Circulating NK-cell subsets in renal allograft recipients with anti-HLA donor-specific antibodies.

Detection of posttransplant donor-specific anti-HLA antibodies (DSA) constitutes a risk factor for kidney allograft loss. Together with complement activation, NK-cell antibody-dependent cell mediated cytotoxicity (ADCC) has been proposed to contribute to the microvascular damage associated to humoral rejection. In the present observational exploratory study, we have tried to find a relationship of circulating donor-specific and non donor-specific anti-HLA antibodies (DSA and HLA non-DSA) with peripheral blood NK-cell subsets and clinical features in 393 renal allograft recipients. Multivariate analysis indicated that retransplantation and pretransplant sensitization were associated with detection of posttransplant DSA. Recipient female gender, DR mismatch and acute rejection were significantly associated with posttransplant DSA compared to HLA non-DSA. In contrast with patients without detectable anti-HLA antibodies, DSA and HLA non-DSA patients displayed lower proportions of NK-cells, associated with increased CD56(bright) and NKG2A(+) subsets, the latter being more marked in DSA cases. These differences appeared unrelated to retransplantation, previous acute rejection or immunosuppressive therapy. Although preliminary and observational in nature, our results suggest that the assessment of the NK-cell immunophenotype may contribute to define signatures of alloreactive humoral responses in renal allograft recipients.

CCL5/RANTES chemokine gene promoter polymorphisms are not associated with atopic and nonatopic asthma in a Spanish population.

CCL5/RANTES, a member of the C-C chemokine family, is a potent eosinophil, monocyte, basophile and lymphocyte chemo-attractant at the site of inflammation. Recent studies revealed that a functional mutation at the -403 position in the promoter may have significance for atopic dermatitis, bronchial asthma, sarcoidosis, rheumatoid arthritis and HIV infection, and others. Another polymorphism in the -28 position has been reported. Our objective was to investigate the possible influence of the CCL5/RANTES promoter polymorphisms in the different types of bronchial asthma. CCL5/RANTES genotyping was performed by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) in 306 asthmatic patients with non-atopic (n = 145) and atopic (n = 161) asthma and 242 controls. The 81.9% of the atopic asthma patients for -403G/A had the G allele and the A allele frequency was 18%. Of the non-atopic asthma patients, the G allele frequency was 79.7% and the A allele was 20.3%. Concerning the -28C/G polymorphism, the frequency of the CCL5/RANTES -28G allele in our patients is 2.8%, which is similar to Spanish adult population. After comparing patients with asthma, atopic patients, non-atopic patients and control population, we found no significant deviation in the distribution of the alleles or genotypes of CCL5/RANTES promoter polymorphisms in any tested comparison. Therefore, human CCL5/RANTES gene promoter polymorphisms are not associated with the different types of bronchial asthma in Spanish population.

Donor Selection Based on Killer Cell Immunoglobulin-Like Receptor (KIR) Genotype May Improve Outcome After T-Cell-Replete Haploidentical Transplantation.

BACKGROUND: In haploidentical stem cell transplantation (SCT), the "ideal donor" selection is not performed in a standardized way according to killer cell immunoglobulin-like receptor (KIR) genotype expressed by potential donors. The aim of this study was to evaluate the relevance of KIR genotype in a series of patients submitted to haploidentical SCT in our center. METHODS: We retrospectively analyzed 30 patients that were prepared with the use of a conditioning regimen including thiotepa-busulfan-fludarabine with high doses of post-transplantation cyclophosphamide (CyPT) and tacrolimus as graft-versus-host disease (GVHD) prophylaxis. We analyzed the impact of the KIR genotype variables (donor AA/Bx haplotype, donor B content, KIR inhibitor mismatches, and mismatching in KIR ligands in the graft-versus-host direction and the host-versus-graft direction) on overall survival, GVHD-free survival, and event-free survival. RESULTS: Statistical significance was found for the presence of mismatches on KIR ligands in the graft-versus-host direction in relation to the diagnosis of chronic GVHD (54% vs 100%; P = .004). Significance was also found for the effect of the donor presence AA or Bx haplotype in relation to the diagnosis of chronic GVHD (50% vs 86%; P = .033). CONCLUSIONS: KIR genotyping can provide useful information that can help us with the right donor choice for haploidentical SCT without T-cell depletion with high doses of CyPT. Donors with Bx haplotype that do not show KIR ligand incompatibilities in the graft-versus-host direction may provide a lower risk of GVHD.

CXCR3+ Monocytes Increase Significantly in Graft Blood Compared to Peripheral Blood in Patients With Stable Kidney Graft Function.

INTRODUCTION: We have recently reported that some lymphocyte populations do not maintain the same proportion in kidney graft blood as in peripheral blood, despite a stable function of the transplanted kidney. These results suggest that a comparative study between leukocyte cells from graft blood and those obtained from peripheral blood could provide information about the inflammatory state of the transplanted organ. In this work we selected the population of CD4+ lymphocytes and monocytes expressing CXCR3 to test this hypothesis. MATERIAL AND METHODS: The study was performed by flow cytometry during month 3, 6, and 12 after transplantation in 58 patients who received an isolated kidney transplant and the same immunosuppressive regimen. The peripheral blood sample was obtained by venipuncture and the graft blood by fine needle aspiration. RESULTS: We found a significant percentage decrease in CXCR3+ monocytes throughout the first year of transplantation in peripheral blood (15.9 ± 20.7 vs. 12.6 ± 12.4 vs. 6.3 ± 9.0, at 3, 6, and 12 months, respectively; P = .001), whereas the percentage of CXCR3+ monocytes in graft blood did not change over this period. This situation resulted in a significant percentage difference between the CXCR3+ monocytes from the graft blood and those from the peripheral blood at the sixth (15.8 ± 8.1 vs. 12.6 ± 12.4, respectively; P = .008) and 12th months (12.9 ± 8.1 vs. 6.3 ± 9.0, respectively; P < .001). CONCLUSIONS: Therefore, we can conclude that the significant percentage increase of CXCR3+ monocytes in graft blood with respect to peripheral blood suggests the presence of inflammatory activity despite renal function being stable during the second half of the first year post-transplantation.

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.

Effect of partial HLA class I match on acute rejection in viral pre-infected human liver allograft recipients.

BACKGROUND: Acute rejection in liver transplants is one of the commonest causes of liver dysfunction in the early postoperative period. However, the factors involved in liver graft rejection are still unknown. Our study was aimed at ascertaining whether the degree of HLA class I and class II compatibility or pretransplant viral infection have any influence on early acute liver graft rejection. METHODS: We reviewed clinical and laboratory data in 190 consecutive patients who underwent a liver transplant. HLA-A, HLA-B, and HLA-DR typing for the establishment of an HLA match score was performed by a standard microcytotoxicity method. The existence of pretransplant viral infection was investigated in sera and biopsy tissue by serologic (hepatitis B virus, hepatitis C virus) and polymerase chain reaction (cytomegalovirus) techniques, respectively. The influence of these two factors in acute rejection and the interaction between them was also analyzed. RESULTS: A strong association between viral infection and acute rejection in the group with partial class I matching was found (odds ratio=7.75; P<0.0009), whereas no correlation was observed in the group with zero class I matching (odds ratio=0.98; P=0.81). The rejection percentage in the group in which partial class I match and viral infections coexisted was 60%, whereas in the partially class I-matched group without pretransplant viral presence it was 16%. CONCLUSIONS: These findings suggest a participation of partial HLA class I compatibility in triggering acute rejection in recipients suffering preoperative viral infections and support the idea that HLA class I antigen matching could play a role as a linking element between the MHC-restricted T cell-mediated response to viral infection and the allogenic response in liver transplantation.

CD28 expression on peripheral blood T lymphocytes after orthotopic liver transplant: upregulation in acute rejection.

Despite immunosuppressive treatments, acute rejection remains a significant cause of graft loss. Efficient allorecognition implicates cognate T-cell interactions and requires costimulatory signals such as those delivered via CD28. Therefore, we have studied CD28 peripheral blood T-cell expression, analyzing its possible implications in liver allograft acute rejection. Fifty-five CsA-immunosuppressed orthotopic liver recipients, with or without acute rejection (AR and NAR) were immunocytometrically monitored after transplant and thirty healthy volunteers were studied as controls. In liver recipients the absolute number of CD28+ cells fell sharply immediately after transplant, but no significant differences were detected between the AR and NAR groups either in the absolute number or in the percentage of CD28+ lymphocytes. By contrast, both CD4+CD28+ and CD8+CD28+ T-cell subsets displayed a significant increase in CD28 intensity expression in AR recipients, whereas CD28 expression was significantly downregulated in the NAR recipients. This data suggests that CD28 molecule can be important in the immunologic events preceding acute rejection and that CD28 up- or downregulation could become a useful predictive marker for acute rejection or tolerance development in liver recipients.

HLA-DRB1 and -DQB1 polymorphism in liver recipients: relationship between HLA-DQB1*0302 allele frequency and acute rejection.

Polymorphism of HLA-DRB1 and HLA-DQB1 loci was performed in fifty-three orthotopic liver graft recipients as well as in 108 unrelated healthy controls. Nonradioactive SSOPs were used to study PCR-amplified DNA from peripheral blood lymphocytes and biopsied material. The comparison frequency for DQB1 alleles did not reveal any significant differences between the total group of liver recipients and controls. However, when the liver recipients were subgrouped according to their rejection episode manifestations, increased and significant frequencies were observed for HLA-DQB1*0302 allele in patients showing acute rejection episodes compared to healthy controls or patients without acute rejection. This relationship did not appear influenced by the amino acid beta alanine residue in the 57th position. On the other hand, the study of the DRB1 allele frequencies did not show significant differences in any study. These results suggest that HLA-DQB1 genes could be important in the liver graft alloresponses, opening a way to a better understanding of the special tolerance state, normally observed in this type of transplant, leading us to consider the possible HLA-DQB1*0302 allele effect on tolerance rupture.

Implication of soluble and membrane HLA class I and serum IL-10 in liver graft acceptance.

Membrane HLA class-I expression (mHLA-I), soluble HLA class-I antigens (sHLA-I) and interleukin (IL)-10 are different factors implicated in the special acceptance of liver allograft. In this study, pre- and post-operative levels of mHLA-I in peripheral blood lymphocytes (PBL) and serum sHLA-I were analyzed in 86 liver transplants, immunosuppressed with Cyclosporine-A, methylprednisolone and azathioprine, and classified into acute-rejection (AR, n = 28) and non-acute-rejection (NAR, n = 58) groups. Serum IL-10 was studied in 47 recipients (AR-group, n = 16 and NAR-group, n = 31). Pre-transplant values of mHLA-I and sHLA-I showed a bimodal distribution (high/low) in NAR-recipients, but in AR-patients were mainly included in the low expression/secretion zone (mHLA-I, p < 0.02 and sHLA-I, p < 0.05). Consequently, average pre-transplant mHLA-I (868 +/- 109 versus 998 +/- 123, p < 0.05) and sHLA-I (1.3 +/- 0.4 versus 2.02 +/- 0.7 microg/ml, p < 0.01) was lower in the AR- than in the NAR-group. After transplant both parameters decreased in the NAR-group, but increased in AR-recipients previous to and on rejection diagnosis day. Additionally, serum IL-10 levels were significantly higher (p < 0.01) in the NAR than in the AR-group during the first 24 h post-transplant. In conclusion, low pre-transplant mHLA-I and sHLA-I levels pre-dispose liver recipients to acute rejection, whereas early post-transplant increases of serum IL-10 appear to be related to a good liver allograft acceptance.

CD28/CTLA-4 and CD80/CD86 costimulatory molecules are mainly involved in acceptance or rejection of human liver transplant.

CD28/CTLA-4 interactions with their specific B7-ligands (CD80 and CD86) have decisive roles in antigenic and allogenic responses. Recently, experimental transplant studies demonstrated that donor-specific tolerance is achieved by blocking these interactions. The present study analyzes the expression of these co-stimulatory molecules in peripheral blood cells from 74 liver recipients and in 16 liver biopsies, which were classified into acute-rejection (AR, n = 27) and nonacute-rejection (NAR, n = 47) groups, as well as their influence on the in vitro response of in vivo allosensitized cells. The results clearly indicate that in human liver transplant too, B7 and CD28/CTLA-4 expression on B and CD4(+) peripheral lymphocytes respectively, contributes to graft acceptance or rejection, and appears to be of crucial importance in modulating the host alloresponse and specific-CTL generation. In the NAR-group, costimulatory molecule expression remained at basal levels after transplant, whereas in the AR-group these molecules were significantly upregulated on days of AR. CTLA-4 was observed in the infiltrating lymphocytes in most of the biopsies, but CD80 or CD86 were not. Moreover, specific cytotoxicity from the in vivo primed cells was clearly suppressed in the NAR-patients with low co-stimulatory molecule expression, whereas this activity was not modified but rather stimulated in the AR-group. Together, these findings indicate that intervention of CD28/CTLA-4/B7 signaling could be therapeutically useful in clinical transplantation.

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.

STR data for the AmpFlSTR Profiler Plus loci from Murcia region (SE Spain).

Frequency data of the nine STRs included in the AmpFlSTR Profiler Plus Kit were determined in a sample of 114 unrelated individuals from Murcia region (SE Spain).

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.

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.

Effect of CYP3A51/3 polymorphism on blood pressure in renal transplant recipients.

BACKGROUND: The cytochrome P450 3A5 (CYP3A5) enzyme has been implicated to determine blood pressure (BP) in humans. Different results have been reported concerning CYP3A5 gene polymorphisms and posttransplantation hypertension in kidney recipients. Our objective was to investigate whether CYP3A5 1/3 polymorphism was associated with ambulatory BP among a population of renal transplant recipients receiving the calcineurin inhibitor tacrolimus for immunosuppression. METHODS: Sixty primary kidney transplant recipients undergoing treatment with tacrolimus were genotyped for the CYP3A5 1/3 polymorphism. We analysed the association of the CYP3A5 alleles with ambulatory systolic and diastolic BP measured at 6 and 24 months posttransplantation. RESULTS: We observed that 23.3% of the patients were CYP3A5 1 carriers and 76.7% were homozygous for CYP3A5 3. CYP3A5 1 carriers showed higher adjusted systolic BP and diastolic BP at 6 and 24 months posttransplantation, and they were prescribed more antihypertensive drugs compared with non CYP3A5 1 carrier patients, albeit not significant. No significant differences were found comparing the distribution of the hypertension classes. CONCLUSION: We did not observe a significant association of CYP3A5 1/3 polymorphism with posttransplantation hypertension, although there were some differences in BP associated with the presence of the CYP3A5 1 allele.

Lack of association of immune cell function test with rejection in kidney transplantation.

BACKGROUND: The Cylex Immuknow assay provides a rapid assessment of global immune function in immunocompromised patients by measuring the global immune responses of CD4 T cells from a whole-blood sample. It may help to monitor the immune status of immunosuppressed transplant patients. However, earlier studies have shown that there is no consensus on the utility of the Immuknow assay in renal transplant rejection. METHODS: T-cell activation was determined by measuring an increase of intracellular adenosine triphosphate (iATP) from CD4 cells in 227 samples from 116 kidney transplant patients. The results were analyzed regarding patient clinical status, namely, rejection, infection, or stability. In addition, we measured the immunologic response of 108 healthy control subjects. RESULTS: There were 24 infectious and 36 rejection episodes. iATP concentrations differed significantly between stable and infected patients (180.5 ± 55.2 vs 375.3 ± 140.1 ng/mL; P < .001) and between infected patients and control subjects (180.5 ± 55.2 vs 436.5 ± 112 ng/mL; P < .001). No correlation was observed between patients suffering an acute rejection episode with this response. CONCLUSIONS: Our results confirmed that the Immuknow assay identified transplant patients at risk for infection. It may provide information to guide immunosuppressive therapy, but the assay did not seem to have the potential to differentiate subjects experiencing rejection.