scholarly journals Natural Killer (NK) Cells Are Functionally Abnormal and NK Cell Progenitors Are Diminished in Granulocyte Colony-Stimulating Factor–Mobilized Peripheral Blood Progenitor Cell Collections

Blood ◽  
1997 ◽  
Vol 90 (8) ◽  
pp. 3098-3105 ◽  
Author(s):  
Jeffrey S. Miller ◽  
Felipe Prosper ◽  
Valarie McCullar

Abstract Granulocyte colony-stimulating factor (G-CSF)–mobilized peripheral blood progenitor cell (PBPC) collections are increasingly emerging as the graft of choice in many centers for autologous transplantation, and with increasing frequency for allogeneic transplantation. However, the role of myeloid cytokines in lymphoid function, lymphoid progenitors, and immune-mediated antitumor responses is not known. We studied PBPC collections from normal donors mobilized with G-CSF (10 μg/kg). CD56+/CD3− natural killer (NK) cells sorted from PBPC products exhibited a diminished ability to kill tumor targets, were less responsive in acquiring increased cytolysis with interleukin-2 (IL-2), and proliferated less than NK cells from normal unprimed peripheral blood. This abnormality was not explained by a change in phenotype of NK cells normally circulating in the blood after G-CSF administration. We could not demonstrate any direct suppressive effect on normal unprimed NK cell proliferation or cytotoxicity by culture with pharmacologic concentrations of G-CSF. We next evaluated the effects of G-CSF on CD34+ NK cell progenitors. CD34+/CD2+, CD34+/CD7+, and CD34+/CD10+ progenitors were markedly diminished in G-CSF–mobilized PBPC products. CD34+ cells cultured in limiting dilution assays showed a sixfold decrease in NK cell progenitors when derived from G-CSF–mobilized CD34+ PBPCs compared with CD34+ cells derived from unprimed marrow. The finding of decreased NK cell function, inhibited proliferation, and diminished cloning frequency after treatment with G-CSF could be mimicked in vitro by culture of primitive marrow progenitors (CD34+, lineage-negative, HLA-DR−) on stromal layers in the presence of exogenous G-CSF. The findings presented here show that G-CSF administration to normal donors decreases NK cell function and the relative frequency of NK cell progenitors within the CD34+ progenitor population. Overcoming this diminished lymphoid capacity may be important to facilitate early posttransplant immunotherapy. Our in vitro model will be used in future studies to determine the mechanism of the G-CSF–induced suppression of NK cell progenitors, which may occur early in the differentiation process.

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2044-2044
Author(s):  
Pomeroy Emily ◽  
Hunzeker John ◽  
Kluesner Mitchell ◽  
Crosby Margaret ◽  
Laura Bendzick ◽  
...  

Abstract Natural Killer (NK) cells are cytotoxic lymphocytes capable of immune surveillance and represent an excellent source of cells for cancer immunotherapy for numerous reasons: 1) they mediate direct killing of transformed cells with reduced or absent MHC expression, 2) they can carryout antibody-dependent cell-mediated cytotoxicity (ADCC) on cells bound by appropriate antibodies via CD16, 3) they are readily available and easy to isolate from peripheral blood, 4) they can be expanded to clinically relevant numbers in vitro. Moreover, as NK cells do not cause graft versus host disease, they are inherently an off-the-shelf cellular product, precluding the need to use a patient's own NK cells to treat their cancer. In light of these attributes, NK cells have been used in many clinical trials to treat a number of cancer types; however, the results have not been as successful as other cellular based immunotherapies, such as CAR-T. In light of this, many groups have taken approaches to augment NK cell function, such as high dose IL15, CARs and Bi- or Tri-specific killer engagers. A synergistic or even alternative approach to these technologies is the use of CRISPR/Cas9-based genome editing to disrupt or manipulate the function of NK genes to improve their utility as an immunotherapeutic agent. In order to enhance the immunotherapeutic efficacy of NK cells we have implemented the CRISPR/Cas9 system to edit genes and deliver CARs. To this end, we have developed methods for high efficiency nucleic acid delivery to NK cells using electroporation. First, primary human NK cells are immunomagnetically isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors. Purified NK cells are then activated and expanded using artificial antigen presenting cells (aAPCs) expressing membrane bound IL21 and 41BB for 7 days and subsequently electroporated (Figure 1A). Using this approach with EGFP encoding mRNA, we achieve high rates of transfection (>90%) and high viability (>90%) (Figure 1B). We next developed gRNAs targeting PD1, CISH, and ADAM17. PD1 is a negative regulator of NK cell function and its cognate receptor, PD-L1, is upregulated in a number of cancers. ADAM17 mediates CD16 cleavage on NK cells to negatively regulate their ability to perform ADCC. CISH is a recently described negative regulator of NK cell activation and integrates cytokine signals, including IL-15. We consistently achieved high rates (up to 90%) of gene inactivation in primary human NK cells across multiple donors (Figure 1C). Importantly, these gene edits do not affect expansion potential and are stable over several rounds of expansion (Figure 1D, E). Moreover, ADAM17 KO NK cells are highly resistant to CD16 cleavage upon activation (Figure 2A-E) and PD1 KO NK cells demonstrate significantly enhanced function against PD-L1 expressing cancer cell lines in vitro and in vivo (Figure 2F-J). These data demonstrate that high efficiency gene editing of NK cells can significantly enhance their function while maintaining in vitro expansion. In an effort to engineer NK cell specificity for cancer immunotherapy, we recently developed CAR molecules designed for use in NK cells (Li et al., 2018, Cell Stem Cell 23, 1-12). To this end, we engineered and tested 10 mesothelin CAR molecules with NK specific transmembrane domains (CD16, NKp44, NKp46, or NKG2D) and intracellular signaling domains (2B4, DAP10, DAP12, CD3ζ, and/or CD137). Utilizing several cancer models, we identified an architecture that significantly enhanced NK activation compared to T-CAR architectures (CAR4: scFv-NKG2D-2B4-CD3ζ). Moreover, NK-CAR4 cells demonstrated increased in vivo expansion, improved activity, and reduced toxicity compared to CAR-T cell therapy. In our studies to develop novel NK CARs, CARs were delivered to iPSC derived test NK cells (iNKs) using the PiggyBac transposon system. In order to deliver NK-CAR4 to peripheral blood NK cells we developed methods for high frequency, site specific integration. To this end, we utilized CRISPR/Cas9 combined with non-integrating recombinant Adeno-Associated Virus (rAAV) DNA donor for homologous recombination. Using an EGFP reporter we were able to optimize this process and deliver EGFP reporter to the AAVS1 safe harbor site with efficiencies >80% in NK cells. We are now utilizing our optimized gene editing approaches to generate multiplex edited CAR-NK cells and results from these studies will be presented. Disclosures Webber: BEAM Therapeutics: Consultancy; B-MoGen Biotechnologies: Employment, Equity Ownership. Felices:GT Biopharma: Research Funding. Moriarity:BEAM Therapeutics: Consultancy; B-MoGen Biotechnologies: Employment, Equity Ownership.


Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2795-2801 ◽  
Author(s):  
GE Tjonnfjord ◽  
R Steen ◽  
SA Evensen ◽  
E Thorsby ◽  
T Egeland

Abstract Primed peripheral blood hematopoietic stem cells (PBSC) generate and sustain lymphohematopoiesis in myeloablated animals, and recent reports indicate that allogeneic transplantation using PBSC grafts may be feasible in humans. A major concern with the use of PBSC transplants is that permanent engraftment may be limited because of lack of sufficient numbers of primitive progenitor cells in the graft. In the present study, in vitro colony formation and immunophenotype of CD34+ cells in PB of healthy adults during short-term granulocyte colony-stimulating factor (G-CSF) administration were compared with that of CD34+ cells in normal bone marrow (BM). The number of CD34+ cells mobilized to PB peaked at day 4 or 5 of G-CSF administration. The phenotypic profile of CD34+ PB cells showed a substantial increase in the percentage of CD34+CD13+ and CD34+CD33+ cells (myeloid progenitors) and a corresponding decrease in the percentage of CD34+CD10+ and CD34+CD19+ cells (B lymphoid progenitors) compared with CD34+ BM cells. The other subsets studied, including CD34+CD38- and CD34+HLA-DR- cells, were present in both compartments in similar proportions. Furthermore, primed CD34+ PB cells were enriched for colony-forming cells (CFC) and displayed an increased clonogenicity when compared with their counterparts in BM. A comparison between a postulated PBSC graft and an average BM graft is presented, showing that such PBSC grafts will be enriched for CD34+ cells as a whole, CD34+CD33+ cells, and colony- forming cells (CFC), factors which have been shown to correlate to acceleration of hematologic reconstitution and reduction in requirements for supportive care in autografting. Hence, we predict that allogeneic transplantation using G-CSF-primed PBSC grafts will result in a more rapid hematologic reconstitution after myeloablative conditioning than BM grafting. The question of whether PBSC allografting will result in permanent engraftment and clinical benefits as observed in autografting has to be determined in prospective clinical studies.


Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2795-2801 ◽  
Author(s):  
GE Tjonnfjord ◽  
R Steen ◽  
SA Evensen ◽  
E Thorsby ◽  
T Egeland

Primed peripheral blood hematopoietic stem cells (PBSC) generate and sustain lymphohematopoiesis in myeloablated animals, and recent reports indicate that allogeneic transplantation using PBSC grafts may be feasible in humans. A major concern with the use of PBSC transplants is that permanent engraftment may be limited because of lack of sufficient numbers of primitive progenitor cells in the graft. In the present study, in vitro colony formation and immunophenotype of CD34+ cells in PB of healthy adults during short-term granulocyte colony-stimulating factor (G-CSF) administration were compared with that of CD34+ cells in normal bone marrow (BM). The number of CD34+ cells mobilized to PB peaked at day 4 or 5 of G-CSF administration. The phenotypic profile of CD34+ PB cells showed a substantial increase in the percentage of CD34+CD13+ and CD34+CD33+ cells (myeloid progenitors) and a corresponding decrease in the percentage of CD34+CD10+ and CD34+CD19+ cells (B lymphoid progenitors) compared with CD34+ BM cells. The other subsets studied, including CD34+CD38- and CD34+HLA-DR- cells, were present in both compartments in similar proportions. Furthermore, primed CD34+ PB cells were enriched for colony-forming cells (CFC) and displayed an increased clonogenicity when compared with their counterparts in BM. A comparison between a postulated PBSC graft and an average BM graft is presented, showing that such PBSC grafts will be enriched for CD34+ cells as a whole, CD34+CD33+ cells, and colony- forming cells (CFC), factors which have been shown to correlate to acceleration of hematologic reconstitution and reduction in requirements for supportive care in autografting. Hence, we predict that allogeneic transplantation using G-CSF-primed PBSC grafts will result in a more rapid hematologic reconstitution after myeloablative conditioning than BM grafting. The question of whether PBSC allografting will result in permanent engraftment and clinical benefits as observed in autografting has to be determined in prospective clinical studies.


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