scholarly journals Radiation sensitivity of resting and activated nonspecific cytotoxic cells of T lineage and NK lineage

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1615-1621 ◽  
Author(s):  
D Zarcone ◽  
AB Tilden ◽  
VG Lane ◽  
CE Grossi
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Radiation Sensitivity ◽  
Lak Cells ◽  
Activated T Cells ◽  
Effector Cells ◽  
Blood Lymphocytes ◽  
Nonspecific Cytotoxicity ◽  
Cytotoxic Function

Abstract Natural killer (NK) cell-mediated killing of tumor cells is a radiation- sensitive function that in most subjects is completely abrogated by treatment of the effector cells with 3,000 cGy. The radiation sensitivity of LAK (lymphokine-activated killer) cells and their precursors, the bulk of which are NK cells, is undetermined. In this study, functional cytotoxicity assays and electron microscopy were used to determine the effect of radiation on the cytotoxic function of NK cells, LAK cells (generated by three-day culture of peripheral blood lymphocytes with IL-2), and LAK cell precursors (lymphocytes irradiated prior to culture with IL-2). For comparison, we analyzed the radiation sensitivity of lectin-dependent cell-mediated cytotoxicity (LDCC), which is primarily a function of CD3+ CD8+ granular lymphocytes. We also analyzed the radiation sensitivity of nonspecific cytotoxicity mediated by mitogen-activated T cells (AK activity). Following 3,000 cGy irradiation, NK cells retained their ability to bind to tumor cell targets but, as shown by both morphologic and functional analyses, they did not undergo activation after conjugate formation, and were unable to release the content of their granules. In order to evaluate LDCC, lymphocytes were depleted of CD16+ cells and tested in a cytotoxicity assay in the presence of Con A. The radiation sensitivity curve was comparable to that of NK cell-mediated cytotoxicity. IL-2-treated lymphocytes (LAK cells) were relatively radioresistant as compared with untreated NK cells, and their cytotoxic function was not abrogated until treatment with greater than 10,000 cGy. Cells receiving such radiation doses displayed cytoplasmic blebbing and damage of their cytoskeletal structures, with disruption of centrioles and microtubules, and disarray of the intermediate filaments. As was shown with NK cells, irradiated LAK cells formed conjugates with tumor targets but failed to degranulate. The radiation sensitivity of nonspecific cytotoxicity mediated by mitogen-activated T cells was identical to that of LAK effector cells. Doses up to 2,000 cGy did not prevent generation of LAK cells from blood lymphocytes, but 3,000 cGy did so. Blast transformation similar to that observed in IL-2- stimulated controls occurred when lymphocytes irradiated with 3,000 cGy were cultured with IL-2. These transformed cells were not cytotoxic and displayed a normal cytoskeletal apparatus but did not bear electron- dense granules.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Radiation sensitivity of resting and activated nonspecific cytotoxic cells of T lineage and NK lineage

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1615-1621 ◽  
Author(s):  
D Zarcone ◽  
AB Tilden ◽  
VG Lane ◽  
CE Grossi
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Radiation Sensitivity ◽  
Lak Cells ◽  
Activated T Cells ◽  
Effector Cells ◽  
Blood Lymphocytes ◽  
Nonspecific Cytotoxicity ◽  
Cytotoxic Function

Natural killer (NK) cell-mediated killing of tumor cells is a radiation- sensitive function that in most subjects is completely abrogated by treatment of the effector cells with 3,000 cGy. The radiation sensitivity of LAK (lymphokine-activated killer) cells and their precursors, the bulk of which are NK cells, is undetermined. In this study, functional cytotoxicity assays and electron microscopy were used to determine the effect of radiation on the cytotoxic function of NK cells, LAK cells (generated by three-day culture of peripheral blood lymphocytes with IL-2), and LAK cell precursors (lymphocytes irradiated prior to culture with IL-2). For comparison, we analyzed the radiation sensitivity of lectin-dependent cell-mediated cytotoxicity (LDCC), which is primarily a function of CD3+ CD8+ granular lymphocytes. We also analyzed the radiation sensitivity of nonspecific cytotoxicity mediated by mitogen-activated T cells (AK activity). Following 3,000 cGy irradiation, NK cells retained their ability to bind to tumor cell targets but, as shown by both morphologic and functional analyses, they did not undergo activation after conjugate formation, and were unable to release the content of their granules. In order to evaluate LDCC, lymphocytes were depleted of CD16+ cells and tested in a cytotoxicity assay in the presence of Con A. The radiation sensitivity curve was comparable to that of NK cell-mediated cytotoxicity. IL-2-treated lymphocytes (LAK cells) were relatively radioresistant as compared with untreated NK cells, and their cytotoxic function was not abrogated until treatment with greater than 10,000 cGy. Cells receiving such radiation doses displayed cytoplasmic blebbing and damage of their cytoskeletal structures, with disruption of centrioles and microtubules, and disarray of the intermediate filaments. As was shown with NK cells, irradiated LAK cells formed conjugates with tumor targets but failed to degranulate. The radiation sensitivity of nonspecific cytotoxicity mediated by mitogen-activated T cells was identical to that of LAK effector cells. Doses up to 2,000 cGy did not prevent generation of LAK cells from blood lymphocytes, but 3,000 cGy did so. Blast transformation similar to that observed in IL-2- stimulated controls occurred when lymphocytes irradiated with 3,000 cGy were cultured with IL-2. These transformed cells were not cytotoxic and displayed a normal cytoskeletal apparatus but did not bear electron- dense granules.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Granulocyte Colony-Stimulating Factor Effectively Mobilizes TCR γδ and NK Cells Providing an Allograft Potentially Enhanced for the Graft-Versus-Leukemia Effect for Allogeneic Stem Cell Transplantation

2021 ◽  
Vol 12 ◽  
Author(s):  
Lia Minculescu ◽  
Henrik Sengelov ◽  
Hanne Vibeke Marquart ◽  
Lars Peter Ryder ◽  
Anne Fischer-Nielsen ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Cell Compartment ◽  
Effector Cells ◽  
Graft Versus Leukemia ◽  
Hla Dr

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potential cure for patients with hematological malignancies but substantial risks of recurrence of the malignant disease remain. TCR γδ and NK cells are perceived as potent innate effector cells in HSCT and have been associated with post-transplant protection from relapse in clinical studies. Immunocompetent cells from the donor are crucial for patient outcomes and peripheral blood stem cells (PBSC) are being increasingly applied as graft source. G-CSF is the preferential mobilizing agent in healthy donors for PBSC grafts, yet effects of G-CSF on TCR γδ and NK cells are scarcely uncovered and could influence the graft composition and potency of these cells. Therefore, we analyzed T and NK cell subsets and activation markers in peripheral blood samples of 49 donors before and after G-CSF mobilization and—for a subset of donors—also in the corresponding graft samples using multicolor flowcytometry with staining for CD3, CD4, CD8, TCRαβ, TCRγδ, Vδ1, Vδ2, HLA-DR, CD45RA, CD197, CD45RO, HLA-DR, CD16, CD56, and CD314. We found that TCR γδ cells were mobilized and harvested with an efficiency corresponding that of TCR αβ cells. For TCR γδ as well as for TCR αβ cells, G-CSF preferentially mobilized naïve and terminally differentiated effector (TEMRA) cells over memory cells. In the TCR γδ cell compartment, G-CSF preferentially mobilized cells of the nonVδ2 types and increased the fraction of HLA-DR positive TCR γδ cells. For NK cells, mobilization by G-CSF was increased compared to that of T cells, yet NK cells appeared to be less efficiently harvested than T cells. In the NK cell compartment, G-CSF-stimulation preserved the proportion of CD56dim NK effector cells which have been associated with relapse protection. The expression of the activating receptor NKG2D implied in anti-leukemic responses, was significantly increased in both CD56dim and CD56bright NK cells after G-CSF stimulation. These results indicate differentiated mobilization and altering properties of G-CSF which could improve the effects of donor TCR γδ and NK cells in the processes of graft-versus-leukemia for relapse prevention after HSCT.

Immunomodulation of human natural killer cell cytotoxic function by organochlorine pesticides

2004 ◽  
Vol 23 (10) ◽  
pp. 463-471 ◽  
Author(s):  
Adrian Reed ◽  
Leticia Dzon ◽  
Bommanna G Loganathan ◽  
Margaret M Whalen
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Tumor Cells ◽  
Organochlorine Pesticides ◽  
Mononuclear Cells ◽  
Viral Infections ◽  
Nk Cell ◽  
Cell Preparation ◽  
Lytic Function ◽  
Cytotoxic Function

Organochlorine pesticides are used worldwide. To our knowledge there have been no studies dealing with the effects of these agents under in vitro conditions on human natural killer (NK) cell cytotoxic function. NK cells play a central role in immune defense against tumor development and viral infections. Thus, any agent that interferes with the ability of NK cells to lyse their targets could increase the risk of tumor incidence and/or viral infections. In this study, we examined the effects of organochlorine pesticides and some of their breakdown products on the ability of human NK cells to lyse tumor cells. A total of 11 compounds were tested. The compounds were tested in both purified NK cells as well as a cell preparation that contained other mononuclear cells (predominantly T cells) and NK lymphocytes (referred to as T/NK cells). Lymphocytes were exposed to the compounds for periods of time ranging from 1 hour to 6 days. Exposure of highly purified NK cells to 5 μ M α-chlordane, γ-chlordane, 4,4'-DDT, heptachlor, oxychlordane, or pentachlorophenol (PCP) inhibited their ability to destroy K562 tumor-cells by 88±5, 92±8, 61±13%, 64±10%, 69±11%, 76±12%, respectively, after a 24h exposure. The loss of cytotoxic function seen with α-and γ-chlordane remained essentially constant out to 6 days, while that seen with 4,4'-DDT, oxychordane and PCP increased with longer exposures (6 d). PCP was the most effective of the compounds tested at decreasing NK function. Of the compounds that caused decreased lytic function when tested in purified NK cells, only PCP and oxychordane decreased the lytic function of the T/NK cell preparation after any exposure. The results provide evidence of relative toxic potential for the 11 compounds and their immunomodulatory effects on other mononuclear cells (such as T-cells, B-cells, and monocytes) as well as NK lymphocyte function.

Lentiviral Transduction of Ex Vivo Expanded Natural Killer Cells with a CD19 Chimeric Antigen Receptor Induces Cytotoxicity against Resistant B Cell Malignancies

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3540-3540
Author(s):  
Muthalagu Ramanathan ◽  
Su Su ◽  
Andreas Lundqvist ◽  
Maria Berg ◽  
Aleah Smith ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Cytotoxicity ◽  
Gfp Expression ◽  
B Cell Malignancies ◽  
Activated T Cells ◽  
Nk Cell Cytotoxicity

Abstract NK cells play an important role in innate immunity against tumors and viral infection. NK cell cytotoxicity is suppressed by self-HLA molecules that bind and activate inhibitory killer immunoglobulin like receptors (KIRs). Expression of a CD19 chimeric receptor on NK cells could induce target specific activating signals that overcome KIR-mediated inhibition, enhancing autologous NK cell cytotoxicity against B-cell malignancies. Although HIV-1 based lentiviral vectors (LVs) have been used to efficiently transfer genes into human T-cells, little data exists on the use of LV vectors to transduce NK cells. In this study, we designed a HIV-based LV vector encoding both a CD19 chimeric antigen receptor (CAR) and green fluorescence protein (GFP) transgenes controlled by a MSCV-LTR promoter (CD19CAR LV vector) to transduce CD3−CD56+ ex vivo expanded human NK cells. The CAR consists of a single chain Fv portion of a mouse mAb against human CD19 fused to the signaling intracellular domain of a CD3 zeta subunit. CD3−CD56+NK cells were expanded ex vivo using irradiated EBV-LCL feeder cells and IL-2 containing media for 7 to 10 days. NK92 cells or expanded NK cells underwent 2 rounds of transduction with the CD19CAR LV vector in the presence of protamine sulfate using retronectin-coated plates. GFP expression measured by flow cytometry 3–4 days following LV transduction was used to assess transduction efficiencies (TE). GFP expression was detected in a mean 41% (range 27–56%) of NK92 cells and a mean 15% (range 6–40%) of ex vivo expanded NK cells. NK cell viability assessed up to 1 week following LV transduction was similar to non transduced NK cells. Following transduction, NK cells continued to expand in culture similar to non-transduced NK cells; seven days following their transduction, transduced NK cells expanded a median 30 fold while non transduced NK cells expanded a median 27 fold (p=n.s.). Cytotoxicity assays showed EBV-LCLs were resistant to killing by IL-2 activated T cells and in vitro expanded NK cells. In contrast, CD19CAR LV vector transduced NK cells were highly cytotoxic against EBV-LCLs; at 10:1 effector to target ratio (E:T), 43% of EBV-LCLs were killed by CD19CAR LV transduced NK cells versus 6% killing by non transduced NK cells (p=0.0002). NK cytotoxicity of K562 targets was not altered by CD19CAR LV transduction; at a 10:1 E: T ratio, LV transduced NK cells lysed 80% of K562 cells vs. 84% lysis by non transduced NK cells (p=n.s.). We next transduced IL-2 activated T-cells with the CD19CAR LV vector to compare their cytotoxicity to transduced NK cells against CD19+ LCLs. At a 10:1 E: T ratio, 11 % vs 1% of LCLs were killed by transduced vs non transduced T cells respectively (p=0.002). Although the TE of IL-2 activated T-cells was higher than NK cells (mean TE of 38 % vs 15% in T-cells and NK cells respectively, p=0.02), LV transduced NK cells were more cytotoxic to EBV-LCLs than transduced T-cells at the same E: T ratios. In conclusion, we show successful transduction of ex vivo expanded NK cells with a CD19CAR can be achieved using a LV vector, with CD19CAR transduced NK cells exhibiting enhanced antigen specific cytotoxicity. These findings provide both a method and rationale for clinical trials exploring the antitumor effects of adoptively infused CD19CAR LV transduced NK cells in patients with refractory B cell malignancies.

Fusion Proteins of Ligands for NKG2D and CD20-Directed ScFvs Sensitize Lymphomas for NK Cell-Mediated Lysis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2843-2843
Author(s):  
Christian Kellner ◽  
Daniela Hallack ◽  
Pia Glorius ◽  
Matthias Staudinger ◽  
Sahar Mohseni Nodehi ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Mhc Class I ◽  
Fusion Proteins ◽  
Nk Cell ◽  
Effector Cells ◽  
Healthy Donors

Abstract Abstract 2843 Natural killer group 2 member D (NKG2D) is an important activating receptor controlling cytotoxicity of natural killer (NK) cells and T cells and plays an important role in immune surveillance against tumors. For redirecting NK cells to B-lymphoid tumor cells two recombinant bifunctional antibody-based fusion proteins were designed in order to coat malignant cells with ligands for NKG2D and attract NK cells. Therefore, a human CD20-directed single-chain fragment variable (scFv) was fused to NKG2D-specific ligands, either MHC class I chain-related protein A (MICA) or unique long 16-binding protein 2 (ULBP2). These two fully human fusion proteins, designated MICA:CD20 and ULBP2:CD20, respectively, were expressed in eukaryotic cells and purified to homogeneity. Size exclusion chromatography revealed that both purified proteins predominantly formed monomers. MICA:CD20 and ULBP2:CD20 specifically and simultaneously bound to CD20 and NKG2D and efficiently mediated lysis of lymphoma cell lines with mononuclear cells from healthy donors as effector cells. Analysis of the activation status of NKG2D-positive T cells and NK cells revealed that MICA:CD20 and ULBP2:CD20 activated resting NK cells, but not T cells, indicating that NK cells were the relevant effector cell population for the two molecules. In cytotoxicity assays using human NK cells from healthy donors, both agents sensitized lymphoma cell lines as well as fresh tumor cells for NK cell-mediated lysis. MICA:CD20 and ULBP2:CD20 induced lysis at low nanomolar concentrations with half maximum effective concentrations between 1 and 4 nM depending on target cells. Interestingly, ULBP2:CD20 exhibited a higher cytolytic potential than MICA:CD20 in terms of maximum lysis. Importantly, MICA:CD20 and ULBP2:CD20 induced lysis of 13/13 tested primary tumor cell samples from patients with different B cell malignancies including chronic lymphocytic leukemia, mantle cell lymphoma and marginal zone lymphoma. Interestingly, cell surface expression of endogenous MICA and ULBP2 was low or not detectable on fresh tumor cells. In addition, ULBP2:CD20 was also capable of inducing lysis of tumor cells in cytotoxicity experiments using autologous patient-derived NK cells as effector cells, indicating that the triggering signal was sufficient to overcome inhibition by interactions between killer cell immunoglobulin-like receptors and MHC class I molecules. Moreover, both MICA:CD20 and ULBP2:CD20 synergistically enhanced antibody-dependent cellular cytotoxicity (ADCC) by the monoclonal antibody daratumumab directed against CD38 which is co-expressed together with CD20 on certain B cell lymphomas. This approach of simultaneously triggering ADCC and natural cytotoxicity by these bifunctional fusion proteins may represent a promising strategy to achieve stronger NK cell-mediated antitumor responses. Disclosures: de Weers: Genmab : Employment. van De Winkel:Genmab: Employment. Parren:Genmab: Employment.

scholarly journals Receptor-induced death in human natural killer cells: involvement of CD16.

1995 ◽  
Vol 181 (1) ◽  
pp. 339-344 ◽  
Author(s):  
J R Ortaldo ◽  
A T Mason ◽  
J J O'Shea
Keyword(s):  
T Cells ◽  
Cell Death ◽  
Nk Cells ◽  
Natural Killer ◽  
Tyrosine Kinases ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Activated T Cells ◽  
Human Natural Killer Cells ◽  
Induced Apoptosis

Propriocidal regulation of T cells refers to apoptosis induced by interleukin 2 (IL-2) activation with subsequent antigen receptor stimulation. We examined whether natural killer (NK) cells exhibited cytokine- and ligand-induced death similar to activated T cells. Peripheral NK cells were examined for ligand-induced death using antibodies to surface moieties (CD2, CD3, CD8, CD16, CD56), with and without prior activation of IL-2. Only those NK cells stimulated first with IL-2 and then with CD16 exhibited ligand-induced death; none of the other antibody stimuli induced this phenomenon. Next we examined various cytokines (IL-2, IL-4, IL-6, IL-7, IL-12, IL-13, interferon alpha and gamma) that can activate NK cells and determined if CD16-induced killing occurred. Only IL-2 and IL-12 induced NK cell death after occupancy of this receptor by aggregated immunoglobulin or by cross-linking with antireceptor antibody. The CD16-induced death was inhibited by herbimycin A, indicating that cell death was dependent upon protein tyrosine kinases. Identical to T cells, the form of cell death for NK cells was demonstrated to be receptor-induced apoptosis. Overall these data indicate that highly activated NK cells mediate ligand-induced apoptosis via signaling molecules like CD16. Whereas the propriocidal regulation of T cells is antigen specific, this is not the case for NK cells due to the nature of the receptor. The clinical implications of this finding are considered.

Antibody-dependent cell-mediated cytotoxicity through Natural Killer (NK) cells: unlocking NK cells for future immunotherapy

2021 ◽  
Vol 22 ◽  
Author(s):  
Ding Sheng Chin ◽  
Crystale Siew Ying Lim ◽  
Fazlina Nordin ◽  
Norsyahida Arifin ◽  
Tye Gee Jun
Keyword(s):  
Nk Cells ◽  
Cancer Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Therapeutic Potential ◽  
Cell Activity ◽  
Activated T Cells ◽  
Effector Cells ◽  
Adcc Assay ◽  
Dependent Cell

Background: Natural killer (NK) cells have potent effector functions that can be further improved for therapeutic purposes through antibody-dependent cell-mediated cytotoxicity (ADCC). Specific killing of virus-infected cells and cancer cells is modulated through target specific antibodies that subsequently recruit NK cells for ADCC. NK cells produce cytokines similar to activated T cells, but is less persistent as NK cells have short-lived responses. These features benefit the development of customisable and more individualised cell-based therapies. Objectives: Preclinical studies with NK cells were promising and several clinical studies are ongoing to investigate their use in antibody therapies. However, more reliable ADCC assays are required for evaluating NK cell activity to optimise therapeutic antibodies. The therapeutic potential of NK cell therapy could then be improved by harnessing ADCC. Methods: This review discuss recent studies on key components of NK cell-mediated ADCC, current clinical trials involving NK cells, ADCC assay developments and various techniques to improve ADCC. Results: Improvements can be made to NK-mediated ADCC through modifications of antibodies, effector cells and target antigens. Different aspects of antibodies were studied extensively, including modifying glycosylation patterns, novel production methods, combination regiments, bispecific antibodies, and conjugated antibodies. Modification of NK cells and tumour surface markers could improve ADCC of even treatment-resistant cancer cells. Additives such as cytokines and other immunomodulatory agents can further augment ADCC to supplement NK cell-based therapies.

Perforin Gene Mutations in Patients with Acquired Aplastic Anemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 998-998
Author(s):  
Elena E. Solomou ◽  
Federica Gibellini ◽  
Stephen J. Chanock ◽  
Daniela Malide ◽  
Maria Berg ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Bone Marrow Failure ◽  
Gene Mutations ◽  
Activated T Cells ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes ◽  
Prf1 Gene

Abstract 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.

Donor’s NK Cells May Influence the Engraftment in Pediatrics Patients after T-Cell Depleted Haploidentical Stem Cell Transplant for Thalassemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4595-4595
Author(s):  
Antonella Isgro ◽  
Marco Marziali ◽  
Pietro Sodani ◽  
Javid Gaziev ◽  
Paola Polchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Hla Class I ◽  
Mixed Chimerism ◽  
Effector Cells ◽  
Post Transplant

Abstract In haploidentical hematopoietic transplantation, donor-versus-recipient NK cell alloreactivity derives from a mismatch between donor NK clones bearing inhibitory Killer Cell Ig-like Receptors (KIRs) for self HLA class I molecules and their HLA class I ligands (KIR ligands) on recipient cells. The mechanism whereby alloreactive NK cells exert their benefits in transplantation has been elucidated. The infusion of alloreactive NK cells ablates recipient T cells which reject the graft, and ablates recipient dendritic cells (DCs) which trigger GvHD, thus protecting from GvHD (Ruggeri et al., Science 2002). NK cell alloreactivity also boosts very rapid rebuilding of donor adaptive immunity to infections. In this study we analysed the potential role of NK cells after haploidentical transplant in b-thalassemia patients. T and B cell depletion was carried out with CD34+ coated magnetic microbeads and the CliniMACS device (Miltenyi Biotec©) from peripheral blood and bone marrow of donors (the mothers) and resulted in grafts consisting of stem cells and effector cells (NK cells, monocytes) with the addition of bone marrow mononuclear cells (BMMNCs 3 × 105/kg of the recipient). A total of 11 pediatric patients with b-thalassemia received T and B cell depleted transplants from their haploidentical mothers with a median number of 15 ×106 CD34 stem cells. To analyse the mechanisms involved in immunological reconstitution post transplant, we analysed T cell subsets by flow cytometry, particularly NK sets (CD3- CD56+, CD3− CD16+ and CD56+CD16+ NK cells) at day + 20 and + 60 post transplant. Day + 20 post transplant, the patients had significantly lower CD4+ T cells in comparison to the controls (1.9 ± 1.4% vs. 47.5 ± 6% respectively), whereas CD8+ T cells numbers did not statistically differ between patients and controls (24.2 ± 33.7% vs. 20 ± 7%). NK cells were among the first lymphocytes to repopulate the peripheral blood, and up to 70% of these cells were CD3-CD56+bright cells. Interestingly, a direct correlation has been observed between the percentages of CD56+CD16+ NK subset and the BM engraftment (in mean 71 ± 21% CD56+CD16+ in the four patients with full engraftment, 27 ± 28% in the three patients with a stable mixed chimerism after BM transplant (70–80% of donor cells) and 1.4 ± 1% in the four patients with rejection). In all the patients the origin of the NK subsets was from the mothers. Day + 60 post transplant an increase in the percentages of CD4+ T cells, naïve CD4+ cells and in thymic naïve Th cells were observed (3 ± 1.2%, 2.9 ± 2.1%, 2.7 ± 1%, respectively). CD8+ T cells were also increased (in mean 35 ± 27.5%), in parallel with the increase of the CD3-CD16+ NK cells (potent cytotoxic effector cells) especially in the patients with full engraftment (in mean 47 ± 20% vs. 28 ± 31% in mixed chimerism) NK CD56+bright cells develop more rapidly than other lymphocytes, but CD16+ NK cells (with cytotoxic potential) require more prolonged exposure to maturation factor (IL-2) in the bone marrow. Interestingly we observed higher percentages of NK subsets just twenty days post transplant in the patients with full engraftment respect the mixed chimerism and the rejection, suggesting a role of donor NK cells on improved engraftment and on prevention of the rejection with the attack of the host lympho-hematopoietic cells. These observations may suggest the importance of NK subsets analyses at the first time of the transplant as an useful parameter for the outcome of the transplant and/or the use of donor’s alloreactive NK cells especially in haploidentical recipients.

scholarly journals The Role of DNAM-1/Tigit Axis of NK Cells in the Regulation of Alloreactive T Cell Responses and the Potential Mechanism

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4530-4530
Author(s):  
Lixia Sheng ◽  
Guifang Ouyang ◽  
MU Qitian ◽  
He Huang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Activated T Cells ◽  
Over Expression ◽  
Lentiviral Transduction ◽  
Cell Responses ◽  
Alloreactive T Cell

Abstract Objective: Previous studies has showed the important role of NK cell in the regulation of alloreactive T cell response and negative regulation of GVHD. The paired receptors DNAM-1 and TIGIT, which bind the same ligands but have opposite effects on NK cell function, might present as potential targets for the GVHD treatment. This study is designed to explore the role of TIGIT/DNAM-1 balance of NK cells in the regulation of alloreactive T cell responses and the potential mechanism. Methods: Antibodies blocking of TIGIT or DNAM-1, over-expression of TIGIT or DNAM-1 via lentiviral transduction and knockdown of TIGIT or DNAM-1 by lentiviral shRNA were used to manipulate the TIGIT/DNAM-1 balance on NK cell. Cytotoxicity assay using alloantigen activated T cells as targets were used to evaluate the regulating function of NK cell on alloreactive T cell responses. Western blot and small molecule inhibitors against PI3K were combined to investigate whether the PI3K-Akt-ERK signaling cascade is involved in the signal transduction process following TIGIT/DNAM-1-PVR engagement. RESULTS: Blocking of DNAM-1 by an anti-DNAM-1 antibody and knockdown of DNAM-1 expression by lentiviral shRNA both resulted in deceased cytotoxicity of NK cells against alloantigen activated T cells, while over-expression of DNAM-1 via lentiviral transduction resulted in enhanced cytotoxicity. Blocking of TIGIT by an anti-TIGIT antibody and knockdown of TIGIT expression by lentiviral shRNA both resulted in increased cytotoxicity of NK cells against alloantigen activated T cells, while over-expression of TIGIT via lentiviral transduction resulted in decreased cytotoxicity. Increases in NK cytotoxicity against activated T cells through TIGIT knockdown could be overcome by blocking DNAM-1 signaling. Simultaneously, over-expression of DNAM-1 or knockdown of TIGIT expression resulted in an increase of the phosphorylation levels of Akt and ERK1/2 in NK cells after contacted with activated T cells, which could be overcome by pretreating NK cells with anti-DNAM-1 or PI3K small molecule inhibitor. Pretreating alloantigen activated T cells with anti-PVR also resulted in deceased cytotoxicity and Akt and ERK1/2 phosphorylation in DNAM-1 over-expression NK cells. Conclusion: The paired receptor DNAM-1/TIGIT on the surface of NK cells compete the same PVR ligand on the surface of activated T cells and the DNAM-1/TIGIT axis is involved in the regulation of cytotoxicity of NK cells on alloantigen activated T cells through PI3K-Akt-ERK cascade phosphorylation. The DNAM-1/TIGIT expression balance may present as biomarkers for aGVHD and potential targets for aGVHD therapy. Disclosures No relevant conflicts of interest to declare.

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