Modulation of Mesenchymal Stem Cell MHC-I Complex Increases Engraftment In Vivo.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1457-1457
Author(s):  
Melisa Soland ◽  
Evan J Colletti ◽  
Mariana Bego ◽  
Chad Sanada ◽  
Christopher D Porada ◽  
...  
Keyword(s):  
Nk Cells ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Large Animal ◽  
Specific Lysis ◽  
Fetal Sheep ◽  
Mhc I ◽  
Npt Ii

Abstract Abstract 1457 Mesenchymal stem cells (MSC) are good candidates for cell therapies due to their immunomodulatory properties, ability to home to/engraft damaged tissues, and potential to differentiate into different cell types. However, when transplanted (Tx) in an allogeneic setting, MSC can elicit an immune response, activating the recipient's cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells, resulting in rejection of the Tx cells and reduced therapeutic efficacy. Human cytomegalovirus (HCMV, has developed several strategies to evade CTL and NK cell recognition. HCMV avoids CTL attack by producing proteins that downregulate MHC-I surface expression. These proteins are coded for by the unique short regions (US) 2, 3, 6 and 11 of HCMV's genome. We have previously shown that when MSC are transduced with retroviral vectors encoding each one of these US proteins, US6 and US11 were the most effective in reducing MSC's HLA-I surface expression and allogeneic CTL recognition and proliferation. However, HLA-I downregulation may render MSC transduced with US6 (MSC-US6) and US11 (MSC-US11) more susceptible to NK killing, undermining MSC's inherent ability to inhibit function of allogeneic NK cells. Here, we first investigated the role of US6 or US11 on MSC allorecognition by NK cells, and on MSC in vivo engraftment capability. NK killing assays demonstrated that US11 generated the most protective effect at the highest NK concentration (E:T ratio 20:1) (% specific lysis for MSC-US6: 60.4 ± 5.7 %; MSC-US11: 45.5 ± 2.4 % vs. MSC: 88.5 ± 3.4 % respectively). However, at an E:T ratio of 10:1 and 5:1 US11 produced the same degree of protection as US6 (E:T ratio of 10:1; % specific lysis for MSC-US6: 30.1 ± 5.6 %; MSC-US11: 26.3 ± 1.9 % vs. MSC: 54.7 ± 1.9 %); (E:T ratio 5:1; % specific lysis for MSC-US6: 11.9 ± 4.2; MSC-US11: 13.4 ± 2.3; vs. MSC: 25.5 ± 4 respectively). Only at an E:T ratio of 1:1 were US6 and US11 similar to untransduced MSCs (% specific lysis for MSC-US6: 4.7 ± 1.6; MSC-US11: 2.1 ± 0.5; vs. MSC: 4.9 ± 1.8; respectively) in terms of inhibition of NK killing. We also studied the role of US6 and 11 on the expression of beta-2-microglobulin (b2m) and other HLA-I molecules, and we found that US6 reduced b2m by 87± 2 % and HLA-G1 by 44±4.7 %, while US11 reduced b2m by 70± 0.6 % but increased HLA-G1 expression by 176.6±1.9 %. Therefore, the increase in HLA-G1 expression induced by US11 may explain the decrease in NK killing observed in the MSC-US11 cells. Furthermore, we investigated whether US6 or US11 could play a role in mediating complement resistance. While US6 increased the expression of CD59 in transduced cells (Mean fluorescence intensity (MFI) increased by 123.3±1), US11 increased the number of cells expressing CD59 by 121.4 ± 0.8 %, but did not modify their MFI. We next compared the in vivo engraftment potential of MSC, MSC-US6 and MSC-US11 by Tx 5.6×10^4 of each cell population into fetal sheep at 60 days of gestation (n=6). Since we have previously reported the ability of MSC to generate liver cells, we first investigated whether the expression of US6 and 11 would allow higher levels of liver engraftment and hepatocyte formation when compared to MSC (MSC-E) transduced with a retroviral vector encoding only NPT-II. Two months after Tx, liver tissues were collected and stained with NPT-II antibody. This revealed that US6 and US11 increased engraftment efficiency by 241% for MSC-US6 and 277% for MSC-US11 (MSC-E: 5.3 ± 0.4 %, MSC-US6: 12.8 ± 0.9 % and MSC-US:11 14.7 ± 0.8 %). Despite the higher level of liver engraftment seen with MSC-US6 and MSC-US11, co-expression of NPT-II and albumin (MSC-US6: 57% MSC-US1: 50% MSC-E: 75%) or NPT-II and Ov-6 was found at significantly lower levels in MSC-US11 and MSC-US6 Tx animals than in those Tx with MSC-E. Nevertheless, similar numbers of NPT-II/CD34 double-positive cells were found in the liver of MSC-US6 and MSC-US11 Tx animals when compared to MSC-E alone. In conclusion, engineering MSC to over-express US6 or US11 is an effective way to reduce CTL proliferation, NK killing and destruction of engrafted cells by the complement membrane attack complex. In agreement with the in vitro studies, transplantation of these cells into a large animal sheep model resulted in significantly higher levels of overall cell engraftment, but not differentiation towards a hepatocytic phenotype. Studies are underway to determine the mechanism by which HCMV proteins are interfering with MSC differentiation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A herpesvirus encoded Qa-1 mimic inhibits natural killer cell cytotoxicity through CD94/NKG2A receptor engagement

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Xiaoli Wang ◽  
Sytse J Piersma ◽  
Christopher A Nelson ◽  
Ya-Nan Dai ◽  
Ted Christensen ◽  
...  
Keyword(s):  
Nk Cells ◽  
Rna Splicing ◽  
Killer Cell ◽  
Surface Expression ◽  
Inhibitory Receptors ◽  
Mhc I ◽  
Natural Killer Cell Cytotoxicity ◽  
Self Recognition ◽  
Missing Self

A recurrent theme in viral immune evasion is the sabotage of MHC-I antigen presentation, which brings virus the concomitant issue of ‘missing-self’ recognition by NK cells that use inhibitory receptors to detect surface MHC-I proteins. Here, we report that rodent herpesvirus Peru (RHVP) encodes a Qa-1 like protein (pQa-1) via RNA splicing to counteract NK activation. While pQa-1 surface expression is stabilized by the same canonical peptides presented by murine Qa-1, pQa-1 is GPI-anchored and resistant to the activity of RHVP pK3, a ubiquitin ligase that targets MHC-I for degradation. pQa-1 tetramer staining indicates that it recognizes CD94/NKG2A receptors. Consistently, pQa-1 selectively inhibits NKG2A+ NK cells and expression of pQa-1 can protect tumor cells from NK control in vivo. Collectively, these findings reveal an innovative NK evasion strategy wherein RHVP encodes a modified Qa-1 mimic refractory to MHC-I sabotage and capable of specifically engaging inhibitory receptors to circumvent NK activation.

VAMP-7 Interacts With The Actin Cytoskeleton To Control Platelet Spreading and Mediate α-Granule Exocytosis During Thrombus Formation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1058-1058
Author(s):  
Secil Koseoglu ◽  
Jennifer L Fitch-Tewfik ◽  
Christian G. Peters ◽  
Lydia Danglot ◽  
Thierry Galli ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Surface Expression ◽  
Wild Type ◽  
Granule Exocytosis ◽  
Granule Secretion ◽  
Platelet Spreading

Abstract Platelet granule secretion is important not only for hemostasis and thrombosis, but also for a variety of physiological processes including inflammation, angiogenesis and malignancy. Vesicle Associated Membrane Proteins (VAMPs) are a group of v-SNARE proteins resident on the platelet granule surface that participate in granule secretion. Platelets contain several VAMP isoforms including VAMP-2, VAMP-3, VAMP-7, and VAMP-8. VAMP-7 is unique in that it contains an N-terminal profilin-like longin domain. Previous work by our group demonstrated spatial segregation of granules expressing different VAMPs during platelet spreading. Granules expressing VAMP-3 and VAMP-8 localized to the granulomere of spreading platelets, while those expressing VAMP-7 moved towards the periphery. Based on this observation, we proposed that VAMP-7+ granules move to the periphery of the spreading platelet to add membrane to growing actin structures. To assess this hypothesis, platelets from VAMP-7 null mice were used to analyze the role of VAMP-7 in platelet spreading, aggregation and secretion. VAMP-7 null platelets were normal in size, shape, and number. When compared to wild-type platelets, VAMP-7 null platelets did not show any defects in aggregation upon exposure to increasing doses of the PAR4 agonist peptide, AYPGKF, or collagen. In contrast, the surface area of VAMP-7 null platelets following 15 min of spreading on poly-L-lysine was only 51% that of wild-type of platelets (P < 0.05). To assess mechanisms of the movement of VAMP-7 to the platelet periphery, the association of VAMP-7 to the Triton X-100-insoluble platelet cytoskeleton was evaluated and results showed that VAMP-7 associated with the actin cytoskeleton. Moreover, VAMP-7 null platelets showed impaired P-selectin surface expression and PF4 secretion at low concentrations of AYPGKF. TIMP-2 and VEGF localize to VAMP-7 expressing granules in the periphery of spread platelets. We therefore evaluated the secretion of TIMP-2 and VEGF from VAMP-7 null platelets. Secretion of TIMP-2 and VEGF was reduced even at saturating doses of agonist (300 mM AYPGKF). To examine the role of VAMP-7 in a-granule exocytosis during platelet activation in vivo, PF4 release was monitored following laser-induced injury of cremaster arterioles. Platelet accumulation at sites of laser injury was identical in wild-type and VAMP-7 null mice. In wild-type mice, PF4 was secreted by activated platelets and bound back to activated endothelium and platelets producing a localized concentration of PF4 that accumulated over 15 min following injury. PF4 release from platelets lacking VAMP-7 was decreased to 47% of that of control. These results demonstrate that VAMP-7 interacts with the actin cytoskeleton and functions selectively in a-granule exocytosis. VAMP-7 associates with the actin cytoskeleton and functions during platelet spreading, adding further support to the premise that membrane fusion occurring during granule secretion is an essential component of normal platelet spreading. This VAMP-7 mediated, actin-dependent mechanism of secretion is not important for platelet thrombus formation, but rather functions in the release of particular granular contents, such as PF4, at sites of vascular injury. Disclosures: No relevant conflicts of interest to declare.

Modulation of Mesenchymal Stem Cell Immunogenicity through Forced Expression of Human Cytomegalovirus Proteins

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2416-2416
Author(s):  
Melisa Soland ◽  
Mariana Bego ◽  
Christopher D Porada ◽  
Esmail D. Zanjani ◽  
Stephen St Jeor ◽  
...  
Keyword(s):  
Immune System ◽  
Human Cytomegalovirus ◽  
Nk Cell ◽  
Membrane Attack Complex ◽  
Surface Expression ◽  
Large Animal ◽  
Cell Recognition ◽  
Mhc I ◽  
Nk Cell Recognition

Abstract Mesenchymal stem cells (MSC) are promising candidates for cell replacement therapy since they have the ability to differentiate, under appropriate conditions, into a broad range of specialized cell types. Furthermore, MSC have low inherent immunogenicity, immunomodulatory properties, and preferentially home to/engraft damaged tissues. However, they are not invisible to the immune system, and upon allogeneic transplantation, MSC can elicit an immune response, that results in activation of the recipient’s cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells and hence rejection of the transplanted cells. Human cytomegalovirus (HCMV), a virus ubiquitously present in humans, has developed several strategies to evade CTL and NK cell recognition. HCMV avoids CTL attack by producing proteins coded for by the unique short region (US) of the genome that downregulate MHC-I surface expression. Moreover, expression of HCMV UL18 protein, which is an MHC-I homolog or decoy, renders CMV-infected cells resistant to NK lysis. Thus, we hypothesized that simultaneous expression of HCMV US protein US2, US3, US6, or US11 with UL18 would render MSC less susceptible to CTL and NK cell recognition and killing. To this end, we started by transducing MSC with the retroviral vectors encoding: US2; US3; US6; or US11, and tested the effect of each of these proteins on expression of MHC-I and CD59, a complement membrane attack complex inhibitor whose expression has been shown to prevent destruction of transplanted cells. MSC expressing US3, US2, US11 and US6 showed substantial MHC-I downregulation of 69%, 74%, 78 % and 95% respectively, when compared to untransduced MSC. Moreover, MSC expressing either US6 or US2 proteins displayed a significant up-regulation of CD59, with US6 causing a 2.5 and US2 a 3.9 fold increase, when compared to untransduced MSC (Mean Fluorescence Intensity: 869.8; 1331.19; 344.99, respectively). Each of the US transduced MSC were then tested for their capability to activate MHC-I mismatched CTL proliferation in both an allogeneic and a xenogeneic setting, using MSC transduced with an empty retrovirus as a control. MSC expressing US2 or US6 reduced CTL proliferation by the same degree, whether CTL were mismatched human- or sheep-derived. Using MSC-US2 we obtained a reduction in CTL proliferation by 18% and 23% in human-or sheep-CTL, respectively, while using MSC-US6 resulted in a reduction in human and sheep CTL proliferation of 60% and 53%. Expression of US11 on MSC was more efficient at abrogating the sheep CTL proliferation than that of mismatched human CTL (reduction in proliferation by 53% vs 31%, respectively). Finally, expression of US3 on MSC only reduced proliferation of human CTL by 21.5%, but had no effect on sheep CTL. In conclusion, engineering MSC to over-express US6 seems to be the most effective way to enable MSC to evade the host immune system, resulting in efficient downregulation of MHC-I surface expression, significant reduction in CTL proliferation, and a reduced formation of the membrane attack complex at the end of the complement cascade, which will reduce destruction of MSC by this system. In vivo studies using a large animal sheep model are underway to demonstrate whether the results obtained in vitro using US6 HCMV protein expression will translate into improved engraftment in vivo.

Impact of β2 integrin deficiency on mouse natural killer cell development and function

Blood ◽  
2011 ◽  
Vol 117 (10) ◽  
pp. 2874-2882 ◽  
Author(s):  
Karine Crozat ◽  
Céline Eidenschenk ◽  
Baptiste N. Jaeger ◽  
Philippe Krebs ◽  
Sophie Guia ◽  
...  
Keyword(s):  
Cell Surface ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Surface Expression ◽  
Cell Maturation ◽  
Cell Surface Expression ◽  
Mcmv Infection ◽  
Β2 Integrin

Abstract Natural killer (NK) cells are innate immune cells that express members of the leukocyte β2 integrin family in humans and mice. These CD11/CD18 heterodimers play critical roles in leukocyte trafficking, immune synapse formation, and costimulation. The cell-surface expression of one of these integrins, CD11b/CD18, is also recognized as a major marker of mouse NK-cell maturation, but its function on NK cells has been largely ignored. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated a mouse carrying an A → T transverse mutation in the Itgb2 gene, resulting in a mutation that prevented the cell-surface expression of CD18 and its associated CD11a, CD11b, and CD11c proteins. We show that β2 integrin–deficient NK cells have a hyporesponsive phenotype in vitro, and present an alteration of their in vivo developmental program characterized by a selective accumulation of c-kit+ cells. NK-cell missing-self recognition was partially altered in vivo, whereas the early immune response to mouse cytomegalovirus (MCMV) infection occurred normally in CD18-deficient mice. Therefore, β2 integrins are required for optimal NK-cell maturation, but this deficiency is partial and can be bypassed during MCMV infection, highlighting the robustness of antiviral protective responses.

scholarly journals Soluble and Exosome-Bound α-Galactosylceramide Mediate Preferential Proliferation of Educated NK Cells with Increased Anti-Tumor Capacity

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 298
Author(s):  
Arnika K. Wagner ◽  
Ulf Gehrmann ◽  
Stefanie Hiltbrunner ◽  
Valentina Carannante ◽  
Thuy T. Luu ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cancer Immunotherapy ◽  
Nk Cell ◽  
Mouse Strains ◽  
Target Cells ◽  
Class I Mhc ◽  
Mhc I ◽  
Cell Responses ◽  
Missing Self

Natural killer (NK) cells can kill target cells via the recognition of stress molecules and down-regulation of major histocompatibility complex class I (MHC-I). Some NK cells are educated to recognize and kill cells that have lost their MHC-I expression, e.g., tumor or virus-infected cells. A desired property of cancer immunotherapy is, therefore, to activate educated NK cells during anti-tumor responses in vivo. We here analyze NK cell responses to α-galactosylceramide (αGC), a potent activator of invariant NKT (iNKT) cells, or to exosomes loaded with αGC. In mouse strains which express different MHC-I alleles using an extended NK cell flow cytometry panel, we show that αGC induces a biased NK cell proliferation of educated NK cells. Importantly, iNKT cell-induced activation of NK cells selectively increased in vivo missing self-responses, leading to more effective rejection of tumor cells. Exosomes from antigen-presenting cells are attractive anti-cancer therapy tools as they may induce both innate and adaptive immune responses, thereby addressing the hurdle of tumor heterogeneity. Adding αGC to antigen-loaded dendritic-cell-derived exosomes also led to an increase in missing self-responses in addition to boosted T and B cell responses. This study manifests αGC as an attractive adjuvant in cancer immunotherapy, as it increases the functional capacity of educated NK cells and enhances the innate, missing self-based antitumor response.

scholarly journals Resistance to Acute Babesiosis Is Associated with Interleukin-12- and Gamma Interferon-Mediated Responses and Requires Macrophages and Natural Killer Cells

2003 ◽  
Vol 71 (4) ◽  
pp. 2002-2008 ◽  
Author(s):  
Irma Aguilar-Delfin ◽  
Peter J. Wettstein ◽  
David H. Persing
Keyword(s):  
Nk Cells ◽  
Gamma Interferon ◽  
Interleukin 12 ◽  
No Production ◽  
Cytokine Responses ◽  
Early Production ◽  
Ifn Γ ◽  
Crucial Part

ABSTRACT We examined the role of the cytokines gamma interferon (IFN-γ) and interleukin-12 (IL-12) in the model of acute babesiosis with the WA1 Babesia. Mice genetically deficient in IFN-γ-mediated responses (IFNGR2KO mice) and IL-12-mediated responses (Stat4KO mice) were infected with the WA1 Babesia, and observations were made on the course of infection and cytokine responses. Levels of IFN-γ and IL-12 in serum increased 24 h after parasite inoculation. The augmented susceptibility observed in IFNGR2KO and Stat-4KO mice suggests that the early IL-12- and IFN-γ-mediated responses are involved in protection against acute babesiosis. Resistance appears to correlate with an increase in nitric oxide (NO) production. In order to assess the contribution of different cell subsets to resistance against the parasite, we also studied mice lacking B cells, CD4+ T cells, NK cells, and macrophages. Mice genetically deficient in B lymphocytes or CD4+ T lymphocytes were able to mount protective responses comparable to those of immunosufficient mice. In contrast, in vivo depletion of macrophages or NK cells resulted in elevated susceptibility to the infection. Our observations suggest that a crucial part of the response that protects from the pathogenic Babesia WA1 is mediated by macrophages and NK cells, probably through early production of IL-12 and IFN-γ, and induction of macrophage-derived effector molecules like NO.

scholarly journals Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice

2011 ◽  
Vol 300 (5) ◽  
pp. L781-L789 ◽  
Author(s):  
Szabolcs Bertok ◽  
Michael R. Wilson ◽  
Anthony D. Dorr ◽  
Justina O. Dokpesi ◽  
Kieran P. O'Dea ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Adhesion Molecules ◽  
Surface Expression ◽  
Tnf Receptor ◽  
Wild Type ◽  
Tnf Receptors ◽  
Pulmonary Endothelial Cells ◽  
Microvascular Inflammation

TNF plays a crucial role in the pathogenesis of acute lung injury. However, the expression profile of its two receptors, p55 and p75, on pulmonary endothelium and their influence on TNF signaling during lung microvascular inflammation remain uncertain. Using flow cytometry, we characterized the expression profile of TNF receptors on the surface of freshly harvested pulmonary endothelial cells (PECs) from mice and found expression of both receptors with dominance of p55. To investigate the impact of stimulating individual TNF receptors, we treated wild-type and TNF receptor knockout mice with intravenous TNF and determined surface expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1) on PECs by flow cytometry. TNF-induced upregulation of all adhesion molecules was substantially attenuated by absence of p55, whereas lack of p75 had a similar but smaller effect that varied between adhesion molecules. Selective blockade of individual TNF receptors by specific antibodies in wild-type primary PEC culture confirmed that the in vivo findings were due to direct effects of TNF receptor inhibition on endothelium and not other cells (e.g., circulating leukocytes). Finally, we found that PEC surface expression of p55 dramatically decreased in the early stages of endotoxemia following intravenous LPS, while no change in p75 expression was detected. These data demonstrate a crucial in vivo role of p55 and an auxiliary role of p75 in TNF-mediated adhesion molecule upregulation on PECs. It is possible that the importance of the individual receptors varies at different stages of pulmonary microvascular inflammation following changes in their relative expression.

scholarly journals PD-1 expression on NK cells can be related to cytokine stimulation and tissue residency

2021 ◽  
Author(s):  
Arnika K Wagner ◽  
Nadir Kadri ◽  
Chris Tibbitt ◽  
Koen van de Ven ◽  
Sunitha Bagawath-Singh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Mhc Class I ◽  
Sequencing Analysis ◽  
Single Cell Rna Sequencing ◽  
Cytokine Stimulation ◽  
Deficient Mice

ABSTRACTAlthough PD-1 was shown to be a hallmark of T cells exhaustion, controversial studies have been reported on the role of PD-1 on NK cells. Here, we found by flow cytometry and single cell RNA sequencing analysis that PD-1 can be expressed on MHC class I-deficient tumor-infiltrating NK cells in vivo. We also demonstrate distinct alterations in the phenotype of PD-1-deficient NK cells which in part could be attributed to a decrease in tumor-infiltrating NK cells in PD-1-deficient mice. NK cells from PD-1-deficient mice exhibited a more mature phenotype which might reduce their capacity to migrate and kill in vivo. Finally, our results demonstrate that PD-L1 molecules in membranes of PD-1-deficient NK cells migrate faster than in NK cells from wildtype mice, suggesting that PD-1 and PD-L1 form cis interactions with each other on NK cells.

scholarly journals A Novel Method to Study the in Vivo Trafficking and Homing of Adoptively Transferred NK Cells in Rhesus Macaques and Humans

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 659-659 ◽  
Author(s):  
Jan Davidson-Moncada ◽  
Noriko Sato ◽  
Robert F Hoyt ◽  
Robert N Reger ◽  
Marvin Thomas ◽  
...  
Keyword(s):  
Nk Cells ◽  
Adoptive Transfer ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Raji Cell ◽  
K562 Cells ◽  
Ct Imaging ◽  
Pet Ct ◽  
Hematological Cancers

Abstract Adoptive transfer of allogeneic or autologous natural killer (NK) cells is now being developed for therapy of both hematological and solid malignancies. The efficacy of NK immunotherapy to mediate anti-tumor effects will ultimately be dependent on their ability to traffic and home to the tumor microenvironment. Recent data suggest expanded NK cells are ineffective at homing to the bone marrow (BM) and lymph nodes (LN) where hematological malignancies reside. A variety of techniques to maintain and/or enforce expression of homing receptors in NK cells are now being explored in preclinical models to improve their localization to the BM and LN. Historically, xenogeneic human into mouse or mouse into mouse models have been utilized for preclinical development of adoptive NK transfer. These experiments often use fluorescent dye-labeled NK cells and require repeated invasive biopsies, which can be confounded by sampling error, or the requirement for post mortem analysis. Here we present a method to track in real time and in vivo adoptively infused zirconium-89 (89Zr) labelled NK cells by PET imaging. A rhesus macaque (RM) model was used for these preclinical experiments as RM and human NK cells have similar expansion kinetics, and have greater similarity than mice in their phenotype, function, and homing receptors and ligands. PBMCs collected from the PB of 13 RMs were enriched for NK cells by CD3+ T-cell depletion and were then expanded for 14 days by culturing with irradiated human EBV-LCL cells in X-VIVO 20 media containing 10% human AB serum and 500 IU/μl of human IL-2. RM NK cells expanded a mean 145±41 fold and contained >99% pure CD3- and CD56+ cells. The phenotype and tumor cytotoxicity of RM NK cells were similar to NK cells expanded from humans (n=3) using similar expansion cultures; at a 10:1 E:T ratio, 67% and 73% of K562 cells were lysed by RM and human NK cell respectively. To label NK cells, 89Zr was conjugated to oxine, which readily permeabilized the cellular membrane and was retained in the cells. Expanded NK cells from both humans and RM showed no changes in CD16 or CD56 expression for up to 6 days following radiolabeling. Human and RM NK cell viability 0 to 24 hours following radiolabelling was 60-100% then declined to 20-30% after 6 days. 89Zr retention by both human and RM NK cells was 75-80% in the first 24 hours of culture but gradually declined with time, decreasing to 20-30% after 7 days of culture. Culturing radiolabeled human NK cells for 24-36 hours with different cellular populations including Ramos and Raji cell lines and normal human PBMCs revealed no significant transfer of radioactivity (max 2% above baseline), establishing that 89Zr was not transferred from labeled to unlabeled cells. Oxine labeling did not alter the cytotoxicity of human or RM NK cells vs K562 cells compared to unlabeled controls. 89Zr-oxine labeling of expanded RM NK cells is currently being used to quantify NK cell trafficking and survival following adoptive transfer in autologous macaques. In these experiments, RM recipients of adoptively infused 89Zr labeled NK cells receive concurrent deferoxamine to chelate and then enhance renal excretion of any free 89Zr that is released from dead cells. In the experiments shown below, 13 x 107 autologous ex vivo expanded 89Zr-labeled RM NK cells were injected IV into a 5.7 kg RM and tracked by sequential PET/CT imaging for 7 days. Up to 1-hour post infusion, most NK cell activity was restricted to the lungs. By 4 hours, NK cells began to traffic from the lungs to the liver and spleen. By 2 days, NK cells were no longer detectable in the lungs and resided largely in the liver and spleen, where they remained for the remainder of the 7 day imaging period. During the entire observation period, little to no NK cell radioactivity was detected in the LN or BM. In conclusion, 89Zr oxine labelling of NK cells followed by PET/CT imaging represents a powerful tool to track the in vivo fate of adoptively transferred NK cells. The RM model presented here provides a method to evaluate and optimize various strategies aimed at altering the phenotype of NK cells, with the goal of improving their homing to the BM and LN where hematological cancers reside. These preclinical in vitro and in vivo data suggest this technology could be safely extended to humans and could be applied to other cellular populations besides NK cells. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals MEC-2 and MEC-6 in the Caenorhabditis elegans Sensory Mechanotransduction Complex: Auxiliary Subunits that Enable Channel Activity

2008 ◽  
Vol 131 (6) ◽  
pp. 605-616 ◽  
Author(s):  
Austin L. Brown ◽  
Zhiwen Liao ◽  
Miriam B. Goodman
Keyword(s):  
Caenorhabditis Elegans ◽  
Single Channel ◽  
Divalent Cations ◽  
Surface Expression ◽  
Homologous Proteins ◽  
Auxiliary Subunits ◽  
Single Channel Currents ◽  
Cholesterol Binding

The ion channel formed by the homologous proteins MEC-4 and MEC-10 forms the core of a sensory mechanotransduction channel in Caenorhabditis elegans. Although the products of other mec genes are key players in the biophysics of transduction, the mechanism by which they contribute to the properties of the channel is unknown. Here, we investigate the role of two auxiliary channel subunits, MEC-2 (stomatin-like) and MEC-6 (paraoxonase-like), by coexpressing them with constitutively active MEC-4/MEC-10 heteromeric channels in Xenopus oocytes. This work extends prior work demonstrating that MEC-2 and MEC-6 synergistically increase macroscopic current. We use single-channel recordings and biochemistry to show that these auxiliary subunits alter function by increasing the number of channels in an active state rather than by dramatically affecting either single-channel properties or surface expression. We also use two-electrode voltage clamp and outside-out macropatch recording to examine the effects of divalent cations and proteases, known regulators of channel family members. Finally, we examine the role of cholesterol binding in the mechanism of MEC-2 action by measuring whole-cell and single-channel currents in MEC-2 mutants deficient in cholesterol binding. We suggest that MEC-2 and MEC-6 play essential roles in modulating both the local membrane environment of MEC-4/MEC-10 channels and the availability of such channels to be gated by force in vivo.

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