Three new HLA class I alleles with synonymous mutations: HLA-A*03:01:62, -C*07:02:82 and -C*12:03:42.

Three new HLA class I alleles with synonymous mutations were identified.

Pretransplant CD28 Biomarker (Levels of Expression and Quantification of Molecules per Cell) in Peripheral CD4+ T Cells Predicts Acute Rejection Episodes in Liver and Kidney Recipients.

BACKGROUND: Acute rejection (AR) remains a significant cause of graft loss. Better approaches to predict AR are being investigated. Surface CD28 protein is essential for T-cell proliferation and survival as well as cytokine production. PATIENTS AND METHODS: Pretransplant CD4+CD28+ peripheral T cells were examined in 30 liver recipients (LRs) and 31 kidney recipients (KRs) by flow cytometry. RESULTS: Pretransplant CD4+CD28+ T cells in LRs were significantly lower in rejectors than nonrejectors (P = .002). Furthermore, the total number of CD28 molecules per cell in LRs (P = .02) as well as KRs (P = .047) was significantly lower in rejectors than nonrejectors. The healthy group did not display differences when compared with patients with end-stage liver disease or renal failure; however, stratification analysis displayed higher levels of CD4+CD28+ when compared with rejected LRs (P = .04) but not KRs. CD28 levels <41.94% were able to discriminate LRs at high risk of AR (P = .003). Similarly, a total number of CD28 molecules ≤8359 (P = .031) in LRs and ≤7669 (P = .046) in KRs correlated with high risk of AR. CONCLUSION: The preliminary results presented herein exhibit a fast and noninvasive method that assists clinicians to prevent AR by monitoring CD4+CD28+ peripheral T cells.

Report From the First and Second Spanish Killer Immunoglobulin-Like Receptor Genotyping Workshops: External Quality Control for Natural Killer Alloreactive Donor Selection in Haploidentical Stem Cell Transplantation.

An important factor affecting the success in the setting of related haploidentical hematopoietic stem cell transplantation (HSCT) is the graft-versus-leukemia effect mediated by natural killer (NK) cells when the donor displays NK alloreactivity versus the recipient. NK cell function is regulated by killer immunoglobulin-like receptors (KIR) and it has been described that donor KIR genotype influences transplantation outcome. This has led to a requirement of laboratories to have a quality assurance program for validation and control of their KIR genotyping methods. The goal of the 1st and 2nd Spanish KIR Genotyping Workshops was to provide an external proficiency testing program in KIR genotyping for Spanish immunology and transplant laboratories. These workshops were conducted during the years 2014-2016 and consisted of 17 participating laboratories typing a set of 20 samples. The presence/absence of 16 mandatory KIR loci (2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 2DP1, 3DL1, 3DL2, 3DL3, 3DS1, and 3DP1) was evaluated per sample. Methods for KIR genotyping included polymerase chain reaction with the use of sequence-specific primers and sequence-specific oligoprobes. Consensus typing was reached in all samples, and the performance of laboratories in external proficiency testing was satisfactory in all cases. The polymorphism detected in the small sample studied in both workshops is indicative of an ample variety of KIR gene profiles in the Spanish population.

Complement component C6 deficiency in a Spanish family: implications for clinical and molecular diagnosis.

Complement component C6 deficiency is a genetic disease presenting as increased susceptibility to invasive Neisseria meningitidis infections. This disorder has rarely been diagnosed in the Spanish population. In this work we report the immunochemical and molecular characterization of complement C6 deficiency in a Spanish patient showing no detectable functional activity of either the classical or alternative complement pathways and reporting a history of several episodes of meningococcal meningitis. The levels of individual complement components C3, C4, C5, C7, C8 and C9 were within the normal range. However, C6 level was low in the patient's serum as measured by radial immunodiffusion. Exon-specific polymerase chain reaction and sequencing of the C6 gene revealed a previously described homozygous single base deletion in exon 6 (c.821delA), leading to a shift in the reading frame that caused the generation of a downstream stop codon, which, in turn, provoked the truncation of the C6 protein (p.Gln274fs). To our knowledge, this is the first report on the c.821delA mutation in the Spanish population, which has previously only been identified in individuals of African ancestry. Characterization of this mutation was thought interesting in order to elucidate its source and help understand the molecular basis of this uncommon deficiency in our population. Moreover, this report highlights the importance of complement screening in cases of repeated meningococcal infections in order to establish its involvement and to consider adequate clinical recommendations such as prophylactic antibiotics or meningococcal vaccines and, subsequently, for genetic counselling.

Post-transplant increase in soluble human leukocyte antigen-G associated with non-severe cardiac allograft vasculopathy.

Cardiac allograft vasculopathy (CAV) is the single most important long-term limitation to heart transplantation. This study aimed to assess the value of monitoring soluble human leukocyte antigen-G (sHLA-G) during the first year post-transplantation to predict the severity of CAV, in 21 out of 77 heart recipients assessed by intravascular ultrasound (IVUS). Serum sHLA-G concentration increased after transplant in recipients free of severe CAV, but decreased in recipients suffering from severe CAV, significant differences between these two groups were found 6 to 12 months post-transplantation. The optimal value of the change in post-transplant sHLA-G for identifying severe CAV was ≥0.062%, which maximized sensitivity (80%) and specificity (100%). Importantly, increases in post-transplant sHLA-G were inversely associated with severe CAV, but directly associated with human cytomegalovirus reactivation. In addition, recipients presenting non-severe CAV or an increased sHLA-G post-transplantation, showed higher numbers of CD8(+)CD28(-) T cells and a down-modulation of CD28 on CD4(+) lymphocytes, which typically identifies CD8(+) regulatory T cells and anergic/tolerogenic T helper cells, respectively. In conclusion, quantification of sHLA-G might offer a complementary non-invasive method for identifying recipients at risk of more severe CAV and who might benefit from earlier preventive therapies, although these results need to be confirmed in larger series.

C1q-fixing human leukocyte antigen assay in immunized renal patients: correlation between Luminex SAB-C1q and SAB-IgG.

BACKGROUND: There is no consensus about the impact of thresholds of complement-fixing antibody assays. Recently, a C1q-SAB assay has been developed to identify complement-fixing HLA antibodies with high sensitivity and specificity. Our aim was to determine the correlation between IgG single antigens beads (SAB) and C1q-SAB assay results among patients on the renal waiting list. PATIENTS AND METHODS: Serum samples from immunized renal waiting list patients as well as negative and positive controls were valided by Luminex (LMX). These sera, which were positive for 166 antibody specificities, were tested for HLA class I in parallel by LMX-IgG and LMX-C1q. RESULTS: Comparison of antibody detection revealed no correlation based on median fluorescent intensity (MFI), levels between the IgG SAB and the C1qSAB assay (P > .05). IgG-positive sera with MFIs as low as 700 were able to fix C1q, whereas other sera with MFIs as high 14,500 did not. Furthermore, there appeared to be disparities in the profiles of class I antigens able to fix C1q-SAB. In our series, only 34% class I IgG SAB antibodies were also C1qSAB+. In several patients, we detected C1qSAB+ against IgGSAB- that was surely due to IgM antibodies. So, the C1qSAB assay detected IgM antibodies that fix complement. CONCLUSION: These data suggested that the C1q-SAB assay could be an important method to evaluate pretransplant virtual crossmatch and to define nonpermitted specificities (C1q-fixing) in kidney transplantation.

Evolution of soluble forms of CD86, CD95 and CD95L molecules in liver transplant recipients.

Co-stimulatory factors such as CD86 and apoptotic molecules such as CD95 and CD95L required to start and to turn off the allogenic immune response may also be present as soluble proteins. To determine the role of the soluble forms of CD86 (sCD86), CD95 (sCD95) and CD95L (sCD95L) in the outcome of liver transplants, we analyzed the circulating levels of these molecules in patients subjected to liver transplantation in the pre-operative period and during the first month post-transplantation. Serum samples were obtained from sixty-nine first orthotopic liver transplants (OLT). The patients were classified into acute rejection (AR=24) and not acute rejection (NAR=45), or considering the presence of chronic active hepatitis B or C (VP=30) or other primary liver diseases (VN=39). The levels of sCD86, sCD95 and sCD95L were analyzed by solid phase sandwich enzyme-linked immunoabsorbent assays. Our results first showed that the pre-transplantation serum levels of sCD86 in the AR group were significantly higher than in the NAR group (1007±82U/mL vs. 739±46U/mL, p=0.006), and in the post-transplantation period these levels decreased sharply. Second, the levels of sCD95L and sCD95 in the pre-transplantation period did not point to statistically significant differences between the AR and NAR groups. Considering primary liver disease, the pre-transplantation levels of sCD86 and sCD95L in the VP group were significantly higher than those of the VN group (VP, 977±69U/mL vs. VN, 722±51U/mL, p<0.002, and VP, 482±78pg/mL vs. VN, 221±31pg/mL, p=0.002, respectively). Multivariate analysis revealed that only the pre-transplantation levels of sCD86 were independently associated with the development of episodes of acute rejection (p=0.005, OR=2.1, IC 95%=1.27-3.47). In conclusion, the present work shows that primary liver disease could influence the pre-transplantation levels of sCD86 and sCD95L. High pre-transplantation serum levels of sCD86 could favor the development of episodes of acute rejection.

CD28 and KIR2D receptors as sensors of the immune status in heart and liver transplantation.

Viral infections and cellular acute rejection (AR) condition immunosuppressive therapy and compromise the evolution of allografts. Immune monitoring can be useful for ascertaining rejection and for differentiating allo-reaction from activation induced by infections. This work analyzes the usefulness of monitoring the expression of CD28 and KIR2D receptors in peripheral blood T lymphocytes by flow cytometry, to ascertain the immune response in heart and liver transplant recipients. In both types of transplant, the up-regulation of CD28 in CD4(+) lymphocytes in the periods of greatest AR frequency indicates an effective allo-response, whereas the post-transplantation emergence of circulating CD8(+)CD28(-) and CD8(+)CD28(-)KIR2D(+) T cells correlates with better early clinical results. Cytomegalovirus (CMV) infection, but not hepatitis C virus (HCV) or other infections, abrogated both CD28 up-regulation and CD8(+)CD28(-)KIR2D(+) T-cell expansion. Our results show that monitoring the expression of CD28 and KIR2D receptors on T lymphocytes might be considered as sensors of the immune status of heart and liver recipients.

Divergences in KIR2D+ natural killer and KIR2D+CD8+ T-cell reconstitution following liver transplantation.

Natural killer (NK) and CD8(+) T cells may be active elements in the allograft response, but little is known about their role in liver transplantation. Some of these cells express killer immunoglobulin-like receptors (KIRs), which after binding specific ligands may transmit inhibitory/activating signals. In this study, circulating NK and CD8(+) T cells expressing CD158a/h (KIR2DL1/S1) or CD158b/j (KIR2DL2/3/S(2)) receptors were analyzed in 142 liver recipients by flow cytometry. They were underrepresented in patients before transplantation, but following transplantation, whereas the KIR2D(+) NK subsets experienced a late recuperation (day 365) mainly in C2-homozygous patients developing early acute rejection, recovery of the 2 CD8(+)KIR2D(+) T cells started earlier, showing significant differences on day 365 between patients without acute rejection and those suffering from it (p = 0.004 and p < 0.0001, respectively). These differences were also evident when the human leukocute antigen-C genotypes of the recipient were considered. In conclusion, whereas the late recovery of KIR2D(+) NK cells in C2/C2 patients appears to be linked to acute rejection, the increase in early CD8(+)KIR2D(+) T cells in overall liver recipients correlates with a most successful early graft outcome. Therefore, monitoring of KIR2D(+) cells appears to be a useful tool for liver transplant follow-up.

Expression of HLA molecules on peripheral blood lymphocytes: a useful monitoring parameter in cardiac transplantation.

During the rejection process of cardiac allografts, the expression of HLA antigens increases on various graft tissues, ie, the myocardium and the interstitial structures. However, in this type of transplant there is a paucity of knowledge about HLA expression on recipient cells, such as peripheral blood mononuclear cells. In the present study expression of HLA class I and class II antigens was monitored on peripheral blood lymphocytes prior to and during a 12-month follow-up, using flow cytometry. In our series, the frequency of acute rejection episodes was greater from the fourth to the ninth month after transplantation, coinciding with a reduction in cyclosporine blood levels. At the same time, expression of HLA class I and class II antigens significantly increased among recipients suffering from more severe acute rejection episodes compared with those showing acceptance of their grafts (P < .01). In conclusion, acute rejection episodes in cardiac transplantation were associated with up-regulation of HLA molecules on recipient peripheral blood cells. Monitoring the expression of HLA molecules on peripheral blood lymphocytes may represent an easy, noninvasive practice to individualize immunosuppressive therapy.

Evaluation of CD86 gene polymorphism at +1057 position in liver transplant recipients.

BACKGROUND: Efficient T cell-APC interaction requires the participation of primary and co-stimulatory signals. The main co-stimulatory pathway involves the interaction of CD80 and CD86, expressed on the APCs, with their T cell counter-receptor, CD28 and CTLA-4. Recently, a G to A transition has been described at position +1057 of the CD86 gene, located in their cytoplasmic tail. METHODS: CD86 polymorphism was analyzed by sequence based typing in DNA samples obtained from 205 liver transplant recipients. Acute rejection and chronic rejection were diagnosed based upon conventional clinical, biochemical and histological criteria. RESULTS: The study of CD86 +1057 (G/A) polymorphism revealed that recipients bearing the A allele or the AA genotype have a reduced risk of acute rejection. In fact, the AA genotype was absent in the group of patients showing acute rejection episodes, whereas its frequency in those patients without acute rejection episodes was 8.8% (P=0.009, OR=0.07). This polymorphism did not reveal any association with the incidence of chronic rejection, but patients bearing the AA genotype showed a higher graft survival rate (83.3%) than those bearing the GA genotype (49.3%) or GG genotype (56.5%). CONCLUSIONS: The results of the present report suggest that the CD86 AA genotype at +1057 position could be involved in liver transplant acceptance, given that its presence is related to a decrease of acute rejection frequency and to a graft survival increase.

Relationship between CDC cross-match in liver recipients and antibody screening by flow cytometry.

Several authors have shown that anti-donor antibodies before liver transplantation are associated with decreased graft survival. The aim of this study was to investigate the relationship between anti-donor antibodies detected by the CDC technique or by FlowPRA, and acute or chronic rejection as well as graft survival. Furthermore, we sought to determine whether anti-donor antibodies, detected by the CDC technique, correlated with those discovered by cytometric screening. The acute rejection incidence among patients with complement-dependent cytotoxicity positive CDC cross-match was similar to that for patients with a negative cross-match. None of the patients with a positive cross-match developed chronic rejection. Allograft survival was significantly lower among recipients with a positive T-lymphocyte cross-match. Indeed, the majority of recipients with positive CDC cross-matches displayed graft failures before first posttransplant year. The results of a positive FlowPRA determination were concordant with a positive CDC cross-match in 85.71% of cases. Our data demonstrate that pretransplant FlowPRA correlates with the final CDC cross-match results. This finding suggests that in the future prospective pretransplant antibody screening with FlowPRA or CDC techniques may be useful to identify high-risk recipients.

Impact of HLA-C on acute rejection in liver transplantation.

Although liver transplants show a special tolerogenic behaviour, rejection remains an important problem that involves several immunological mechanisms, some of which are unknown. Our study sought to analyze the influence of HLA-C polymorphism on short-term liver graft acceptance by HLA-C genotyping of 100 orthotopic liver transplant recipient-donor pairs. Recipients were statified according to the occurrence of acute rejection. HLA-Cw*06 allele appeared to be underrepresented among recipients without versus those with acute rejection or those in control groups. With regard to HLA-C allelic compatibility, the frequency of acute rejection or those in episodes decreased with fewer HLA-C mismatches. These findings suggest the participation of HLA-C molecules in liver graft alloresponses, involving HLA-C genotyping, as well as compatibility.

HLA polymorphism in the Murcia population (Spain): in the cradle of the archaeologic Iberians.

Human leukocyte antigen (HLA) study in Murcian individuals was performed in order to provide information of their historical origins and relationships with other Iberian and Mediterranean populations. HLA class I and class II alleles were determined in 173 unrelated Caucasoid donors from Murcia Region in the Southeast of Spain by serologic and DNA based polymerase chain reaction (PCR) typing. Class I antigen and class II allele frequencies of our series were not very different to those found in Spaniards. The analysis of extended haplotypes showed that the three haplotypes most frequent in our population were respectively, A29-B44-Cwb-DRB1*0701-DRB4*0101-DQA1*0201-DQB1*0202, A1-B8-Cw7-DRB1*0301-DRB3*0101-DQA1*0501-DQB1*0201 and A30-B18-Cw5-DRB1*0301-DRB3*0101-DQA1*0501-DQB1*0201. They were followed by A26-B38-Cwb-DRB1*1301-DRB3*0202-DQA1*0103-DQB1*0603, which could point to an ancestral relationship between Murcian and Portuguese Iberian populations, and by A2-B7-Cw7-DRB1*1501-DRB5*0101-DQA1*0102-DQB1*0602 also present in all Iberian Peninsula populations. Allelic frequencies, populations distance dendrogram and correspondence analysis were used to study the relationships between Murcian and other populations. The closest relation was observed with Spaniards and Portuguese, followed in decreasing order by French, Italians, Algerians, Germans, Catalans, Basques, Cretans, Sardinians, and Greeks. Thus, Murcian population seems to belong to the European genetic pool, revealing a lesser genetic distance with the North Africans and the rest of populations from the Iberian Peninsula.