Overexpression of KIR inhibitory ligands (HLA-I) determines that immunosurveillance of myeloma depends on diverse and strong NK cell licensing.

Missing self recognition makes cancer sensitive to natural killer cell (NKc) reactivity. However, this model disregards the NKc licensing effect, which highly increases NKc reactivity through interactions of inhibitory killer cell immunoglobulin-like receptors (iKIR) with their cognate HLA-I ligands. The influence of iKIR/HLA-ligand (HLA-C1/C2) licensing interactions on the susceptibility to and progression of plasma cell (PC) dyscrasias was evaluated in 164 Caucasian patients and 286 controls. Compared to controls, myeloma accumulates KIR2DL1-L2+L3- genotypes (2.8% vs. 13.2%, p < 0.01, OR = 5.29) and less diverse peripheral repertoires of NKc clones. Less diverse and weaker-affinity repertoires of iKIR2D/HLA-C licensing interactions increased myeloma susceptibility. Thus, the complete absence of conventional iKIR2D/HLA-C licensing interactions (KIR2DL1-L2+L3-/C2C2, 2.56% vs. 0.35%; p < 0.05; OR = 15.014), single-KIR2DL3+/C1+ (20.51% vs. 10.84%; p < 0.05; OR = 2.795) and single-KIR2DL2+/C1+ (12.82% vs. 4.9%; p < 0.01; OR = 5.18) interactions were over-represented in myeloma, compared to controls. Additionally, KIR2DL1-L2+L3- (20% vs. 83%, p < 0.00001) as well as KIR3DL1- (23% vs. 82%, p < 0.00001) genotypes had a dramatic negative impact on the 3-y progression-free survival (PFS), particularly in patients with low-tumor burden. Remarkably, myeloma-PCs, compared to K562 and other hematological cancers, showed substantial over-expression of HLA-I ("increasing-self" instead of missing-self), including HLA-C, and mild expression of ligands for NKc activating receptors (aRec) CD112, CD155, ULBP-1 and MICA/B, which apparently renders myeloma-PCs susceptible to lysis mainly by licensed NKc. KIR2DL1-L2+L3-/C2C2 patients (with no conventional iKIR2D/HLA-C licensing interactions) lyse K562 but barely lyse myeloma-PCs (4% vs. 15%; p < 0.05, compared to controls). These results support a model where immunosurveillance of no-missing-self cancers, e.g., myeloma, mainly depends on NKc licensing.

Epidemiology, Evolution, and Long-Term Survival of Alcoholic Cirrhosis Patients Submitted to Liver Transplantation in Southeastern Spain.

BACKGROUND: Alcoholic cirrhosis (AC) is a common cause of death among individuals abusing alcohol. In the last resort, liver transplantation (LT) is considered the only solution to save the patient's life, generating socioeconomic and public health problems. Clinical and sociodemographic characteristics, rejection frequency, and short- and long-term graft survival are not well known in end-term AC patients undergoing LT. The aim was to determine the sociodemographic and clinical characteristics, their incidence in LT, main pre- and posttransplant complications, and short- and long-term post-transplant graft survival in AC patients in southeastern Spain. METHODS: The medical records of 1,026 patients who underwent LT over the last 23 years were retrospectively reviewed, and demographic data and posttransplant survivals were analyzed and compared. Biochemical characteristics, major pre- and posttransplant complications and short- and long-term survivals were analyzed in a total of 398 male patients with AC undergoing LT. RESULTS: AC and viral cirrhosis are the main indications for LT in our study. Mostly represented in our study are AC men without associated viral infections with a mean age of 53.06 years. Main pretransplant complications in AC patients are ascites (78.3%) and encephalopathy (43.5%), while acute graft rejection is the most common liver posttransplant complication (26.6%), nevertheless with low graft loss frequency (1.1%). AC and autoimmune cirrhosis show the best posttransplant survival in both the short and long term. Patients with AC included on the waiting list for LT were Child-Pugh class B (52.1%) and Model for End-Stage Liver Disease score of 10 to 19 (71.2%). The highest percentage of AC patient survival was observed at 1 year posttransplant (81.2%) and progressively decreased over time up to 10 years posttransplant (69.6%). Pretransplant complications such as ascites and encephalopathy did not have an influence on the percentage of posttransplant survivals, although better survival rates were observed in nonviral AC patients. CONCLUSIONS: AC without viral infections is the main indication for LT in southeastern Spain although its frequency has decreased in last decade. AC is a good indication for LT for its high survival rate and few posttransplant complications. Despite having a high percentage of pretransplant complications (ascites and encephalopathy) but does not appear to influence survivals being observed posttransplant survival rates above those expected. Conversely, viral infections in the patient with AC decrease patient survivals. The main future goals are design new strategies to detect, treat, and reduce AC frequency in our population and know alcoholic recidivism rate posttransplant in our population.

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.

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.

Hemophagocytic lymphohistiocytosis: Overview and diagnostic procedure. A case induced by anexpansion of monoclonal EBV-negative NK cells

Las causas más comunes de linfohistiocitosis hemofagocítica (HLH) sonexpansiones clonales de células NK y T, inducidas por EBV, así como las alteraciones genéticas que comprometen la actividad asesina de las NKs. Generalmente, HLH se desencadena por una disfunción inmune en la que se desarrolla hipercitoquinemia. En este trabajo se resumen las causas más comunes de HLH y se presenta un caso en el que una expansión monoclonal decélulas NK, EBV-negativas, se asocia a HLH en una paciente aquejada de Síndrome de Griscelli tipo-2 (GS2). Se trata de una niña de 17 meses con unamutación de nueva descripción en RAB27A, con albinismo parcial, fiebre persistente, hepatoesplenomegalia, adenopatías y citopenias al diagnóstico. Nose detectaron evidencias de infecciones virales activas, incluida EBV. Se detectó una expansión de células NKs (5300/µl) CD2+CD7+CD8+CD16+CD56+CD94+CD158a/h+CD158b/j–Perforin+Granzyme-B+. Tras el tratamiento (Protocolo HLH-2004: Cyclosporina, Etoposido y Dexametasona), la cifra de células NK se redujo a 850/µl y que aumentaron progresivamente hasta alcanzar niveles similares al diagnóstico. El ensayo de inactivación del cromosoma X demostró monoclonalidad de células NK. Dichas células manteníanintacta su actividad asesina y secretaban grandes cantidades de IFN-γ. Aldiagnóstico los niveles séricos de sIL-2R (36.8 ng/ml) e IFN-γ (400 pg/ml)estaban elevados. En conclusión, se describe un caso de una expansión monoclonal de células NK, EBV-negativas, que secretan grandes cantidades deIFN-γ como la causa más probable del episodio de HLH en una paciente conGS2. Tras el trasplante de médula ósea de su hermana HLA-idéntica, las cifrasy el fenotipo de las células NK recobraron la normalidad (AU)

Clonal natural killer (NK) and T cell expansions induced by EpsteinBarr virus (EBV) and genetic alterations compromising NK cell killing arethe most common causes of hemophagocytic lymphohistocytosis (HLH).Generally, HLH is induced by an immune dysfunction where hypercytokinemia develops into reactive hemophagocytosis. In this work wereview the causes of HLH and describe a case of a monoclonal expansionof EBV-negative NK cells associated to HLH in a seventeen-month-oldgirl suffering of Griscelli syndrome type-2 with novel RAB27A mutation and showing partial albinism, persistent fever, hepatosplenomegaly,adenopaties and cytopenias. At diagnosis, no evidence of active viral infections, including EBV, was found. Expansion of NK cells (5300/µl in peripheral blood) CD2+CD7+CD8+CD16+CD56+CD94+CD158a/h+CD158b/j–Perforin+Granzyme B+ was found. After treatment (HLH-2004 protocol:Cyclosporin, Etoposide and Dexamethasone), NK cell count fell to 850/µland progressively increased to pre-therapy levels by week 28. X-chromosome inactivation assay demonstrated NK cell monoclonality. NK cellssustained a strong killing and secreted high amounts of IFN-γ. At diagnosis, serum levels of sIL-2R (36,8 ng/ml) and IFN-γ (400 pg/ml) wereelevated. In conclusion, we describe a monoclonal expansion of EBV-negative NK cells highly secretory of IFN-γ as the most probable cause of HLHepisode in a patient with Griscelli syndrome type-2. NK cell count recovered normal levels and phenotype after bone marrow transplantationfrom her HLA identical sister (AU)

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.

Association of monoclonal expansion of Epstein-Barr virus-negative CD158a+ NK cells secreting large amounts of gamma interferon with hemophagocytic lymphohistiocytosis.

We report the first case of hemophagocytic lymphohistiocytosis (HLH) induced by the monoclonal expansion of Epstein-Barr virus (EBV)-negative NK cells. Consanguinity of the patient's parents made it necessary to discard familial HLH in the patient and her sister with identical HLA markers and demonstrate that no cause other than the expansion of NK cells, which secrete high levels of gamma interferon, was inducing HLH in this patient.

Increased number of cytotoxic CD3+ CD28- gammadelta T cells in peripheral blood of patients with cutaneous malignant melanoma.

BACKGROUND: A rise of CD3+ TCRgammadelta+ CD28- T cells has previously been observed after an in vitro long-lasting activation or even during viral infection. OBJECTIVE: The aim of this study was to investigate the expression of CD28 on lymphocytes bearing CD3/TCRgammadelta receptors in cancer, i.e. cutaneous melanoma. METHODS: TCRgammadelta lymphocytes were analysed in 41 Caucasian melanoma patients and 39 healthy individuals by flow cytometry. Patients were stratified according to the American Joint Committee on Cancer (AJCC) clinical stage. RESULTS: The number of circulating CD3+ TCRgammadelta+ T cells was significantly increased in both AJCC stages I-II and AJCC stage III patients compared with healthy individuals. This increase was mediated by an accumulation of the CD3+ TCRgammadelta+ CD28- T-cell subset, which expressed a high amount of perforin both in normal individuals and melanoma patients. CONCLUSION: This work shows, for the first time, a rise of the cytotoxic CD3+ TCRgammadelta+ CD28- T-cell population in melanoma patients, which may be important in anticancer surveillance.

Impact of recipient HLA-C in liver transplant: a protective effect of HLA-Cw*07 on acute rejection.

The involvement of the human leukocyte antigen (HLA) in liver graft acceptance is controversial, but the frequency of acute rejection (AR) remains high in spite of the use of the modern immunosuppressive agents. The present study was aimed at determining whether an association exists between liver recipient HLA-C polymorphism and AR development that could influence graft acceptance. Four hundred and forty-six liver recipients and 473 controls were studied within the framework of a collaborative study carried out by the Spanish Transplant Immunotolerance Group (RED-GIT). HLA-A and -B were typed by the standard microlymphocytotoxicity technique, and HLA-C by polymerase chain reaction-sequence-specific oligonucleotide probes (PCR-SSOP). A statistically significant decrease in the HLA-Cw*07 allele frequency was found in liver recipients suffering AR episodes compared to those without AR (NAR). Studies regarding the possible influence of the Asn80 and Lys80 epitopes showed that the Asn80 epitope also could be associated with AR. However, further analysis considering Asn80 alleles others than HLA-Cw*07, confirmed that the apparent protective effect of the Asn80 epitope was actually from the HLA-Cw*07 allele. In conclusion, the HLA-Cw*07 allele carried by the liver recipient is negatively associated with AR development, and could be considered a predictive factor for liver graft acceptance.

Analysis of KIR2D receptors on peripheral blood lymphocytes from liver graft recipients.

KIR2D receptors are killer cell immunoglobulin-like receptors (KIRs) specific for HLA-C epitopes, that are expressed on NK cells as well as on minor peripheral blood T-cell subsets, and are able to control NK and T cells activity. The present work explores NK, and particularly CD8(+) T cells expressing KIR2D2L1/S1 (CD158a) or KIR2D2L2/3/S2 (CD158b) receptors in liver graft alloresponse. Flow cytometry was used to analyse peripheral blood mononuclear cells stained with anti-CD158a and anti-CD158b antibodies from 110 liver recipients and 46 healthy controls, previous to and along the first month after transplantation. Pre-transplantation data shows that both CD158a and CD158b molecules can be detected on NK and T cells from all patients and controls, but both KIR2D(+)NK cells are significantly under-represented in patients respect to controls (P<0.001), and CD3(+)CD8(+)CD158a(+) cells decreased particularly in patients suffering from acute rejection (4.03+/-1.33 cells/microL) compared with controls (7.8+/-2.4 cells/microL). Following transplantation, KIR2D(+)CD8(+) T-cell repertoires increased through the first month, mainly in recipients with a good graft acceptance. In summary, monitoring of KIR2D(+)CD8(+) T cells, particularly KIR2DL1/S1(+)CD8(+) T cells at pre-transplant, and both KIR2DL1/S1(+) and KIR2DL2/3/S2(+) T-cell subsets at early post-transplant period, could offer useful information for clinical follow-up of liver grafts.

Natural killer receptors on CD8 T cells and natural killer cells from different HLA-C phenotypes in melanoma patients.

PURPOSE: Because immune mechanisms involved in cutaneous melanoma have not been fully elucidated, efforts have been made to achieve prognosis markers and potential targets for immune therapies, but they have not been entirely fruitful thus far. Therefore, the goal of this study was to investigate the involvement of early changes in CD8 T cells and CD56 natural killer (NK) cells expressing NK receptors in different HLA-C dimorphism groups of melanoma patients. EXPERIMENTAL DESIGN: CD8 T cells and CD56 NK cells were analyzed in 41 patients and 39 sex- and age-matched controls with different HLA-C genotypes by flow cytometry. HLA-C dimorphism at position 80 was tested by PCR sequence-specific primers and PCR sequence-specific oligonucleotide to examine whether it could mediate in the emergence of cells expressing killer cell immunoglobulin-like receptors. RESULTS: Thirty-five of 41 patients had benign sentinel node, and showed an imbalance in the absolute number of CD8(+)DR(+) or CD8(+)CD161(+) peripheral blood T cells according to the CD28 coexpression compared with controls. CD8(+)CD28(-)CD158a(+) T and CD56(+)CD158a(+) NK cells were significantly increased in HLA-C(Lys80) homozygous nonmetastatic patients, whereas only CD56(+)CD158a(+) NK cells increased in heterozygous ones. An up-regulation of the CD158a KIR receptor was also seen on NK cells but not in T cells of patients at advanced disease stages. CONCLUSIONS: This work provides, for the first time, evidence of immune activation in early stages of cutaneous melanoma, together with an increase of cells expressing CD158a in patients bearing the corresponding HLA-C ligand, which may be important to evaluate the disease progression and to use individualized immune therapeutic approaches.

HLA class I and class II frequencies in patients with cutaneous malignant melanoma from southeastern Spain: the role of HLA-C in disease prognosis.

Available data have led to a controversy on the relationship between human leukocyte antigen (HLA) and cutaneous malignant melanoma susceptibility or prognosis. Moreover, the influence of HLA-C on melanoma has not yet been well established. Therefore, the aim of the current study was to analyze the possible influence of the HLA system on melanoma susceptibility and prognosis in the Spanish population. For this purpose, HLA-A and HLA-B serotyping and HLA-C, HLA-DRB1, and HLA-DQB1 genotyping by polymerase chain reactions using sequence-specific oligonucleotide (PCR-SSO) and sequence-specific primer (PCR-SSP) were performed in 174 melanoma patients and 227 ethnically matched controls. The number of controls was increased up to 356 for HLA-C typing. Patients were stratified according to the histological subtypes of melanoma, sentinel lymph node status, tumor thickness, and ulceration of primary lesion. No HLA-A, HLA-B, HLA-DRB1, or HLA-DQB1 relationship with melanoma was observed for susceptibility or disease prognosis. However, the analysis of HLA-C locus showed that individuals homozygous for HLA-C(Lys80) were significantly more frequent within the patient than the control group. Remarkably, individuals homozygous for group 2 HLA-C alleles (HLA-C(Lys80)) seem to be associated with metastatic progression of melanoma. In contrast, we found a negative association between group 1 HLA-C alleles (HLA-C(Asn80)) and disease susceptibility or metastasis development. In conclusion, although an association with HLA-A, HLA-B, HLA-DRB1, or HLA-DQB1 was not demonstrated, the study of the HLA-C locus revealed that the analysis of the dimorphism at position 80 in the alpha1 helix may help to evaluate the risk and prognosis of melanoma in our population.

Flow cytometric quantification of apoptosis and proliferation in mixed lymphocyte culture.

BACKGROUND: The one-way mixed lymphocyte culture (MLC) is the classic culture used for studying the allogenic immunoresponse in vitro, but stimulator and responder cell identifications and quantification of apoptotic or proliferative responder cells are unreliable. METHODS: Peripheral blood mononuclear cells were labeled with 5- (and 6-) carboxy fluorescein diacetate succinimidyl ester (CFSE) and stimulated with allogenic unlabeled irradiated cells in unidirectional cultures. Apoptosis was determined by the 7-aminoactinomycin D technique, and the absolute number of each cell population was calculated by adding a fixed number of cells stained with propidium iodide as the reference standard for each test. RESULTS: CFSE labeling of cells under different cultures did not affect the results of proliferation or apoptosis. Data of apoptosis obtained with this method were comparable to those of the monoclonal antibody technique, and the proliferation level determined by [(3)H]-thymidine incorporation or counting the number of proliferative living cells, as proposed in this method, showed a good correlation. CONCLUSIONS: The method presented in this report allows the simultaneous determination of apoptosis and proliferation in MLCs and the analysis of cell phenotype, thereby avoiding the use of radioactivity. This assay opens new perspectives for a better understanding of the mechanisms implied in the establishment or break of tolerance to the graft in solid organ transplants.