophils and neutrophils. Today I will focus on the role eventually of IL-17 in these pathways of acute rejection. Slide 5 So as you all will probably know, naïve T cells may differentiate into different types of
polarised T cells. For example, upon exposure in vitro to IFN-γ and IL-12 these naïve cells can actually differentiate into the so-called TH-1 T cells that are producing IFN-γ and other cytokines. Upon IL-4 and IL-2 exposure, these naive T cells might differentiate into TH-2 which is important for fighting parasites and also play a role in allergy and asthma. More recently, it has also been shown that these naive T cells in vitro upon exposure to TGF-β and IL-2 can differentiate into so-called regulatory T cells, which are characterised by the transcription factor FoxP3, and these cells are major players in immune tolerance and are secreting among other cytokines TGF-β and IL-10. More recently, even these naive T cells have been shown that upon exposure to TGF-β and IL-6, IL-21 and IL-23 might actually differentiate into the socalled TH-17 type of T cells which are major pro-inflammatory T cells which are important to fight extracellular bacteria, fungi and are playing also a role in autoimmunity and as recently has been shown also an important role in allograft rejection. Slide 6 IL-17 might be produced after activation of two different types of receptors. I will not go into the details but most importantly IL-17 might act on different cell types among which neutrophils, endothelial cells, keratinocytes and fibroblasts leading really to a spectrum of activity, recruitment of neutrophils leading to a lot of inflammation and also --- cells leading to an increase of vascular permeability but also angiogenesis. IL-17 has also been shown to induce metalloproteinases. So it's a major pleiotropic and pleomorphic I would say cytokine. Slide 7
Importantly IL-17 is a family of cytokines of 6 members, A to F among which A and F are the best characterised and studied. A and F are massively produced by the so-called TH-17. But most importantly, these cytokines might also be produced by other cell types among which CD8, natural cursors, gamma delta, neutrophils but also mastcells. It's important to remember that IL-17 A and F share among 50% of aminoacids. Slide 8
So what's known about IL-17 and allograft rejection? As I told you, there is nothing published at least about these cytokines and pulmonary renal disease but it's about 15 years ago already that Cees van Kooten from Leiden described that IL-17 might activate human renal epithelial cells in vitro and that these cytokines were expressed during allograft rejection. After there was kind of nothing and then about 5 years later the first publication came out studying the mrna levels of IL-17 in different settings of transplantation including renal transplantation, lung, liver and cardiac transplantation. But in all these studies the clinical relevance of IL-17 as well as the cellular source of these cytokines was not evaluated. Slide 9 So we aimed to evaluate the predictive value eventually of this IL-17 expression in allograft rejection upon acute rejection and also the cellular source of IL-17. Slide 10
For that we studied 3 different cohorts. First of all, the test cohort including 50 patients, a validation cohort also with 50 patients and control patients and 10 protocol renal biopsy were taken 6 months after transplantation without any significant histological features. Regarding acute rejection we had 40 pure I would say acute T cell-mediated rejections and 8 combined antibody-mediated rejections and T cells-mediated rejection. So we performed the BANFF classification and we performed double staining for IL-17 and T cells using CD3, mastcells for tryptase, neutrophils CD-15 and importantly we used a polyclonal antibody from R&D that was really extensively validated in our lab. These antibodies recognised both IL-17 A and F. We looked mostly for the response to conventional antirejection therapy and renal outcome in this setting. Slide 11 The 3 next slides will give you a characteristic expression I would say of IL-17 in the different settings. So here, we have the double staining for CD3 and IL-17. IL-17 being in blue and CD3 in red. As you see, upon acute rejection there are a lot of T cells invading the kidney. Here you have glomeruli where there is no counterstaining, a few single blue cells which are IL-17 positive cells. You can appreciate that in this infiltrate we could hardly find double positive cells such as these ones which are also here turning to purple, we have red and blue, turning to purple but they were really only a few cells that expressed both CD 3 and IL- 17.
Slide 12 In contrast when we stained for tryptase, a marker for mastcells and IL-17, we could find quite a lot of purple cells such as these ones, so meaning that quite a lot of mastcells were also expressing IL-17. Slide 13
This was also true for the neutrophil marker CD15 showing that a lot of these IL-17 positive cells were also neutrophils. Slide 14 So first, we looked at the potential to discriminate between protocol and acute rejection looking at these IL-17 positive cells. As you can see, hardly any IL-17 positive cells were found in protocol renal biopsy without histological changes after transplantation at 6 months. But in contrast, when we put all acute rejections together, we had a major increase in IL-17 positive cells invading the graft. You can see that it discriminates really well between no ejection and acute rejection. Slide 15
When we looked at the distribution of these IL-17 positive cells, we were quite surprised I would say to see that the majority of these IL-17 positive cells turned out to be neutrophils. That's the blue. About 25% were mastcells. Less than 1% of all IL-17 positive cells were T cells. So we also discarded I would say these double positive cells from further analysis because it was just one or two cells per renal biopsy so probably not really clinically relevant. Slide 16 So we focused on the mastcells and neutrophils and IL-17. This is the correlation between IL- 17 positive cell types and the BANFF score and clinical parameters at the time of renal biopsy. As you can see for all IL-17 positive cells there is a correlation with the total and inflammatory infiltrates meaning that IL-17 just parallels the inflammation in the renal biopsy and there is a negative correlation with the renal function at the time of biopsy. Interestingly, when we looked at the mastcells positive for IL-17, there was also a clear correlation with the tubular atrophy and interstitial fibrosis meaning that it might be that these mastcells producing IL-17 might be involved in fibrosis. But this is just a hypothesis that we have to test further. Slide 17
Importantly, we looked at the eventual role of potential prediction value of these IL-17 in response to therapy but we couldn't see any association between IL-17 positive cells and response to anti-rejection therapy and in line also no correlation between IL-17 positive cells, infiltration and CD4 positivity, so a marker for antibody-mediated rejection. Slide 18 So we performed univariate analysis. I will skip that but from this univariate analysis it turned out that 3 parameters were associated with the occurrence of late graft dysfunction defined as a graft function less than 30 ml/minute at the last follow-up or return to dialysis. These 3 parameters were the time from transplantation to biopsy. So late rejection was associated in the univariate analysis to late outcome as known in the literature and also the C4d positivity, so antibody-mediated rejection in this univariate analysis was associated with late graft dysfunction. But when we put the 3 parameters in the multivariate analysis, we found that one lady IL-17 positivity was associated with late renal outcome and it was true for poor renal function and return to dialysis. Slide 19
So we wanted to validate these findings in an independent cohort. So we included almost 50 additional renal biopsies and we performed the same staining. So in red you have the validation cohort and on the black line is the test cohort and as you can see, it's about the same showing that indeed the total IL-17 positive infiltrates correlate with a poor renal function at the last follow-up. So there is a clear correlation between a lot of IL-17 positive cells and poor late function and it is was also true for return to dialysis. After we looked at the best cut-off for the number of IL-17 positivity regarding sensitivity and specificity and we found that 4 IL-17 positive cells for each --- was the best cut-off in our study and using that we found in this Kaplan-Meier curve that indeed the renal biopsy with more than four IL-17 positive cells was associated with higher priority to graft dysfunction at last follow-up. The grey area is the 95% confidence interval. Slide 20 So clearly from this study we could say that yes IL-17 positivity parallels the inflammation in renal allograft upon acute rejection. That in our hands at least major sources of intragraft IL- 17 were neutrophils and mast cells, that we couldn't find any predictive value regarding the anti-rejection therapy response but that IL-17 infiltration in the renal biopsy independently predicts poor renal outcome.
Slide 21 In this study they had more or less the same conclusion but what they did is they included about 56 patients among which 40 were acute T cell-mediated rejections and 16 were combined T cell and antibody-mediated rejections. But in this study they also performed staining for FoxP3 disease, this transcription factor is characteristic for the regulatory T cells, so inducing tolerance and IL-17. They looked at response to therapy and 1- and 5-year graft survival. Slide 22 They performed just a single staining and this is an example of this publication showing single FoxP3 positive cells and a few IL-17 positive cells. In this study using this ratio between FoxP3, so tolerance inducer and IL-17 a potentially inflammatory inducer, they found that indeed the renal biopsy with a lot of FoxP3 and low IL-17 had a good response to steroids after rejection. But in contrast, they had a biopsy with a high ratio of IL-17 compared to FoxP3 had a poor response to steroid treatment. Also in this study combing FoxP3 and IL-17
they showed that renal biopsy with a high FoxP3 ratio had a good follow-up of allograft survival after 1 and 5 years after transplantation. In contrast and significantly the renal biopsy with a lot of IL-17 and low FoxP3 had poor prognosis. Slide 23 In this study, they also looked at the correlation eventually with the marker for antibodymediated rejection, C4d but they couldn't find any correlation between IL-17 and FoxP3 for this marker and indeed, they found that this high IL-17 FoxP3 ratio significantly correlated with steroid resistant rejection and also correlated with an allograft outcome in a multivariate analysis. So more or less comparable to our study. Slide 24 But in contrast, there was at the same time another study also looking at the same I would say namely the IL-17 expression in allografts upon acute rejection showing quite different results that I will discuss with you. In this study, they included 20 renal biopsies with acute
antibody-mediated rejection. Slide 25 10 with T cells and they used another antibody and I would just like to go through these results showing that regarding T cell-mediated rejection we could find an increase in IL-17 cells. But interestingly in this study on antibody- mediated rejection they found mostly an expression of IL-17 in tubular epithelial cells and they performed also double staining showing that 80% of these IL-17 positive cells were T cells in contrast to our results showing that it's less than 1%. Slide 26
So a big discrepancy. Slide 27
I won't go into the details of the in vitro study which are really interesting but go to the conclusions of this study showing that most of these IL-17 positive T cells are CD14 cells in acute T cell-mediated rejection. That upon antibody-mediated rejection tubules are the major source of IL-17 and that complement may induce IL-17 gene and protein in tubular epithelial cells. Slide 28 I think I have now just some issues showing what is finally the major source of IL-17 upon acute renal rejection, what might be the different expression of IL-17 in acute and antibodymediated rejection and we can discuss that also. Slide 29 I haven't found any available data on real time Slide 30
Pcr for IL-17 in renal biopsy so far. Slide 31 I had some slides about the discussion Slide 32
but we can just use it for the discussion eventually. Slide 33 I would like to finish by thanking all these people mentioning that this work has been done mostly by Unsal Yapici in the lab with also the support of the Dutch Kidney Foundation and I would like to thank you for your attention. Thank you. Slide 34
- So thank you very much Sandrine, I think a very nice overview and very exciting results. While people come to the microphone actually the main question is if you take the IL-17 family, there are 6 members and for instance, from the work of the TGF for instance, TGF-β and TGF-α, we know that only a minor change, let's say 1 aminoacid change, determines whether it's α or β and the functional activities of these two molecules are so very much different. So the question is actually whether looking at this full spectrum, I assume your antibody sees the full spectrum of all family members, is there any possibility to distinguish between the 6 family members? - Ok thank you, indeed that's really an important point that I think that there is no actual antibody discriminating between the 6 members of this family and the antibodies which are working are all polyclonal antibodies so they recognize both IL-17 A and F. So we cannot discriminate between both. You are perfectly right that it might be really important for the function of these cytokines and discriminating might give more insight into the pathogenesis of the role of IL-17 in allograft rejection. - So my co-chair Piero Ronco from Paris also has a question. Please go ahead. - Thank you Mo and thank you for a nice presentation. You showed that IL-17 was associated with late graft dysfunction but how does it work? There is this paper by Cees van Kooten showing that IL-17 affects, alters proximal tubular epithelial cells. Could you speculate a little bit, very briefly? - Yes, using antibody IL-17 expression on tubular epithelial cells in contrast to the other study. So I think that the most important thing is that IL-17 in our hands at least, is produced by mast cells and neutrophils. And as you know, mast cells are major profibrotic cells that might then induce late renal fibrosis leading to poor renal outcome. So I think it's the combination of neutrophils and mast cells producing this IL-17 leading to remodelling of the matrix through activation of metalloproteinases and so on that might lead finally to poor outcome in terms of renal fibrosis and tubular atrophy. Because response to conventional therapy in our small cohort did not show any difference so I think it might be much later leading to the scarring of your graft. - Thank you.
- Thank you very much Sandrine. Unfortunately, we don't have more time to go into more detail.