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.

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.

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.

High-resolution characterization of allelic and haplotypic HLA frequency distribution in a Spanish population using high-throughput next-generation sequencing.

Next-generation sequencing (NGS) at the HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1 and -DRB3/4/5 loci was performed on 282 healthy unrelated individuals from different major regions of Spain. High-resolution HLA genotypes defined by full sequencing of class I loci and extended coverage of class II loci were obtained to determine allele frequencies and also to estimate extended haplotype frequencies. HLA alleles were typed at the highest resolution level (4-field level, 4FL); with exception of a minor deviation in HLA-DPA1, no statistically significant deviations from expected Hardy Weinberg Equilibrium (HWE) proportions were observed for all other HLA loci. This study provides new 4FL-allele and -haplotype frequencies estimated for the first time in the Spanish population. Furthermore, our results describe extended haplotypes (including the less frequently typed HLA-DPA1 and HLA-DQA1 loci) and show distinctive haplotype associations found at 4FL-allele definition in this Spanish population study. The distinctive allelic and haplotypic diversity found at the 4FL reveals the high level of heterozygosity and specific haplotypic associations displayed that were not apparent at 2-field level (2FL). Overall, these results may contribute as a useful reference source for future population studies, for HLA-disease association studies as a healthy control group dataset and for improving donor recruitment strategies of bone marrow registries. HLA genotyping data of this Spanish population cohort was also included in the 17th International Histocompatibility and Immunogenetics Workshop (IHIW) as part of the study of HLA diversity in unrelated worldwide populations using NGS.

CD28 biomarker quantification and expression level profiles in CD4+ T-lymphocytes in solid organ transplantation.

The introduction of anti-calcineurin-based therapies has led to an increase in the one-year survival as well as graft function rates in patients undergoing solid organ transplantation (SOT). Nonetheless, early cellular acute rejection (EAR) incidence still remains a major challenge that irrevocably heads to poor outcomes. The mechanisms underlying CD4 T cell activation in SOT are still under research. In this sense, CD28 co-stimulatory molecule plays a pivotal role triggering CD4 T cell activation as well as survival maintenance. Previous own studies stated the role that CD4+CD28+ circulating T lymphocytes plays before and during EAR episodes. We assessed the percentage as well as the absolute number of CD28 molecules on CD4+ T cells as predictive surrogate biomarker of EAR in a prospective cohort of liver and kidney transplant recipients. Quantitative analysis of CD28 was carried out on whole peripheral blood samples by flow cytometry. Decreased pre-transplant expression of CD28 was associated with EAR in both study groups. Furthermore, the expression of CD28 within the rejected group, experimented an up-regulation upon transplantation. These preliminary results suggest that patients undergoing liver or kidney transplant can be stratified at high risk of EAR according to their CD28 molecule expression on peripheral CD4+ T lymphocytes.

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.

Humoral Response in Liver Allograft Transplantation: A Review of the Role of Anti-Human Leukocyte Antigen (HLA) Antibodies.

Antibody-mediated rejection (AMR) is relatively uncommon in AB0-compatible liver grafts; however, recent data suggest that antibody-mediated mechanisms may play a role in the differential pathogenesis of liver graft rejection. In this sense, it has been shown that staining for diffuse C4d deposition (in endothelium or stroma of portal tracts or sinusoids) could be used as a tissue biomarker of humoral allograft rejection and fibrosis in biopsies obtained in post-transplantation period, in addition to the determination of donor specific antibodies (DSA). Therefore, more stringer criteria have to be established in order to define real antibody-mediated injury (acute rejection, chronic rejection or fibrosis development) in liver graft transplant. These criteria should intend to correlate allograft dysfunction associated with compatible histological findings, presence of DSAs and C4d positivity and should be clarified with respect to rejection injury frequency, its severity and its response to antibody-depleting immunosuppression in the future. This could improve our progressive understanding of the clinical-pathological characteristic and diagnostic approaches of AMR and fibrosis.

HLA Epitopes and Shared Molecular Eplet Characterization and Their Implication on Transplant Outcome: The Experience of One Center.

We report three interesting cases concerning antibody-mediated rejection (AMR), associated or not with anti-donor-specific antibodies, and detection of implicated molecular epitopes. The first report presents a case of intra-allele sensitization. The second case presents an interesting case concerning Luminex mean fluorescence intensity (MFI) levels considered to be low risk antibodies (<1000), but producing AMR. The third case occurred after a second kidney transplantation mediated by antibodies directed against HLA-C antigens (MFI<1000) in the previous transplantation (which was considered to be an indicator of low-risk of AMR).

Informe del Taller Ibérico de Histocompatibilidad 2013. Componente de análisis de situación de procedimiento de pruebas cruzadas en guardias de trasplante de órganos / Report of the 2013 Iberian Histocompatibility Workshop of 2013. Component analysis of the proceedings of cross-matching procedures in emergency organ transplantation

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HLA-C antibodies are associated with irreversible rejection in kidney transplantation: Shared molecular eplets characterization.

We report an interesting case concerning an irreversible antibody-mediated rejection (AMR), associated with anti-HLA-C DSA, which occurred after a second kidney transplantation despite having determined a low number of antibodies directed against HLA-C antigens (MFI<1000) in the previous transplantation (which was then considered to be an indicator of low risk of AMR). A 63-year-old woman was re-transplanted with pre-transplant (PrT) sensitization. On day 7 post-transplantation, oligoanuria occurred and increased MFIs for the detected PrT antibodies and other antibodies (non-detected or detected with very low PrT MFI) were observed. SAB assay also showed antibodies against the second donor HLA-C mismatches and other HLA-C antigens. Nephrologists suspected AMR and the patient was therefore treated with methylprednisolone/plasmapheresis/IVIG/anti-CD20 without improvement, which led to transplantectomy. Histologic analysis confirmed acute AMR. Interestingly, it was possible to define exactly the potential immunizing epitopes whose recognition determines the specific antibody production. So, 1st donor DSAs (detected PrT with low MFI), 2nd donor DSAs (detected PTP), and non-DSA detected PTP have several shared eplets, being the 11AVR eplet the only one present on all alleles. Thus, the recognition of 11AVR eplet in the first transplant modeled the patient's antibody response. Therefore, we propose that donor HLA-C typing should always be performed for recipients with anti-HLA-C antibodies, and specific shared-eplets should be investigated in order to determine previous transplant mismatches.

KIR gene mismatching and KIR/C ligands in liver transplantation: consequences for short-term liver allograft injury.

BACKGROUND: Killer immunoglobulin-like receptors (KIRs) bind human leukocyte antigen (HLA) class-I (HLA-I) ligands and regulate functions of natural killer cells and subsets of T cells. KIR/HLA-I interactions allow predicting natural killer cell alloreactivity in hematopoietic stem cell transplantation and in HLA-compatible kidney transplants, but its meaning in liver transplantation remains controversial. METHODS: KIR and HLA genotypes were studied in 402 liver transplants, using sequence-specific oligonucleotides and primer methods. Recipients and donor KIRs, HLA-C genotypes, KIR gene mismatches (MMs) between recipient-donor pairs, and KIR/HLA-ligand combinations were analyzed in overall transplantations, in the acute rejection (AR; n=110) and non-AR (n=292) groups. RESULTS: KIR gene MMs between recipients and donors, mainly in activating KIRs, and KIR2DL3 and KIR2DS1 of recipients in the presence of donor C2 ligands, significantly enhanced early AR rate (P<0.05), with KIR2DL3 and KIR2DS1 exhibiting a synergic effect in dependence of the donor C2 ligand number (χ2=7.662, P=0.022). KIR2DL3, KIR2DS1, and also KIR2DS4 significantly influenced short-term graft survival, with a benefit for transplantations combining KIR2DL3 recipients and donors having C1 ligands (log rank, P<0.019 at 1 year; hazards ratio [HR], 0.321; 95% confidence interval [CI], 0.107-0.962; P=0.042), whereas KIR2DS1 and KIR2DS4 recipients combined with donors lacking C1 ligands (C2/C2) exhibited a worse graft survival (log rank, P=0.035 at 6 months; HR, 7.713; 95% CI, 2.156-27.369; P=0.002 for KIR2DS1; and log rank, P=0.006 at 1 year; HR, 3.794; 95% CI, 1.267-11.365; P=0.017 for KIR2DS4). CONCLUSIONS: This study shows that KIR gene-gene MMs increase AR and that KIRs/C ligands associated to AR and KIR2DS4/C ligands also influence short-term graft survival.

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.

Low median fluorescence intensity could be a nonsafety concept of immunologic risk evaluation in patients with shared molecular eplets in kidney transplantation.

Human leukocyte antigen (HLA) antibodies are usually "epitope" and not "antigen" specific. This work presents an interesting case concerning Luminex median fluorescence intensity (MFI) levels in antibodies considered low risk (<1,000), but producing humoral rejection. These low-titer antibodies could play an important role in transplantation. A 42-year-old woman was retransplanted with a deceased donor with negative complement-dependent cytotoxicity cross-matching. Our patient was pretransplant (PrT) sensitized to HLA antigens (single antigens (SA) = 31%) for 1 previous transplant. Thus, the formerly detected sensitized antigens were A32, A30, A31, cross-reacting group 5C, and DQ3 with a MFI(max) ≈ 4,127. In the posttransplantation period (PTP), the patient exhibited important instability in renal function and we detected an increased SA percentage (61%) with MFI(max) = 15,029 (A*32) with other antigens (detected with a low PrT MFI [<1,000]) as anti-A*03 (MFI(max) = 13,301) and anti-A*11 (MFI(max) = 13,714) specificities. Anti-A*03 was a donor-specific antibody (DSA). Renal biopsy was compatible with humoral rejection. The patient was pulsed with methylprednisolone, plasmapheresis, and intravenous immunoglobulin without improvement. Thus, we added anti-CD20 and the initial clinical response was highly favorable. Biopsies resulted in suggestive rejection reversion. MFI A*03 DSA decreased to 6,908 and later to MFI(max) = 5,505. After a 6-month PTP, the patient is well with MFI(max) = 3,124. It was possible to define exactly the potential immunizing epitope eplets whose recognition determined the specific antibody production. A*32:01, A*30:01, A*31:01 (detected PrT), A*11:01, and A*03:01 (detected PTP) alleles have several shared eplets (62QE, 70AQS, and 76VGT), with 62QE being the only eplet present on all alleles. In conclusion, low MFI levels in antibodies considered low risk could be important in posttransplant humoral rejection, although the patient's renal function can be restored. Thus, specific shared eplets should always be investigated with respect to previous transplant mismatches.

Influence of human leukocyte antigen mismatching on rejection development and allograft survival in liver transplantation: is the relevance of HLA-A locus matching being underestimated?

The influence of HLA matching on liver transplant is still controversial, as studies have failed to demonstrate an adverse effect of HLA mismatching on transplant outcome. We examined the effect of HLA mismatching on transplant outcome in a series of 342 consecutive liver transplants (224 finally analyzed). HLA typing was performed by serological and molecular methods. HLA-A matching was associated with an increased chronic rejection incidence (P=0.04). Indeed, HLA-A match also demonstrated a significant impact on allograft survival (P=0.03), confirming previous observation concerning to rejection, as complete HLA-A mismatching favored a better liver transplant outcome. Analysis of HLA-A+B+DR matching also demonstrated a significant impact on graft survival (P<0.05). Multivariate Cox regression analysis confirmed the effect of HLA-A and DPB1 matching as independent risk factors for graft loss. Another independent factor was a positive pre-transplant crossmatch. In conclusion, liver transplant outcome has not been found to be improved by HLA matching, however a poorer HLA compatibility favored a better graft survival and decreased rejection incidence, with a special relevance for HLA-A matching.

HLA-C matching and liver transplants: donor-recipient genotypes influence early outcome and CD8+KIR2D+ T-cells recuperation.

BACKGROUND: Fully human leukocyte antigens (HLA)-mismatched liver grafts are well accepted, but the HLA influence on acceptance or rejection is unclear and much less so the impact of HLA-C, which may be conditioned by the fact that HLA-C-encode molecules are the major ligands for killer cell immunoglobulin-like receptors (KIR). METHODS: The HLA-C allele compatibility and the effect of donor and recipient HLA-C genotype on early liver graft acceptance and on CD8KIR T-cells recuperation were analyzed in a series of 431 primary liver transplants. Standard polymerase chain reaction PCR-SSO was used for HLA-C typing and flow cytometry to identify T cells KIR positives. Transplants were classified into two groups: acute rejection and nonacute rejection, and individual HLA-C genotypes as C1/C1, C2/C2, and C1/C2. RESULTS: A favorable effect of HLA-C allelic compatibility on early liver graft acceptance was found because acute rejection significantly increased in transplants performed with 2 HLA-C allele mismatches (P=0.02). Considering the HLA-C groups, it was observed that C1/C2 heterozygous donors were best accepted in C1/C1 patients than in C2/C2 recipients, who experienced a high rate of acute rejection (P<0.004 and P<0.005, respectively). In addition, after transplantation CD3CD8KIR2D T-cells repertoires significantly increased in C1/C1 and C1/C2, but not in C2/C2 patients. CONCLUSIONS: This study confirms the benefit of HLA-C allele matching on early liver transplant outcome and shows that donor HLA-C heterozygosis influences the alloresponse of C1 and C2 homozygous patients and the recuperation of CD3CD8KIR2D T cells, suggesting an involvement in liver graft tolerance.