Ph.D. Program in Biomedical Sciences and Oncology

A relevant question in HLA immunization, not yet well understood, is why only a fraction of all subject challenging allogeneic stimuli produce antibodies. Until now, the humoral response has been evaluated with classical complement-dependent cytotoxicity (CDC) methods only. As the new technique could better define all types of antibody response, we are now able to consider why the anti-HLA response might be elicited only in a subgroup of patients. We can expect that two different orders of reasons might explain this phenomenon. The first is in relationship with the immunogenicity of the epitope challenge: the same HLA antigen could be immunogenic only in those patients having certain HLA genotypes. Alternatively, the ability to grow an antibody response could be due to the particular genetic background of each exposed individual.

At this purposes we intend to analyse at least 100 immunized patients before and after transplantation. The immunogenetic challenges (and particularly the previous HLA mismatches) will be compared with the humoral response and categories of HLA produced antibodies. When a humoral response is detected, a correlation between HLA genotype of patients and type of antibody produced will be done. We can hypothesize that certain mismatch might evoke a humoral response only in patients with particular HLA genotypes. Moreover, polymorphism in the HLA region, or in other genetic regions relevant for immune response, may influence the humoral response. For this reason we aim to correlate antibody response with HLA-A, B, C, DR and DQ alleles, as their relevance in allogeneic response. Moreover, polymorphisms in HLA-G, IL-10, IL2, IL4, IL2R, IFNG, TNFA will be typed and correlated with the humoral response, since it is well known that the individual reactivity also depends on genotypes at this loci.              

The ability to recognize the antigenic HLA mismatches that could be more immunogenic, or the immunological condition of high or low humoral response, could have an important impact on the selection of candidates for a certain donor

Chronic hepatitis C virus (HCV) infection is the most common cause of liver cirrhosis leading to liver transplantation (LT) in Italy accounting for 46% of patients (pts). Recurrence of HCV hepatitis after LT occurs in the vast majority of cases determining worse results as compared to non-HCV pts. Several new factors have been recently correlated to the prognosis of recurrence. Some are related to the donor (donor age, HLA-DR, IL28B polymorphism, HBcAb status), others to the recipient (MELD score, HLA-DR, IL28B polymorphism). We hypothesize that allocation of grafts taking into account donor-recipient genetic and virological characteristics (INTENTIONAL MATCH, IM) could improve the results of LT in HCV pts. Aim of the research project is to develop innovative algorithms of allocation, supposed to be effective to: 1.increase response to antiviral therapy and improve graft and patient survivals in HCV pts (primary endpoint); 2a.postpone recurrence; 2b.stop/reduce fibrosis progression; 2c.avoid/postpone evolution to cirrhosis; 2d.discriminate other donor and recipient risk co-factors (secondary endopoints).HCV pts aged 18 to 69 years will be included. 2 phases are planned. In the retrospective phase (3 months, N=500 pts), data from transplants performed in HCV pts at Turin LT Center will be analyzed (2001-2010). Donor and recipient HLA typing are already known, IL28B genotype analysis will be obtained from recipient and donor DNAs extracted from blood. Donor HBcAb data are already present. In the prospective phase (30 months, N=150 pts), HCV pts will be typed for IL28B and HLA-DR. Donors will be characterized for IL28B, HLA-DR and HbcAb during the brain death observation period. Algorithms of IM previously developed will be tested prospectively. Interim analyses will be done at 12 and 18 months. IM will be stopped in case of results inferior to those observed in the retrospective phase. Amelioration of graft survival in HCV pts of at least 8-10% at 3 years is expected.

The identification of markers of graft tolerance is a goal in solid organ transplantation to avoid the risk of immunosuppressive (IS) related adverse events including infectious complications, malignancies and metabolic disorders that contribute deeply to morbidity and mortality among transplanted patients. It is clearly established that even T cells have a predominant role in allograft cell infiltration during acute cellular rejection and to orchestrate the immune responses to infections, also donor specific antibodies (DSA) play a pivotal role in the outcome of allograft.

In the last ten years, Piemonte Kidney Transplant Centres performed 1860 transplants from deceased donor, of which 224 were retransplants. Actually, 1540 (83%) of these patients have a stable graft function. Notably, 25% of the active list of patients waiting for a kidney are older patients, and more than 38% of transplanted patients in the past years were older than 60 years. Moreover, patients with panel of reactive antibodies (PRA)> 50 are 14%, and 10% have a PRA>80. In this view, graft survival of aged >60 recipients is not statistically different in respect to younger:  in older recipients graft rejection less frequently are responsible of failure while infections and tumors are more responsible of morbidity and mortality transplant-related. Therefore, the more accurate evaluation of immunological status induced by IS therapy is essential, especially to avoid infections, in the elderly population.

The aim of our study is to determine whether some immune and genetic parameters could be useful to identify an age-related immunological profile that could be instrumental to address the most efficacious IS regime, to better manage graft function and therapy side effects after kidney transplant.

Since HLA matching play a pivotal role in the outcome of kidney transplant, we analyzed the number of total (HLA A, B, DR) mismatches (MM) in our patients: 9% had less than 2 MM, 51% had 2/3 MM, 40% had more than 4 MM, independently of age. Only HLA DR number of MM were: 0 MM, 44%; 1 MM, 54%; 2 MM, 2%, respectively.

The study will involve in a retrospective way stable kidney patients that underwent to transplant in our centres. In order to assess their immunological profile, patients will be divided in four classes in according to their age at transplant: 18-39 years, 40-59 years, 60-69 years and over 70 years old. In each group 100 patients with at least 2-years stable graft function will be characterized for a total of 400 patients involved. We will plan to analyze:

• the strength of immune response by Cylex Immuknow assay;
• markers of renal function, such as serum creatinine, proteinuria and soluble CD30 levels, and clinical features, including frequencies of rejection episodes, tumor and infections incidence;
• proinflammatory cytokines and chemokines, Treg-Th17-Th1 balance in the peripheral blood;
• BK and CMV specific rensponse by analyzing the frequencies of IFN-g and IL-10 producing cells and the correlation between HLA A, B, DR alleles and  the presence of viruses
• HLA specific antibodies.

We introduced the ImmuKnow (IK) assay to follow the outcome of allograft in kidney transplanted patients to monitor the infection/rejection risk. This is a new method to measure the immune response in the peripheral blood, as the capability of PHA-stimulated CD4⁺ T cells to produce intracellular adenosine triphosphate (ATP). Immune response is categorized as strong (≥525ng/ml), moderate (226-524) or low (≤225) according to the Kowalski’s validation study. We tested ATP 3, 6, 12 and 18 months after kidney transplantation observing a decrease of ATP activity after 3 months in all patients. Interestingly, after 3 months ATP level is significantly lower (p<0.005) in recipients over 60 years of age compared to those that are under 60. After 6, 12 and 18 months the reduction of ATP was confirmed, addressing to a stable lower response in patients over 60, suggesting an age-related difference in the immune cellular response to allograft. Notably, at any time tested there was a significant lower difference in the ATP values in patients over 60 and viral infectious free respect to the younger, but no significant differences were found in the IS therapy. This preliminary data demonstrate that ATP level evaluation could be instrumental to identify the potentially more tolerant patients. This allows us to characterize the immune status of these patients, assuming the possibility to modulate IS therapy in an age-related way.