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.

SpeS: A Novel Superantigen and Its Potential as a Vaccine Adjuvant against Strangles.

Bacterial superantigens (sAgs) are powerful activators of the immune response that trigger unspecific T cell responses accompanied by the release of proinflammatory cytokines. Streptococcus equi (S. equi) and Streptococcus zooepidemicus (S. zooepidemicus) produce sAgs that play an important role in their ability to cause disease. Strangles, caused by S. equi, is one of the most common infectious diseases of horses worldwide. Here, we report the identification of a new sAg of S. zooepidemicus, SpeS, and show that mutation of the putative T cell receptor (TCR)-binding motif (YAY to IAY) abrogated TCR-binding, whilst maintaining interaction with major histocompatibility complex (MHC) class II molecules. The fusion of SpeS and SpeSY39I to six S. equi surface proteins using two different peptide linkers was conducted to determine if MHC class II-binding properties were maintained. Proliferation assays, qPCR and flow cytometry analysis showed that SpeSY39I and its fusion proteins induced less mitogenic activity and interferon gamma expression when compared to SpeS, whilst retaining Antigen-Presenting Cell (APC)-binding properties. Our data suggest that SpeSY39I-surface protein fusions could be used to direct vaccine antigens towards antigen-presenting cells in vivo with the potential to enhance antigen presentation and improve immune responses.

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.

NK Cell Education in Tumor Immune Surveillance: DNAM-1/KIR Receptor Ratios as Predictive Biomarkers for Solid Tumor Outcome.

Natural killer cell (NKc)-based therapies offer promising outcomes in patients with tumors, but they could improve with appropriate selection of donors and optimization of methods to expand NKcs in vitro Education through licensing interactions of inhibitory killer cell immunoglobulin-like receptors (iKIR) and NKG2A with their cognate HLA class-I ligands optimizes NKc functional competence. This work has evaluated the role of licensing interactions in NKc differentiation and the survival of cancer patients. We have analyzed KIR and KIR-ligand genes, and the expression of activating (CD16 and DNAM-1/CD226) and inhibitory (NKG2A and iKIRs) receptors on peripheral blood NKcs in 621 healthy controls and 249 solid cancer patients (80 melanoma, 80 bladder, and 89 ovarian). Licensing interactions upregulated the expression of activating CD226, reduced that of iKIR receptors, and shifted the CD226/iKIR receptor ratio on NKc membranes to activating receptors. A high tumor burden decreased CD226 expression, reduced the ratio of CD226/iKIR, and negatively affected patient survival. The progression-free survival (38.1 vs. 67.0 months, P < 0.002) and overall survival (56.3 vs. 99.6 months, P < 0.00001) were significantly shorter in patients with lower expression of CD226 on NKcs. Hence, transformed cells can downmodulate these licensing-driven receptor rearrangements as a specific mechanism to escape NKc immune surveillance. Our results suggest the importance of the CD226/iKIR receptor ratio of NKcs induced by licensing interactions as critical determinants for solid cancer immune surveillance, and may provide predictive biomarkers for patient survival that may also improve the selection of donors for NKc immunotherapy.

The Immunity Gap Challenge: Protection against a Recent Florida Clade 2 Equine Influenza Strain.

Vaccination is one of the most effective tools for limiting the impact of equine influenza (EI). The humoral immunity established following a primary vaccination course can decrease significantly between the second (V2) and third immunisations (V3), leaving some horses insufficiently protected for several weeks. This so-called "immunity gap" poses a challenge to all EI vaccines. During this period, the EI infection of vaccinated animals may be followed by marked clinical signs and virus shedding. However, several EI vaccines have been shown to stimulate equine influenza virus (EIV)-specific cell-mediated immunity, which is likely to play a role in protection against EIV infection and/or mitigate the clinical and virological signs of EI. Reducing the interval between V2 and V3 has been shown to be counterproductive to longer-term immunity. Further research is needed to define and address the "immunity gap" in horses. This study aimed to measure the level of protection induced by a whole inactivated, ISCOMatrix adjuvanted, EI and tetanus vaccine (Equilis Prequenza-Te) when challenged during the immunity gap (i.e., immediately before the recommended boost immunisation, more than 5 months after V2) using infection with a recent heterologous Florida Clade 2 (FC2) equine influenza virus (EIV) strain. This vaccine was tested in a Welsh mountain pony model. A group of seven ponies was vaccinated twice, 4 weeks apart. The protective antibody response was measured and ponies were challenged, along with 5 unvaccinated control ponies, by experimental infection with the FC2 A/eq/Northamptonshire/1/13 EIV strain, 158 days (around 5.2 months) after V2 and their clinical signs and virus shedding were monitored. EI serology was measured by single radial haemolysis (SRH) and haemagglutination inhibition (HI). Clinical signs and virus shedding (measured by qRT-PCR and hen's egg titration) were compared with controls. All vaccinates had detectable, low SRH antibody titres and most had detectable, low HI titres. Significant clinical and virological protection was observed in vaccinates (p < 0.05), supporting the good performance of this vaccine against a recent EIV strain. In this study, the impact of the immunity gap in ponies was limited after primary vaccination with this whole inactivated, ISCOMatrix adjuvanted EI and tetanus vaccine (Equilis Prequenza-Te) when infected several months after V2 with a recent FC2 strain, which is representative of EIV circulating in the EU.

Refinement of the equine influenza model in the natural host: A meta-analysis to determine the benefits of individual nebulisation for experimental infection and vaccine evaluation in the face of decreased strain pathogenicity.

Equine Influenza (EI) is an important respiratory disease of horses caused by H3N8 equine influenza viruses (EIV). Vaccination is a key strategy to prevent or control this disease. However, EIV undergoes continuous antigenic drift and whilst numerous EI vaccines are commercially available worldwide, an accurate evaluation of their efficacy is frequently required through clinical trials conducted in the natural host. Room nebulisation is one of the chosen methods to challenge horses during EI vaccine studies. A potential decreased pathogenicity observed with recent Florida Clade 2 (FC2) EIV isolates have increased the heterogeneity of the clinical response and virus shedding measured after infection by room nebulisation, which reduced the statistical power of studies. Our objectives were to compare clinical and virological parameters following experimental infection with several different EIV strains and to confirm that individual nebulisation is a model refinement that prevents an increase of the number of animals per group. This study is a retrospective comparison and meta-analysis of clinical and virological results collected from 9 independent EIV infection studies in the natural host. Naïve Welsh mountain ponies were experimentally infected by room or individual nebulisation with FC2 EIV strains, including A/equine/Richmond/1/07 (R/07), A/equine/East Renfrewshire/11 (ER/11), A/equine/Cambremer/1/2012 (C/12) and A/equine/Northamptonshire/1/13 (N/1/13). The retrospective meta-analysis confirmed a decreased pathogenicity of the EIV ER/11 and C/12 strains when compared with R/07. Experimental infection by individual nebulisation improved the clinical and virological parameters induced by recent FC2 strains, when compared with conventional room nebulisation. In conclusion, individual nebulisation offers a better control of the challenge dose administered and a greater homogeneity of the response measured in control animals. This in turn, helps maintain the number of animals per group to the minimum necessary required to obtain meaningful results in vaccine efficacy studies, which adheres to the 3Rs (Replacement, Reduction and Refinement) principles.

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.

Allele-specific recognition by LILRB3 and LILRA6 of a cytokeratin 8-associated ligand on necrotic glandular epithelial cells.

The LILRs are a family of receptors that regulate the activities of myelomonocytic cells. We found that specific allelic variants of two related members of the LILR family, LILRB3 and LILRA6, interact with a ligand exposed on necrotic glandular epithelial cells. The extracellular domains of LILRB3 and LILRA6 are very similar and their genes are highly polymorphic. A commonly occurring allele, LILRB3*12, displayed particularly strong binding of these necrotic cells and further screening of the products of LILRB3 alleles identified motifs that correlated with binding. Immunoprecipitation of the ligand from epithelial cell lysates using recombinant LILRB3*12, identified cytokeratins 8, 18 and 19. Purified proteins obtained from epithelial cell lysates, using anti-cytokeratin 8 antibodies, were able to activate LILRB3*12 reporter cells. Knock-down of cytokeratin 8 in epithelial cells abrogated expression of the LILRB3 ligand, while staining with recombinant LILRB3*12 showed co-localisation with cytokeratin 8 and 18 in permeabilised breast cancer cells. Necrosis is a common feature of tumours. The finding of a necrosis-associated ligand for these two receptors raises the possibility of a novel interaction that alters immune responses within the tumour microenvironment. Since LILRB3 and LILRA6 genes are highly polymorphic the interaction may influence an individual's immune response to tumours.

Copy number and nucleotide variation of the LILR family of myelomonocytic cell activating and inhibitory receptors.

Leukocyte immunoglobulin-like receptors (LILR) are cell surface molecules that regulate the activities of myelomonocytic cells through the balance of inhibitory and activation signals. LILR genes are located within the leukocyte receptor complex (LRC) on chromosome 19q13.4 adjacent to KIR genes, which are subject to allelic and copy number variation (CNV). LILRB3 (ILT5) and LILRA6 (ILT8) are highly polymorphic receptors with similar extracellular domains. LILRB3 contains inhibitory ITIM motifs and LILRA6 is coupled to an adaptor with activating ITAM motifs. We analysed the sequences of the extracellular immunoglobulin domain-encoding regions of LILRB3 and LILRA6 in 20 individuals, and determined the copy number of these receptors, in addition to those of other members of the LILR family. We found 41 polymorphic sites within the extracellular domains of LILRB3 and LILRA6. Twenty-four of these sites were common to both receptors. LILRA6, but not LILRB3, exhibited CNV. In 20 out of 48 human cell lines from the International Histocompatibility Working Group, LILRA6 was deleted or duplicated. The only other LILR gene exhibiting genomic aberration was LILRA3, in this case due to a partial deletion.

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.

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.

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.

HBV and HCV infections and acute rejection differentially modulate CD95 and CD28 expression on peripheral blood lymphocytes after liver transplantation.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) generally reinfect liver graft early posttransplantation and lead to poorer graft and patient survivals. In the present study the influence of acute rejection (AR), HBV and HCV infections, and human leukocyte antigen (HLA) class-I compatibility on the expression of CD28 (in 237 liver recipients) and CD95 (in 114 liver recipients) on peripheral blood cells were evaluated by flow cytometry during the first month after transplantation. HBV/HCV infections induced strong CD95 upregulation on CD3+ lymphocytes. Maximal CD95 upmodulation was found in infected recipients showing partial HLA class-I compatibility. AR and virus reinfection could be distinguished because CD28 was upregulated on CD4+ lymphocytes only in recipients with AR, irrespective of their status regarding HBV/HCV infections. In conclusion, cytometric co-evaluation of CD95 and CD28 expression on peripheral blood lymphocytes could be useful to discriminate AR from cellular activation induced by viral reinfection of the liver graft.

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.

TGF-beta1 gene polymorphism in liver graft recipients.

Cytokines are known to be important mediators during liver graft outcome and their gene polymorphism could affect the overall expression and secretion of cytokines. In this retrospective study, we analyzed the effect of TGF-beta1 polymorphism in 150 liver allograft recipients. Genotyping PCR-SSP were performed for TGF-beta1 gene (codon 10T/C and 25C/G). TGF-beta1 polymorphism at codon 10 and 25 correlate borderline with liver graft acceptance and when the combination between codon 10 and 25 was analyzed, it revealed that T/T G/C genotype and the TC haplotype were significantly associated with graft acceptance (p<0.05). TGF-beta1 high secretor phenotype was also increased in the acute rejection group close to significance (p=0.06). In conclusion, these findings show a correlation between TGF-beta1 gene polymorphism and liver graft acceptance.