Donor NK and T Cells in the Periphery of Lung Transplant Recipients Contain High Frequencies of Killer Cell Immunoglobulin-Like Receptor-Positive Subsets.

Introduction: For end-stage lung diseases, double lung transplantation (DLTx) is the ultimate curative treatment option. However, acute and chronic rejection and chronic dysfunction are major limitations in thoracic transplantation medicine. Thus, a better understanding of the contribution of immune responses early after DLTx is urgently needed. Passenger cells, derived from donor lungs and migrating into the recipient periphery, are comprised primarily by NK and T cells. Here, we aimed at characterizing the expression of killer cell immunoglobulin-like receptors (KIR) on donor and recipient NK and T cells in recipient blood after DLTx. Furthermore, we investigated the functional status and capacity of donor vs. recipient NK cells. Methods: Peripheral blood samples of 51 DLTx recipients were analyzed pre Tx and at T0, T24 and 3wk post Tx for the presence of HLA-mismatched donor NK and T cells, their KIR repertoire as well as activation status using flow cytometry. Results: Within the first 3 weeks after DLTx, donor NK and T cells were detected in all patients with a peak at T0. An increase of the KIR2DL/S1-positive subset was found within the donor NK cell repertoire. Moreover, donor NK cells showed significantly higher frequencies of KIR2DL/S1-positive cells (p<0.01) 3wk post DLTx compared to recipient NK cells. This effect was also observed in donor KIR+ T cells 3wk after DLTx with higher proportions of KIR2DL/S1 (p<0.05) and KIR3DL/S1 (p<0.01) positive T cells. Higher activation levels of donor NK and T cells (p<0.001) were detected compared to recipient cells via CD25 expression as well as a higher degranulation capacity upon activation by K562 target cells. Conclusion: Higher frequencies of donor NK and T cells expressing KIR compared to recipient NK and T cells argue for their origin in the lung as a part of a highly specialized immunocompetent compartment. Despite KIR expression, higher activation levels of donor NK and T cells in the periphery of recipients suggest their pre-activation during the ex situ phase. Taken together, donor NK and T cells are likely to have a regulatory effect in the balance between tolerance and rejection and, hence, graft survival after DLTx.

Expression of Natural Killer Cell Inhibitory Receptors is Associated with Significant Liver Injury in Chronic Hepatitis C in Children.

INTRODUCTION AND AIM: Natural Killer (NK) cells play an important role in innate immune response to viral infections and their high proportion is situated in the liver. The aim of this study was to analyze possible relation between the expression of NK cell receptors and varied intensity of liver lesions in chronic hepatitis C (CHC) in children. MATERIAL AND METHODS: Study included 105 children with CHC - 54 boys and 51 girls, age 13.62 ± 3.48 years. Blood specimens were taken at the day of the liver biopsy. Histological evaluation was performed according to METAVIR scoring system. Circulating NK cells were evaluated by flow cytometry. The results were shown as a proportion of cells expressing evaluated receptor and its' mean fluorescent intensity (MFI). RESULTS: In 58 children with CHC (55.2%) significant liver fibrosis was observed ( ≥F2). Higher proportion of cells expressing CD158e inhibitory receptors was observed in the group of children with ALT > 2UNL (21.11 ± 14.60 vs. 12.22 ± 8.99%; p = 0.037). While higher proportion of cells expressing inhibitory CD158b receptor was observed in children with significant fibrosis (F ≥ 2) compared to minimal fibrosis (F < 2) - (34.14 ± 12.44 vs. 27.48 ± 8.71%; p = 0.049). Children with advanced fibrosis (F ≥ 3) had higher MFI of NK cell CD 158b receptor than children with fibrosis scored F < 3 - (5344.20 ± 3407.49 vs. 2979.67 ± 1190.64; p = 0.049). Proportion of NK cells expressing CD158b was found a predictor of significant fibrosis in univariate analysis - [OR 1.065; 95%CI (1.07-1.15); p = 0.046]. CONCLUSIONS: Higher proportion of NK cells expressing inhibitory CD158b and CD158e receptors is associated with significant liver injury.

KIR3DL1+HLA-B Bw4Ile80 and KIR2DS1+HLA-C2 combinations are both associated with ankylosing spondylitis in the Iranian population.

Contribution of killer cell immunoglobulin-like receptors (KIR) and their human leucocyte antigen (HLA) class I ligands in the pathogenesis of autoimmune diseases has been shown in several studies. In this study, the possible association of KIR genes, their known HLA ligands and compound KIR/HLA genotypes with ankylosing spondylitis (AS) was assessed. Combined KIR/HLA ligand genotyping was performed by a polymerase chain reaction-sequence-specific primers assay in 35 Iranian patients with AS, and genotypes were compared to those in 200 healthy individuals. The frequencies of telomeric cluster genes KIR2DL5A, KIR2DS1 and KIR3DS1 were significantly increased in AS patient group (P(c) = 0.0082, P(c) = 0.0195 and P(c) = 0.0328, respectively). Conversely, HLA-Bw4 ligand (the presence of one or more -B Bw4(Ile80) , -B Bw4(Thr80) and -A Bw4 epitopes) (P(c) = 0.0004) and HLA-B Bw4(Ile80) (P(c) = 0.053) were less frequent in these patients. Meanwhile, compound KIR/HLA genotype analyses revealed lower frequency of KIR3DL1+HLA-B Bw4(Ile80) (P(c) = 0.0343) and higher frequency of KIR2DS1+HLA-C2 (P(c) = 0.0308) combinations in patients with AS than in controls. In addition, the genotypes iKIR+HLA > aKIR+HLA (P(c) = .0308) and iKIR+HLA > aKIR (P(c) = 0.0258) were statistically less common, and genotypes iKIR+HLA = aKIR+HLA (P(c) = 0.0081) and iKIR+HLA < aKIR (P(c) = 0.077) were more common in patient group. Our findings suggest a role for excessive or inappropriate NK cell activation through 'KIR/HLA' system in AS disease.

Evaluation of degranulation and cytokine production in natural killer cells from spondyloarthritis patients at single-cell level.

BACKGROUND: A pathogenetic role of natural killer (NK) cells has been hypothesized in spondyloarthritides (SpA), but still conflicting data exist. Aim of this study was to investigate, in a cohort of SpA patients, the peripheral blood NK cell number, the cytokine production (IFNγ and TNFα), and the cytotoxic activity on target cell stimulation. Moreover, we aimed to evaluate the expression of KIR3DL1, an inhibitory receptor binding HLA class I alleles, including HLA-B27 strongly associated with SpA, between different NK cell subsets. METHODS: We enrolled 21 SpA patients and 21 healthy controls. Multicolor flow cytometry was used to analyze the cytokine levels triggered by activating receptors, the degranulation as CD107a surface expression on target cell stimulation, the number and KIR3DL1 expression on peripheral blood NK cells. RESULTS: When stimulated with P815 cells preincubated with antibodies against activating receptors, NK cells produced IFNγ to a lesser extent respect to healthy controls (P = 0.0012). No differences were found in TNFα production and NK cell degranulation in patients and controls. We observed a higher frequency of KIR3DL1-positive NK cells from SpA patients than in controls (P = 0.02). Similar results were obtained in NK cell subsets. CONCLUSIONS: In our SpA patients, we observed a reduced IFNγ production, which may be explained by the elevated frequency of KIR3DL1-expressing NK cells, while the other parameters were similar to those of healthy controls. These results may support the role of NK cells in the pathogenesis of SpA, although more data are needed to substantiate these observations.

Altered natural killer (NK) cell frequency and phenotype in latent autoimmune diabetes in adults (LADA) prior to insulin deficiency.

Approximately 10% of the patients diagnosed with type 2 diabetes (T2D) have detectable serum levels of glutamic acid decarboxylase 65 autoantibodies (GADA). These patients usually progress to insulin dependency within a few years, and are classified as being latent autoimmune diabetes in adults (LADA). A decrease in the frequency of peripheral blood natural killer (NK) cells has been reported recently in recent-onset T1D and in high-risk individuals prior to the clinical onset. As NK cells in LADA patients have been investigated scarcely, the aim of this study was to use multicolour flow cytometry to define possible deficiencies or abnormalities in the frequency or activation state of NK cells in LADA patients prior to insulin dependency. All patients were GADA-positive and metabolically compensated, but none were insulin-dependent at the time blood samples were taken. LADA patients exhibited a significant decrease in NK cell frequency in peripheral blood compared to healthy individuals (P=0.0018), as reported previously for recent-onset T1D patients. Interestingly, NKG2D expression was increased significantly (P<0.0001), whereas killer cell immunoglobulin-like receptor (KIR)3DL1 expression was decreased (P<0.0001) within the NK cell population. These observations highlight a defect in both frequency and activation status of NK cells in LADA patients and suggest that this immunological alteration may contribute to the development of autoimmune diabetes by affecting peripheral tolerance. Indeed, recent evidence has demonstrated a regulatory function for NK cells in autoimmunity. Moreover, the decrease in NK cell number concords with observations obtained in recent-onset T1D, implying that similar immunological dysfunctions may contribute to the progression of both LADA and T1D.