Growth of MCF-7 human breast carcinoma in severe combined immunodeficient mice: Growth suppression by recombinant interleukin-2 treatment and role of lymphokine-activated killer cells

Summary The severe combined immunodeficient (SCID) mouse, lacking functional T and B lymphocytes, has been considered by many groups to be a prime candidate for the reconstitution of a human immune system in a laboratory animal. In addition, this immuno-deficient animal would appear to have excellent potential as a host for transplanted human cancers, thus providing an exceptional opportunity for the study of interactions between the human immune system and human cancer in a laboratory animal. However, because this animal model is very recent, few studies have been reported documenting the capability of these mice to accept human cancers, and whether or not the residual immune cells in these mice (e.g. natural killer, NK, cells; macrophages) possess antitumor activities toward human cancers. Thus, the purpose of this study was (a) to determine whether or not a human breast carcinoma cell line (MCF-7) can be successfully transplanted to SCID mice, (b) to determine whether or not chronic treatment of SCID mice with a potent lymphokine (recombinant interleukin-2, rIL-2) could alter MCF-7 carcinoma growth, and (c) to assess whether or not rIL-2-activated NK cells (LAK cells) are important modulators of growth of MCF-7 cells in SCID mice. To fulfill these objectives, female SCID mice were implanted s.c. with MCF-7 cells (5 × 10⁶ cells/mouse) at 6 weeks of age. Six weeks later, some of the mice were injected i.p. twice weekly with rIL-2 (1 × 10⁴ U mouse^–1 injection^–1). Results clearly show that MCF-7 cells can grow progressively in SCID mice; 100% of the SCID mice implanted with MCF-7 cells developed palpable measurable tumors within 5–6 weeks after tumor cell inoculation. In addition, MCF-7 tumor growth was significantly (P <0.01) suppressed by rIL-2 treatment. rIL-2 treatment was non-toxic and no effect of treatment on body weight gains was observed. For non-tumor-bearing SCID mice, splenocytes treated in vitro with rIL-2 (lymphokine-activated killer, LAK, cells) or splenocytes derived from rIL-2-treated SCID mice (LAK cells) had significant (P <0.01) cytolytic activity toward MCF-7 carcinoma cells in vitro. In contrast, splenocytes (LAK cells) derived from tumor(MCF-7)-bearing rIL-2-treated SCID mice lacked cytolytic activities toward MCF-7 cells in vitro. No significant concentration of LAK cells in MCF-7 human breast carcinomas was observed nor did rIL-2 treatment significantly alter growth of MCF-7 cells in vitro. Thus, while rIL-2 treatment significantly suppressed growth of MCF-7 breast carcinomas in SCID mice, the mechanism of this growth suppression, albeit clearly not involving T and B lymphocytes, does not appear to be mediated via a direct cytolytic activity of LAK cells toward the carcinoma cells. However, rIL-2-activated SCID mouse splenocytes (LAK cells) do possess the capability of significant cytolytic activity toward MCF-7 human breast carcinoma cells. Thus, treatment of SCID mice with a potent lymphokine (rIL-2) induces a significant antitumor host response, a response that does not involve T and B lymphocytes and appears not to involve NK/LAK cells. This host response must be considered in future studies designed to investigate the interactions of reconstituted human immune systems and human cancers within this highly promising immuno deficient experimental animal model.     

NOD/SCID小鼠模型在实验血液学研究中的应用

NOD/SCID（非肥胖糖尿病/重症联合免疫缺陷）小鼠是在SCID（重症联合免疫缺陷）小鼠的基础上与非肥胖性糖尿病小鼠（NOD/Lt）品系回交的免疫缺陷鼠。NOD/SCID小鼠既有先天免疫缺陷,又有T和B淋巴细胞缺乏,各种肿瘤细胞可以植入,且较少发生排斥反应及移植物抗宿主病（GVHD）,所以NOD/SCID小鼠逐渐成为血液学实验研究的有用工具。本文从NOD/SCID小鼠的生物学特性、建立人类白血病模型、干细胞移植、药物研究及NOD/SCID小鼠应用中存在的不足和改良等方面综合述评。     

Intra-bone marrow transplantation of human CD34+ cells into NOD/LtSz-scid IL-2rγnull mice permits multilineage engraftment without previous irradiation

Background aimsNon-irradiated immunodeficient recipients provide the best physiologic setting for revealing hematopoietic stem cell (HSC) functions after xenotransplantion. An approach that efficiently permits the detection of human hematopoietic repopulating cells in non-irradiated recipients is therefore highly desired.MethodsWe compared side-by-side the ability to reconstitute hematopoiesis via intra-bone marrow transplantation (IBMT) in three commonly used mouse strains avoiding previous irradiation.ResultsNon-irradiated NOD/SCID and NOD/SCID (β2m?/? mouse strains prevent engraftment even after IBMT. In contrast, combining the robustness of the NOD/SCID IL-2Rγ?/? recipient with the sensitivity of IBMT facilitates the detection, without previous host irradiation, of human SCID-repopulating cells 10 weeks after transplantation. The level of chimerism averaged 14% and multilineage engraftment (lymphoid dominant) was observed consistently in all mice. Analysis of injected and non-injected bones, spleen and peripheral blood demonstrated that engrafting cells were capable of in vivo migration and expansion.ConclusionsCombining the robustness of the NOD/SCID IL-2Rγ?/? mouse strain with the sensitivity of IBMT strongly facilitates long-term multilineage engraftment and migration for human CD34⁺ cells without the need for previous irradiation.     

Humanized Rag1-/- γc-/- mice support multilineage hematopoiesis and are susceptible to HIV-1 infection via systemic and vaginal routes

Several new immunodeficient mouse models for human cell engraftment have recently been introduced that include the Rag2(-/-)γc(-/-), NOD/SCID, NOD/SCIDγc(-/-) and NOD/SCIDβ2m(-/-) strains. Transplantation of these mice with CD34(+) human hematopoietic stem cells leads to prolonged engraftment, multilineage hematopoiesis and the capacity to generate human immune responses against a variety of antigens. However, the various mouse strains used and different methods of engrafting human cells are beginning to illustrate strain specific variations in engraftment levels, duration and longevity of mouse life span. In these proof-of-concept studies we evaluated the Balb/c-Rag1(-/-)γ(-/-) strain for engraftment by human fetal liver derived CD34(+) hematopoietic cells using the same protocol found to be effective for Balb/c-Rag2(-/-)γc(-/-) mice. We demonstrate that these mice can be efficiently engrafted and show multilineage human hematopoiesis with human cells populating different lymphoid organs. Generation of human cells continues beyond a year and production of human immunoglobulins is noted. Infection with HIV-1 leads to chronic viremia with a resultant CD4 T cell loss. To mimic the predominant sexual viral transmission, we challenged humanized Rag1(-/-)γc(-/-) mice with HIV-1 via vaginal route which also resulted in chronic viremia and helper T cell loss. Thus these mice can be further exploited for studying human pathogens that infect the human hematopoietic system in an in vivo setting.     

Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce differential organ engraftment

In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34⁺ cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation.Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34⁺ cord blood stem cell preparations.     

Cellular source of soluble interleukin 2 receptors in serum of mice after recombinant interleukin 2 administration.

Previous studies have shown that peripheral blood mononuclear cells activated in vitro not only express cell-associated interleukin 2 receptors (IL2R) but also release a soluble form of this receptor. In this study, we demonstrate that administration of human recombinant IL 2 (rIL 2) to mice results in increased spleen weights, splenic natural killer (NK) cell cytolytic activity, and serum levels of soluble IL2R. However, compared with rIL 2-treated heterozygote controls, beige mice treated with rIL 2 displayed similar elevations in serum soluble IL2R but significantly less splenic NK activity. Likewise, administration of anti-asialo GM1 antiserum to rIL 2-treated mice resulted in a dramatic reduction in splenic NK cytolytic activity, but no reduction in serum soluble IL2R. Conversely, while rIL 2 treatment of BALB/c mice produced increased splenic NK activity and serum soluble IL2R, similar treatment of BALB/c nude mice resulted in elevation of only splenic NK activity. These studies demonstrate that administration of rIL 2 to normal mice can elevate both serum IL2R levels and splenic NK cytolytic activity. However, the results suggest that T cells are likely to be the source of elevated serum IL2R after rIL 2 administration.     

An improved protocol for efficient engraftment in NOD/LTSZ-SCIDIL-2Rγnull mice allows HIV replication and development of anti-HIV immune responses

Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rγ(null) (NSG) and NOD/SCID/IL2Rγ(null) (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials.     

Donor NK Cells and IL-15 Promoted Engraftment in Nonmyeloablative Allogeneic Bone Marrow Transplantation

Donor NK cells could promote engraftment by suppressing host alloreactive responses during allogeneic bone marrow transplantation (allo-BMT). The biological activity of NK cells could be significantly enhanced by IL-15. The current study attempted to evaluate the effect of donor NK cells and IL-15 administration on engraftment and immune reconstitution in a murine nonmyeloablative allo-BMT model. Mice infused with donor NK cells and treated with IL-15 during nonmyeloablative allo-BMT resulted in increased donor engraftment compared with either treatment alone. The number of donor-derived cell subsets also increased in the spleen of the recipient mice with combination treatment. The alloreactivity to donor type Ags was significantly reduced in the recipient mice with donor NK cell infusion and IL-15 treatment, which was manifested by decreased proliferation and IL-2 secretion of splenocytes from recipient mice in response to donor type Ags in MLR and decreased capacity of the splenocytes killing donor type tumor targets. We subsequently exposed recipient mice to reduced irradiation conditioning and showed that donor NK cell infusion and hydrodynamic injection-mediated IL-15 expression could synergistically promote donor engraftment and suppress alloreactivity during nonmyeloablative allo-BMT. Infusion of CFSE-labeled donor CD45.1(+) NK cells demonstrated that IL-15 could enhance the infused donor NK cell proliferation and function in vivo. IL-15 treatment also promoted donor bone marrow-derived NK cell development and function. Thus, donor NK cell infusion and IL-15 treatment could synergistically promote the engraftment and the development of donor-derived cell subsets and suppress the host alloresponse in a murine nonmyeloablative allo-BMT model.     

Human B cell growth and differentiation in the spleen of immunodeficient mice

Human mAbs (HumAbs) have therapeutic potential against infectious diseases and cancer. Heretofore, their production has been hampered by ethical constraints preventing the isolation of Ag-specific activated B cells by in vivo immunization. Alternatively, severe combined immune deficient (SCID) mice, transplanted i.p. with human (Hu)-PBLs, allow the in vivo stimulation of human Ab responses without the usual constraints. Unfortunately, human B cells only represent a minor fraction of the surviving graft, they are scattered all over the animal body, and thus are hard to isolate for subsequent immortalization procedures. To prevent this dispersion and to provide the human B cells with a niche for expansion and maturation, SCID mice were engrafted with Hu-PBL directly into the spleen. Simultaneously endogenous murine NK cell activity was depleted by treatment with an anti-mouse IL-2 receptor beta-chain Ab. During engraftment, human B lymphocytes became activated, divided intensely, and differentiated into plasmacytoid cells. In vivo exposure to a recall Ag after cell transfer induced expansion of Ag-specific B cell clones. One week after inoculation, human B cells were abundant in the spleen and could easily be recovered for fusion with a heteromyeloma line. This resulted in the formation of stable hybridoma cell lines that secreted Ag-specific HumAbs. Thus transplantation of human lymphoid cells in the spleens of immune deficient mice represents a model for the study of human T cell-dependent B cell activation and proves to be an excellent tool for the successful production of HumAbs.     

The nonclassical major histocompatibility complex molecule Qa-2 protects tumor cells from NK cell- and lymphokine-activated killer cell-mediated cytolysis

The cytotoxic activity of NK cells is regulated by class I MHC proteins. Although much has been learned about NK recognition of class I autologous targets, the mechanisms of NK self-tolerance are poorly understood. To examine the role of a nonpolymorphic, ubiquitously expressed class Ib Ag, Q9, we expressed it on class I-deficient and NK-sensitive B78H1 melanoma. Presence of this Qa-2 family member on tumor cells partially protected targets from lysis by bulk lymphokine-activated killer (LAK) cells. H-2K(b)-expressing B78H1 targets also reduced LAK cell activity, while H-2D(b) offered no protection. Importantly, blocking with F(ab')(2) specific for Q9 or removal of this GPI-attached molecule by phospholipase C cleavage restored killing to the level of vector-transfected cells. Experiments with LAK cells derived from H2(b) SCID and B6 mice established that NK1.1(+)TCR(-) NK and NK1.1(+)TCR(+) LAK cells were the prevalent cytolytic populations inhibitable by Q9. Treatment of mice with poly(I:C) also resulted in generation of Q9-regulated splenic cytotoxicity. LAK cells from different mouse strains responded to Q9, suggesting that the protective effect of this molecule is not detectably influenced by Ly49 polymorphisms or the presence/absence of Q9 in NK-harboring hosts. We propose that Q9 expressed on melanoma cells serves as a ligand for yet unidentified NK inhibitory receptor(s) expressed on NK1.1(+) NK/T cells.     

The Use of SCID Mice in Biotechnology and as a Model for Human Disease

Abstract

The use of SCID (severe combined immunodeficient) mice in medical research and biotechnology has increased tremendously in recent years. This review outlines the major characteristics of these animals and the impediments that they pose to the engraftment of human cells and tissues. The development of our SCID mice pretreatment protocol (anti-asialo GM1 antisera and radiation) is described, and the results of xenotransplantation studies of human cells and tissues in these pretreated animals are outlined. Wherever possible, data from transplantation studies (of human tissues and cells) in pretreated and nonpretreated animals are compared. The potential of our pretreated SCID mice for medical research and biotechnology is discussed.     

Effects of adriamycin on the activity of mouse natural killer cells

Adriamycin, a widely employed anti-neoplastic agent, was found to have either inhibitory or stimulatory effects on NK activity, depending on the site examined. A single i.p. administration of ADM resulted in a rapid increase of cytolytic activity by PEC of various mouse strains. The effector cells appeared to be NK cells, being nonadherent and nonphagocytic; they expressed low amounts of Thy 1.2 antigen and had the same pattern of specificity as splenic NK cells. In contrast to the stimulatory effects of NK activity of PEC, ADM caused a transient dose-dependent depression of NK activity in the spleen, with a peak reduction at day 3 and recovery within a few days thereafter. The depressed NK activity could be reversed by removal of adherent cells by passage through a nylon column. Moreover, ADM induced cytostatic activity against tumor cells by macrophages, suggesting that activated macrophages may be responsible for suppression of splenic NK activity. The possible modulation of the levels of NK activity by ADM-induced macrophages was supported by mixture experiments, in which plastic adherent spleen cells from ADM-treated mice, but not from normal mice, inhibited the NK activity of normal spleen cells.     

Evidence that lymphokine-activated killer cells and natural killer cells are distinct based on an analysis of congenitally immunodeficient mice

The in vitro incubation of lymphoid cells in RIL 2 results in the generation of LAK cells that are broadly lytic to autologous, syngeneic, and allogeneic fresh tumor cells, but which do not lyse fresh, normal cells. Strains of mice with congenital immunodeficiencies were tested both for the presence of NK cells and for their capacity to generate LAK cells after in vitro incubation with IL 2. Splenocytes obtained from two immunodeficient mouse strains (NIH-Beige-Nude and NIH-Beige-Nude-XID) failed to generate LAK cells, but displayed significant activity. Splenocytes from another immunodeficient mouse strain (NIH-Beige-XID) generated LAK cells but did not display NK cell activity. This dissociation of activation of LAK cells from NK cells among the immunodeficient strains indicates that the LAK and NK cell lytic systems are distinct.     

Primary HIV-1 infection of human CD4+ T cells passaged into SCID mice leads to selection of chronically infected cells through a massive fas-mediated autocrine suicide of uninfected cells

We have recently shown that a human CD4+ T cell line (CEM-SS) acquires the permissiveness to M-tropic strains and primary isolates of HIV-1 after transplantation into SCID mice. This permissiveness was associated with the acquisition of a memory (CD45RO+) phenotype as well as of a functional CCR5 coreceptor. In this study, we have used this model for invest-igating in vivo the relationships between HIV-1 infection, apoptosis and T cell differentiation. When an in vivo HIV-1 infection was performed, the CEM cell tumors grew to a lower extent than the uninfected controls. CEM cells explanted from uninfected SCID mice (ex vivo CEM) underwent a significant level of spontaneous apoptosis and proved to be CD45RO+, Fas+ and Fas-L+, while Bcl-2 expression was significantly reduced as compared to the parental cells. Acute HIV-1 infection markedly increased apoptosis of uninfected ex vivo CEM cells, through a Fas/Fas-L-mediated autocrine suicide/fratricide, while parental cells did not undergo apoptosis following viral infection. The susceptibility to apoptosis of ex vivo CEM cells infected with the NSI strain of HIV-1, was progressively lost during culture, in parallel with the loss of Fas-L and marked changes in the Bcl-2 cellular distribution. On the whole, these results are strongly reminiscent of a series of events possibly occurring during HIV-1 infection. After an initial depletion of bystander CD4+ memory T cells during acute infection, latently or chronically infected CD4+ T lymphocytes are progressively selected and are protected against spontaneous apoptosis through the development of an efficient survival program. Studies with human cells passaged into SCID mice may offer new opportunities for an in vivo investigation of the mechanisms involved in HIV-1 infection and CD4+ T cell depletion.     

Immunodeficient mice have elevated numbers of NK cells in non-lymphoid tissues

Natural killer (NK) cells are an important part of the innate immune response. They have the ability to recognize and kill many types of tumor cells and promote immunity against intracellular pathogens. In this study, we analyzed the in situ localization of NK cells within wildtype and immunodeficient mice using a novel in situ analysis method. We have identified NK cells in tissues of B6 and B6.Rag1(-/-) mice and demonstrated an increase in the percentage of NK cells and the total number of NK cells in the lung and liver of immunodeficient mice. This increase was not due to an increase in NK cell activation. This study describes a means to identify NK cells within complex tissue environments, and the increase in NK cells in non-lymphoid tissues may explain much of the increased NK cell activity observed in T-cell-deficient mice.     

hu-PBL-SCID小鼠体内人淋巴细胞状况及其对人肺癌移植瘤转移的影响

将人外周血淋巴细胞经腹腔移植于T、B细胞严重联合免疫缺陷的SCID小鼠后,成功地建立了hu - PBL- SCID小鼠模型。在移植后4 周,其小鼠血清中存在人免疫球蛋白,其脾淋巴细胞中,所检测的8 种免疫表型人淋巴细胞亚群均存在,表明人淋巴细胞在SCID小鼠体内出现了增殖或重建。但hu- PBL- SCID小鼠及未免疫重建的SCID小鼠体内人肺巨细胞癌PLA- 801DL的生长及转移情况未见明显差异,提示hu- PBL- SCID小鼠体内的人淋巴细胞不具有明显的抗肿瘤活性。     

Down-regulation of natural killer cell activity in MoLV leukemogenesis: evidence for tumor-cell-mediated suppression

The role of natural killer (NK) cells in retrovirus-induced leukemogenesis was studied. These cells which do not require prior sensitization are considered as a part of the body's defense system against tumor development and spread. Neonate BALB/c mice infected with Moloney murine leukemia virus (MoLV) develop leukemia within 3-6 months. The MoLV-infected mice showed a progressive loss of endogenous and augmented NK activity, correlated with the development of the leukemic state. Mixing of spleen cells from tumor-bearing mice with NK-augmented splenocytes resulted in suppression of NK activity. In addition, mixing of T cell lines isolated from MoLV-induced tumors with augmented splenocytes also resulted in the down-regulation of NK cell activity. The present study demonstrates that tumor cells from leukemic organs and leukemic T cell lines can actively suppress NK cell function. It is postulated that after MoLV infection the progression of virus-transformed T cells to a fully developed tumor depends on the ability of these cells to down-regulate NK cell activity and thus escape immune surveillance.     

Modulation of NK cell activity by moderate intensity endurance training and chronic ethanol consumption.

Chronic ethanol consumption can suppress natural killer (NK) cell activity. Exercise after ethanol administration may enhance blood ethanol clearance, which may benefit the immune response. This study examined the effects of moderate intensity endurance training and chronic ethanol consumption (20% wt/vol) on splenic NK cell activity. Mice were assigned to one of four groups: sedentary, water drinking (SED-H2O); sedentary, ethanol consuming (SED-EtOH); trained, water drinking (TR-H2O), and trained, ethanol consuming (TR-EtOH). TR groups ran 60 min/day, 5 days/wk, at 12 m/min for 10 wk. Mice were killed 48 h after exercise. Baseline NK cell activity was suppressed 30% in TR and EtOH groups compared with SED-H2O controls. Activation with recombinant human interleukin-2 increased cytolytic activity in all groups four- to fivefold. These results indicate that training did not abrogate the effects of chronic ethanol consumption on NK cell activity. Furthermore, moderate endurance training may contribute to suppressed nylon wool-enriched NK cell activity in murine splenocytes for as long as 48 h after exercise.     

The tumorigenicity of mouse embryonic stem cells and in vitro differentiated neuronal cells is controlled by the recipients' immune response

Embryonic stem (ES) cells have the potential to differentiate into all cell types and are considered as a valuable source of cells for transplantation therapies. A critical issue, however, is the risk of teratoma formation after transplantation. The effect of the immune response on the tumorigenicity of transplanted cells is poorly understood. We have systematically compared the tumorigenicity of mouse ES cells and in vitro differentiated neuronal cells in various recipients. Subcutaneous injection of 1x10(6) ES or differentiated cells into syngeneic or allogeneic immunodeficient mice resulted in teratomas in about 95% of the recipients. Both cell types did not give rise to tumors in immunocompetent allogeneic mice or xenogeneic rats. However, in 61% of cyclosporine A-treated rats teratomas developed after injection of differentiated cells. Undifferentiated ES cells did not give rise to tumors in these rats. ES cells turned out to be highly susceptible to killing by rat natural killer (NK) cells due to the expression of ligands of the activating NK receptor NKG2D on ES cells. These ligands were down-regulated on differentiated cells. The activity of NK cells which is not suppressed by cyclosporine A might contribute to the prevention of teratomas after injection of ES cells but not after inoculation of differentiated cells. These findings clearly point to the importance of the immune response in this process. Interestingly, the differentiated cells must contain a tumorigenic cell population that is not present among ES cells and which might be resistant to NK cell-mediated killing.