STAT3 Is Activated By EBV in T or NK Cells Leading to Development of EBV-T/NK-Lymphoproliferative Disorders

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3026-3026
Author(s):  
Honami Komatsu ◽  
Ken-Ichi Imadome ◽  
Haruna Shibayama ◽  
Tomotaka Yada ◽  
Momoko Yamada ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Sh2 Domain ◽  
Lymphoproliferative Disorders ◽  
Clinical Samples ◽  
Ebv Infection ◽  
Xenograft Models ◽  
T Cell Lines

Abstract Introduction Epstein–Barr virus (EBV) genome is positive not only in B-, but also T- or NK-lymphoid neoplasms: extranodal NK/T-cell lymphoma (ENKL), aggressive NK-cell leukemia, and EBV-positive T- or NK-cell lymphoproliferative disorders (EBV-T/NK-LPDs). EBV-T/NK-LPDs are disorders formerly called chronic active EBV infection presenting sustained inflammation, such as infectious mononucleosis-like symptoms, hypersensitivity to mosquito bites, or hydroa vacciniforme-like eruption accompanied by clonal proliferation of EBV-infected T or NK cells. EBV makes the infected B-cells immortal leading to B-cell lymphomas. However, why and how EBV infects T or NK cells and the mechanism of action responsible for the development of EBV-T/NK-neoplasms has not been elucidated yet. STAT3 is a transactivation factor which mediates proliferation and anti-apoptotic signaling. It was reported that a large variety of primary tumor cells as well as tumor-derived cell lines from patients harbored constitutively activated STAT3. In addition, tyrosine 705 (Y705) of STAT3 was constitutively phosphorylated in ENKL cells (Leukemia, 23, p1667, 2009). Objectives We designed this study to investigate STAT3 activation and its contribution to EBV-T/NK-LPDs development. Materials and Methods EBV-positive T-cell lines, SNT8, SNT15, SNT16, and NK-cell lines, SNK1, SNK6, SNK10, were examined. The EBV-negative T-cell lines HPB-ALL, Jurkat, MOLT4 and peripheral blood mononuclear cells (PBMCs) from healthy donors were used as the negative controls. Clinical samples were obtained from EBV-T/NK-LPDs patients who were diagnosed according to the previously described criteria (Blood, 119, p.673, 2012). To detect and isolate EBV-infected cells, T and NK cells were separated using magnetic beads from PBMCs. STAT3 phosphorylation in EBV-T/NK-LPDs cells were examined in clinical samples and xenograft models of EBV-T/NK-LPDs generated by transplantation of PBMCs from the EBV-T/NK-LPDs patients to NOD/Shi-scid/IL-2Rγnull mice. Mutation of STAT3 was examined by direct sequencing. For in vitro EBV infection, EBV was prepared from the culture medium of B95-8 cells and added to MOLT4 cells (Proc Natl Acad Sci, 100, p7836, 2003). Results First, we investigated the activation of STAT3 in EBV-positive T- or NK-cell lines. Phosphorylation of STAT3 on Y705 and serine 727 (S727) was more clearly detected in comparison with that in EBV-negative cell lines by western blotting under their maintenance condition. STAT3 was localized in the nucleus in the EBV-T/NK-cells. These results indicated STAT3 was constitutively activated in EBV-T/NK-cells. We validated the results in EBV-infected T or NK cells derived from 5 EBV-T/NK-LPDs patients (infected cell types: CD4, 1; CD8, 2; and CD56, 2). Phosphorylation of Y705 and S727 of STAT3 was detected in EBV-infected T or NK cells in them. Immunohistological staining also detected the phosphorylation of EBV-positive cells in the tissue of the xenograft models. In these EBV-T/NK-cells, gene mutation was not identified in SH2 domain of STAT3. Next, we examined the direct effect of EBV on STAT3 activation by in virto EBV infection on MOLT4 cells. Immunofluorescence staining detected that STAT3 moved to the nucleus after the infection. Finally, STAT3 specific inhibitor STA-21 suppressed the proliferation of EBV-T/NK-cells. Conclusions STAT3 is activated by EBV leading to growth promoting effects on EBV-T/NK-LPDs. STAT3 can be an attractive molecular target of the treatment for the disorders. Disclosures No relevant conflicts of interest to declare.

A Novel HIV Envelope Bi-Specific Killer Engager Enhances Natural Killer Cell Mediated ADCC Responses Against HIV-Infected Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2517-2517 ◽  
Author(s):  
Zachary B. Davis ◽  
Todd Lenvik ◽  
Louis Hansen ◽  
Martin Felices ◽  
Sarah Cooley ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Nk Cell ◽  
Mhc I ◽  
Variable Heavy ◽  
Infected Cells ◽  
T Cell Lines ◽  
Hiv Envelope

Abstract Natural Killer (NK) cells, a critical component of the immune response to viral infection, recognize and destroy cells with diminished expression of major histocompatibility class-I (MHC-I) molecules and expression of ligands for activating NK receptors such as NKG2D. Down-modulation of MHC-I is a hallmark of viral infection, as it allows infected cells to evade a CD8 T-cell response. Stalling of the cell cycle to enhance viral replication induces NK activation ligands such as the NKG2D ligands unique long binding proteins (ULBP)-1 and -2 which could trigger NK destruction of infected cells. Unfortunately, incomplete down-modulation of MHC-I by HIV leaves HLA-C on the cell surface, which inhibits the majority of NK cells from killing infected targets. CD16, the low affinity Fc receptor, is the most potent NK cell activating receptor. It mediates antibody dependent cell-mediated cytotoxicity (ADCC), and can override inhibition by MHC-I. We designed a series of bi-specific killer-engager (BiKE) constructs to direct NK cell ADCC against an HIV-infected target. We linked the Fab portions of broadly neutralizing (bn)Abs to a novel llama-derived nanobody EF91 that binds CD16 at high affinity and signals strong activation. We chose to use EF91 as its structure is unique compared to the use of a single chain variable fragment (scFv). Rather than being composed of a variable heavy (VH) and variable light (VL) chain, the nanobody is composed of a single variable heavy (VHH) domain. A distinct advantage to using a CD16 nanobody over a scFv is in the purity of the generated product. During protein folding it is not uncommon for the wrong VH to associate with the wrong VL; the result of which is a nonfunctional product. Since the nanobody is single VHH, and does not require association with another domain, there is less risk of a misfolded product. Nanobodies are also known to have similar, if not increased, affinity for their target molecules. In the case of EF91, this may result in more robust activation of NK cells than with a traditional scFv. We tested a BiKE constructed with the bnAb, VRC01, which recognizes the CD4 binding domain of HIV-Env. The specificity of our novel anti-CD16 nanobody was demonstrated by binding of our BiKE construct to CD16+ NK cells (Figure 1A). Function of our BiKE construct was tested by incubating it with chronically infected T-cell lines (HIV-IIIB and ACH-2) or with their respective uninfected counterparts (H9 and CEM). We only observed binding to infected cells (Figure 1B), demonstrating HIV-Env binding specificity to the HIV strains ACH-2 (LAI strain) and HIV-IIIB. The ability of the anti-Env BiKE construct to mediate ADCC and IFNγ production was tested against two uninfected CD4 T-cell lines or their infected counterparts. While NK cells degranulated when incubated with the infected cell lines (50% against HIV-IIIB and 20% against LAI), this response was markedly enhanced when co-incubated with the HIV-Env specific BiKE (80% against HIV-IIIB and 60% against LAI) (Figure 1C). Furthermore, the HIV-Env BiKE enhanced IFNγ production against HIV-infected T-cell lines compared to responses in the absence of BiKE (28% against HIV-IIIB compared to 36% with BiKE; 15% against ACH-2 compared to 37% with BiKE) (Figure 1D). Our data demonstrate that a BiKE construct containing the Fab of an HIV bnAb and an anti-CD16 component can eliminate HIV-infected targets that express the HIV-envelope on their surface. The reservoir of latently infected CD4 T cells lack expression of any recognizable virus protein on the cell surface, we plan to combine our BiKE strategy with cellular activation using IL-15. Alternatively, we can construct a tri-specific engager (TriKE) with an IL-15 segment that may activate CD4 T cells while enhancing NK cell killing. Disclosures Cooley: Fate Therapeutics: Research Funding. Vallera:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

Epstein–Barr Virus Induces Activation-Induced Cytidine Deaminase Expression in T or NK Cells Leading to Mutagenesis and Development of Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1765-1765 ◽  
Author(s):  
Ayako Arai ◽  
Masato Horino ◽  
Ken-Ichi Imadome ◽  
Kohsuke Imai ◽  
Honami Komatsu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Lymphoma ◽  
Jurkat Cells ◽  
Clinical Samples ◽  
Ebv Infection ◽  
Infected Cells

Abstract Introduction Epstein–Barr virus (EBV) infects B cells and rarely T or NK cells, causing EBV-positive T/NK-cell lymphoproliferative neoplasms (EBV-T/NK-neoplasm), such as extranodal NK/T-cell lymphoma, aggressive NK-cell leukemia, and EBV-positive T- or NK-cell lymphoproliferative disorders (EBV-T/NK-LPDs). EBV-T/NK-LPDs are fatal disorders presenting sustained inflammation, such as infectious mononucleosis-like symptoms, hypersensitivity to mosquito bites, or hydroa vacciniforme-like eruption accompanied by clonal proliferation of EBV-infected T or NK cells. EBV infects B cells making them immortal and resulting in B-cell lymphomas. However, why and how EBV infects T or NK cells and the mechanism of action responsible for the development of these EBV-induced malignancies has not been elucidated yet. Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation and class switch recombination of immunoglobulin genes. Deregulated AID expression acts as a genomic mutator leading to the development of B-cell lymphoma. In addition, EBV infection induces AID expression in B cells. These findings indicate that AID has a role in EBV-induced lymphomagenesis in B cells. However, of interest, AID transgenic mice developed T-cell lymphoma (J. Exp. Med., 197, p.1173, 2003). Recently, Nakamura et al. reported that AID expression was upregulated in the peripheral blood mononuclear cells (PBMCs) of EBV-T/NK-LPDs patients (Eur. J. Dermatol., 21, p.780, 2011). Therefore, we hypothesized that EBV infection induces AID expression in T or NK cells contributing to the tumor development. Objectives We designed this study to investigate AID expression and its contribution to EBV-T/NK-neoplasms development. Materials and Methods EBV-positive T- and NK-cell lines, SNT8, SNK6, SNT13, SNT15, and SNT16, were examined. The EBV-negative T-cell line HPB-ALL, Jurkat and human primary T cells derived from cord blood were used as the negative controls. Clinical samples were obtained from EBV-T/NK-LPDs patients who were diagnosed according to the previously described criteria (Blood, 119, p.673, 2012). To detect and isolate EBV-infected cells, T and NK cells were separated using magnetic beads from PBMCs. AID expression in tissue lesions were examined in clinical samples and xenograft models of EBV-T/NK-LPDs generated by transplantation of PBMCs from the EBV-T/NK-LPDs patients to NOD/Shi-scid, IL-2R γKO mice. For in vitro EBV infection, EBV was prepared from the culture medium of B95-8 cells and added to HPB-ALL or Jurkat cells (Proc. Natl. Acad. Sci., 100, pp.7836-40, 2003). To determine AID-induced mutation of the gene, DNA was extracted from SNT13, SNK6, or EBV-infected Jurkat cells, and c-myc gene was cloned by TA cloning, which was then sequenced. We compared the number of the mutation with that of Jurkat cells. Results We detected AID expression in EBV-positive T- or NK-cell lines by RT-PCR, western blotting, and immunological staining using confocal microscopy, whereas it was not detected in the control. Furthermore, we validated the results in EBV-infected T or NK cells derived from 12 EBV-T/NK-LPDs patients (infected cell types: CD4, 5; CD8, 3; and CD56, 4). Quantitative RT-PCR demonstrated that AID expression was upregulated in EBV-infected T or NK cells compared with the control. Expression was confirmed by immunological staining using confocal microscopy in 5 patients. We also detected AID expression by histopathological staining in EBV-infected cells in lesions of 4 patients and the EBV-T/NK-LPDs xenograft models. Subsequently, we examined the role of EBV for AID expression in T cells. It was demonstrated that AID expression was induced by in vitro EBV infection in the T-cell line HPB-ALL and primary T cells. Moreover, it was demonstrated by the luciferase assay that the viral protein LMP1 upregulated AID promoter activity in HPB-ALL cells. Finally, we found that SNT13, SNK6, and EBV-infected Jurkat cells had increased number of the mutation of c-myc gene compared with Jurkat cells. Conclusions We previously reported that EBV infection of T or NK cells enhances survival of the infected cells by the activation of NF-κB. The results of the present study suggest that EBV contributes to the development of EBV-T/NK-neoplasms through NF-κB-induced cell survival and AID-induced mutagenesis leading to clonal evolution and expansion. Disclosures: No relevant conflicts of interest to declare.

Gene Expression Profiling Reveals Activation of Multiple Oncogenic Pathways and Over-Expression of Survivin in the Pathogenesis of Extranodal Nasal-Type NK/T Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3931-3931
Author(s):  
Wee-Joo Chng ◽  
Viknesvaran Selvarajan ◽  
Gaofeng Huang ◽  
Jianbiao Zhou ◽  
Mark Law ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Nasal Type ◽  
Over Expression ◽  
Natural Killer T Cell ◽  
Oncogenic Pathways

Abstract Abstract 3931 Poster Board III-867 Extranodal nasal-type Natural Killer/T-cell lymphoma (NKTCL) is a distinct clinicopathologic entity most commonly affecting Asians and Central and South Americans, and characterized by a clonal proliferation of NK or T cells with a cytotoxic phenotype. There is a strong association with Epstein-Barr Virus. The tumor is aggressive with patient usually surviving short duration even with chemotherapy. We performed the first comprehensive genome-wide gene expression profiling (GEP) of extranodal nasal-type Natural Killer/T-cell lymphoma (NKTCL) using formalin-fixed paraffin embedded (FFPE) tissue (n=25) and NK cell lines (n=5) and compared the results to the GEP of normal NK cells using the Illumina DASL whole genome array, with the objective of understanding the oncogenic pathways involved in the pathogenesis of NKTCL and to identify potential therapeutic targets. Quantitative-PCR validation of the GEP findings revealed over-expression of candidate genes BIRC5 (survivin), EZH2 and STMN1 in NK cell lines compared to normal NK cells, consistent with the GEP data. We then extracted a list of genes that are differentially expressed between NKTCL and normal NK cells and tissue controls. We than subjected this list of genes to pathway and network analysis using Metacore. This revealed a significant enrichment for cell cycle related genes and pathways. Furthermore, the network analysis results demonstrated a pro-proliferative and anti-apoptotic phenotype in NKTCL characterized by activation of Myc and nuclear factor kappa B (NF-KB), and deregulation of p53. This was further corroborated using immunohistochemistry on tissue microarray of NKTCL (n=33, including all cases with GEP performed). We observed a significant percentage of NKTCL showing overexpression for c-Myc (45.4%), p53 (87.9%) and NF-KB p50 (67.7%) on immunohistochemistry. Notably, overexpression of survivin was observed in 97% of the cases. Based on our findings, we propose a model of NKTCL pathogenesis where deregulation of p53 together with activation of MYC and NF-KB, possibly driven by EBV LMP-1, result in the cumulative upregulation of survivin. When KHYG and NKYS cell lines were treated with a compound IDR E804 which inhibited survivin, there is significant inhibition of cell growth as assess by MTS assay and induction of apoptosis as measured using Annexin V staining by flow cytometry. This suggests that compounds inhibiting survivin may be a potentially useful novel therapeutic approach in NKTCL. Disclosures: No relevant conflicts of interest to declare.

EZH2 Mediates Resistance To Apoptosis In Nktl By Activating Nfkb Signaling Through Repression Of TNFAIP3/A20 By H3K27 Trimethylation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1278-1278 ◽  
Author(s):  
Koh Tze Loong
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Target Genes ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Strong Association ◽  
Histone Methyltransferase ◽  
Clinical Samples ◽  
P Value ◽  
Chi Square

Abstract Extranodal nasal-type natural killer/T-cell lymphoma (NKTL) is a rare and aggressive form of lymphoma with poor outcome. A better understanding of the molecular pathophysiology of the disease is needed to development better treatment and improve patient’s outcome. We previously showed that EZH2, a histone methyltransferase, is abnormally over-expressed in NK cell lines as compared to normal NK cells, and may therefore be important for the pathogenesis and treatment of NKTL. 3- Deazaneplanocin A (DZNep) is a drug that can deplete EZH2 levels. Downregulation of EZH2 by DZNep induces apoptosis in a panel of NKTL cell lines but not normal NK cells. We perform gene expression studies to identify possible mechanism by which DZNep induce apoptosis in NKTL. There is significant enrichment of NFKB target genes amongst the genes downregulated after DZNep treatment. In addition, 2 inhibitors of the NFKB pathways, TNFAIP3/A20 and NFKBIA were upregulated. The downregulation of these genes were validated by PCR. We further found that NFKB2 was downregulated after DZNep treatment on western blot suggesting that NFKB signaling was downregulated. Similar results were obtained when we silence EZH2 using siRNA suggesting that much of the effect of DZNep is mediated through downregulation of EZH2. When TNFAIP3/A20 was expressed in untreated NKTL cells, apoptosis was induced. Conversed with TNFAIP3/A20 was knocked down in cells treated by DZNep, DZNep induced apoptosis was reduced. These data suggest in NKTL, EZH2 downregulate TNFAIP3/A20 and activate NFKB leading to resistance to apoptosis. We next investigated if EZH2 directly regulate TNFAIP3/A20 expression at its promoter. Using chromatin immunoprecipitation, we found that there is high occupancy of EZH2 and H3K27me3 at the TNFAIP3/A20 promoter in NKTL. Upon EZH2 knockdown, EZH2 and H3K27me3 is markedly reduced at the promoter and there is concurrent increase in TNFAIP3 expression. Consistent with cell lines data, in clinical samples, there is a strong association between H3K27me3 and nuclear p50 expression on a tissue microarray of NKTL (Chi-square p-value 0.01). We have therefore uncovered a novel mechanism by which EZH2 activates the NFKB pathway through its histone methyltransferase activity on the promoter of TNFAIP3/A20 resulting in resistance of NKTL to apoptosis. Disclosures: No relevant conflicts of interest to declare.

CAR-Engineered T Cells Specific for the Elotuzumab Target SLAMF7 Eliminate Primary Myeloma Cells and Confer Selective Fratricide of SLAMF7+ Normal Lymphocyte Subsets

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 115-115 ◽  
Author(s):  
Sophia Danhof ◽  
Tea Gogishvili ◽  
Silvia Koch ◽  
Martin Schreder ◽  
Stefan Knop ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Lymphocyte Subsets ◽  
Normal Lymphocyte ◽  
Car T Cells ◽  
Car T ◽  
T Cell Lines

Abstract Background: SLAMF7 (CS1, CD319) is uniformly and highly expressed in multiple myeloma (MM) where it promotes adhesion and survival of malignant plasma cells (mPCs) in the bone marrow niche. It is absent on normal solid organ tissues but known to be expressed on lymphocyte subsets (T, B and NK cells). Clinical evaluation of the anti-SLAMF7 monoclonal antibody (mAb) Elotuzumab (huLuc63) has resulted in marked reversible lymphodepletion and conferred potent anti-MM efficacy in combination therapy. Here, we evaluated the potential to generate SLAMF7-directed chimeric antigen receptor (CAR) modified T cells from previously treated MM patients and analyzed their potency against autologous mPCs as well as fratricidal activity against normal lymphocyte subsets. Methods: Flow cytometric analyses for SLAMF7 expression on mPCs and normal lymphocyte subsets of MM patients (n=67) and healthy donors (n=20) was performed using specific mAbs and matched isotype controls. A SLAMF7-specific CAR was constructed using the VH/VL targeting domains of mAb huLuc63, fused to an Ig-Fc spacer and a signaling module of CD3ζ and CD28. Lentiviral gene transfer was performed into CD3/CD28-bead stimulated bulk CD4+ and CD8+ T cells of MM patients (n=7). CAR transgene positive T cells were enriched using an EGFRt transduction marker and expanded for functional analyses. Results: We confirmed high SLAMF7 expression levels on mPCs in all analyzed samples and detected SLAMF7 expression on a fraction of normal lymphocytes obtained from peripheral blood of MM patients, including naïve and memory CD4+ (95% CI: 33-59%) and CD8+ T cells (75-95%), B cells (25-35%) and NK cells (94-98%). Remarkably, the proportion of SLAMF7+ cells was significantly higher in MM patients compared to healthy donors in all corresponding lymphocyte subsets (p<0.05). Despite high level SLAMF7 expression on the input T cell population, functional CD4+ and CD8+ T cells expressing the SLAMF7-CAR could be readily generated in all 7 MM patients, and expanded to therapeutically relevant doses in a single expansion cycle following enrichment (>107 cells). We analyzed the kinetics of SLAMF7 expression on CD4+ and CD8+ CAR T cells during the manufacturing process and detected rapid disappearance of SLAMF7+ T cells in T cell lines modified with the SLAMF7-CAR. By contrast unmodified T cells and T cell lines expressing a CD19-CAR retained a significant proportion of SLAMF7+ T cells, suggesting that expression of the SLAMF7-CAR induced killing of SLAMF7+ T cells. In vitro functional testing of SLAMF7-CAR CD4+ and CD8+ T-cell lines confirmed potent specific lysis of SLAMF7+ MM cell lines including MM1.S and OPM-2 and stable SLAMF7-transfectants of K562, as well as antigen specific IFNγ secretion and productive proliferation. In a flow cytometry based cytotoxicity assay, co-incubation of mPCs with autologous (or allogeneic) SLAMF7-CAR T cells resulted in elimination of >90% of mPCs after a 4-hour incubation period, whereas CD19-CAR or unmodified T cells had no discernible effects. Moreover, in an in vivo xenograft MM model (NSG/MM1.S) a single administration of SLAMF7-CAR T cells resulted in complete MM clearance and long-term survival, whereas mice treated with CD19-CAR or unmodified T cells rapidly expired from progressive disease. Finally, we analyzed the fratricidal potential of SLAMF7-CAR T cells to predict hematologic toxicities that might occur in a clinical setting. Co-incubation of purified CD4+ and CD8+ primary T cells, B cells and NK cells with SLAMF7-CAR T cells resulted in rapid and specific elimination of only SLAMF7+ subsets, whereas SLAMF7- cells remained viable and functional as confirmed for CD4+ and CD8+ T cells by inducible IFNγ secretion. Conclusion: Our data demonstrate that SLAMF7-specific CAR T cells can be reproducibly generated from MM patients and exert remarkable anti-myeloma efficacy in pre-clinical models in vitro and in vivo. Lymphocytic fratricide does not preclude the manufacture of SLAMF7-CAR T cells but might be associated with acute (cytokine storm) or chronic (viral infections) side effects in a clinical setting. However, such toxicities may be prevented e.g. by preparative lymphodepletion and antiviral prophylaxis and enable the implementation of SLAMF7-CAR T cell therapy as a safe and effective modality in the treatment of MM. Disclosures Knop: Celgene Corporation: Consultancy. Einsele:Novartis: Consultancy, Honoraria, Speakers Bureau; Amgen/Onyx: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau.

The human antimicrobial and chemotactic peptides LL-37 and α-defensins are expressed by specific lymphocyte and monocyte populations

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3086-3093 ◽  
Author(s):  
Birgitta Agerberth ◽  
Jehad Charo ◽  
Joachim Werr ◽  
Berit Olsson ◽  
Farah Idali ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Primary Cultures ◽  
Γδ T Cells ◽  
Chemotactic Activity ◽  
Antibacterial Peptides ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
T Cell Lines

We identified antibacterial components in human T and natural killer (NK) cells by using freshly isolated lymphocytes enriched for T and NK cells as starting material. After growing these lymphocytes for 5 days in the presence of interleukin (IL)–2, we isolated and characterized several antibacterial peptides/proteins from the supernatant—α-defensins (HNP 1-3), LL-37, lysozyme, and a fragment of histone H2B—although other active components were also present. We then used reverse transcriptase–polymerase chain reaction to search for expression of the gene coding for LL-37 in several B-cell lines, γδ T-cell lines, NK clones, and one monocytic cell line, with positive results, but found no expression in several αβ T-cell lines. The α-defensins (HNP 1-3) were also found to be expressed in several of these cell lines. To confirm the presence of these antibacterial peptides in lymphocytes, we localized them to NK, γδ T cells, B cells, and monocytes/macrophages by using double-staining immunohistochemical analysis of freshly isolated lymphocytes. We also found that primary cultures of lymphocytes transcribe and secrete LL-37 and that these processes are affected by IL-6 and interferon-γ. In addition, we demonstrated that LL-37 has chemotactic activity for polymorphonuclear leukocytes and CD4 T lymphocytes, whereas others have shown chemotactic activity for human α-defensins (HNP 1-2). These findings suggest that microbicidal peptides are effector molecules of lymphocytes and that antibacterial activity previously shown to be derived from T and NK cells may be partly mediated by the antibacterial peptides LL-37 and HNP 1-3.

Enhanced T-cell response to mosquito extracts by NK cells in hypersensitivity to mosquito bites associated with EBV infection and NK cell lymphocytosis

2005 ◽  
Vol 96 (8) ◽  
pp. 519-526 ◽  
Author(s):  
Yoshiki Tokura ◽  
Hiroyuki Matsuoka ◽  
Chizuko Koga ◽  
Hideo Asada ◽  
Naohiro Seo ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Cell Response ◽  
Nk Cell ◽  
T Cell Response ◽  
Ebv Infection

scholarly journals Epstein-Barr virus (EBV)-carrying and -expressing T-cell lines established from severe chronic active EBV infection

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1446-1457 ◽  
Author(s):  
S Imai ◽  
M Sugiura ◽  
O Oikawa ◽  
S Koizumi ◽  
M Hirao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Gene Rearrangement ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr ◽  
T Cell Lines

Four novel Epstein-Barr virus (EBV)-carrying T-cell lines, designated SIS, AIK-T8, AIK-T4, and SKN, were established from peripheral blood lymphocytes (PBL) of patients with severe chronic active EBV infection, in the presence of interleukin-2 and 4-deoxyphorbol ester. AIK-T8 and - T4 were derived from a single patient. Cell marker and genotype analyses showed that SIS, AIK-T8, and AIK-T4 had mature T-cell phenotypes with clonally rearranged T-cell receptor (TCR) genes, whereas SKN had an immature T-cell phenotype without TCR gene rearrangement. None of the cell lines expressed B, natural killer, or myeloid antigens or had Ig gene rearrangement. All lines carried EBV genomes in a single episomal form. SIS, AIK-T8, and SKN showed the same phenotype, TCR gene configuration, and/or EBV clonotype as their source or biopsied materials; therefore, they represented EBV-infected T cells proliferating in the patients. TCR gene and EBV episomal structures similar to those of AIK-T4 were not found in its source PBL, probably due to the few parental clones in vivo. All lines expressed EBV-encoded small RNA (EBER) 1, nuclear antigen (EBNA) 1, and latent membrane protein (LMP) 1, -2A, and -2B, but not other EBNAs that could be recognized by EBV-specific immune T cells. EBV replicative antigens were rarely expressed or induced. Such EBV latency reflects the in vivo situation, in which the T cells may evade immune surveillance and be insensitive to antiherpesvirus drugs. Collectively, the data suggest that EBV can target and latently infect T cells at any stage of differentiation in vivo, thus potentially causing uncontrolled T-cell proliferation. These cell lines will facilitate further analyses of possible EBV-induced oncogenicity in T cells.

scholarly journals Antiproliferative effect of antilymphocyte globulins on B cells and B- cell lines

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2164-2170
Author(s):  
N Bonnefoy-Berard ◽  
M Flacher ◽  
JP Revillard
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Immunosuppressive Agents ◽  
Lymphoproliferative Disorders ◽  
Hla Dr ◽  
T Cell Lines ◽  
Vitro Effect

Antithymocyte and antilymphocyte globulins (ALG) are currently used as immunosuppressive agents in organ transplantation and for the treatment of acute graft-versus-host disease and aplastic anemia. Since any type of immunosuppressive treatment is known to carry the risk of developing B-cell lymphoproliferative disorders, we investigated the in vitro effect of ALG on human B-cell activation and proliferation. The data demonstrate that whatever the source of lymphocytes used for ALG preparation (thymocytes, thoracic duct lymphocytes, B- or T-cell lines), (1) ALG react with both B- and T-cell lines, and (2) ALG contain antibodies specific for B cells (eg, CD21) or common to T and B cells (eg anti-beta 2-microglobulin, anti-HLA-DR, CD18, CD11a) in addition to T-cell-specific antibodies. Unlike all other T-cell mitogens tested (Concanavalin A [Con A], Pokeweek mitogen [PWM], CD3 and CD2 antibodies), ALG do not trigger B-cell differentiation into immunoglobulin-secreting cells at concentrations which induce maximum T- cell proliferation. This effect could be attributed to a direct interaction of ALG with B lymphocytes as shown by the capacity of ALG to block the response of purified B cells to a variety of activators. Furthermore, all the ALG tested were shown to inhibit the proliferation of six of the seven Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and six of the seven Burkitt's lymphoma cell lines studied. This selective B-cell antiproliferative property of ALG was not reproduced with CD11a, CD18, CD21, CD24, or anti-HLA-DR monoclonal antibodies (MoAbs). These results suggest that, although suppressing T- cell responses, ALG treatment may directly control B cell proliferation to some extent, in keeping with the relatively low risk of posttransplant lymphoproliferative disorders reported with ALG.

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