Successful adoptive immunotherapy with OK432-inducible activated natural killer cells in tumor-bearing mice

We had demonstrated that the NK cell mediated cytotoxicity of murine spleen cells could be augmented byin vivo priming and subsequentin vitro challenge with a streptococcal preparation OK432, and the cell surface phenotype of induced killer cells was Thy-1⁺, asialo GM1⁺, suggesting that the activated cells were of NK lineage (OK-NK cell). We had also clarified that IL-2 played a major role in inducing the OK-NK cells via the production of IFN-. In this study, we examined the effect of adoptive transfer of OK-NK cells on syngeneic tumors in mice. Mice were implanted with SP2 myeloma cells intraperitoneally (i.p.), or C26 colon adenocarcinoma cells subcutaneously to make the models of peritonitis carcinomatosa or solid tumor, and the OK-NK cells were transferred i.p. or intratumorally, adoptively. By the adoptive transfer of OK-NK cells, 92% of mice bearing SP2-tumor had be cured. The tumor growth of C26-solid tumor was inhibited, and the survival rate of mice bearing C26-tumor was significantly increased. The intratumoral remnants of¹²⁵I-labelled OK-NK cells were 61, 27 and 8% at 4, 12 and 36h after intratumoral transfer, respectively. By multiple transfer of OK-NK cells, the antitumor effect was more effectively augmented than that of a single transfer. Results in this study suggested that OK-NK cells could be useful for the therapy of cancer patients.     

Characterization of human natural killer cells for therapeutic use

As a part of the innate immune system, natural killer (NK) cells are cytotoxic lymphocytes that can exert cytotoxic activity against infected or transformed cells. Furthermore, due to their expression of a functional Fc receptor, they have also been eluded as a major effector fraction in antibody-dependent cellular cytotoxicity. These characteristics have led to multiple efforts to use them for adoptive immunotherapy against various malignancies.  There are now at least 70 clinical trials testing the safety and efficacy of NK cell products around the world in early-phase clinical trials. NK cells are also being tested in the context of tumor retargeting via chimeric antigen receptors, other genetic modification strategies, as well as tumor-specific activation strategies such as bispecific engagers with or without cytokine stimulations. One advantage of the use of NK cells for adoptive immunotherapy is their potential to overcome HLA barriers. This has led to a plethora of sources, such as cord blood hematopoietic stem cells and induced pluripotent stem cells, which can generate comparatively high cytotoxic NK cells to peripheral blood counterparts. However, the variety of the sources has led to a heterogeneity in the characterization of the final infusion product. Therefore, in this review, we will discuss a comparative assessment strategy, from characterization of NK cells at collection to final product release by various phenotypic and functional assays, in an effort to predict potency of the cellular product.     

A new human pancreatic carcinoma cell line developed for adoptive immunotherapy studies with lymphokine-activated killer cells in nude mice

Summary A human tumor cell line designated SU.86 has been established from a moderate-to-poorly differentiated pancreatic carcinoma of ductal origin specifically for adoptive immunotherapy studies. This line was characterized as to its ability to be lysed in vitro by autologous and allogeneic lymphokine-activated killer (LAK) and natural killer cells and to grow in nude mice. SU.86 has been growing continuously in cell culture for more than 100 passages since 22 September 1986. Transplantation orthotopically and heterotopically into athymic Swiss nude mice showed that tumor take was 100% in the orthotopic position when young (4 to 6 wk old) mice were used and 0% when adult (8 wk old) mice were used (P=0.004). In the heterotopic position (subcutaneous), tumor take was 100% in neonate (2 to 3 wk old) and young mice and 50% in adults. The rate of tumor growth was inversely correlated with age (P<0.001). The histologic pattern is similar to that observed in most human pancreatic carcinomas with pseudoglandular structures and frequent mitotic figures. SU.86 has a doubling time of 77 h in vitro and produces carcinoembryonic antigen, 594 ng/10⁶ cells in 3 d. Chromosomal analysis shows heterogeneity with two notable cell subpopulations. The cell line is moderately sensitive to lysis by LAK cells in a standard, 4-h chromium-51 release assay (35.4±4.0%). When grown together with LAK cells in vitro, it is lysed completely in culture in 8 to 15 d, depending on the serum concentration.     

A regimen of surgical adjuvant immunotherapy for cancer with interleukin 2 and lymphokine-activated killer cells

We designed a unique regimen of adoptive immunotherapy with lymphokine-activated killer (LAK) cells and recombinant interleukin 2 (rIL-2) for application with surgical adjuvant therapy of cancer. The regimen features the prolonged (6 consecutive days) s.c. administration of low-dose rIL-2 and the transfer ofex vivo generated LAK cells from regional lymph node lymphocytes, obtained at the time of surgical operation. According to this regimen, 5 patients with primary lung cancer received immunotherapy about 2 weeks after surgery (pulmonary lobectomy). Clinical toxicities included fever(5/5), fatigue(5/5), slight(< 5%) weight gain(5/5), increase of pleural effusion at the lobectomy site(2/5), and edema formation(1/5). All toxicities reversed within 4 days after the completion of therapy. Rebound lymphocytosis after therapy ranged from 2.4 to 5.5-fold (mean, 4.3-fold) over the baseline. Peripheral blood lymphocytes obtained during this lymphocytosis exhibitedin vitro LAK activity in 4 of 5 patients. Thus, the regimen is considered to be well-tolerable and immunologically active in regard to the postoperative state of the patients.     

Intraperitoneal delivery of human natural killer cells for treatment of ovarian cancer in a mouse xenograft model

Background aimsThere is an urgent need for novel therapeutic strategies for relapsed ovarian cancer. Dramatic clinical anti-tumor effects have been observed with interleukin (IL)-2 activated natural killer (NK) cells; however, intravenous delivery of NK cells in patients with ovarian cancer has not been successful in ameliorating disease. We investigated in vivo engraftment of intraperitoneally (IP) delivered NK cells in an ovarian cancer xenograft model to determine if delivery mode can affect tumor cell killing and circumvent lack of NK cell expansion.MethodsAn ovarian cancer xenograft mouse model was established to evaluate efficacy of IP-delivered NK cells. Tumor burden was monitored by bioluminescent imaging of luciferase-expressing ovarian cancer cells. NK cell persistence, tumor burden and NK cell trafficking were evaluated. Transplanted NK cells were evaluated by flow cytometry and cytotoxicity assays.ResultsIP delivery of human NK cells plus cytokines led to high levels of circulating NK and was effective in clearing intraperitoneal ovarian cancer burden in xenografted mice. NK cells remained within the peritoneal cavity 54 days after injection and had markers of maturation. Additionally, surviving NK cells were able to kill ovarian cancer cells at a rate similar to pre-infusion levels, supporting that in vivo functionality of human NK cells can be maintained after IP infusion.ConclusionsIP delivery of NK cells leads to stable engraftment and antitumor response in an ovarian cancer xenograft model. These data support further pre-clinical and clinical evaluation of IP delivery of allogeneic NK cells in ovarian cancer.     

“Differentiation Induction” culture of human leukemic myeloid cells stimulates high production of macrophage differentiation inducing factor

We recently developed a new culture system based on dialysis perfusion (designated JCC-device) for the generation and expansion of human lymphokine-activated killer (LAK) cells (Murata et al., 1990). More recently we have scaled up the volume of the culture vessel of the JCC-device from 100 ml to 400 ml for clinical use. In the present study, using this new 400 ml JCC-device, we cultured human lymph node lymphocytes (LNL) obtained from 8 surgical patients with primary lung cancer, and investigated the cellular characteristics in comparison with a conventional batchwise culture system using tissue culture dishes. With the JCC-device, the cell density reached a maximum 2.7×10⁷ cells/ml with greater than 90% viability by the appropriate exchange of perfusion medium and by making additions at the appropriate intervals for recombinant interleukin-2 (rIL-2). The expansion fold of LNL with the JCC-device, ranging 6.6- to 19.2-fold (mean 13.8-fold), was not significantly different from that in dish cultures. There was no marked difference in cell surface phenotypes between the two culture systems in 7 out of 8 cases. As for LAK activity of LNL, the JCC culture was either superior or equal in 4 out of 8 cases, but inferior in the other 4 cases to the conventional dish cultures. In the latter cases, the usage of serum for the JCC culture was limited, which might have resulted in the low LAK activity. The JCC-device was able to reduce the consumption of basal medium, rIL-2 and serum by 20%, 84% and 96%, respectively compared to the conventional tissue culture systems. The JCC-device improved the routine performance of adoptive immunotherapy with LAK cells and rIL-2.Abbreviations LAK lymphokine-activated killer - rIL-2 recombinant interleukin-2 - LNL lymph node lymphocytes - BM basal medium - CM complete medium - HBSS Hanks balanced salt solution - JRU Japan reference unit     

母胎界面自然杀伤细胞的研究进展

自然杀伤(natural killer,NK)细胞是母胎界面丰度最高的免疫细胞,在妊娠早期的子宫蜕膜中大量积聚.研究表明母胎界面NK细胞具有独特表型和功能,在妊娠期免疫耐受调节、子宫内膜蜕膜化、滋养细胞侵袭、子宫螺旋动脉重塑、胎盘形成和胎儿生长、发育等多方面都发挥关键作用,但是其在妊娠中的功能及其作用机制还有待深入研究...     

Effect of exposure to interleukin-21 at various time points on human natural killer cell culture

Background aimsInterleukin-21 (IL-21) can enhance the effector function of natural killer (NK) cells but also limits their proliferation when continuously combined with IL-2/IL-15. Paradoxically, membrane-bound (mb)-IL-21 has been shown to improve human NK cell proliferation when cultured with IL-2/mb-IL-15. To clarify the role of IL-21, we investigated the effect of the timing of IL-21 addition to NK cell culture.MethodsIL-2/IL-15–activated NK cells were additionally treated with IL-21 according to the following schedules; (i) control (without IL-21); (ii) first week (day 0 to day 7); (iii) intermittent (the first 3 days of each week for 7 weeks); (iv) after 1 week (day 8 to day 14); and (v) continuous (day 0 to day 49). The expression of NK receptors, granzyme B, perforin, CD107a, interferon-γ, telomere length and NK cell death were measured by flow cytometry.ResultsCompared with the control (2004.2-fold; n = 10 healthy donors) and intermittent groups (2063.9-fold), a strong proliferative response of the NK cells on day 42 was identified in the “first week” group (3743.8-fold) (P < 0.05). NK cells treated with IL-21 in the “first week” group showed cytotoxicity similar to that in control cells. On day 28, there was a significant increase in cytotoxicity of “first week” NK cells that received IL-21 treatment for an additional 2 days compared with the “first week” NK cells (P < 0.05).ConclusionsThese data suggest that controlling temporal exposure of IL-21 during NK cell proliferation can be a critical consideration to improve the yields and cytotoxicity of NK cells.     

Role of uterine natural killer cells in angiogenesis of human decidua of the first-trimester pregnancy

Decidualization is accompanied by extensive angiogenesis, which is an essential step in the maturation of new blood vessels in mammalian pregnancy. The purpose of this study was to determine a distribution of uNK cells (CD56 uNK or CD56bright cells) in human decidua of the first-trimester pregnancy, and investigate whether uNK cells in human decidua could express vascular endothelial growth factor (VEGF-A) and/or angiopoietin2 (Ang2). Our immunohistochemical staining results demonstrated that a great amount of uNK (CD56 ) cells scattered throughout the decidual stroma and near endometrial gland and spiral vessels in human decidua. The protein expression of VEGF-A and Ang2 was detected in decidual stroma cells, capillary endothelial cells and glandular cells in tissue specimens. There was a positive correlation between microvessel density (MVD) and the number of the CD56-positive uNK cells in decidual stroma, and also between the number of the CD56-positive uNK cells and VEGF-A protein expression in the tissue. In addition, we found that uNK cells in human decidua could express VEGF-A mRNA, but not Ang2 mRNA, in isolated uNK cells in human decidua of the first-trimester gestation by combination of LCM and Nested-PCR. Our study indicated that uNK cells, through expressing VEGF-A, may play an important role in the angiogenic response at the time of human decidualization and early placenta development.     

Kinetics of interleukin-2 induced changes in rigidity of human natural killer cells

The success of adoptive immunotherapy is dependent in part on the successful delivery of effector cells to the tumor and the expression of cellular activities, such as adhesion, extravasation, and cytotoxic activity of the effector cells in the tumor. The structural rigidity of the effector cell is an important determinant of these functions. The present study was designed to quantify the changes in cellular rigidity and cytotoxic activity of human natural killer (NK) cells in the presence or absence of interleukin-2 (IL-2). Micropipet aspiration was used to measure the resistance of NK cells to an imposed external deformation. Homogeneous suspensions of NK cells were activated with 1000 U/mL of recombinant IL-2 in vitro and tested for cellular rigidity from 0 to 96 h post stimulation. The IL-2 activated cells increased their rigidity within 24 h and maintained it at this level for 96 h. Prolonged incubation of cells in IL-2 (14 d) resulted in a consistently high rigidity, which was further increased on starvation of the cells from IL-2. The increased rigidity of these cells was maintained throughout 96 h of IL-2 deprivation, although significant relaxation of rigidity was observed between 48 and 96 h. The relaxation of rigidity was associated with an increase in the number of nonviable cells. Reintroduction of IL-2 for 24 h to a culture of NK cells depleted of IL-2 for 48 h did not restore the cells to the predepletion level of rigidity. Cytotoxic activity of the activated NK cells following removal of IL-2 decreased to about 60% of the control activity within 24 h and continued through 72 h postdeprivation. These findings suggest that the initial activation of human NK cells by IL-2 will produce a relatively rapid increase in rigidity that may cause entrapment of these cells in small capillaries in vivo and that removal of IL-2 will produce an additional increase in rigidity, which is associated with decreased functional activity.     

Opportunities and limitations of natural killer cells as adoptive therapy for malignant disease

Although natural killer (NK) cells can be readily generated for adoptive therapy with current techniques, their optimal application to treat malignant diseases requires an appreciation of the dynamic balance between signals that either synergize with or antagonize each other. Individuals display wide differences in NK function that determine their therapeutic efficacy. The ability of NK cells to kill target cells or produce cytokines depends on the balance between signals from activating and inhibitory cell-surface receptors. The selection of NK cells with a predominant activating profile is critical for delivering successful anti-tumor activity. This can be achieved through selection of killer immunoglobulin-like receptor–mismatched NK donors and by use of blocking molecules against inhibitory pathways. Optimum NK cytotoxicity may require licensing or priming with tumor cells. Recent discoveries in the molecular and cellular biology of NK cells inform in the design of new strategies, including adjuvant therapies, to maximize the cytotoxic potential of NK cells for adoptive transfer to treat human malignancies.     

Generation of lymphokine activated killer cells in a new high density dialyzing culture apparatus

In order to establish an efficient culture system for the generation of lymphokine activated killer (LAK) cells, we have developed a new device which is essentially based on a continuous dialyzing culture vessel. LAK cells grown in such a system showed higher cytotoxicity than those grown under conventional culture conditions. By using this new apparatus with continuous regulation of infused interleukin 2, nutritional medium, and pO₂ and pCO₂, yields of 2×10⁷ cells/ml were achieved and maintained for more than 21 days. These cells also showed a significant increase of LAK activity on a per cell basis.     

Surface glycoprotein of human natural killer cells recognized by wheat germ agglutinin

We analyzed surface glycoproteins of human natural killer (NK) cells by utilizing lectins. Among the lectins tested, wheat germ agglutinin (WGA) was found to bind preferentially to CD16(Leu11)-positive lymphocytes as determined by two-colour flow cytometry. Analysis of glycoproteins in the lysate prepared from NK cells with sodium dodecyl sulfate (SDS) gel electrophoresis followed by Western blotting and¹²⁵I labeled WGA staining revealed that a glycoprotein with anM _r of 65 kDa was strongly bound to the lectin, but no corresponding glycoprotein was detected in the lysate of T lymphocytes. This glycoprotein (GP65) gave several spots in the pI range 4.1–4.6 on 2-dimensional gel electrophoresis. Sialidase treatment of GP65 resulted in a single spot on the 2-dimensional gel, suggesting that GP65 is heterogeneous in the degree of sialylation. GP65 was shown to be exposed on the cell surface, since it was radiolabeled with¹²⁵I by the lactoperoxidase-catalyzed method. We next isolated GP65 from human peripheral blood lymphocytes by a combination of chromatography on a cation-exchange column and a WGA-agarose column and preparative SDS gel electrophoresis. It is suggested that GP65 is a novel surface glycoprotein on human NK cells.     

Effect of irradiation-induced intercellular adhesion molecule-1 expression on natural killer cell-mediated cytotoxicity toward human cancer cells

Background aims

Irradiation enhances the adhesion between natural killer (NK) cells and target cells by up-regulating intercellular adhesion molecule-1 (ICAM-1) on target cells. Therefore, we investigated the effect of irradiation-induced ICAM-1 expression on human cancer cells on NK cell–mediated cytotoxicity.

Engineering of human mesenchymal stem cells resistant to multiple natural killer subtypes

Mesenchymal stem cells (MSCs) as a therapeutic promise are often quickly cleared by innate immune cells of the host including natural killer (NK) cells. Efforts have been made to generate immune-escaping human embryonic stem cells (hESCs) where T cell immunity is evaded by defecting β-2-microglobulin (B2M), a common unit for human leukocyte antigen (HLA) class I, and NK cells are inhibited via ectopic expression of HLA-E or -G. However, NK subtypes vary among recipients and even at different pathologic statuses. It is necessary to dissect and optimize the efficacy of the immune-escaping cells against NK subtypes. Here, we first generated B2M knockout hESCs and differentiated them to MSCs (EMSCs) and found that NK resistance occurred with B2M^-/- EMSCs expressing HLA-E and -G only when they were transduced via an inducible lentiviral system in a dose-dependent manner but not when they were inserted into a safe harbor. HLA-E and -G expressed at high levels together in transduced EMSCs inhibited three major NK subtypes, including NKG2A⁺/LILRB1⁺, NKG2A⁺/LILRB1^-, and NKG2A^-/LILRB1⁺, which was further potentiated by IFN-γ priming. Thus, this study engineers MSCs with resistance to multiple NK subtypes and underscores that dosage matters when a transgene is used to confer a novel effect to host cells, especially for therapeutic cells to evade immune rejection.     

Lactococcus lactis subsp. lactis JCM 5805 activates natural killer cells via dendritic cells

Lactococcus lactis subsp. lactis JCM 5805 (JCM5805) has been shown to stimulate plasmacytoid dendritic cells (pDC). Here, we investigated the effect of JCM5805 on NK cells. In vitro studies suggested that JCM5805 activated natural killer (NK) cells via dendritic cells including pDC. Furthermore, the oral administration of JCM5805 enhanced the cytotoxic activity of NK cells     

Studies on NK cell purification by negative selection in human peripheral blood

We have attempted to improve negative selection procedures for the large scale purification of human CD _in3 ^– CD56⁺ NK cells. In a series of experiments, purifications of NK cells from 10⁸ PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3⁺ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10⁸ cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10⁹ PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior toin vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.     

Targeting autophagy blocks melanoma growth by bringing natural killer cells to the tumor battlefield

Solid tumors are able to establish and sustain an immune suppressive microenvironment, which prevents the infiltration of cytotoxic effector immune cells into the tumor bed. We showed that genetic targeting of the macroautophagy/autophagy gene Becn1/Beclin1 in B16-F10 tumors inhibits their growth by inducing a massive infiltration of functional natural killer (NK) cells into the tumor bed. Such infiltration is primarily due to the ability of BECN1-defective tumor cells to overexpress and release CCL5 cytokine in the tumor microenvironment by a mechanism involving the activation of the MAPK8/JNK-JUN/c-Jun signaling pathway. Clinically, we reported a strong positive correlation between the expression of NK cell marker and CCL5 in human melanoma tumors and more importantly, a significant increased survival is found in melanoma patients expressing a high level of CCL5. Overall, these findings highlight the impact of targeting autophagy in breaking the immunosuppressive tumor microenvironment barrier, thus allowing the trafficking of cytotoxic NK cells into the tumor bed. This study underscore the importance of autophagy inhibition in tumors as a novel therapeutic strategy to fully exploit NK cells antitumor properties in clinical settings.     

Strategies for improving antitumor activity utilizing IL-2: Preclinical models and analysis of antitumor activity of lymphocytes from patients receiving IL-2

Conclusions Considerable enthusiasm remains for the successful utilization of the immune system for the immunotherapy of human cancers. Immunotherapeutic maneuvers have been able to mediate impressive antitumor responses for some patients with advanced and refractory malignancies. Unfortunately, the number of patients who benefit from current immunotherapies is low, while the toxicity for many of the patients receiving these treatments is high. It is becoming quite clear that the development of successful immunotherapeutic strategies will involve a carefully chosen combination of immunotherapeutic modalities or of immunotherapy combined with either surgery, radiation therapy, or chemotherapy. The use of an IL-2 based regimen which is clinically tolerable and can provide significant immune activation continues to remain central to many of these treatment approaches. Preclinicalin vitro and animal model systems can evaluate promising treatment strategies, including combination approaches. As an effective immunotherapeutic approach will likely require use of a combination of biologically active agents, the scheduling of these therapies may have profound importance both for optimal antitumor responses as well as clinical tolerance.     

α‐galactosylceramide generates lung regulatory T cells through the activated natural killer T cells in mice

Our previous study showed that intraperitoneal injection of α‐galactosylceramide (α‐GalCer) has the ability to activate lung iNKT cells, but α‐GalCer‐activated iNKT cells do not result in airway inflammation in wild‐type (WT) mice. Many studies showed that iNKT cells had the capacity to induce Treg cells, which gave rise to peripheral tolerance. Therefore, we examined the influence of intraperitoneal administration of α‐GalCer on the expansion and suppressive activity of lung Treg cells using iNKT cell‐knockout mice and co‐culture experiments in vitro. We also compared airway inflammation and airway hyperresponsiveness (AHR) after α‐GalCer administration in specific anti‐CD25 mAb‐treated mice. Our data showed that intraperitoneal injection of α‐GalCer could promote the expansion of lung Treg cells in WT mice, but not in iNKT cell‐knockout mice. However, α‐GalCer administration could not boost suppressive activity of Treg cells in WT mice and iNKT cell‐knockout mice. Interestingly, functional inactivation of Treg cells could induce airway inflammation and AHR in WT mice treated with α‐GalCer. Furthermore, α‐GalCer administration could enhance iNKT cells to secrete IL‐2, and neutralization of IL‐2 reduced the expansion of Treg cells in vivo and in vitro. Thus, intraperitoneal administration of α‐GalCer can induce the generation of lung Treg cells in mice through the release of IL‐2 by the activated iNKT cells.