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Immune-monitoring in kidney transplantation

The use of novel immune-monitoring tools enabling an accurate knowledge of antigen-specific cellular immune responses against donor or viral antigens opens a new perspective in the field of diagnostics of organ transplantation

Oriol Bestard MD PhD
Head of Kidney Transplant Unit, 
Nephrology department, 
Bellvitge University Hospital, 
Barcelona University, IDIBELL, Spain
Monitoring antigen-specific immune responses against both main pathogens and donor antigens in organ transplantation would be the ideal approach to guide clinicians to individualise immunosuppression and therefore improve allograft outcome. The enzyme-linked immunosorbent spot (ELISPOT) assay has been shown to be an accurate and sensitive immune assay to assess antigen-specific cellular response, showing high predictive capacity to predict the advent of allograft rejection and main viral infections. Its validation in the context of international multicentre trials is warranted and currently underway.
Significant progress has been made in prolonging one-year kidney allograft survival in the last decade, basically due to the advent of potent immunosuppressive drugs that have directly favoured a significant reduction of acute rejection rates. Indeed, The advent of calcineurin inhibitor (CNI) based immunosuppression resulted in the one-year renal allograft failure rate dropping from around 50% twenty years ago to less than 10% in more recent times. 
Despite a massive improvement in renal allograft survival in the first year following transplantation 10-year graft survival can be as low as 50%. Nevertheless, long-term allograft and patient main outcomes have not improved as expected, due to chronic immunosuppressive-related side effects and to the development of chronic allograft rejection.1,2
While it is very well known that T cells play a key role initiating and spreading the alloimmune response, and are main actors protecting transplant recipients from viral infections such as cytomegalovirus (CMV), Polyomavirus (BKV) or Epstein-Barr virus (EBV),3,4 today the risk of allograft rejection or infection is basically based on the assessment of the humoral effector pathway of the adaptive immunity. In fact, only circulating anti-HLA or anti-viral antibodies, as well as the use of clinical and pharmacokinetic variables that indirectly suggest the potential risk of allograft rejection or infection after transplantation are currently evaluated. 
An additional approach trying to assess the impact of the alloimmune response on allograft outcome is based on the histological graft examination through surveillance biopsies in order to determine the degree of organ preservation. However, an allograft biopsy is an invasive procedure that implies patient hospitalisation, is costly, is subject to sample error and is not capable of anticipating alloimmune events.5 Altogether, no adequate measure of the overall donor/viral-specific T cell immunity is obtained. Obviously, this is far behind what modern medicine is based, which is sustained on the use of accurate markers accurately illustrating the underlying biological process. 
Remarkably, different to experimental transplantation in animal models, a hallmark of human transplantation is represented by the presence of antigen-specific memory T cells, which can trigger rapid and efficient effector responses after a subsequent secondary antigen exposure. Of note, such memory T cell immune response is highly influenced by heterologous immunity as well as by the use of T cell depleting agents, leading to homeostatic cellular proliferation,6 two main features occurring after organ transplantation in humans. Therefore, the assessment of viral-specific as well as donor-specific memory/effector T cell responses using uninvasive, sensitive and reliable immune assays would be ideal tools for monitoring the risk of infection and allograft rejection, respectively, in transplant patients. 
The enzyme-linked immunosorbent spot (ELISPOT) assay is a highly sensitive immune technique used to detect frequencies of cytokine-secreting lymphocytes at a single cell level when challenged by a given specific antigen.7 Cells that are cultured in the presence of a given stimuli in wells coated with a specific antigen and incubated for a short period of time (18–24 hours), to allow only preformed antigen-specific T cells to react and release effector cytokines. Every spot detected is correlated with a cell that has been primed. Computerised plate readers have been developed to increase reproducibility of the ELISPOT assay. 
Tracking frequencies of circulating antigen-specific memory/effector T cells
Immune-monitoring the frequency of alloreactive donor-specific memory/effector T cells using the IFN-γ ELISPOT assay both before and after kidney transplantation, has been shown to correlate with relevant post-transplant outcomes after kidney transplantation such as increased incidence of T cell mediated acute rejection (TCMR) or worse allograft function progression.8,9 In a recent study, we were able to show that kidney transplant patients displaying high frequencies of donor-specific T cells prior to transplantation were more likely to develop acute TCMR, particularly within the initial period of time after transplantation. 
This effect was especially evident among younger transplant recipients and those transplant recipients not receiving T cell depletion as induction therapy.10 Taking into account all this background, and as a first attempt to use this assay to prospectively titrate the burden and type of immunosuppression after transplantation, our group evaluated in a pilot study the pre-transplant anti-donor cellular alloresponse of 60 consecutive kidney transplant patients to receive either a CNI-based or CNI-free immunosuppressive regimen depending on the presence or absence of detectable cellular response before transplantation.11 Interestingly, we showed that patients without detectable anti-donor T cell responses displayed better allograft function progression and absence of subclinical TCM subclinical rejection in six-month protocol biopsies. 
Of note, in the context of the European consortium RISET (Reprogramming the Immune-system for the establishment of Tolerance), this assay was cross-validated across different European centres showing very high reproducible results for measuring donor-specific T cell alloresponses.12 Importantly, a large prospective international study aiming at validating this assay for allowing immunosuppression minimisation is currently undergoing in the context of the European consortium BIO-DRIM (Biomarker-driven Personalised Immunosuppression) (
Likewise to measuring alloimmune responses, the remarkable sensitivity of the ELISPOT assay has made this technique an advantageous tool to also measure T cell immune responses against specific viral antigens.4 Indeed, in the context of kidney transplantation, cytomegalovirus (CMV) infection is associated with significantly worse patient and graft survival, due to chronic exposure to immunosuppressants mainly targeting T cells.13 Taking into account this background, the characterisation of the immune T cell function of transplant patients against CMV antigens has also been deeply evaluated to determine the individual immune risk to develop CMV infection. 
In this regard, using the IFN-γ ELISPOT assay we analysed the frequencies of CMV-specific memory/effector T cell responses against main immunogenic CMV antigens such as IE-1 and pp-65 in a large retrospective cohort of kidney transplant patients both before and after transplantation, all of them being serologically positive against CMV (IgG+) and receiving a seropositive kidney allograft (IgG+). We observed that patients with low or undetectable preformed anti-CMV T cell frequencies, particularly against IE-1 CMV antigens rather than against pp65, were found to be at significantly increased risk of developing post-transplant CMV infection or disease than patients showing a robust and detectable high anti-IE-1 T cell immune response.14
In addition, when some seronegative kidney transplant recipients were also assessed for the presence of CMV-specific T cell responses using this same assay, a relevant proportion of patients did similarly show high frequencies of CMV-specific T cell responses thus, being at lower risk of CMV infection after transplantation.15 Currently, a prospective, randomised multicentre trial (RESPECT) is evaluating the capacity of this immune assay to predict the risk of CMV infection among kidney transplant patients.
Tracking frequencies of circulating antigen-specific memory/effector B cells
On the other hand, even though the assessment of anti-HLA antibodies in the serum of transplant patients has significantly improved with the advent of novel highly sensitive assays, the sole focus on circulating anti-HLA antibodies to determine the allosensitisation state of a given transplant patient may underestimate the magnitude of the complete humoral immune response as it excludes the detection of the entire memory B cells (mBC) pool. 
Therefore, and similarly to the assessment of antigen-specific T cell responses using the IFN-γ ELISPOT, our group focused efforts on the development of a novel B cell ELSIPOT assay approach in order to track alloreactive and viral-specific circulating mBC capable of secreting antigen-specific (IgG) antibodies. This technique consists on an antigen-independent polyclonal cellular stimulation culture method based on the TLR 7/8 agonist R848 and IL-2 to convert circulating antigen-specific mBC onto antibody-secreting cells (ASC) capable of secreting alloantibodies, without modifying their antigen repertoire clonality.
Using this assay, with the aim of assessing the presence of circulating anti-viral mBC in kidney transplant patients, we initially used a CMV-sp B cell ELISPOT and analysed in a subset of kidney transplant recipients, either seropositive (IgG+) or seronegative (IgG–) for CMV prior to transplantation, the presence of circulating CMV-specific mBC and its impact protecting from viral infection. Interestingly, we showed that in a subset of seronegative patients, high frequencies of CMV-sp mBC could be clearly detected using this assay and furthermore, that these patients displayed significantly lower incidence of post-transplant CMV infection, despite not showing circulating anti-CMV antibodies in the serum.15
While tracking alloreactive mBC circulating in peripheral blood, we investigated the impact of circulating alloreactive mBC responses using the HLA-specific B cell ELSIPOT assay in a large group of 70 highly HLA sensitised patients on the waiting list for transplantation, as well as in a subset of kidney transplant individuals.16 In contrast to previously reported HLA B cell ELISPOT methods, our approach allowed us to use peripheral blood mononuclear cells rather than sorted B cells, thus simplifying the assay and in addition, as frequencies of HLA-sp mBCs are relatively low, multimerised class I and class II HLA molecules were used, enabling clear detection of very low IgG-producing mBC frequencies. 
In this study, we confirmed that mBC frequencies against both class I and class II HLA antigens may be detected in peripheral blood in patients with obvious allogeneic sensitisation background, even in the absence of detectable circulating antibodies. Remarkably, we described that high frequencies of donor-specific alloreactive mBCs are present in kidney transplant patients during antibody-mediated rejection and also before transplantation, reflecting an active baseline antidonor sensitisation state not always shown by circulating donor-specific alloantibodies (DSA). Of note, the higher donor-specific mBC frequency, the more severe rejection type was observed. 
In summary, sensitive new cell-based immune-monitoring tools providing accurate information about the anti-donor and anti-viral immune responses in transplant patients such as the T cell and B cell ELSIPOT assays are currently under development, showing promising data regarding its predictive value to discriminate patients at risk of allograft rejection and infection.
The endeavour of bringing these immune assays into day-to-day clinical practice is challenging, but will be the key to achieving individualised immunosuppression in the era of personalised medicine.  Notably, the impact of these new tools extends beyond transplantation, since they could provide key information also pertinent for the management of patients with autoimmune diseases. 
The development and validation of these assays should be primarily done in prospective observational studies in the context of international networks with the direct participation of the biotech industry.
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  2. Orandi BJ et al. Survival benefit with kidney transplants from HLA-incompatible live donors. N Engl J Med 2016;374:940–50.
  3. Crespo E, Bestard O. Biomarkers to assess donor-reactive T-cell responses in kidney transplant patients. Clin Biochem 2016;49(4–5):329–37.
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  7. Heeger PS et al. Pretransplant frequency of donor-specific, IFN-gamma-producing lymphocytes is a manifestation of immunologic memory and correlates with the risk of posttransplant rejection episodes. J Immunol 1999;163(4):2267–75.
  8. Nickel P et al. Enzyme-linked immunosorbent spot assay for donor-reactive interferon-gamma-producing cells identifies T-cell presensitization and correlates with graft function at 6 and 12 months in renal-transplant recipients. Transplantation 2004;78(11):1640–6. 
  9. Hricik DE et al. Enzyme linked immunosorbent spot (ELISPOT) assay for interferon-gamma independently predicts renal function in kidney transplant recipients. Am J Transplant 2003;3(7):878-84. 
  10. Crespo E et al. Pre-transplant donor-specific t-cell alloreactivity is strongly associated with early acute cellular rejection in kidney transplant recipients not receiving T-cell depleting induction therapy. PLoS One 2015;10(2):e0117618.
  11. Bestard O et al. Prospective assessment of antidonor cellular alloreactivity is a tool for guidance of immunosuppression in kidney transplantation. Kidney Int 2013;84(6):1226–36.
  12. Bestard O et al. Cross-validation of IFN-γ Elispot assay for measuring alloreactive memory/effector T cell responses in renal transplant recipients. Am J Transplant 2013;13(7):1880–90. 
  13. Sagedal S et al. The impact of cytomegalovirus infection and disease on rejection episodes in renal allograft recipients. Am J Transplant 2002;2(9):850–6.
  14. Bestard O et al. Pretransplant immediately early-1-specific T cell responses provide protection for CMV infection after kidney transplantation. Am J Transplant 2013;13(7):1793–805.
  15. Lúcia M et al. Preformed frequencies of cytomegalovirus (CMV)-specific memory T and B cells identify protected CMV-sensitized individuals among seronegative kidney transplant recipients. Clin Infect Dis 2014;59(11):1537–45.
  16. Lúcia M et al. Preformed circulating HLA-specific memory B cells predict high risk of humoral rejection in kidney transplantation. Kidney Int 2015;88(4):874–87.