Monitoring of Serological, Cellular and Genomic Biomarkers in Transplantation, Computational Prediction Models and Role of Cell-Free DNA in Transplant Outcome.

The process and evolution of an organ transplant procedure has evolved in terms of the prevention of immunological rejection with the improvement in the determination of immune response genes. These techniques include considering more important genes, more polymorphism detection, more refinement of the response motifs, as well as the analysis of epitopes and eplets, its capacity to fix complement, the PIRCHE algorithm and post-transplant monitoring with promising new biomarkers that surpass the classic serum markers such as creatine and other similar parameters of renal function. Among these new biomarkers, we analyze new serological, urine, cellular, genomic and transcriptomic biomarkers and computational prediction, with particular attention to the analysis of donor free circulating DNA as an optimal marker of kidney damage.

Killer Cell Immunoglobulin-like Receptors (KIR) and Human Leucocyte Antigen C (HLA-C) Increase the Risk of Long-Term Chronic Liver Graft Rejection.

Chronic liver rejection (CR) represents a complex clinical situation because many patients do not respond to increased immunosuppression. Killer cell immunoglobulin-like receptors/Class I Human Leukocyte Antigens (KIR/HLA-I) interactions allow for predicting Natural Killer (NK) cell alloreactivity and influence the acute rejection of liver allograft. However, its meaning in CR liver graft remains controversial. KIR and HLA genotypes were studied in 513 liver transplants using sequence-specific oligonucleotides (PCR-SSO) methods. KIRs, human leucocyte antigen C (HLA-C) genotypes, KIR gene mismatches, and the KIR/HLA-ligand were analyzed and compared in overall transplants with CR (n = 35) and no-chronic rejection (NCR = 478). Activating KIR (aKIR) genes in recipients (rKIR2DS2+ and rKIR2DS3+) increased CR compared with NCR groups (p = 0.013 and p = 0.038). The inhibitory KIR (iKIR) genes in recipients rKIR2DL2+ significantly increased the CR rate compared with their absence (9.1% vs. 3.7%, p = 0.020). KIR2DL3 significantly increases CR (13.1% vs. 5.2%; p = 0.008). There was no influence on NCR. CR was observed in HLA-I mismatches (MM). The absence of donor (d) HLA-C2 ligand (dC2-) ligand increases CR concerning their presence (13.1% vs. 5.6%; p = 0.018). A significant increase of CR was observed in rKIR2DL3+/dC1- (p = 0.015), rKIR2DS4/dC1- (p = 0.014) and rKIR2DL3+/rKIR2DS4+/dC1- (p = 0.006). Long-term patient survival was significantly lower in rKIR2DS1+rKIR2DS4+/dC1- at 5-10 years post-transplant. This study shows the influence of rKIR/dHLA-C combinations and aKIR gene-gene mismatches in increasing CR and KIR2DS1+/C1-ligands and the influence of KIR2DS4+/C1-ligands in long-term graft survival.

Killer immunoglobulin-like receptor repertoire analysis in a Caucasian Spanish cohort with inflammatory bowel disease.

Immunological molecules are implicated in inflammatory disorders, including inflammatory bowel disease (IBD; Crohn disease [CD] and ulcerative colitis [UC]). Killer cell immunoglobulin-like receptors (KIRs) are also genetically variable proteins involved in immune function. They are expressed by NK cells and certain T lymphocytes, regulate specificity and function by interaction with HLA Class I molecules, may be either inhibitory or activating and are polymorphic both in terms of alleles and haplotype gene content. Genetic associations between activating KIRs and certain autoimmune and inflammatory diseases have been reported; however, a possible association between KIR and IBD remains unclear. The aim of this study was to determine the relationship between KIR repertoire and IBD pathologies in a Spanish cohort. KIR variability was analyzed using PCR-sequence specific oligonucleotide probes (SSOP). Inhibitory KIR2DL5 was found more frequently in UC and IBD patient groups than in healthy controls (P = 0.028 and P = 0.01, respectively), as was activating KIR2DS1 (P = 0.02, Pc > 0.05, UC vs. Controls; P = 0.001, Pc = 0.01, IBD vs Controls; P = 0.01, Pc > 0.05, Controls vs CR), KIR2DS5 (P = 0.0028, Pc = 0.04, Controls vs UC; P = 0.0001, Pc = 0.0017, Controls vs IBD; P = 0.01, Pc > 0.05, Controls vs CD) and KIR3DS1 (P = 0.012, Pc > 0.05, Controls vs IBD). Our data suggest that imbalance between activating and inhibitory KIR may partially explain the different pathogeneses of these IBDs and that there is a hypothetical role for the telomeric B region (which contains both KIR2DS5 and KIR2DS1) in these diseases.

KIR gene variability in cutaneous malignant melanoma: influence of KIR2D/HLA-C pairings on disease susceptibility and prognosis.

Natural killer and CD8(+) T cells are believed to be involved in the immune protection against melanoma. Their function may be regulated by a group of receptors defined as killer immunoglobulin-like receptors (KIRs) and their cognate HLA class I ligands. In this study, we analyzed the influence of KIR genes and KIR/HLA-I combinations on melanoma susceptibility and/or prognosis in a Spanish Caucasian population. For this purpose, KIR genotyping by PCR-SSP and HLA-C genotyping by reverse PCR-SSO were performed in 187 melanoma patients and 200 matched controls. We found a significantly low frequency of KIR2DL3 in nodular melanoma (NM) patients (P = 0.001) and in ulcerated melanoma patients (P < 0.0001). Similarly, the KIR2DL3/C1 combination was significantly decreased in melanoma patients (Pc = 0.008) and in patients with sentinel lymph node (SLN) melanoma metastasis (Pc = 0.002). Multivariate logistic regression models showed that KIR2DL3 behaves as a protective marker for NM and ulcerated melanoma (P = 0.02, odds ratio (OR) = 0.14 and P = 0.04, OR = 0.28, respectively), whereas the KIR2DL3/C1 pair acts as a protective marker for melanoma (P = 0.017, OR = 0.54), particularly superficial spreading melanoma (P = 0.02, OR = 0.52), and SLN metastasis (P = 0.0004, OR = 0.14). In contrast, the KIR2DL3(-)/C1C2 genotype seems to be correlated with NM and ulceration. We also report that the KIR2DL1(+)/S1(-)/C2C2 genotype is associated with susceptibility to melanoma and SLN metastasis. Altogether, the study of KIR2D genes and HLA-C ligands may help in assessing cutaneous melanoma risk and prognosis.

Higher Expression of Activated CD8+ T Lymphocytes (CD8+CD25+, CD8+CD69+ and CD8+CD95+) Mediate Early Post-Transplant Acute Tubular Injury in Kidney Recipients.

BACKGROUND: Acute kidney injury (AKI) is a leading cause of early post-transplant kidney damage. Furthermore, acute tubular necrosis (ATN) is appointed as the most prevalent form of AKI, a frequent multifactorial process associated with high morbidity and mortality, yet giving rise to delayed graft function (DGF) and, ultimately, allograft dysfunction. Common factors such as prolonged cold ischemia time, advanced donor age, cadaveric versus living donor, donor history of hypertension, as well as donation after cardiac death have all been deemed risk factors for ATN. With the increasing number of older cadaveric and cardiac donors in the donation process, ATN could have a detrimental impact on patient welfare. Therefore understanding the underlying process would benefit the transplant outcome. We aimed to prospectively monitor several T cell subsets in a cohort of kidney transplant recipients (KTrs) to investigate whether there is an adaptive immune-mediated involvement in the ATN process. METHODS: Peripheral blood was collected from 31 KTrs at different time points within the first-year post-transplantation for in vitro stimulation with Concanavalin-A (Con-A) in a humidified 5% CO2 incubator at 37 °C for 72 hours. Upon cell stimulation, flow cytometry was applied to quantify the surface expression through the median fluorescence intensity (MFI) of CD4+CD25+, CD8+CD25+, CD4+CD38+, CD8+CD38+, CD4+CD154+, CD8+CD154+, CD4+CD69+, CD8+CD69+, CD4+CD95+, and CD8+CD95+ T cells. Statistical analysis was carried out with SPSS Statistics IBM v.25 (IBM Corp, Armonk, NY, USA). MFIs values were compared using a univariate analysis by a nonparametric U-Mann Whitney test. ROC analysis was applied to define cut-off values most capable of stratifying patients at high risk of ATN. Spearman's rank-order coefficient test was applied to correlate biomarkers with allograft function. Multivariate regression independently validated CD8+ T lymphocytes as surrogate biomarkers of ATN. A p-value < 0.05 was considered statistically significant. RESULTS: KTrs who developed ATN upon transplantation had significantly higher expression of CD25, CD69, and CD95 on CD8+ and lower expression of CD95 on CD4+ T lymphocytes than patients with stable graft function. ROC curve analysis showed that MFIs ≥1015.20 for CD8+CD25+, ≥2489.05 for CD8+CD69+, ≥4257.28 for CD8+CD95+, and ≤1581.98 for CD4+CD95+ were capable of stratifying KTrs at high risk of ATN. Furthermore, patients with an MFI below any cut-off were significantly less likely to develop ATN than those with other values. The allograft function was correlated with the CD4+CD95+/CD8+CD95+ ratio in KTrs who developed ATN. The multivariate analysis confirmed that, within the first-month post-transplant, MFI values of CD8+CD25+, CD4+CD95+, and CD8+CD95+ T lymphocytes, along with donor age, serum creatinine, and GFR were independent risk factors to ATN. Moreover, we were also able to corroborate previous immune factors of importance in immune-mediated response to the allograft, such as the patient's maximum panel reactive antibody (PRA) or the maintenance immunosuppression therapy. CONCLUSIONS: Our results demonstrate evidence for the implication of CD8+ T lymphocytes in the development of ATN early in the post-transplant phase. Post-transplant monitoring of activated CD8+ T lymphocytes may help identify which patients require further clinical intervention to prevent graft damage.

Evaluation of Antibodies Directed Against Two GPCRs, Anti-AT1R and Anti-ETAR, on Kidney Transplant Outcome.

BACKGROUND: The role of an alloimmune response against non-self-antigens is established in organ transplantation. HLA incompatibilities are mainly responsible for this recognition between donor and recipient, but they may also be involved in the reactivity against other alloantigens expressed on the allograft resulting from an autoimmune response developed against selfantigens. OBJECTIVE: Our study aimed to determine the presence of non-anti-HLA antibodies (anti-AT1R and anti-ETAR) in sera from patients with end-stage renal disease, who underwent kidney transplantation in pre- and post-transplantation samples to study their influence on the development and evolution of acute humoral rejections and DSAs. METHODS: Antibodies (Abs) against two G protein-coupled receptors (GPCRs), angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR), have been detected in the sera of transplant recipients, who experience allograft dysfunction, patients with coronary heart disease, marginal hypertension and refractory, vascular lesions, myocardial hypertrophy and chronic inflammatory diseases, such as atherosclerosis or sclerosis. RESULTS: Kidney graft recipients were monitored for anti-ETAR, -AT1R, and -HLA Abs in pre-and post-transplant evolution, and anti-AT1R and/or -ETAR Abs were detected in 24% of recipients (22.4% with anti-AT1R Abs and 9.8% with anti-ETAR Abs). Due to acute humoral rejection, Graft loss was detected in 6.4% of patients with anti-GPCRs non-HLA Abs, and 3.2% had DSA anti-HLA Abs. In this research, we have described how the function of the anti-GPCRs autoAbs and how these Abs that activate GPCRs could influence graft outcome. CONCLUSION: In conclusion, there is a high association of non-HLA anti-GPCRs Abs levels with reduced kidney function after transplantation, especially in the presence of DSA anti-HLA Abs. Although more studies are needed, anti-AT1R and anti-ETAR antibodies may be helpful biomarkers that allow the risk of graft loss to be assessed.

A high concentration of TGF-ß correlates with opportunistic infection in liver and kidney transplantation.

Transforming growth factor-ß (TGF-ß) has been associated with numerous human infections, but its role in the occurrence of opportunistic infection (OI) after solid organ transplantation remains unexplored. This study aimed to assess the utility of the TGF-ß following in vitro stimulation of whole peripheral blood (WPB) as a surrogate biomarker of post-transplant OI in a cohort of liver and kidney recipients. Thirty liver and thirty-one kidney transplant recipients were recruited to be prospectively monitored for one-year post-transplantation. Enzyme-linked immunosorbent assay (ELISA) was performed to calculate IFN-γ, IL-17, IL-10 and TGF-ß concentration in the supernatant from the activated WPB. Recipients showed higher TGF-ß concentrations compared to IFN-γ, IL-17, IL-10 at baseline, although these differences were not significant between INF and NoINF. However, recipients who developed an OI within the first sixth months had a higher concentration of TGF-ß than those without OI. A concentration of TGF-ß > 363.25 pg/ml in liver and TGF-ß > 808.51 pg/ml in kidney recipients were able to stratify patients at high risk of OI with a sensitivity and specificity above 70% in both types of solid organ transplantations. TGF-ß could provide valuable information for the management of liver and kidney recipients at risk of post-transplant infection.

Immunological Risk Stratification of Bladder Cancer Based on Peripheral Blood Natural Killer Cell Biomarkers.

BACKGROUND: Bladder cancer (BC) is highly immunogenic. Bacillus Calmette-Guérin (BCG) immunotherapy offers the best results in non-muscle-invasive BC (NMIBC). Natural killer cells (NKcs) play decisive roles in BCG-mediated immune response and in general cancer immune-surveillance. OBJECTIVE: To analyze killer-cell immunoglobulin-like receptors (KIRs), their human leukocyte antigen class-I (HLA-I) ligands, and the expression of DNAX Accessory Molecule-1 (DNAM-1/CD226) on peripheral blood (PB) NKcs, to identify useful predictive biomarkers in BC. DESIGN, SETTING, AND PARTICIPANTS: KIR/HLA-ligand genotypes were compared between 132 BC, 201 other solid cancers, 164 plasma cell disorders, and 615 healthy Caucasoid controls. CD226 expression was evaluated by flow cytometry. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: KIR/HLA-I interactions and CD226 expression on NKcs (CD226high or CD226low) were compared across study groups, cancer stages, treatments, and progression-free and overall survival of patients, using chi-square, analysis of variance/post hoc, Kaplan-Meier/log-rank, and regression analyses. RESULTS AND LIMITATIONS: Three immunological risk groups were identified: low risk (KIR2DL1-L2+L3-/C1C1- and KIR2DL1+L2+L3+/C1C1+), intermediate risk (rest), and high risk (KIR2DL5+/HLA-C*16+ and KIR2DL1+L2+L3-), which displayed different 10-yr progression-free rates (83.3%, 48.6%, and 0%, respectively; p<0.001) and survival rates (83.3%, 54.3%, and 6.2%, respectively; p<0.001) for muscle-invasive T2/T4, and 10-yr progression-free rates (100%, 81.6%, and 50%, respectively; p<0.05) for NMIBC-T1 treated with BCG. Immunological risk stratification had an independent prognostic value to just histological staging for survival (hazard ratio=2.93, p<0.00001, Harrell C-statistic=0.779). CD226 expression on PB NKcs improved immunological stratification in intermediate-risk T1-T4 BC patients, with survival rates of 94.1% and 66.7% for CD226high and CD226low (p<0.05), respectively. CONCLUSIONS: Immunological risk stratification will complement BC histopathology to improve risk stratification and guide the selection of personalized treatments. Understanding of the molecular mechanisms of NKc tumor immune surveillance will enable the development of future NKc-based therapies. PATIENT SUMMARY: This work describes a peripheral blood test that aids in our understanding of the immune defense mechanisms against bladder cancer, is useful for classifying patient risk, and will guide personalized treatments.

Influence of Preformed Antibodies in Liver Transplantation.

The significance of human leukocyte antigen (HLA) matching and preformed donor-specific antibodies (DSAs) in liver transplantation remains unclear. The aim of this study was to analyze the presence of DSAs in a large cohort of 810 liver recipients undergoing liver transplant to determine the influence on acute (AR) or chronic liver rejection (CR), graft loss and allograft survival. DSAs were identified using complement dependent cytotoxicity crossmatch (CDC-CM) and multiplexed solid-phase-based flow cytometry assay (Luminex). CDC-CM showed that a 3.2% of liver transplants were positive (+CDC-CM) with an AR frequency of 19.2% which was not different from that observed in negative patients (-CDC-CM, 22.3%). Only two patients transplanted with +CDC-CM (7.6%) developed CR and suffered re-transplant. +CDC-CM patients showed a significantly lower survival rate compared to -CDC-CM patients (23.1% vs. 59.1%, p = 0.0003), developing allograft failure within the first three months (p < 0.00001). In conclusion, we have demonstrated a relationship between the presence of preformed DSAs and the low graft liver survival, indicating the important role and the potential interest of performing this analysis before liver transplantation. Our results could help to detect patients with an increased risk of graft loss, a better choice of liver receptors as well as the establishment of individualized immunosuppressive regimens.

Activating KIRs on Educated NK Cells Support Downregulation of CD226 and Inefficient Tumor Immunosurveillance.

Therapies using NK cells (NKc) expanded/activated ex vivo or stimulated in vivo with new immunostimulatory agents offer alternative opportunities for patients with recurrent/refractory tumors, but relevant biomarkers to guide the selection of patients are required for optimum results. Overall survival of 249 solid cancer patients was evaluated in relation to the genetics and/or the expression on peripheral blood NKcs of inhibitory and activating killer-cell immunoglobulin-like receptors (iKIR and aKIR, respectively), HLA class I ligands, CD226 (also known as DNAM-1), and NKG2A. Compared with patients with higher expression, patients with low expression of CD226 on total NKcs showed shorter mean overall survival (60.7 vs. 98.0 months, P < 0.001), which was further reduced in presence of telomeric aKIRs (KIR2DS1-DS5 and/or KIR3DS1, 31.6 vs. 96.8 months, P < 0.001). KIR2DL2/S2+, KIR3DL1+, KIR2DL1+, and KIR2DL3+ NKc subsets in the presence of their cognate ligands primarily contributed to shortening patients' overall survival by increasing the sensitivity to CD226 downmodulation in aKIR-rich telomeric genotypes. In patients with high tumor burden who died during the follow-up period, aKIR-rich telomeric genotypes were associated with: (i) specific downmodulation of CD226 on educated NKcs but not on CD8+ T cells or uneducated NKcs, (ii) lower expression of CD226 and higher expression of NKG2A on aKIR+ NKcs, and (iii) lower numbers of total CD56dim NKcs. The reduced expression of CD226 on NKcs with aKIR-rich genotypes may be a biomarker indicative of NKc hyporesponsiveness in patients that could benefit from new NKc immune-stimulatory therapies.