Expansion Of Human Natural Killer Cells Research Articles

Sequential Exposure to IL21 and IL15 During Human Natural Killer Cell Expansion Optimizes Yield and Function.

Natural killer (NK) cells are frequently expanded for the clinic using irradiated, engineered K562 feeder cells expressing a core transgene set of membrane-bound (mb) IL15 and/or mbIL21 together with 41BBL. Prior comparisons of mbIL15 to mbIL21 for NK expansion lack comparisons of key attributes of the resulting NK cells, including their high-dimensional phenotype, polyfunctionality, the breadth and potency of cytotoxicity, cellular metabolism, and activity in xenograft tumor models. Moreover, despite multiple rounds of K562 stimulation, studies of sequential use of mbIL15- and mbIL21-based feeder cells are absent. We addressed these gaps and found that using mbIL15- versus mbIL21-based feeder cells drove distinct phenotypic and functional profiles. Feeder cells expressing mbIL15 alone drove superior functionality by nearly all measures, whereas those expressing mbIL21 alone drove superior yield. In combination, most attributes resembled those imparted by mbIL21, whereas in sequence, NK yield approximated that imparted by the first cytokine, and the phenotype, transcriptome, and function resembled that driven by the second cytokine, highlighting the plasticity of NK cell differentiation. The sequence mbIL21 followed by mbIL15 was advantageous in achieving significant yields of highly functional NK cells that demonstrated equivalent in vivo activity to those expanded by mbIL15 alone in two of three xenograft models. Our findings define the impact of mbIL15 versus mbIL21 during NK expansion and reveal a previously underappreciated tradeoff between NK yield and function for which sequential use of mbIL21-based followed by mbIL15-based feeder cells may be the optimal approach in many settings.

A three-dimensional immune-oncology model for studying in vitro primary human NK cell cytotoxic activity.

Immunotherapy has emerged as a promising therapeutic approach for treating several forms of cancer. Adoptive cell transfer of immune cells, such as natural killer (NK) cells, provides a powerful therapeutic potential against tumor cells. In the past decades, two-dimensional (2D) tumor models have been used to investigate the effectiveness of immune cell killing. However, the 2D tumor models exhibit less structural complexity and cannot recapitulate the physiological condition of the tumor microenvironment. Thus, the effectiveness of immune cells against tumor cells using these models cannot fully be translated to clinical studies. In order to gain a deeper insight into immune cell-tumor interaction, more physiologically relevant in vivo-like three-dimensional (3D) tumor models have been developed. These 3D tumor models can mimic the dynamic cellular activities, making them a much closer representation of the in vivo tumor profiles. Here, we describe a simple and effective protocol to study the cytotoxic activity of primary human NK cells toward the 3D tumor spheroids. Our protocol includes isolation and expansion of human NK cells, labeling and formation of tumor spheroids, co-culture of NK cells and tumor spheroids, and evaluation of cytotoxic activity using a confocal microscope. This protocol is also applicable to other types of tumors and immune cells.

Abstract LB141: Assessment of natural killer (NK) cell activity for immunotherapy using a novel flow cytometry-based killing assay

Abstract Natural killer (NK) lymphocytes exhibit potent responses against transformed and pathogen-infected cells. NK cells can act rapidly as they monitor the bloodstream and tissues for tumorigenic and infected cells that lack “self” markers of MHC class I. Upon recognition of a foreign invader or tumor cell, NK cells release cytotoxic proteins such as interferon-gamma and granzyme B. Because of their ability to recognize stressed cells lacking “self” markers, human NK cells have shown promise as therapeutics in clinical trials to reduce disease burden in patients with therapy-resistant or advanced-stage blood cancers. With more than one hundred NK cell- and induced NK cell-based clinical trials ongoing for blood cancers and refractory solid tumors, there is an ever-increasing need for NK cell therapies to treat cancer and immune diseases. We sought to develop novel, clinically relevant, and robust methods for human NK cell expansion. In addition, to simplify the processing of cells for characterization, we developed a flow cytometry based killing assay to monitor NK activity. Therefore, within the same experiment, we can analyze NK cell phenotype profiles with specific antibody panels while also assessing NK cell killing activity. Using optimized serum- and xeno-free conditions with minimal cell manipulations, we have expanded highly purified NK cells up to 500-fold in 14 days from human peripheral blood mononuclear cells (PBMCs) using optimized cytokines and Cloudz CD2/NKp46 microspheres. To characterize the resulting NK cell population, we used a unique antibody panel to phenotype the NK cells by flow cytometry. Within the same experiment, NK killing assays against K562 cells can be performed using a flow cytometry-based killing assay, which has the advantage of using one instrument for all analyses, shortened processing times, and reproducibility. Using this workflow, we can identify different culture conditions that adversely affect NK activity. Together, this data presents a comprehensive and clinically relevant workflow for the expansion of highly purified NK cells and assessing their killing potential. Furthermore, this workflow can be used for screening the activity of chimeric antigen receptor (CAR) expressing immune cells against specific cancer antigens. Citation Format: Jody Bonnevier, Christine Goetz, Li Peng, Jamie Van Etten, Bora Faulkner, Christopher Hammerbeck, Ariel Miller, Gabriella Perell, Christopher Johnson, Joseph Lomakin, David Hermanson, Kevin Flynn. Assessment of natural killer (NK) cell activity for immunotherapy using a novel flow cytometry-based killing assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB141.

Expansion of Human NK Cells Using K562 Cells Expressing OX40 Ligand and Short Exposure to IL-21.

Background: Natural Killer (NK) cell-based immunotherapy used to treat cancer requires the adoptive transfer of a large number of activated NK cells. Here, we report a new effective method to expand human NK cells ex vivo using K562 cells genetically engineered (GE) to express OX40 ligand (K562-OX40L) in combination with a short exposure to soluble IL-21. In addition, we describe a possible mechanism of the NK cell expansion through the OX40 receptor-OX40 ligand axis which is dependent on NK cell homotypic interaction.Methods: K562-OX40L cells were generated by lentiviral transduction and were used as feeder cells to expand and activate NK cells from PBMCs in the presence of IL-2/IL-15. Soluble IL-21 was also added in various concentrations only once at the beginning of the culture. NK cells were expanded for 4–5 weeks, and the purity, expansion rate, phenotype and function (cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine production, CD107a degranulation) of these expanded NK cells were compared to those generated by using K562 feeder cells.Results: The culture of NK cells with K562-OX40L cells in combination with the transient exposure to IL-21 highly enhanced NK cell expansion to approximately 2,000-fold after 4 weeks of culture, compared to a 303-fold expansion using the conventional K562 cells. Mechanistically, the OX40-OX40L axis between the feeder cells and NK cells as well as the homotypic interaction between NK cells through the OX40-OX40L axis were both necessary for NK cell expansion. The short exposure of NK cells to IL-21 had a synergistic effect with OX40 signaling for NK cell expansion. Apart from their enhanced expansion, NK cells grown with K562-OX40L feeder cells were similar to those grown with conventional K562 cells in regard to the surface expression of various receptors, cytotoxicity, ADCC, cytokine secretion, and CD107 degranulation.Conclusion: Our data suggest that OX40 ligand is a potent co-stimulant for the robust expansion of human NK cells and the homotypic NK cell interactions through the OX40-OX40L axis is a mechanism of NK cell expansion.

X-ray as Irradiation Alternative for K562 Feeder Cell Inactivation in Human Natural Killer Cell Expansion.

γ-Irradiation has been proven to be the most effective method to inactivate K562 cells, but γ-irradiators are not available in some institutes. This study was designed to compare the effects of X-ray and γ-irradiation on K562 cells in natural killer (NK) cell expansion. To expand NK cells, isolated peripheral blood mononuclear cells (PBMCs) were co-cultured with γ-irradiated or X-ray-treated K562 cells plus IL-2 and IL-15. Characteristics of expanded NK cells were identified by flow cytometry. NK cell expansion rate tended be to lower in the X-ray-treated group (68.9±32.6) than the γ-irradiated group (78±28.7), but the difference was not significant (p=0.39). Furthermore, NK cell functions or receptor expression were similar in the two groups. Our results suggest that X-ray treatment can be used as an alternative to γ-irradiation for K562 cells inactivation in human NK cell expansion.

Human natural killer cell expansion in vitro using mitomycin-C treatment as an alternative to irradiation of modified K562 feeder cells.

Abstract Introduction Current methods for in vitro expansion of Natural Killer (NK) cells require feeder cell irradiation, which is not readily available in many biomedical research or clinical facilities. Here we evaluated mitomycin-C (MMC) treatment of feeder cells as an alternative to irradiation for the purpose of expanding NK cells in human peripheral blood mononuclear cell (PBMC) cultures. Methods Using PBMCs from 4 healthy blood donors and genetically modified K562 feeder cells (K562-mb21-41BBL cells) that preferentially stimulate NK cells, we evaluated the efficacy of MMC treatment in limiting the proliferation of the feeder cells at a range of doses (0 ug/mL, 2 ug/mL, 10 ug/mL, 20 ug/mL, 30 ug/mL, and 200 ug/mL) with 3-hour incubation. We used 7AAD dye and CFSE proliferation dye to measure viability and cell proliferation, respectively. We then compared the percentages of NK cells (CD3neg, CD56pos) in PBMCs cultured for 14 days in the presence of MMC-treated feeder cells or irradiated feeder cells using multiparametric flow cytometry. Results We found that 10 ug/mL dose of MMC for 3-hrs achieved a significantly higher percentage of viable cells compared to higher doses (p<0.05, ANOVA), and also significantly limited proliferation compared to untreated feeder cells. Furthermore we found no significant difference in NK cell expansion by 10 ug/mL MMC treated feeder cells and 100 grays irradiated feeder cells. Discussion In conclusion, we found that 10 ug/mL mitomycin-C treatment of modified K562 feeder cells was optimal for limiting their proliferation for NK cell expansion that was equivalent to using irradiated feeder cells.

Ex Vivo Expansion of Human NK Cells Using K562 Engineered to Express Membrane Bound IL21.

Natural killer (NK) cells have gained significant attention for adoptive immunotherapy of cancer due to their well-documented antitumor function. In order to evaluate the therapeutic efficacy of NK cell adoptive immunotherapy in preclinical models with a potential for clinical translation, there is a need for a reliable platform for ex vivo expansion of NK cells. Numerous methods are reported in literature using cytokines and feeder cells to activate and expand human NK cells, and many of these methods are limited by low-fold expansion, cytokine dependency of expanded NK cells or expansion-related senescence. In this chapter, a robust NK cell expansion protocol is described using K562 cell line gene modified to express membrane bound IL21 (K562 mb.IL21). We had previously demonstrated that this platform enables the highest fold expansion of NK cells reported in the literature to date (>47,000-folds in 21 days), and produces highly activated and pure NK cells without signs of senescence, as determined by telomere shortening.

Natural killer cells expanded on membrane-bound IL15 or IL21 activate different miRNA transcriptional profiles with functional consequences

MicroRNA (miRNA) are non-coding regulatory RNA that are key posttranscriptional regulatory machinery of cells. miRNA play important role in several physiological functions during embryonic development, differentiation, cancer progression as well as hematopoiesis, and regulation of immune responses. miRNA regulate development of various immune cells including T and Natural killer (NK) cells. Owing to significant interest in adoptive immunotherapy, numerous methods for expanding NK cells have been developed in recent years. We have previously developed a robust platform for ex-vivo expansion of human NK cells using K562 expressing membrane-bound IL21 (K562.mbIL21). The study of the role of miRNA in NK cells biology is an emerging area of research in development, homeostasis and immune responses. However, the role and effect of expansion of NK cells on miRNA and consequent impact on post-transcriptional regulation of protein expression in expanded NK cells remain to be studied. In this study we investigated the role of distinctive miRNA expression profiles that could influence post-transcriptional regulation of gene expression, and consequently differential protein expression in NK cells expanded on K562.mbIL21 platform compared to primary unexpanded NK cells. We had previously shown that K562.mbIL21 platform enables significantly higher expansion of NK cells compared to K562 expressing membrane bound IL15 (K562 mbIL15), therefore in this study we also compared miRNA expression profiles of NK cells expanded on K562.mbIL15 and K562.mbIL21. We used primary protein and miRNA obtained from, human NK cells derived from 4 healthy donors, Pre and post-expansion on K562.mbIL15 and K562.mbIL21 platforms. miRNA expression profiles were analyzed using Nanostring's nCounter. Initial screening of our dataset revealed 13 miRNAs that are differentially expressed by at least 16 folds between fresh and expanded NK cells or mbIL15 and mbIL21. Comparison between the mbIL15 and mbIL21 expanded cells revealed differential expression of miRNAs critical for regulation of cell cycle proteins as well as key cytokines (TNF-a and interferon-g) such as miR-124-3p and miR 146a-5p (76x and 32x lower in mbIL21 expanded cells respectively). Also, analysis of protein expression by RPPA revealed upregulation of cell cycle proteins cyclin D1 and AMPK, and downregulation of pro-apoptotic protein Bim in mbIL21 expanded NK cells compared with mbIL15 and/or primary unexpanded cells. We are currently correlating miRNA expression and proteomic profiles of expanded and unexpanded cells to identify possible targets of miRNA regulation and subsequently study specific roles of these miRNA in this expansion system by overexpression o/blockade of target miRNA.

Insulin-like growth factor-1 receptor (IGF-1R) inhibition promotes expansion of human NK cells which maintain their potent antitumor activity against Ewing sarcoma cells.

Patients with primary metastatic or relapsed Ewing sarcomas (EwS) have a poor prognosis. While inhibitory insulin-like growth factor 1 receptor (IGF-1R)-specific antibodies have shown single agent activity in some patients with refractory disease, effective therapeutic targeting will rely on optimal combinations with conventional or innovative therapies. Specifically, combination of inhibitory IGF-1R antibodies with adoptive transfer of activated natural killer (NK) cells may have therapeutic benefit in EwS without adding toxicity. We investigated the in vitro effects of IGF-1R targeting on the immunological profile of EwS cells and on the survival and tumor targeting capacity of K-562-activated NK cells. IGF-1R inhibition reliably reduced EwS cell viability without affecting expression of immune-modulatory and MHC molecules. In NK cells, we observed a significant superior expansion following in vitro activation in the presence of IGF-1R-specific antibodies, while expression of differentiation markers and activating receptors remained unaffected. Activated NK cells coincubated with EwS cells showed potent degranulation responses unaffected by IGF-1R inhibition. These findings were reproducible in a stimulator cell-free NK cell expansion system, suggesting that direct effects of IGF-R1 antibodies on the IGF-R1 pathway in NK cells induce their activation and expansion. Activated human NK cells respond to IGF-1R inhibition with superior expansion kinetics while maintaining potent antitumor responses against EwS. Combination of adoptive NK cell transfer with IGF-1R targeting may be an efficient means to eliminate minimal residual disease after conventional therapy and thereby rescue patients at the highest risk of relapse.

CXCL10-induced migration of adoptively transferred human natural killer cells toward solid tumors causes regression of tumor growth in vivo.

Adoptive infusion of natural killer (NK) cells is being increasingly explored as a therapy in patients with cancer, although clinical responses are thus far limited to patients with hematological malignancies. Inadequate homing of infused NK cells to the tumor site represents a key factor that may explain the poor anti-tumor effect of NK cell therapy against solid tumors. One of the major players in the regulation of lymphocyte chemotaxis is the chemokine receptor chemokine (C-X-C motif) receptor 3 (CXCR3) which is expressed on activated NK cells and induces NK cell migration toward gradients of the chemokine (C-X-C motif) ligand (CXCL9, 10 and 11). Here, we show that ex vivo expansion of human NK cells results in a tenfold increased expression of the CXCR3 receptor compared with resting NK cells (p = 0.04). Consequently, these NK cells displayed an improved migratory capacity toward solid tumors, which was dependent on tumor-derived CXCL10. In xenograft models, adoptively transferred NK cells showed increased migration toward CXCL10-transfected melanoma tumors compared with CXCL10-negative wild-type tumors, resulting in significantly reduced tumor burden and increased survival (median survival 41 vs. 32 days, p = 0.03). Furthermore, administration of interferon-gamma locally in the tumor stimulated the production of CXCL10 in subcutaneous melanoma tumors resulting in increased infiltration of adoptively transferred CXCR3-positive expanded NK cells. Our findings demonstrate the importance of CXCL10-induced chemoattraction in the anti-tumor response of adoptively transferred expanded NK cells against solid melanoma tumors.

Way to go to exploit NK cells' versatile talents for cancer immunotherapy.

Way to go to exploit NK cells' versatile talents for cancer immunotherapy.

NK cell responses to cytomegalovirus infection lead to stable imprints in the human KIR repertoire and involve activating KIRs

AbstractHuman natural killer (NK) cells are functionally regulated by killer cell immunoglobulin-like receptors (KIRs) and their interactions with HLA class I molecules. As KIR expression in a given NK cell is genetically hard-wired, we hypothesized that KIR repertoire perturbations reflect expansions of unique NK-cell subsets and may be used to trace adaptation of the NK-cell compartment to virus infections. By determining the human “KIR-ome” at a single-cell level in more than 200 donors, we were able to analyze the magnitude of NK cell adaptation to virus infections in healthy individuals. Strikingly, infection with human cytomegalovirus (CMV), but not with other common herpesviruses, induced expansion and differentiation of KIR-expressing NK cells, visible as stable imprints in the repertoire. Education by inhibitory KIRs promoted the clonal-like expansion of NK cells, causing a bias for self-specific inhibitory KIRs. Furthermore, our data revealed a unique contribution of activating KIRs (KIR2DS4, KIR2DS2, or KIR3DS1), in addition to NKG2C, in the expansion of human NK cells. These results provide new insight into the diversity of KIR repertoire and its adaptation to virus infection, suggesting a role for both activating and inhibitory KIRs in immunity to CMV infection.

Expanded Natural Killer Cells for Cellular Therapy of Acute Myeloid Leukemia.

Approximately half of the patients with acute myeloid leukemia (AML) harbor occult disease during therapy, leading to overt relapse. Novel treatments are needed to advance cure rates. AML cells are sensitive to natural killer (NK) cell cytotoxicity if they express HLA Class I molecules that do not bind killer-inhibitory receptors (KIR) on NK cells. The demonstration that haploidentical NK cells can expand in vivo and exert anti-AML activity when infused after non-myeloablative conditioning (Miller et al., Blood 105: 3051, 2005), provided impetus to further explore their clinical potential and develop ways to increase their efficacy. The success of NK cell therapy depends on: i) mismatch in recipient HLA and donor KIR phenotype, allowing NK cell alloreactivity; ii) infusion of sufficient numbers of NK cells to achieve an effector: target (E:T) ratio that produces a significant leukemia cytoreduction. We found that K562 cells genetically modified to express membrane-bound IL-15 and 4-1BB ligand (K562-mb15-41BBL) induced expansion of human NK cells (Imai et al., Blood 106: 376, 2005). In the present study, we first tested the stimulatory capacity of irradiated K562-mb15-41BBL in 34 additional healthy donors: CD56+ CD3− NK cell expansion after 7–10 days of culture was 5–87 fold (median, 22); after 21 days, NK cells could expand >1000 fold. CD3+ T cells expanded minimally or not at all. NK cells derived from 12 healthy donors were tested against the AML cell lines K562, KG-1, U937 and HL-60. Expanded NK cells were consistently cytotoxic at low E:T ratios. Thus, mean (± SD) cytotoxicity after 4 hrs at 4: 1 was 85.1% ± 8.7% for K562, 83.7% ± 9.4% for KG-1, 78.8% ± 15.2% for U937 and 94.8% ± 5.1% for HL-60. Expanded NK cells were effective even when outnumbered by target cells: at a 0.5: 1 ratio, cytotoxicities were 34.1% ± 14.7% with K562, 51.5% ± 16.5% with KG-1, 24.5% ± 14.8% with U937 and 52.1% ± 9.8% with HL-60. We next tested cytotoxicity of expanded NK cells from 10 donors against primary cells obtained from the bone marrow of 9 newly diagnosed patients with AML. Median cytotoxicity after 4 hrs of culture at a 4: 1 ratio was high, although interdonor variability was observed, with cytoxicities ranging from 22% to 90%. When expanded NK cells were cultured for 7 days with primary AML cells in the presence of bone marrow mesenchymal cells (to prevent spontaneous apoptosis of the AML cells) we could detect cytotoxicity at a 0.01:1 E:T ratio. Expanded NK cells were consistently more cytotoxic than primary NK cells from the same donor. Gene expression studies revealed marked changes in expression of adhesion molecules and cytokine transcripts after expansion. Expanded NK cells exerted considerable antileukemic effect in NOD-SCID-IL2Rgammanull mice engrafted with human AML cells, providing a strong rationale for their clinical testing. To this end, the K562-mb15-41BBL stimulatory cell line is currently being made under cGMP conditions and conditions for large-scale NK cell expansion have been established in support of a pilot protocol in which expanded haploidentical NK cells with be administered to patients with refractory AML.

Identical twins discordant for type 1 diabetes show a different pattern of in vitro CD56+ cell activation

Recent studies in animal models indicate a role for natural killer (NK) cells in the protection against type 1 diabetes. In humans, a reduction of NK cell numbers has been reported in identical twins discordant for type 1 diabetes, irrespective of whether they have the disease. Here we have tested whether the activation and expansion of human NK cells with lipopolysaccharide (LPS) reveals differences between these twins. Proportions of CD56(+) NK cells and T-cells and Va24Vb11(+) NK-T cells from diabetic and non-diabetic twins was assessed before and after activation using flow cytometry. NK receptor usage was monitored by PCR and flow cytometry. The profile of the expressed Killer Cell immunoglobulin-like receptor (KIR) repertoire (using mRNA) in freshly isolated NK cells was identical in pairs of identical twins, despite marked variation among individual twins as well as controls. Basal numbers of CD56(+) and CD94(+) (CD3(-) and CD3(+)) cells and Valpha24(+)Vbeta11(+) NK-T cells were similarly strongly correlated between identical twins (p < 0.006 for all correlations). Following LPS stimulation, the pattern of KIR mRNA expression remained unaltered in twins and the proportion of NK cells and Valpha24(+)Vbeta11(+) NK-T cells remained correlated between pairs of twins. However, there was a significant reduction in the proportion of CD56(+) cells and CD94(+) cells (whether defined as CD3(-) or CD3(+)) responding to LPS in the diabetic compared to the non-diabetic twin (p = 0.031 and 0.025, respectively). This reduction in NK cell expansion in response to LPS in patients with type 1 diabetes is consistent with a non-genetically determined alteration in the innate immune response either predisposing to or resulting from the disease.

A new method for in vitro expansion of cytotoxic human CD3 −CD56 + natural killer cells

Adoptive transfer of immunocompetent cells may induce anti-tumor effects in vivo. However, a significant obstacle to the development of successful cellular immunotherapy has been the availability of appropriate cytotoxic cells. Among the immunologic effector cells that are considered mediators of anti-tumor effects, those with the highest per-cell cytotoxic capacity express a natural killer (NK) cell phenotype, i.e., CD56 +CD3 −. However, such cells are normally present only in low numbers in peripheral blood mononuclear cells (PBMCs), lymphokine activated killer (LAK), and cytokine induced killer (CIK) cell preparations. To optimize the expansion of human NK cells, PBMCs were cultured in different serum free medium supplemented with monoclonal anti-CD3 antibodies and interleukin (IL)-2 at varying concentrations. By using Cellgro stem cell growth medium supplemented with 5% human serum and IL-2 (500 U/ml) cells expanded 193-fold (median, range 21–277) after 21 days, and contained 55% (median, range 7–92) CD3 −CD56 + cells. The remaining cells were CD3 + T cells, 22% (median, range 2–68) of which co-expressed CD56. The expanded cell population lysed 26 to 45% of K562 targets in a 1:1 effector to target ratio, signifying substantial cytotoxic efficacy. The described method is a simple and efficient way of expanding and enriching human NK cells. We have termed these high-yield CD3 −CD56 + cells cytokine-induced natural killer (CINK) cells.

Potential mechanisms of human natural killer cell expansion in vivo during low-dose IL-2 therapy

The continuous, in vivo infusion of low-dose IL-2 selectively expands the absolute number of human natural killer (NK) cells after 4-6 weeks of therapy. The mechanism responsible for this expansion is unknown and was examined in this study. NK cells cultured at low concentrations of IL-2, comparable to those found during in vivo therapy, proliferate for 6 days and then exit the cell cycle. However, NK cells in vivo did not traverse the S/G(2)/M phase of the cell cycle during low-dose IL-2 therapy. Low concentrations of IL-2 delay programmed cell death of NK cells but have the same effect on resting T cells that do not expand in vivo. When CD34(+) bone marrow hematopoietic progenitor cells are cultured for 21 days with low concentrations of IL-2, they differentiate into CD56(+)CD3(-) NK cells, not T cells. Thus, the selective expansion of human NK cells during continuous in vivo infusion of low-dose IL-2 likely results from enhanced NK-cell differentiation from bone marrow progenitors, combined with an IL-2-dependent delay in NK-cell death, rather than proliferation of mature NK cells in the periphery.

Human Natural Killer Cell Expansion Is Regulated by Thrombospondin-Mediated Activation of Transforming Growth Factor-β1 and Independent Accessory Cell-Derived Contact and Soluble Factors

Abstract Natural killer cells (NK) were studied to determine factors important in their expansion. Flourescence-activated cell sorter (FACS) purified CD56+/CD3- NK cells cultured alone for 18 days in rIL-2 containing medium (1,000 U/mL) showed enhanced cytotoxicity but only minimal expansion. NK expansion was increased (12.5 +/- 1.6-fold) by coculturing NK with soluble factors produced by irradiated peripheral blood mononuclear cells (PBMNC) in which the two populations were separated by a microporous membrane. However, maximal NK expansion was always observed when NK were cocultured in direct contact with irradiated PBMNC (49.4 +/- 5.9-fold). To determine if marrow stroma, which supports differentiation of primitive NK progenitors, was a better accessory cell population than irradiated PBMNC, NK were cocultured in direct contact with primary marrow stromal layers. NK expansion with marrow stroma was similar to PBMNC. Fibroblast cell lines (M2–10B4, NRK-49F, NIH-3T3) and human umbilical vein endothelial cells (HUVEC), all homogeneous populations and devoid of monocytes, also exhibited a similar contact-dependent increase in NK expansion. Experiments were designed using fixed M2–10B4 stromal cells to separate the contact-induced proliferative stimuli from soluble factors. NK plated directly on ethanol/acetic acid-fixed M2–10B4, which leaves stromal ligands (cell membrane components and ECM) intact, resulted in increased NK expansion compared with medium alone. We further show that the combination of independent contact and soluble factors is responsible for maximal late NK expansion (days 28 through 40) but paradoxically inhibits early NK expansion (day 7). The proliferation inhibitory effects were verified by 3H-thymidine uptake and could be detected at days 2 through 6 but no longer 14 days after the initiation of the culture. We show that both laminin and thrombospondin inhibit early NK proliferation, whereas only thrombospondin was capable of also stimulating late NK expansion. The effect of thrombospondin on early NK proliferation is related to activation of transforming growth factor-beta 1 (TGF-beta) because anti-TGF-beta neutralizing antibody completely abrogated thrombospondin-mediated inhibition of early NK proliferation. Although inhibitory early in culture, active TGF-beta added only at culture initiation increases late NK expansion similar to thrombospondin. TGF-beta was not present in the thrombospondin preparation but latent TGF-beta in serum, or TGF-beta transcripts identified in IL-2-activated NK could explain paracrine or autocrine mechanisms for the regulation of NK proliferation. Finally, anti-TGF-beta neutralizing antibody only minimally affects stroma-mediated inhibition of early NK proliferation suggesting that aside from thrombospondin/TGF-beta, additional contact factors are important for the regulation of NK proliferation.

Human natural killer cell expansion is regulated by thrombospondin- mediated activation of transforming growth factor-beta 1 and independent accessory cell-derived contact and soluble factors

Natural killer cells (NK) were studied to determine factors important in their expansion. Flourescence-activated cell sorter (FACS) purified CD56+/CD3- NK cells cultured alone for 18 days in rIL-2 containing medium (1,000 U/mL) showed enhanced cytotoxicity but only minimal expansion. NK expansion was increased (12.5 +/- 1.6-fold) by coculturing NK with soluble factors produced by irradiated peripheral blood mononuclear cells (PBMNC) in which the two populations were separated by a microporous membrane. However, maximal NK expansion was always observed when NK were cocultured in direct contact with irradiated PBMNC (49.4 +/- 5.9-fold). To determine if marrow stroma, which supports differentiation of primitive NK progenitors, was a better accessory cell population than irradiated PBMNC, NK were cocultured in direct contact with primary marrow stromal layers. NK expansion with marrow stroma was similar to PBMNC. Fibroblast cell lines (M2-10B4, NRK-49F, NIH-3T3) and human umbilical vein endothelial cells (HUVEC), all homogeneous populations and devoid of monocytes, also exhibited a similar contact-dependent increase in NK expansion. Experiments were designed using fixed M2-10B4 stromal cells to separate the contact-induced proliferative stimuli from soluble factors. NK plated directly on ethanol/acetic acid-fixed M2-10B4, which leaves stromal ligands (cell membrane components and ECM) intact, resulted in increased NK expansion compared with medium alone. We further show that the combination of independent contact and soluble factors is responsible for maximal late NK expansion (days 28 through 40) but paradoxically inhibits early NK expansion (day 7). The proliferation inhibitory effects were verified by 3H-thymidine uptake and could be detected at days 2 through 6 but no longer 14 days after the initiation of the culture. We show that both laminin and thrombospondin inhibit early NK proliferation, whereas only thrombospondin was capable of also stimulating late NK expansion. The effect of thrombospondin on early NK proliferation is related to activation of transforming growth factor-beta 1 (TGF-beta) because anti-TGF-beta neutralizing antibody completely abrogated thrombospondin-mediated inhibition of early NK proliferation. Although inhibitory early in culture, active TGF-beta added only at culture initiation increases late NK expansion similar to thrombospondin. TGF-beta was not present in the thrombospondin preparation but latent TGF-beta in serum, or TGF-beta transcripts identified in IL-2-activated NK could explain paracrine or autocrine mechanisms for the regulation of NK proliferation. Finally, anti-TGF-beta neutralizing antibody only minimally affects stroma-mediated inhibition of early NK proliferation suggesting that aside from thrombospondin/TGF-beta, additional contact factors are important for the regulation of NK proliferation.