A functional role of HLA-G expression in human gliomas: an alternative strategy of immune escape

HLA-G is a nonclassical MHC molecule with highly limited tissue distribution that has been attributed chiefly immune regulatory functions. Glioblastoma is paradigmatic for the capability of human cancers to paralyze the immune system. To delineate the potential role of HLA-G in glioblastoma immunobiology, expression patterns and functional relevance of this MHC class Ib molecule were investigated in glioma cells and brain tissues. HLA-G mRNA expression was detected in six of 12 glioma cell lines in the absence of IFN-gamma and in 10 of 12 cell lines in the presence of IFN-gamma. HLA-G protein was detected in four of 12 cell lines in the absence of IFN-gamma and in eight of 12 cell lines in the presence of IFN-gamma. Immunohistochemical analysis of human brain tumors revealed expression of HLA-G in four of five tissue samples. Functional studies on the role of HLA-G in glioma cells were conducted with alloreactive PBMCs, NK cells, and T cell subpopulations. Expression of membrane-bound HLA-G1 and soluble HLA-G5 inhibited alloreactive and Ag-specific immune responses. Gene transfer of HLA-G1 or HLA-G5 into HLA-G-negative glioma cells (U87MG) rendered cells highly resistant to direct alloreactive lysis, inhibited the alloproliferative response, and prevented efficient priming of cytotoxic T cells. The inhibitory effects of HLA-G were directed against CD8 and CD4 T cells, but appeared to be NK cell independent. Interestingly, few HLA-G-positive cells within a population of HLA-G-negative tumor cells exerted significant immune inhibitory effects. We conclude that the aberrant expression of HLA-G may contribute to immune escape in human glioblastoma.     

Potent induction of TNF-α during interaction of immune effectors with oral tumors as a potential mechanism for the loss of NK cell viability and function

The inhibitory role of TNF-α on survival of naïve and IL-2 treated NK cells has been demonstrated in the past. However, its effect on the function of these cells against tumor cells, in particular against oral tumors has not been established. We investigated the significance of secreted TNF-α in death and functional loss of splenocytes and NK cells in ex-vivo cultures with oral tumors. Oral tumors trigger potent secretion of TNF-α by human and murine immune effectors. Absence of TNF-α increases the cytotoxic activity and secretion of IFN-γ by IL-2 treated splenocytes and NK cells in co-cultures with MOK L2D1+/p53?/? oral tumor cells. IL-2 treated splenocytes and NK cells from TNF-α ?/? mice survive and proliferate more when compared to cells from TNF-α +/+ mice. Cell death induced by F. nucleatum, an oral bacteria, in TNF-α ?/? splenocytes are considerably lower than that induced in TNF-α +/+ splenocytes where potent release of TNF-α is reproducibly observed. Addition of exogenous rTNF-α to IL-2 treated splenocytes and NK cells decreased survival and function of splenocytes and NK cells obtained from TNF-α ?/? mice against oral tumors. These findings suggest that potent induction of TNF-α during interaction of immune effectors with oral tumors and/or oral bacteria is an important factor in decreasing the function and survival of cytotoxic immune effectors. Strategies to neutralize TNF-α may be beneficial in the treatment of oral cancers.     

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.     

HLA-G与肿瘤研究进展

人类白细胞抗原-G（human leukocyte antigen G,HLA—G1属于非经典的HLAI类分子,是机体内重要的免疫耐受分子。HLA．G可以与表达在免疫细胞上的受体结合直接发挥免疫抑制功能,同时通过诱导产生调节性T细胞（regulatory Tcells,Treg）或“Trogocytosis”机制参与机体的免疫耐受,以协助肿瘤细胞实现免疫逃逸。近年来研究发现,HLA．G在多种恶性肿瘤中均存在异常表达并抑制宿主的抗肿瘤免疫反应。对HLA-G在肿瘤中的表达及可能的作用机制研究进展作一综述。     

HLA-G*0105N null allele encodes functional HLA-G isoforms

Expression of the nonclassical HLA class I antigen, HLA-G, is associated with immune tolerance in view of its role in maintaining the fetus in utero, allowing tumor escape, and favoring graft acceptance. Expressed on invasive trophoblast cells, HLA-G molecules bind inhibitory receptors on maternal T lymphocytes and NK cells, thereby blocking their cytolytic activities and protecting the fetus from maternal immune system attack. The HLA-G gene consists of 15 alleles, including a null allele, HLA-G*0105N. HLA-G*0105N presents a single base deletion, preventing translation of both membrane-bound (HLA-G1) and full-length soluble isoforms (HLA-G5) as well as of the spliced HLA-G4 isoform. The identification of healthy subjects homozygous for this HLA-G null allele suggests that the HLA-G*0105N allele may generate other HLA-G isoforms, such as membrane-bound HLA-G2 and -G3 and the soluble HLA-G6 and -G7 proteins, which may substitute for HLA-G1 and -G5, thus assuming the immune tolerogeneic function of HLA-G. To investigate this point, we cloned genomic HLA-G*0105N DNA and transfected it into an HLA-class I-positive human cell line. The results obtained indicated that HLA-G proteins were indeed present in HLA-G*0105N-transfected cells and were able to protect against NK cell lysis. These findings emphasize the role of the other HLA-G isoforms as immune tolerogeneic molecules that may also contribute to maternal tolerance of the semiallogenic fetus as well as tumor escape and other types of allogeneic tissue acceptance.     

Inhibiting Stat3 signaling in the hematopoietic system elicits multicomponent antitumor immunity

The immune system can act as an extrinsic suppressor of tumors. Therefore, tumor progression depends in part on mechanisms that downmodulate intrinsic immune surveillance. Identifying these inhibitory pathways may provide promising targets to enhance antitumor immunity. Here, we show that Stat3 is constitutively activated in diverse tumor-infiltrating immune cells, and ablating Stat3 in hematopoietic cells triggers an intrinsic immune-surveillance system that inhibits tumor growth and metastasis. We observed a markedly enhanced function of dendritic cells, T cells, natural killer (NK) cells and neutrophils in tumor-bearing mice with Stat3(-/-) hematopoietic cells, and showed that tumor regression requires immune cells. Targeting Stat3 with a small-molecule drug induces T cell- and NK cell-dependent growth inhibition of established tumors otherwise resistant to direct killing by the inhibitor. Our findings show that Stat3 signaling restrains natural tumor immune surveillance and that inhibiting hematopoietic Stat3 in tumor-bearing hosts elicits multicomponent therapeutic antitumor immunity.     

Immune surveillance properties of human NK cell-derived exosomes

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56(+) and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.     

HLA-G inhibits rolling adhesion of activated human NK cells on porcine endothelial cells.

Human NK cells adhere to and lyse porcine endothelial cells (pEC) and therefore may contribute to the cell-mediated rejection of vascularized pig-to-human xenografts. Since MHC class I molecules inhibit the cytotoxic activity of NK cells, the expression of HLA genes in pEC has been proposed as a potential solution to overcome NK cell-mediated xenogeneic cytotoxicity. HLA-G, a minimally polymorphic HLA class I molecule that can inhibit a wide range of NK cells, is an especially attractive candidate for this purpose. In this study we tested whether the expression of HLA-G on pEC inhibits the molecular mechanisms that lead to adhesion of human NK cells to pEC and subsequent xenogeneic NK cytotoxicity. To this end two immortalized pEC lines (2A2 and PED) were stably transfected with HLA-G1. Rolling adhesion of activated human NK cells to pEC monolayers and xenogeneic cytotoxicity against pEC mediated by polyclonal human NK lines as well as NK clones were inhibited by the expression of HLA-G. The adhesion was partially reversed by masking HLA-G on pEC with anti-HLA mAbs or by masking the HLA-G-specific inhibitory receptor ILT-2 on NK cells with the mAb HP-F1. The inhibition of NK cytotoxicity by HLA-G was only partially mediated by ILT-2, indicating a role for other unknown NK receptors. In conclusion, transgenic expression of HLA-G may be useful to prevent human NK cell responses to porcine xenografts, but is probably not sufficient on its own. Moreover, the blocking of rolling adhesion by HLA-G provides evidence for a novel biological function of HLA molecules.     

Analysis of HLA expression in human tumor tissues

Cancer cells can be detected and destroyed by cytotoxic T lymphocytes in many experimental tumor systems, and--as has been well-documented--in some human tumors. In humans however, most diagnosed tumors are not eliminated by T cells but grow steadily, invading and metastasizing until the host is destroyed. Evidence is accumulating that progressive tumor growth occurs not because the immune system is defective or deteriorated, but because the cancer cell is capable of developing a variety of strategies to escape immune recognition. In addition, cancer cells acquire new biological properties to generate invasive capacity in order to migrate and colonize new tissues. Major histocompatibility complex (MHC) antigens are molecules that are specialized in communicating with the T cell receptor and natural killer (NK) cell ligands. With the former, they use the interaction with peptides derived from processed cellular and exogenous proteins to monitor self and non-self status. With the latter, they determine the degree of activation and killing capacity of NK cells by interacting with NK receptors. Any change in the MHC profile of tumor cells (including classical and nonclassical MHC molecules) may therefore have a profound influence on the immune recognition and immune rejection of cancer cells. We have reviewed the data from our laboratory and other groups, and have presented a standardized procedure for analyzing the MHC profile of human tumors with special emphasis on the quality and laboratory use of the material obtained from microdissected tumor samples. Appropriate tissue processing is of particular relevance, since it is not possible to obtain tumor cell lines from most patients. Oncologists require rapid information on the MHC profile of the tumor if gene therapy is envisaged to restore normal MHC class I gene expression.     

The gp49B1 inhibitory receptor regulates the IFN-gamma responses of T cells and NK cells

The magnitude and diversity of Ag-specific T cell effector activity have been proposed to be controlled by an integration of positive signals transduced by the TCR and negative signals originating from inhibitory cell surface molecules. Although the lectin family of NK cell-associated inhibitory receptors has been reported to regulate the function of murine CTLs, gp49B1, the Ig superfamily member is not known to be expressed on T cells. Moreover, the consequences of the lack of an endogenously expressed NK cell-associated inhibitory receptor on T cell functions are not known. We report that gp49B1 is expressed by nearly all activated CD8 and CD4 T cells in addition to NK cells during an immune response to viral, bacterial, or tumor challenge. Kinetics of gp49B1 expression parallel functional capability and subside in the memory phase. Following vaccinia viral infection, IFN-gamma production by both subsets of T cells and NK cells is enhanced in gp49B1-deficient mice compared with gp49B1(+/+) mice. The stimulation threshold for IFN-gamma production is also lower in gp49B1-deficient T cells. In contrast, no significant differences were observed in the cytotoxic responses. We conclude that gp49B1 is a unique inhibitory receptor that is induced in multiple lineages of innate and adaptive immune cells during an infection and controls their IFN-gamma, but not cytotoxic responses.     

Suppressor factor from tumor-allosensitized spleen cells--its effect on in vitro proliferation of tumor cells and in vivo skin allograft survival

C57BL/6 mice are sensitized ip with allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the sensitized mice are used in the production of suppressor factor or treated with mitomycin and used as suppressor cells. Sensitized spleen cells incubated with the specific alloantigen (DBA/2 m-treated spleen cells) release suppressor factor (SF)² which inhibits cell proliferation in mixed lymphocyte culture (MLC) as well as the in vitro generation of cytotoxic cells (CML). SF is most effective when added eary during MLC. SF also inhibits mitogen responsiveness of normal spleen cells. In addition to inhibiting lymphocyte function in vitro, suppressor cells as well as SF inhibit the in vitro proliferation of tumor cells. This inhibition is specific for the tumor to which the suppressor cells are induced. The inhibition of tumor cell proliferation is not due to the presence of cytotoxic cells in the spleen of the tumor-allosensitized mice. Suppressor cells from neonatal mice do not inhibit the in vitro proliferation of tumor cells. SF injected iv into C57BL/6 mice decreases the mixed lymphocyte reactivity of the host spleen cells and decreases the ability of the host to reject skin allografts. We interpret these data to suggest that tumor-allosensitized spleen cells, and the SF they produce, not only affect lymphocyte function but also inhibit tumor cell proliferation. This dual effect of suppressor cells could be an important part of the immune surveillance against tumors.     

The selection of tumor variants with altered expression of classical and nonclassical MHC class I molecules: implications for tumor immune escape

Tumor immune escape variants can be identified in human and experimental tumors. A variety of different strategies are used by tumor cells to avoid recognition by different immune effector mechanisms. Among these escape routes, alteration of MHC class I cell surface expression is one of the mechanisms most widely used by tumor cells. In this review we focus our attention on the T-cell immune selection of MHC class I–deficient tumor variants. Different altered MHC class I phenotypes that originate from multiple molecular mechanisms can be identified in human tumors. MHC-deficient tumor clones can escape T-cell immune responses, but are in theory more susceptible to NK-cell–mediated lysis. In this context, we also review the controversial issue of the aberrant expression of nonclassical HLA class I molecules, particularly HLA-G, in tumors. This expression may be relevant in tumor cells that have lost the capacity to interact with NK inhibitory receptors—namely, those tumor cells with no HLA-B or HLA-C expression. Most published studies have not analyzed these possibilities and do not provide information about the complete HLA-A, HLA-B, or HLA-C molecule profiles of the tumors studied. In contrast, HLA-E has been reported to be expressed in some tumor cell lines with very low HLA-A, HLA-B, and HLA-C expression, suggesting that HLA-E may indeed, in some cases, play a role by inhibiting NK lysis of cells that otherwise would be destroyed by NK cells. Finally, we provide evidence that the status of the immune system in the tumor-bearing animal is capable of defining the MHC profile of the tumor cells. In other words, MHC class I–negative metastatic colonies are produced in immunocompetent animals, and MHC class I–positive colonies in T-cell immunodeficient individuals.This article forms part of the Symposium in Writing Tumor escape from the immune response, published in Vol. 53.     

Tolérance fœtomaternelle : rôle de la molécule HLA-G dans la protection du fœtus contre l'activité natural killer maternelle

HLA-G is a non-classical major histocompatibility complex class I molecule selectively expressed on extravillous trophoblast cells at the fetal—maternal interface. HLA-G may play an important role in maintaining maternal immune tolerance of the semi-allogenic fetus. In this study, we demonstrate for the first time the protective role of HLA-G during pregnancy. Indeed, cytotrophoblast cells of the fetus are resistant to lytic activity by maternal decidual natural killer cells. In order to precisely characterize the immunological functions of HLA-G products, we have investigated the protective role of the membrane-bound HLA-G1 and HLA-G2 isoforms against NK cell cytotoxicity. For this purpose, HLA-G1 and HLA-G2 cDNAs were transfected into the HLA-class I negative human K562 cell line. We demonstrate that both HLA-G1 and HLA-G2 transfectants inhibit NK cytolysis observed in peripheral blood from 25 donors (males and females). This led us to the conjecture that HLA-G is the public ligand for natural killer inhibitory receptors present in all individuals.     

Alloantigens placed into the anterior chamber of the eye induce specific suppression of delayed-type hypersensitivity but normal cytotoxic T lymphocyte and helper T lymphocyte responses

Intracameral inoculation of allogeneic B16F10 melanoma cells (C57BL/6) into LP/J mice resulted in progressively growing intraocular tumors and impaired delayed-type hypersensitivity (DTH) reactivity. Additional experiments showed that DTH responses were specifically down-regulated by splenic T suppressor cells. By contrast, subcutaneous inoculation of B16F10 melanoma cells induced significant DTH responses to the alloantigens expressed on the tumor cells and stimulated brisk rejection of the subcutaneously injected tumor cells. In spite of the T suppressor cell inhibition of DTH reactivity, significant cytotoxic T lymphocyte activity could be demonstrated in lymphoid cell suspensions from hosts harboring allogeneic intraocular tumors. The demonstrated cytotoxic T lymphocyte activity is particularly noteworthy because it occurs in the face of severely suppressed DTH responsiveness and thus implies that the intracameral presentation of alloantigens evokes a precise immunoregulatory process that selectively and concomitantly modulates specific cellular immune components; one immune process (cytotoxic T lymphocyte function) is stimulated whereas the other (DTH responsiveness) is down-regulated.     

Cancer immunoediting of the NK group 2D ligand H60a

Cancer immunoediting describes the process whereby highly immunogenic tumor cells are removed, or edited, from the primary tumor repertoire by the immune system. In immunodeficient mice, the editing process is hampered, and "unedited" tumor cells can be recovered and studied. In this study, we compared unedited and edited tumors for their expression of NK group 2D (NKG2D) ligands, a family of surface proteins expressed on tumor cells that can activate NK cell cytotoxic activity. We found that the expression of the NKG2D ligand H60a was more heterogeneous in groups of unedited 3'-methylcholanthrene sarcoma cell lines compared with that in edited 3'-methylcholanthrene sarcoma cell lines (i.e., some unedited cell lines expressed very high levels of H60a, whereas other unedited and edited cell lines expressed very low levels). We also found that some highly immunogenic cell lines displayed a bimodal distribution consisting of H60a-hi and H60a-lo cells. In one of these cell lines, the H60a-hi cells could be removed by passaging the cells through RAG2(-/-) mice, resulting in edited cell lines that were poor targets for NK cells and that displayed progressive tumor growth. This editing of H60a-hi cells required NK cells and NKG2D. Our studies show that the expression of H60a on tumors cells can be actively modulated by the immune system, thereby implicating this NKG2D ligand in tumor immunosurveillance.     

Human T Cell Crosstalk Is Induced by Tumor Membrane Transfer

Trogocytosis is a contact-dependent unidirectional transfer of membrane fragments between immune effector cells and their targets, initially detected in T cells following interaction with professional antigen presenting cells (APC). Previously, we have demonstrated that trogocytosis also takes place between melanoma-specific cytotoxic T lymphocytes (CTLs) and their cognate tumors. In the present study, we took this finding a step further, focusing on the ability of melanoma membrane-imprinted CD8⁺ T cells to act as APCs (CD8⁺T-APCs). We demonstrate that, following trogocytosis, CD8⁺T-APCs directly present a variety of melanoma derived peptides to fraternal T cells with the same TCR specificity or to T cells with different TCRs. The resulting T cell-T cell immune synapse leads to (1) Activation of effector CTLs, as determined by proliferation, cytokine secretion and degranulation; (2) Fratricide (killing) of CD8⁺T-APCs by the activated CTLs. Thus, trogocytosis enables cross-reactivity among CD8⁺ T cells with interchanging roles of effectors and APCs. This dual function of tumor-reactive CTLs may hint at their ability to amplify or restrict reactivity against the tumor and participate in modulation of the anti-cancer immune response.     

Suppression of in vitro maintenance and interferon-mediated augmentation of natural killer cell activity by adherent peritoneal cells from normal mice

The ability of adherent peritoneal cells (APC) to inhibit murine natural killer (NK) cell activity was examined. Nylon wool-nonadherent splenic effector cells were incubated overnight with or without different numbers of APC. NK activity was then measured against YAC-1 in a 4-hr 51Cr-release cytotoxicity assay. Proteose peptone-elicited or unstimulated resident APC from normal mice markedly suppressed NK activity of splenic effector cells in the presence or absence of exogenously added interferon. The suppression was dependent on the number of APC added with 10% APC, relative to the number of effector cells, resulting in a greater than 65% inhibition of cytotoxicity. The effector phase of cytotoxicity was not the target of the suppressor cells, because APC did not suppress NK activity when they were present only during the cytotoxicity assay. The addition of APC to alloimmune cytotoxic T cells under similar conditions resulted in no inhibition of cytotoxicity. Both syngeneic and allogeneic APC suppressed NK activity, but several murine macrophage-like cell lines lacked this property. In contrast to APC, incubation of effector cells with adherent spleen cells from normal mice resulted in no inhibition of NK activity. APC from mice injected with C. parvum were less inhibitory for NK activity than normal resident APC. In contrast, C. parvum APC suppressed concanavalin A-induced lymphoproliferation and were directly cytotoxic to tumor target cells in vitro, whereas normal APC lacked these properties. The results indicate that the peritoneum of untreated mice contains suppressor cells that can inhibit the in vitro maintenance and IFN-mediated augmentation of NK activity. In addition, these results indicate a broader spectrum of immune reactivities regulated by APC and suggest that, depending on their level of activation, APC can preferentially inhibit different immune functions.     

Inhibition of nuclear factor kappa B (NFκB) activity in oral tumor cells prevents depletion of NK cells and increases their functional activation

The aim of this study is to identify candidate factors which may be responsible for the functional inactivation and depletion of NK cells by tumor cells. Inhibition of NFκB activity by an IκB super-repressor in HEp2 cells, a cell line commonly used as an oral tumor model, blocked tumor-induced NK cell death, and increased the function of NK cells significantly. Increased expression of CD69 early activation antigen on NK cells as well as augmented proliferation and secretion of IFN-γ by NK cells were observed when these cells were co-incubated with IκB super-repressor transfected HEp2 cells (HEp2-IκB_(S32AS36A)). More importantly, the secretion of IL-6 was significantly inhibited when NK cells were co-cultured with HEp2-IκB_(S32AS36A) cells. In addition, the survival and function of cytotoxic effector cells remained significantly elevated in the presence of IFN-γ-treated HEp2-IκB_(S32AS36A) cells when compared to either untreated or IFN-γ-treated vector-alone transfected HEp2 cells. Similar findings to those obtained using purified peripheral blood NK cells were also observed when non-fractionated peripheral blood mononuclear cells were used in the co-cultures of immune effectors with HEp2 cell transfectants. Addition of recombinant human IL-6 to the co-cultures of immune effectors with the NFκB knockdown HEp2 tumor cells substantially decreased the levels of secreted IFN-γ. Thus, the results presented in this paper suggest that the inhibition of NFκB function in oral tumors may serve to activate and expand the function and numbers of NK cells. Moreover, NFκB-mediated increase in IL-6 secretion by oral tumors may in part be responsible for the observed inactivation and death of the immune effectors.This work was supported by RO1-DE12880 from NIDCR-NIH.     

Cloned "anomalous" killer cells derived from allogeneic mixed leukocyte culture

In addition to allospecific cytotoxic lymphocytes, cytolytic effector cells capable of killing a broad range of targets are generated during mixed leukocyte culture (MLC). These cells, which have been previously called anomalous killer cells, are a distinct functional subset separate from natural killer cells or allospecific cytotoxic lymphocytes but display many characteristics of lymphokine-activated killers. In order to isolate anomalous killer cells for detailed analysis, we generated the cytolytic effectors from an allogeneic MLC using heat-inactivated stimulators. This treatment of the stimulator population abrogated the generation of classical allospecific cytotoxic lymphocytes but allowed the generation of anomalous killer cells which were subsequently cloned via limiting dilution. The clones derived by this method displayed the functional properties of anomalous killers seen in bulk MLCs. The clones demonstrated potent cytolytic activity against both NK-sensitive and NK-resistant tumor targets in vitro and also suppressed tumor growth in vivo. Ultrastructural studies revealed features similar to those of cloned antigen-specific cytolytic cells and clones with NK-like function. The cells expressed surface glycoproteins associated with both NK and T lymphocytes including Thy-1, Ly-2, T200, Qa-5, asialo GM1, and the antigens defined by the NK alloantisera NK-2.1 and NK-3.1. These cells may play an important role during early phases of the immune response, since cytolytic cells of broad specificity may protect the host until classical cytotoxic lymphocytes with restricted specificity are generated.