Normal Natural Killer Research Articles

Natural killer (NK) cell activity during HIV infection: a decrease in NK activity is observed at the clonal level and is not restored after in vitro long-term culture of NK cells.

NK cell activity is impaired in HIV-infected patients. The mechanisms behind the altered NK functions are not clear, and conflicting data concerning NK and antibody-dependent cellular cytotoxicity (ADCC) activity have been reported. In order to investigate whether this impairment is also observed at the clonal level and whether it is related to a defect at the target cell binding and/or the post-binding level, we evaluated highly purified NK cell lines and cloned NK cells obtained from 22 HIV-infected patients at different stages of disease and compared them with normal controls for their ability to: (i) kill K-562 and U-937 cell lines using a 51Cr release assay; (ii) bind and kill K-562 and U-937 cells at the single cell binding level; (iii) release NK cytotoxic factor (NKCF), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma); (iv) kill anti-IgM preincubated Daudi cell line (ADCC activity). This study with cloned NK cells or NK cell lines from HIV-infected individuals showed: (i) a decrease in their lytic capability against target cell lines; (ii) a low ability to form conjugates with K-562 and U-937 cell lines with respect to controls; (iii) a decreased ability to kill bound target cells; (iv) low levels of released NKCF, TNF-alpha and IFN-gamma after incubation with U-937 cells. Taken together, these findings suggest that the impaired NK cell function during HIV infection is also observed at the clonal level and is related to defects both at the target and post-binding levels. However, the precise mechanisms remain to be determined. The inability to restore normal NK activity after long-term culture in the presence of high levels of recombinant IL-2 is in agreement with the hypothesis of a 'general anergic process' during HIV infection.

IL-21 Enhances Antitumor Responses without Stimulating Proliferation of Malignant T Cells of Patients with Sézary Syndrome

IL-21, a common gamma-chain cytokine secreted by activated CD4+ T cells, influences both humoral and cell-mediated immune responses through the regulation of T, B, dendritic, and natural killer (NK) cells. Sézary syndrome is an advanced form of cutaneous T-cell lymphoma, a clonally derived malignancy of CD4+ T cells that is characterized by profound defects in host cellular immune function. As a modulator of both innate and adaptive immune responses, IL-21 could play an important role in augmenting cell-mediated immunity in these patients. Normal donor and Sézary syndrome patient peripheral blood mononuclear cells were cultured with IL-21 and tested for CD8+ T- and NK-cell activation, NK-cell cytotoxicity, and tumor cell proliferation and apoptosis. IL-21 resulted in a modest increase in CD8+ T- and NK-cell activation, associated with a marked increase in cytolytic activity against both K562 and malignant CD4+ T-cell targets. Although IL-21 failed to demonstrate pro-apoptotic effects on the malignant CD4+ T cells, it is noteworthy that it had no demonstrable proliferative effects on these cells. Thus, IL-21 may play an important role in enhancing the host immune response of Sézary syndrome patients through the increased cytolytic activity of T and NK cells.

89: Early Lymphocyte Recovery Predicts Superior Survival after Autologous Stem Cell Transplantation in Non-Hodgkin Lymphoma: A Prospective Study

Background: Day 15 absolute lymphocyte count (ALC-15) ≥500 cells/μl after autologous peripheral blood hematopoietic stem cell transplantation (APHSCT) is a significant predictor for survival. Limitations of previous reports have been their retrospective nature and no ALC-15 lymphocyte subsets analysis. Thus, from 2/2002 until 2/2007, 50 non-Hodgkin lymphoma (NHL) patients were enrolled in a prospective study to address these limitations. Methods: APHSCT eligible NHL patients who signed written consent for additional blood collections participated in the study. The primary end point was to confirm the ALC-15 survival role and to identify the lymphocyte subsets of greatest impact on ALC-15 after APHSCT. Results: The median age of the cohort was 57.7 years (range: 23–73). The groups (ALC-15 ≥500 vs < 500 cells/μl) were balanced to lymphoma type (p = 0.4), gender (p = 0.5), age (p = 0.2), extranodal sites (p = 0.4), lactate dehydrogenase (p = 0.5), performance status (p = 0.5), stage (p = 0.5), international prognostic index (p = 0.9), number of prior chemotherapies (p = 0.1), clinical status at APHSCT (p = 0.2), and infused CD34/kg (p = 0.8). With a median follow-up of 22.2 months (range: 6–63.7 months), patients with an ALC-15 ≥500 cells/μl (n = 35) experienced superior overall survival (OS) and progression-free survival (PFS) compared with those who did not (median OS: not reached vs 19 months, 3 years OS rates of 80% vs 40%, p < 0.0004; median PFS: not reached vs 3.4 months, 3 years PFS rates of 60% vs 14%, p < 0.0001, respectively). By day 15 post-APHSCT 30% of patients achieved a normal CD3, 18% a normal CD4, 38% a normal CD8, 4% a normal CD19, and 76% a normal [CD16+/56+/CD3-] counts. In Univariate analysis CD16+/56+/CD3- natural killer (NK) cells were the only ALC-15 lymphocyte subset that predicted for survival. Patients with a normal absolute NK cell count (≥80 cells/μl, n = 38) experienced superior OS and PFS compared with those who did not (median OS: not reached vs 5 months, 3 years OS rates of 76% vs 36%, p < 0.0001; and median PFS: not reached vs 3 months, 3 years PFS rates of 57% vs 9%, p < 0.0001, respectively). Conclusion: This study confirms the prognostic ALC-15 survival role and identifies NK cells as the key lymphocyte subset affecting clinical outcomes after APHSCT in NHL. This study was supported in part by the Grant CA90628–04A from the National Institute of Health, and the University of Iowa/Mayo Clinic Lymphoma Spore CA97274.

Progress in understanding and managing natural killer‐cell malignancies

The World Health Organization classification of haematolymphoid tumours recognizes three categories of natural killer (NK)-cell neoplasms: blastic NK-cell lymphoma, aggressive NK-cell leukaemia, and extranodal NK/T-cell lymphoma, nasal-type. Recent studies indicate that CD4+CD56+ blastic NK-cell lymphoma is of plasmacytoid dendritic cell origin, and true tumours of precursor NK-cell origin may be present mainly in the CD4-CD56+ subset. Myeloid/NK-cell precursor acute leukaemia may also develop from precursor NK cells. However, because the developmental pathway of normal NK cells is not well understood, tumours of precursor NK-cell origin are not clearly identified. Among mature NK-cell tumours, extranodal NK/T-cell lymphoma is relatively common in Asia and Latin America. In localized disease, chemoradiotherapy seems to be promising, and in advanced disease, new combination chemotherapies are under active investigation. Aggressive NK-cell leukaemia is rare and has a poor prognosis. Because NK-cell neoplasms are rare and difficult to manage, rigorous studies are required for their understanding and management.

Close Resemblance between Chemokine Receptor Expression Profiles of Lymphoproliferative Disease of Granular Lymphocytes and Their Normal Counterparts in Association with Elevated Serum Concentrations of IP-10 and MIG

T-cell large granular lymphocyte (T-LGL) leukemia and chronic natural killer (NK) cell lymphocytosis (CNKL) are major subtypes of lymphoproliferative disease of granular lymphocytes (LDGL). To clarify the mechanism of LGL proliferation and the relationship with the chemokine system in LDGL, we enrolled 22 T-LGL leukemia patients and 8 CNKL cases, analyzed the expression profiles of chemokine receptors, and measured the serum concentrations of the corresponding chemokines. There were no significant differences in chemokine receptor expression profiles between T-LGL leukemia patients and healthy donors. An association of CCR5 and CXCR3 expression levels on LGLs was recognized in T-LGL leukemia patients (r = 0.84; P < .001). Among the chemokines, serum IP-10 and MIG levels were significantly higher in LDGL patients than in healthy donors (P < .05, and P < .001, respectively), and MIG expression was associated with the number of circulating LGLs (r = 0.73; P < .01). The chemokine receptor phenotypes of LDGL cells are essentially similar to those of normal T-cells and NK cells. The roles of IP-10 and MIG in the pathophysiology of LDGL need further examination.

Peritoneal natural killer cells from epithelial ovarian cancer patients show an altered phenotype and bind to the tumour marker MUC16 (CA125)

The ovarian tumour marker MUC16 (CA125) inhibits the cytotoxic responses of human natural killer (NK) cells and down-regulates CD16. Here we show that approximately 10% of the peripheral blood NK cells (PBNK) from the epithelial ovarian cancer (EOC) patients are CD16(-) CD56(br) whereas 40% of the peritoneal fluid NK (PFNK) carry this phenotype, which is usually associated with NK cells from the lymph nodes or human decidua. PBNK from healthy donors exposed to PF show a significant increase in the CD16(-) CD56(br) population. This shift in phenotype is not caused by increased apoptosis of the CD16(+) CD56(dim) cells or selective proliferation of the CD16(-) CD56(br) NK cells. Thus, the terminal differentiation of the CD16(-) CD56(br) NK cells to CD16(+) CD56(dim) subset that occurs during normal NK cell development may actually be a reversible step. A majority of the NK cell receptors (NKp46, NKp44, NKG2D, CD244, CD226, CD158a, CD158b, and CD158e) studied were down-regulated in the PFNK. MUC16 binds selectively to 30-40% of CD16(+) CD56(dim) NK cells in EOC patients indicating that phenotypic alterations in these cells are mediated by tumour-derived soluble factors. Similar to EOC, MUC16 in early pregnancy also binds to NK cells suggesting shared mechanisms of NK cell suppression in feto-maternal tolerance and immune evasion by ovarian cancers.

Functional characterization of natural killer cells in type I leukocyte adhesion deficiency

AbstractIn this study, we analyzed IL-2–activated polyclonal natural killer (NK) cells derived from 2 patients affected by leukocyte adhesion deficiency type I (LAD1), an immunodeficiency characterized by mutations of the gene coding for CD18, the β subunit shared by major leukocyte integrins. We show that LAD1 NK cells express normal levels of various triggering NK receptors (and coreceptors) and that mAb-mediated engagement of these receptors results in the enhancement of both NK cytolytic activity and cytokine production. Moreover, these activating NK receptors were capable of recognizing their specific ligands on target cells. Thus, LAD1 NK cells, similarly to normal NK cells, were capable of killing most human tumor cells analyzed and produced high amounts of IFN-γ when cocultured in presence of target cells. Murine target cells represented a common exception, as they were poorly susceptible to LAD1 NK cells. Finally, LAD1 NK cells could efficiently kill or induce maturation of monocyte-derived immature dendritic cells (DCs). Altogether our present study indicates that in LAD1 patients, 3 important functions of NK cells (eg, cytotoxicity, IFN-γ production, and DC editing) are only marginally affected and provides new insight on the cooperation between activating receptors and LFA-1 in the induction of NK cell activation and function.

Monozygous twins with a microdeletion syndrome involving BTK, DDP1, and two other genes; evidence of intact dendritic cell development and TLR responses

We report for the first time monozygous twins with a microdeletion syndrome involving genes coding for Bruton's tyrosine kinase (Btk) and deafness-dystonia peptide 1 (DDP1), and two other genes. Apart from its essential role in B cell development, Btk is indicated to affect signaling mediated by toll like receptors (TLRs) and development of dendritic cells (DCs) but results are conflictive. The twins revealed normal numbers of plasmacytoid and myeloid DCs (pDCs and mDCs). Moreover, BTK null cells from these patients exhibited robust responses to TLR agonists, normal natural killer (NK) cell activity, and normal pDC functions. Our results do not indicate the essential role of Btk in TLR signaling and DC development.

Natural Killer Cell Functional Activity Suppression By Intravenous Immunoglobulin, Intralipid and Soluble Human Leukocyte Antigen‐G

The purpose of this study was to compare the ability of intravenous immunoglobulin (IVIg), intralipid and soluble human leukocyte antigen (sHLA)-G to suppress natural killer (NK) cell cytotoxicity in an in vitro assay. Blood samples taken from 275 women experiencing reproductive failure were analyzed for NK cytotoxicity and the suppression of NK cytotoxicity by IVIg 4 and 2 mg/mL (n = 275), intralipid 18 and 9 mg/mL (n = 275) and sHLA-G 70 and 35 ng/mL (n = 50) using immunofluorescent labeled K562 cells as targets and flow cytometry. Natural killer cytotoxicity was suppressed in all samples. Among patients with normal NK cell activity, IVIg suppressed NK cytotoxicity by 44.9 +/- 8.1%, intralipid suppressed NK killing by 45.2 +/- 8.3% and sHLA-G suppressed by 49.0 +/- 9.2%. When specimens with abnormal NK activity were observed for suppression of cytotoxicity, IVIg suppressed by 38.9 +/- 5.4%, intralipid suppressed by 39.8 +/- 6.2% and sHLA-G suppressed by 39.9 +/- 5.0%. Intravenous immunoglobulin, intralipid and sHLA-G suppressed NK cell cytotoxicity with equal efficacy in an in vitro assay.

Antitumor NK activation induced by the Toll-like receptor 3-TICAM-1 (TRIF) pathway in myeloid dendritic cells

Myeloid dendritic cells (mDCs) recognize and respond to polyI:C, an analog of dsRNA, by endosomal Toll-like receptor (TLR) 3 and cytoplasmic receptors. Natural killer (NK) cells are activated in vivo by the administration of polyI:C to mice and in vivo are reciprocally activated by mDCs, although the molecular mechanisms are as yet undetermined. Here, we show that the TLR adaptor TICAM-1 (TRIF) participates in mDC-derived antitumor NK activation. In a syngeneic mouse tumor implant model (C57BL/6 vs. B16 melanoma with low H-2 expresser), i.p. administration of polyI:C led to the retardation of tumor growth, an effect relied on by NK activation. This NK-dependent tumor regression did not occur in TICAM-1(-/-) or IFNAR(-/-) mice, whereas a normal NK antitumor response was induced in PKR(-/-), MyD88(-/-), IFN-beta(-/-), and wild-type mice. IFNAR was a prerequisite for the induction of IFN-alpha/beta and TLR3. The lack of TICAM-1 did not affect IFN production but resulted in unresponsiveness to IL-12 production, mDC maturation, and polyI:C-mediated NK-antitumor activity. This NK activation required NK-mDC contact but not IL-12 function in in vivo transwell analysis. Implanted tumor growth in IFNAR(-/-) mice was retarded by adoptively transferring polyI:C-treated TICACM-1-positive mDCs but not TICAM-1(-/-) mDCs. Thus, TICAM-1 in mDCs critically facilitated mDC-NK contact and activation of antitumor NK, resulting in the regression of low MHC-expressing tumors.

Cytokine-Activated Natural Killer Cells Exert Direct Killing of Hepatoma Cells Harboring Hepatitis C Virus Replicons

Hepatitis C virus (HCV)-specific impairments in host immunity have been described at multiple levels of the innate and adaptive response, which may lead to viral persistence in the majority of infections. Understanding of HCV-associated immune defects could lead to novel therapeutic advances. Natural killer (NK) cells, the major effector cells of the innate immune system, are functionally impaired in chronic HCV infection. It has been suggested that this phenotype is a result of virus-specific defects in antigen-presenting cells (APCs) that regulate NK cell activity, as normal NK function is restored when they are stimulated ex vivo. In this study, we used human NK cell cytotoxicity assays to evaluate the activation-induced effects of NK cells on the HCV replicon-containing hepatic cells. We found that cytokine-activated NK cells were capable of inducing an HCV-associated, perforin/granzyme-dependent lysis of human hepatoma cells and that this required direct cellular contact and was independent of MHC class I expression levels. In contrast, on removal of cytokine stimulation, NK cells failed to exert any direct cytolytic effect on replicon targets. These findings suggest an important underlying mechanism by which NK cells control HCV infection and, with appropriate understanding of HCV-associated immune defects, could lead to novel therapeutic advances.

Perforin Gene Mutations in Patients with Acquired Aplastic Anemia.

Perforin is a cytolytic protein expressed mainly in activated cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In T and NK cells perforin is stored in cytoplasmic granules and is essential for killing via non-Fas-mediated mechanisms. Perforin regulates the translocation of granzyme B from cytotoxic cells into target cells; after entering the target-cell granzyme B migrates to the target cell nucleus to participate in triggering apoptosis. Functional perforin is essential for normal CTL and NK cell function; without perforin CTL and NK cells show reduced or no cytolytic effect. Inherited perforin mutations account for 20–40% of familial hemophagocytic lymphohistiocytosis, an autosomal recessive fatal disease of early childhood characterized by uncontrolled accumulation of activated T cells and macrophages in many organs, increased Th1 cytokines and absent functional perforin. Acquired aplastic anemia (AA), the paradigm of immune mediated bone marrow failure syndromes, is characterized by hematopoietic stem cell destruction by activated T cells and Th1 cytokines. We examined whether mutations in Prf1 occur in AA; peripheral blood DNA samples from 75 patients and 302 controls were analyzed. Three novel nonsynonymous Prf1 mutations among five unrelated patients (ages: 21, 31, 33, 75, and 77 years old), not present in controls, were discovered; two polymorphisms were also identified (H300H, A274A). The mutations were in the coding region of Prf1 gene. In exon 2, arginine was replaced by histidine in one patient (CGT/CAT, R4H) and in 3 patients the same A91V mutation was identified (GCG/GTG, alanine to valine substitution). In exon 3, serine was replaced by isoleucine (S388I; AGC/ATC) in one patient. Germ-line origin of the Prf1 mutations was established by their presence also in DNA from buccal mucosa obtained from affected AA patients. Four of five patients with mutations showed some hemophagocytosis in the bone marrow examination when first diagnosed, but there were no other typical features of hemophagocytic syndrome such as hepatosplenomegaly or altered liver function tests. None of the patients with Prf1 mutations experienced hematologic recovery with immunosuppressive treatment. Perforin protein levels in all patients carrying mutations were very low or absent. By confocal microscopy, CD8 cells from patients with Prf1 mutations had complete absence of perforin granules (perforin and cathepsin D showed the expected pattern of co-localization in controls' cytotoxic granules). NK cell killing efficiency from patients carrying mutations in a standard Cr51-release cytolytic assay was significantly decreased compared to controls. Prf1 gene mutations may be related to a more severe phenotype of AA associated with marrow hemophagocytosis and failure to respond to immunosuppression. Mutations in the immune regulatory mechanisms identified in young children can manifest in adults without typically associated clinical findings or a suggestive family history. Mechanistically, Prf1 gene mutations help explain the aberrant proliferation and activation of cytotoxic T cells that are destructive of hematopoietic stem cells in AA and may be useful as predictive factors for responses to immunosuppressive treatments and the decision to rapidly undertake stem cell replacement. Prf1 gene mutations are genetic risk factors for bone marrow failure syndromes.

Analysis of Gene Expression Patterns and Gene Copy Number Changes in Human NK Cell Malignancies.

Natural killer (NK)-cell lymphomas/leukemias are a group of aggressive malignancies distinct from the indolent large granular lymphocyte (LGL) proliferations. In the World Health Organization (WHO) classification, NK-cell malignancies are divided into two groups (1) extranodal NK-cell lymphoma, nasal type (ENKL), and (2) aggressive NK-cell leukemia (ANKL). Only three studies of genome wide alteration have been published in these malignancies. We studied eight NK lymphoma cell lines (NK-92, KHYG1, YT, HANK-1, NK-YS, SNK-6, SNK-3 and KAI3) and seven NK lymphomas/leukemias specimens to:determine DNA gains and losses in comparative genomic hybridizations (CGH) and compare these alterations with those reported literature to define recurring chromosomal abnormalities,obtain transcription profiles of NK malignancies and compare them with normal NK cells(resting or IL-2 stimulated), andcorrelate the genomic profiles with transcriptional profiles to identify possible candidate genes involved in lymphoma development.Whole genome tiling arrays, containing 26,819 bacterial artificial chromosomes (BAC) were employed for CGH study.Our CGH study identified frequent recurrent abnormalities that have been reported in all previous studies. These include gains in 17q11.2–q21 (frequency: 40%); 1q22–q23.1 (frequency: 50 %); and 7q21.13–q22.1(frequency: 50%) and all these regions showed at least a frequency of 17% in other reports. A number of regions were also described in at least one other published reports (e.g. 1q32.2–q41, 6p25.5 and 17q 21.2). Regions of loss with a frequency ≥ 20% including 11q23.1–23.2(20%); 7p22.1–p22.2 (40%); 6q21(50%); 6q25.1–25.3(50%); and 17p13–13.1(50%) showed consistent results with previous reports. 6q21 loss was detected in two ANKL cell lines (NK92 and KHYG1) but not in ENKL cell lines . Gains of 8p12–q21.12, 8q22.1–q24.13, and 8q24.21 were also unique to ANKL cell lines.Transcriptional profiling using affymetrix gene chips on these cell lines and tumor specimen were compared with normal resting and IL-2 activated NK cells. We observed significant down-regulation (6–9 folds) of killer cell Ig-like receptor (KIR) (KIR2DL3, KIR2DS1, KIR3DL1, KIR3DL2) in NK-cell lymphoma as compared to normal NK cells. This may be related to the restricted clonal expression of the KIRs in neoplasms. High expression of pro-proliferative genes (e.g. Cyclin B1, A2, BUB1, CDC2, CDC20, CDCA1, CDCA3, and CDC7) was observed in NK lymphoma. On examining gene signatures/pathways (using BRB-array Tools), we observed significant increased expression of genes (p<0.001) involved in oxidative-phosphorylation /electron transport chain and MYC target signature in NK-cell lymphoma. A number of genes involved in TGF-beta signaling (p<0.0001) were down-regulated in lymphoma samples as compared to normal cells. This may correlate with the deletion of two crucial regulators (TGFBR3 and SMAD7) of this pathway, located at 1p32–33 and 18q21.1 regions respectively. In summary, combination of the genomic and transcriptional profiling by microarrays may lead to the identification of candidate genes in genomic alterations and provide clues to the pathogenesis of NK-cell malignancy.

Complete correction of murine Artemis immunodeficiency by lentiviral vector-mediated gene transfer

Artemis gene mutations are responsible for the development of a severe combined immunodeficiency [radiation-sensitive (RS) SCID] characterized by a severe B and T cell deficiency and a normal natural killer cell population. To establish the feasibility of a gene therapy approach to the treatment of RS-SCID, we generated a series of lentiviral vectors expressing human Artemis from different promoters and used them to transduce highly purified hematopoietic stem cells (HSCs) from Artemis knockout mice. HSCs transduced by the different viruses were transplanted into either lethally irradiated Rag-1-deficient animals or Artemis knockout mice treated with a nonmyeloablative dose of Busulfan. In both models, transplantation of HSCs transduced by a vector that used a murine phosphoglycerate kinase (PGK) promoter led to a complete functional correction of the immunodeficiency. Corrected animals displayed rescue of mature B cells with normal levels of serum immunoglobulins, together with complete rescue of the T cell compartment as evidenced by the presence of mature T lymphocytes in peripheral blood as well as normal values of thymocytes in thymus. Those B and T cells were capable of activation, as shown both by in vitro stimulation responses and in vivo after immune challenge. Overall, the results indicate that a gene therapy approach for RS-SCID involving the transplantation of genetically modified HSCs is indeed feasible. Furthermore, our studies suggest the possibility that nonmyeloablative conditioning regimens might be effectively used to promote engraftment of genetically modified cells in the case of diseases where standard irradiation-based myeloablative bone marrow transplantation protocols may prove problematic.

Concomitant increase of LMP1 and CD25 (IL‐2‐receptor α) expression induced by IL‐10 in the EBV‐positive NK lines SNK6 and KAI3

Extranodal, nasal NK/T-cell lymphomas are regularly Epstein-Barr virus (EBV)-positive, with a type II latency pattern, expressing thus EBNA-1 and LMP1. The contribution of EBV to the tumor development is not known. Similarly to normal natural killer (NK) cells, cell lines derived from malignancies with a NK phenotype require IL-2 for in vitro proliferation. In our effort to explore the contribution of EBV, particularly the role of the LMP1 protein, to the pathogenesis of the NK lymphoma we found that its expression, studied in the NK-lines SNK6 and KAI3, depended on the supply of IL-2 or other cytokines. In the absence of IL-2 other cytokines, such as IL-10 and IFN-gamma, could maintain LMP1, but the cells did not proliferate. When grown in IL-2, the SNK6 cells produced IL-10 and IFN-gamma, and these cytokines mediated the expression of LMP1. IL-10 treatment enhanced, while IFN-gamma receptor blocking antibody reduced, the expression of CD25 and CD54 in the EBV-positive, but not in the EBV-negative lines. IL-10 treated cells required lower amount of IL-2 for proliferation compared to the untreated cells. This effect was seen only with the EBV-positive NK lines in which LMP1 and CD25 were concomitantly upregulated. By this mechanism EBV could have an important role in the development of NK lymphoma since the inflammatory component in the tumor tissue can provide these cytokines.

The chemokine CXCL14 (BRAK) stimulates activated NK cell migration: Implications for the downregulation of CXCL14 in malignancy

The primary function of chemokines is the regulation of leukocyte trafficking by stimulating directional chemotaxis. The chemokine CXCL14 (BRAK) is highly expressed in all normal tissues, but is not expressed in most malignant tissues. The chemotactic activity of CXCL14 has been difficult to characterize. Recently it was reported that CXCL14 is a chemoattractant for activated monocytes and immature dendritic cells. Given that CXCL14 is downregulated upon transition to malignancy, we sought to characterize whether CXCL14 might play a role in NK cell chemotaxis. Human natural killer (NK) cells were isolated from buffy coats obtained from normal volunteers and were activated with lymphocyte conditioned media, IL-2, and ionomycin. Standard transwell chemotaxis assays, proliferation assays, and chromium release cell cytotoxicity assays were performed. CXCL14 was found to stimulate migration of activated human NK cells in transwell chemotaxis assays by 1.4-fold. Similarly, it increased migration of an IL-2-dependent natural killer leukemia (NKL) cell line by 1.9-fold. Antisera against CXCL14 or pertussis toxin blocked this chemotactic effect. However, CXCL14 did not affect the proliferation or cytotoxic activity of normal human NK cells. CXCL14 also stimulated the chemotaxis of immature monocyte-derived dendritic cells. CXCL14 may play a role in the trafficking of NK cells to sites of inflammation or malignancy. In addition, the downregulation of the expression of CXCL14 might be an important step in successful oncogenesis to prevent NK immune surveillance of the malignancy.

May immunization strategies that target dendritic cells really offer advantages for HIV-1?

The recent work by Newton and coworkers [1] that recently appeared on this journal describes how in vitro culture of monocyte-derived dendritic cells (DC) could be suitably used to enhance T-cell proliferative responses in HIV-1 infected patients in adjuvant immunotherapeutic protocols. The work is undoubtedly of high technical value, uses patient groups chosen in an elegant manner, and suitably adopts autologous DC–T cell mixed lymphocyte cultures to explore DC function. The perceived feeling upon reading the work is that based on their normal function and phenotype immunotherapy with monocyte-derived DC could be straightforwardly useful in HIV-1 infection, in addition to HAART, and should be introduced in future plans for in vivo development. Unfortunately, although apparently these conclusions are supported by the excellent work performed, they are not supported by accumulating evidence outside the limited focus of ‘DC phenotype and function’. Indeed, ample evidence cautioning against (or at best questioning) the use of DC ‘vaccination’ for HIV-1 infected patients are published and should encourage additional research and discussion. Cross-talk between DC and natural killer (NK) cells has been shown to influence the quality of adaptive immune responses [3,4]. This takes place through selection of immature DC (iDC) which may be killed by NK cells through interaction with NKp30 natural cytotoxicity receptors (NCR) and through optimal induction of DC maturation [5,6]. It has been known for some time that during HIV-1 infection NK cell function is depressed [7,8]. Mechanisms leading to this defect have been described over recent years and include reduced expression of NCR i.e., NKp30, NKp46 and NKp44 [9,10]. A very similar defect has been shown to occur in patients with acute myelogenous leukaemia (AML), where reduced expression of NKp30 and NKp46 is evident [11]. NK cells expressing a decreased number of NCR molecules are known to display a correlated decreased cytotoxic activity [12]. This decreased activity reflects also on iDC killing and induction of iDC maturation, in which the correct expression of NKp30 plays a pivotal role [3–6]. Finally, with particular regard to iDC ‘vaccination’ of patients with AML it has been shown recently that autologous iDC are not killed by the NK cells as they express decreased NKp30 molecules [13]. Because partial function of NK cells expressing low levels of NKp30, as in AML and HIV-1 infection, could lead to inappropriate survival of iDC with tolerogenic potential, use of antigen-DC stimulation in patients with HIV-1 infection should be viewed as potentially harmful. In fact the work by Newton et al.[1] addresses only T-cell proliferation, and not T-cell function (e.g., antigen-specific cytotoxicity, cytokine production). Should iDC in HIV-1 infected patients support proliferation of T cells with type 2 cytokine patterns, we would have a highly unwanted effect, as suggested by others with the induction of tolerogenic DCs and with lack of selection of optimally active mature DC [2,14]. In conclusion, the final comment by the authors [1] that ‘immunisation strategies that target DC may therefore offer significant advantages in the ability to stimulate HIV-specific protective immune responses’ could be dangerously misleading. We need to take into account also a correct editing of adaptive immune responses in the presence of normal NK cell function, in order to avoid embarking into costly programs that might prove to be flawed from the beginning, leading to a relevant waste of time, money and, more importantly, patient quality of life and health. Sponsorship: ADM is supported by grants awarded by Istituto Superiore di Sanità (Programma Nazionale AIDS #40B.67, Italian Concerted Action for AIDS vaccine (I.C.A.V.), Accordi di Collaborazione Scientifica n. 40D61 e 45D/1.13.

A family with Papillon-Lefèvre syndrome reveals a requirement for cathepsin C in granzyme B activation and NK cell cytolytic activity

Activation of granzyme B, a key cytolytic effector molecule of natural killer (NK) cells, requires removal of an N-terminal pro-domain. In mice, cathepsin C is required for granzyme processing and normal NK cell cytolytic function, whereas in patients with Papillon-Lefèvre syndrome (PLS), loss-of-function mutations in cathepsin C do not affect lymphokine activated killer (LAK) cell function. Here we demonstrate that resting PLS NK cells do have a cytolytic defect and fail to induce the caspase cascade in target cells. NK cells from these patients contain inactive granzyme B, indicating that cathepsin C is required for granzyme B activation in unstimulated human NK cells. However, in vitro activation of PLS NK cells with interleukin-2 restores cytolytic function and granzyme B activity by a cathepsin C-independent mechanism. This is the first documented example of a human mutation affecting granzyme B activity and highlights the importance of cathepsin C in human NK cell function.

Cytolytic function and survival of natural killer cells are severely altered in myelodysplastic syndromes

Natural Killer (NK) cells are critical in host defense against malignant transformation and are potent antileukemic cytotoxic effectors. In the present study, we investigated the peripheral NK function in patients with myelodysplastic syndromes (MDS). We demonstrated that the peripheral NK cell population was quantitatively normal in MDS patients. Furthermore, NK cells displayed an expression of the activating natural cytotoxicity receptors (NCR) NKp46 and NKp30 as well as NKG2D similar to that observed in donors, but exert a highly decreased constitutive cytolytic activity compared to resting normal NK cells. Although activation with IL-2 resulted in the upregulation of NKp46 expression by MDS-NK cells, their cytolytic function remained deeply altered as compared to activated donor NK cells. In addition, MDS NK cells did not proliferate in vitro, and displayed an increased rate of apoptosis in response to IL-2 stimulation although the spontaneous apoptosis was not significantly increased. Interestingly, a proportion of peripheral MDS-NK cells were derived from the MDS clone as the cytogenetic anomaly found in bone marrow karyotype was also detected in 20-50% of circulating NK cells. In conclusion, NK cells' cytolytic function and proliferative capacities in response to activation by cytokines are profoundly altered in MDS.

Effects of surgery on the generation of lymphokine-activated killer cells in patients with breast cancer.

Natural killer (NK) cell activity and the capacity to generate lymphokine-activated killer (LAK) cell activity were studied in 43 patients with operable breast cancer before and after surgery. Results were compared with those from ten healthy subjects. Patients with breast cancer had normal LAK and NK cell activity before surgery. A subgroup of patients with stage III disease had depressed LAK cell activity (P < 0.013). NK cell activity decreased by over 50 per cent on the first day after surgery and did not return to preoperative levels by day 7 (P < 0.0005). Generation of LAK cell activity was unaffected by surgery. The addition of 10 per cent autologous plasma to the culture medium during the induction of LAK cell activity in vitro did not suppress LAK cell activity in patients with breast cancer. These results suggest that perioperative adjuvant immunotherapy based on interleukin 2 and LAK cells is not ruled out by systemic suppressive effects from either cancer or surgery.