Cold Target Inhibition Assays Research Articles

Differential susceptibility of cells expressing allogeneic MHC or viral antigen to killing by antigen-specific CTL.

CD8(+) cytotoxic T lymphocytes (CTLs) generated by immunization with allogeneic cells or viral infection are able to lyse allogeneic or virally infected in vitro cells (e.g., lymphoma and mastocytoma). In contrast, it is reported that CD8(+) T cells are not essential for allograft rejection (e.g., heart and skin), and that clearance of influenza or the Sendai virus from virus-infected respiratory epithelium is normal or only slightly delayed after a primary viral challenge of CD8-knockout mice. To address this controversy, we generated H-2(d)-specific CD8(+) CTLs by a mixed lymphocyte culture and examined the susceptibility of a panel of H-2(d) cells to CTL lysis. KLN205 squamous cell carcinoma, Meth A fibrosarcoma, and BALB/c skin components were found to be resistant to CTL-mediated lysis. This resistance did not appear to be related to a reduced expression of MHC class I molecules, and all these cells could block the recognition of H-2(d) targets by CTLs in cold target inhibition assays. We extended our observation by persistently infecting the same panel of cell lines with defective-interfering Sendai virus particles. The Meth A and KLN205 lines infected with a variant Sendai virus were resistant to lysis by Sendai virus-specific CTLs. The Sendai virus-infected Meth A and KLN205 lines were able to block the lysis of Sendai virus-infected targets by CTLs in cold target inhibition assays. Taken together, these results suggest that not all in vivo tissues may be sensitive to CTL lysis.

Survivin is a shared tumor-associated antigen expressed in a broad variety of malignancies and recognized by specific cytotoxic T cells

AbstractSurvivin, a member of the inhibitor of apoptosis protein family, is expressed in almost all types of malignancies, making this protein a useful tool for the development of broadly applicable vaccination therapies. We used a recently identified HLA-A2 binding peptide and dendritic cells (DCs) from healthy donors to induce survivin-specific cytotoxic T lymphocytes (CTLs) in vitro. These T cells efficiently lysed target cells pulsed with the cognate peptide. Furthermore, survivin-specific CTLs recognized HLA-A2–matched tumor cell lines and primary malignant cells from patients with leukemia in an antigen-specific and HLA-restricted manner as demonstrated with the use of cold target inhibition assays and blocking antibodies. To validate the immunogenicity of survivin we performed the experiments in an autologous setting and used monocyte-derived DCs as targets. Interestingly, we found that DCs up-regulate survivin expression on stimulation with tumor necrosis factor α (TNF-α). However, these mature DCs were not recognized by survivin-specific CTLs, whereas they lysed autologous mature DCs pulsed with the antigenic peptide or transfected with whole tumor RNA purified from a survivin-expressing cell line. To further analyze the possible use of survivin-specific CTLs in cancer therapies, we induced survivin-specific CTLs using peripheral blood mononuclear cells (PBMNCs) and DCs from a patient with chronic lymphocytic leukemia (CLL). The in vitro–generated T cells efficiently recognized autologous malignant CLL cells, whereas they spared autologous-purified nonmalignant B cells or DCs. Our results demonstrate that survivin epitopes are presented on a broad variety of malignancies and can be applied in vaccination therapies.

Transfection of dendritic cells with RNA induces CD4- and CD8-mediated T cell immunity against breast carcinomas and reveals the immunodominance of presented T cell epitopes.

Transfection of dendritic cells (DC) with tumor-derived RNA has recently been shown to elicit tumor-specific CTL capable of recognizing and lysing a variety of tumor cells. In our study we analyzed the induction of HLA class I- and II-restricted T cell responses against MCF-7 breast cancer cells. Using this approach we were able to elicit CD4- and CD8-mediated antitumor responses. The CTL specifically lysed MCF-7 cells and DC electroporated with MCF-7 RNA, but spared control cell lines. The specificity of the cytotoxic activity was confirmed in cold target inhibition assays and using mAbs blocking HLA class I molecules. Interestingly, these polyclonal cytotoxic T cells recognized selectively two epitopes derived from the MUC1 and Her-2/neu tumor Ags. The induced Th cells were found to be entirely HLA class II restricted and showed a significant cross-reactivity to a renal cell carcinoma cell line, similar to the results obtained with cytotoxic T cells.

Regression of extensive pulmonary metastases in mice by adoptive transfer of antigen-specific CD8(+) CTL reactive against tumor cells expressing a naturally occurring rejection epitope.

In this study, we developed a mouse model of adoptive immunotherapy reflecting immune recognition of syngeneic tumor cells naturally expressing an endogenous rejection Ag. Specifically, in a pulmonary metastases model, we examined the potency and maintenance of an antitumor CD8(+) CTL response in vivo, as well as its effectiveness against an "extensive" tumor burden. The approach taken was to first generate tumor-specific CTL from mice challenged with the CMS4 sarcoma coadministered with anti-CTLA4 mAb, which has been shown to facilitate the induction of Ag-specific T cell responses in vivo. An H-2L(d)-restricted nonamer peptide, derived from an endogenous murine leukemia provirus was identified as a CMS4-reactive CTL epitope based upon the following: CTL cross-recognition of another syngeneic tumor cell line (CT26 colon carcinoma) previously characterized to express that gene product; sensitization of Ag-negative lymphoblasts or P815 targets with the peptide; and by cold target inhibition assays. In vivo, the adoptive transfer of CMS4-reactive CTL (> or =1 x 10(6)) resulted in nearly the complete regression of 3-day established lung metastases. Furthermore, mice that rejected CMS4 following a single adoptive transfer of CTL displayed antitumor activity to a rechallenge 45 days later, not only in the lung, but also at a s.c. distal site. Lastly, the adoptive transfer of CTL to mice harboring extensive pulmonary metastases (> 150 nodules) led to a substantial reduction in tumor burden. Overall, these data suggest that the adoptive transfer of tumor-specific CTL may have therapeutic potential for malignancies that proliferate in or metastasize to the lung.

Thalidomide and immunomodulatory derivatives augment natural killer cell cytotoxicity in multiple myeloma

AbstractThe antiangiogenic activity of thalidomide (Thal), coupled with an increase in bone marrow angiogenesis in multiple myeloma (MM), provided the rationale for the use of Thal in MM. Previously, the direct anti-MM activity of Thal and its analogues (immunomodulatory drugs, IMiDs) on MM cells was demonstrated, suggesting multiple mechanisms of action. In this study, the potential immunomodulatory effects of Thal/IMiDs in MM were examined. It was demonstrated that Thal/IMiDs do not induce T-cell proliferation alone but act as costimulators to trigger proliferation of anti-CD3–stimulated T cells from patients with MM, accompanied by an increase in interferon-γ and IL-2 secretion. However, an increase in autologous T-cell killing of patient MM cells could not be demonstrated. A role for natural killer (NK)- and LAK-cell–mediated killing is suggested because IL-2–primed peripheral blood mononuclear cells (PBMCs) treated with Thal/IMiDs demonstrated significantly increased lysis of MM cell lines. Cold target inhibition assays suggested NK- rather than LAK-cell–mediated killing. Furthermore, this killing was not major histocompatibility complex-class restricted, and the depletion of CD56+ cells blocked the drug-induced MM cell lysis. It was significant that increased killing of patient MM cells by autologous PBMCs treated with Thal/IMiDs was also observed. Although the in vivo relevance of NK-cell–mediated MM cell killing is unknown, phenotypic analysis performed in MM patients receiving Thal therapy demonstrated an increase in CD3−CD56+cells in patients responding to therapy. Thus in vitro and in vivo data support the hypothesis that Thal may mediate its anti-MM effect, at least in part, by modulating NK cell number and function.

Dendritic cell-based vaccines in patients with hematological malignancies.

The epithelial mucin MUC1 is overexpressed on many epithelial malignancies as well as on some B-cell lymphomas and multiple myelomas. In the present study, we described MUC1 expression also on primary AML blasts. To analyze the presentation of MUC1-derived HLA-A2 restricted peptides by primary AML blasts, we induced MUC1-specific cytotoxic T-lymphocytes (CTLs) in vitro using peptide pulsed dendritic cells from HLA-A2+ healthy donors as antigen-presenting cells. These CTLs efficiently lysed primary AML blasts that constitutively expressed both MUC1 and HLA-A2. The specificity of the CTLs was confirmed in a cold target inhibition assay. Our data demonstrate that MUC1-derived peptides are tumor antigens in AML which could potentially be used for immunotherapeutic approaches.

Characterization of CD8+ cytotoxic T lymphocyte/tumor cell interactions reflecting recognition of an endogenously expressed murine wild-type p53 determinant.

p53 mutations are frequently found in human cancers and are often associated with the overexpression of wild-type (WT) protein or peptide sequences, supporting the notion that WT p53 epitopes may serve as potential targets for tumor immunotherapy. We have developed a cytotoxic T lymphocyte (CTL)/p53 tumor-associated antigen (TAA) model, based on immune recognition of a WT p53 determinant. WT p53-peptide-specific, major histocompatibility complex (MHC) classI-restricted CTL were produced from immunocompetent C57BL/6 (H-2b) mice after immunization with a previously defined WT p53 peptide (p53(232-240)) Epitope-specific CTL were then employed to identify syngeneic tumor cell populations expressing that antigenic determinant. Two syngeneic tumor cell lines, MC38 colon carcinoma and MC57G fibrosarcoma, were demonstrated to express the endogenous WT p53(232-240) determinant naturally, as defined by CD8 + CTL recognition. Cold-target inhibition assays confirmed that CTL-mediated lysis was due to immune recognition of the p53(232-240) peptide epitope. The p53(232-240)-specific CTL line did not lyse syngeneic normal cells (i.e., mitogen-activated splenocytes) in the absence of exogenous peptide, suggesting that the WT-p53-specific CTL could distinguish between tumor cells expressing self-TAA and normal host cells. We have demonstrated, for the first time, that the adoptive transfer of WT-p53-specific CTL to mice with established pulmonary metastasis resulted in antitumor activity in vivo. The ability to generate MHC-class-I-restricted CD8- CTL lines specific for a non-mutated p53 determinant from normal, immunocompetent mice, which display antitumor activity both in vitro and in vivo (by adoptive transfer), may have implications for the immunotherapy of certain p53-expressing malignancies.

Mapping the HLA-A24-restricted T-cell epitope peptide from a tumour-associated antigen HER2 / neu: possible immunotherapy for colorectal carcinomas.

HER2 / neu is a potential antigen candidate for immunotherapy because of its correlation to a poor prognosis and high expressions in many kinds of epithelial tumours. Especially in the colorectal carcinomas, the higher expression of HER2 / neu is recognized in metastatic regions as well as in primary sites. Several CTL epitopes restricted by HLA-A2.1 and -A3 were identified so far, however epitopes restricted by HLA-A24, that is one of the most common allele in Japanese and Caucasians, have not been identified. In this paper, we showed identification of a CTL epitope peptide of HER2 / neu restricted by HLA-A24. HLA-A24 binding peptides selected by an analysis based on HLA-A24 binding motifs were determined for their binding affinities to HLA-A24 molecules. The peptide with a sequence of RWGLLLALL (position 8–16) named HE1 showed the highest affinity. We induced CTLs from CD8+cells of HLA-A24 healthy donors by stimulation with HE1-pulsed autologous dendritic cells. The CTLs showed cytotoxic activity against not only the peptide-pulsed target cells but also HLA-A24 colorectal tumour cell lines that endogenously overexpressed HER2 / neu. The antigen-specificity was confirmed by cold target inhibition assay using HE1-pulsed target cells. In summary, HER2 / neu peptide, RWGLLLALL, may contribute to the induction of antitumour immunity with the peptide-based immunotherapy for the colorectal carcinomas. © 2001 Cancer Research Campaign http://www.bjcancer.com

HER-2/neu is expressed in human renal cell carcinoma at heterogeneous levels independently of tumor grading and staging and can be recognized by HLA-A2.1-restricted cytotoxic T lymphocytes

The HER-2/neu oncoprotein, a 185 kDa membrane-associated tyrosine kinase with extensive homology to the epidermal growth factor receptor (EGF-R), is overexpressed in breast and ovarian carcinomas. Its overexpression is closely associated with poor prognosis in the course of disease. Here we demonstrate HER-2/neu overexpression in both established cell lines and biopsy material obtained from renal epithelial tumors. Immunohistochemical analysis of human kidney tumor lesions using 2 HER-2/neu-specific antibodies revealed HER-2/neu expression in more than 40% of primary epithelial renal tumors and more than 30% of primary renal cell carcinoma (RCC) specimens. A distinctive HER-2/neu expression pattern was found in different subtypes of kidney tumors with the highest frequency in chromophilic and chromophobic RCC, but neither associated with disease stage nor tumor grade. Eight of 10 RCC cell lines expressed significant levels of HER-2/neu mRNA and protein, but at a lower level compared with HER-2/neu overexpressing ovarian carcinoma cells. To evaluate the immune response against HER-2/neu expressing HLA-A2-positive (HLA-A2(+)) RCC cells, allogeneic HLA-A2-restricted cytotoxic T-lymphocyte (CTL) lines generated by pulsing dendritic cells with 3 different HER-2/neu-derived peptides, (HER-2(9.369), HER-2(9.435) and HER-2(9.689), were utilized in chromium-release assays. Specific lysis of HER-2/neu expressing HLA-A2(+) RCC cell lines was mediated by CTL lines specific for each of these 3 HER-2/neu-derived epitopes. The fine specificity of 2 CTL clones was defined to the epitopes HER-2(9.435) and HER-2(9.689). Their specificity was then confirmed by cold target inhibition assays. In addition, CTL-mediated lysis was enhanced by pulsing tumor cells with exogenous HER-2/neu-specific peptides. Our data suggest that (i) HER-2/neu is heterogeneously expressed in different subtypes of RCC, (ii) HER-2/neu is naturally processed by RCC and (iii) HER-2/neu epitopes presented by RCC can be recognized by HLA-A2-restricted, HER-2/neu-specific CTL.

Two Types of Anti-TL (Thymus Leukemia) CTL Clones with Distinct Target Specificities: Differences in Cytotoxic Mechanisms and Accessory Molecule Requirements

TCRalphabeta CTL clones recognizing mouse thymus leukemia (TL) Ags were established and categorized into two groups: those killing any TL+ target cells (type I) and those killing only TL+ Con A blasts (type II). Cold target inhibition assays showed that the antigenic determinant(s) recognized by type II clones are expressed not only on TL+ Con A blasts but also on other TL+ target cells. The relation of the target specificity to the killing machinery and the accessory molecules involved in cytotoxicity were therefore analyzed using four representative clones selected from each type. Of the target cells tested, Fas was only expressed on Con A blasts, indicating that Fas ligand (FasL)-dependent cytotoxicity is limited to such cells. All four type II and one of four type I clones expressed FasL on the surface, while both types contained perforin in the cytoplasm. Blocking studies using neutralizing anti-FasL mAbs and concanamycin A (CMA), a selective inhibitor of the perforin pathway, suggested that type I clones kill target cells by way of perforin, while type II clones kill TL+ Con A blasts through FasL together with perforin. For their cytotoxicity, type I CTLs require a signal through CD8, while type II require LFA-1/ICAM-1 interactions. Type II clones also need a co-stimulatory signal through an unknown molecule for perforin-dependent cytotoxicity. These results taken together suggest that the difference in the target specificity of anti-TL CTL clones is due to variation in the killing machineries and the dependence on accessory molecules.

The role of calnexin in NK-target cell interaction

In this study, a relationship between target cell sensitivity to natural killing and target cell expression of the molecular chaperon calnexin was assessed. The NK-resistant cell line NKR was originally derived from the NK-sensitive, human T-cell line CEM and does not synthesize calnexin protein or mRNA. The cell lines CEM, NKR and 1B9 (NKR transfected with a calnexin cDNA) were compared in a number of assays. All the lines but CEM were resistant to NK in conventional 4 h cytotoxicity assay, but were highly sensitive to IL-2 activated NK. Incubation of NK cells with CEM but not with the other two lines led to increased expression of the NK cell activation marker CD69. Treatment of effector cells with PGE 2 and TGF- β resulted in an inhibition of NK activity and CD69 expression. The calnexin transfected clone 1B9 clone had intermediate ability to block cytotoxicity in cold target inhibition assay compared to CEM and NKR. Expression of the adhesion molecules CD44 and LFA-1 α was significantly higher on both calnexin positive cell lines compared to NKR. These data suggest that calnexin controls the expression of some, but not all, target structures that are necessary for binding and activation of NK cells.

Cytotoxic T lymphocytes derived from bone marrow mononuclear cells of multiple myeloma patients recognize an underglycosylated form of MUC1 mucin.

MUC1 is a highly immunogenic epithelial mucin and serves as a tumor-associated antigen in breast, pancreatic and ovarian carcinomas. We previously reported the expression of MUC1 on myeloma cells and the establishment of an HLA-unrestricted cytotoxic T lymphocyte (CTL) line TN that recognized MUC1 from peripheral blood mononuclear cells in a multiple myeloma patient. In this study, we attempted to induce such CTL from six other multiple myeloma patients consecutively in order to show that the induction of the CTL line TN had not resulted from some idiosyncrasy of the first patient. Bone marrow mononuclear cells were used to induce CTL, because they contain myeloma cells that might stimulate the autologous lymphocytes. Bulk CTL lines were induced from two out of six patients. The CTL line TS was CD8+ cell dominant and KY was CD4+ cell dominant. Both CTL lines lysed MUC1+ myeloma and breast carcinoma cell lines. The cytotoxicity of the CTL lines was inhibited by anti-CD3, anti-alpha beta TCR and anti-MUC1 mAb. It was also inhibited by a MUC1 transfectant, but not by a mock transfectant in cold target inhibition assays. MUC1 was transfected into a human colonic carcinoma cell line. The reactivity of anti-MUC1 core protein mAb and the cytotoxicity of the CTL against the transfectant was enhanced by the treatment of the cells with an O-glycosylation inhibitor. Thus it is generally accepted that the HLA-unrestricted CTL which directly recognize the underglycosylated from of MUC1 using their TCR could be induced from a certain proportion (approximately 30%) of untreated multiple myeloma patients.

Mechanism of interaction of pH 3.0 treated tumor cells expressing lower levels of class I MHC antigens, with IL-2 activated NK cells

Effect of lowering the levels of class I MHC antigens, on the ability of three murine (YAC, P815, SP 2O) and three human (Molt4, Raji, HR7) tumor cell lines, to compete in cold target inhibition assays, was studied. Specific reduction in class I MHC levels (range 60–76%) was induced by brief exposure to citrate buffer (pH 3.0). Susceptibility of the acid pH treated tumor cells to lysis by IL-2 activated NK cells, was found to be significantly increased in all cases with the exception of YAC cells. Reduction in class I MHC antigen expression by acid pH treatment, had no significant effect on competing ability of the tumor cells. In contrast, tumor cells on which MHC class I antigens were upregulated by interferon treatment, had significantly lower ability to compete. These results suggest that the mechanisms of altered interactions between MHC I up or down regulated target cells may be different. A model compatible with increased NK susceptibility along with an unaltered ability to compete for acid pH treated tumor cells, has been proposed.

Missing self by heterogeneous natural killer cells

Some murine (YAC, P815 and SP20) and human (Molt4, Raji and HR7) tumour cell lines were (i) treated with IFN-γ for inducing enhanced expression of MHC class I antigen, or (ii) given a brief treatment with citrate buffer (pH 3.0), which resulted in denaturation of class I MHC antigens on these tumour cells. IFL-γ or acid treated tumour cells were used as unlabelled competing targets in cold target inhibition assays. The results indicated that the competing ability of acid-treated tumour cells remained unaltered, whereas IFN-γ treated tumour cells competed with significantly less efficiency. These results have been evaluated in light of the current view of NK cell development and the expression of inhibitory receptors for MHC class I molecules (IRMs), on NK cells. A modified view on NK cell heterogeneity based upon IRM expression has been proposed which reconciles several apparently discordant observations about the activity and role of NK cells. Two classes of NK cells have been proposed. Type I NK ceils have target recognition receptors which do not recognize autologous normal cells, lack IRMs, and may participate in first line of defence against transformed cells in vivo. Type II NK cells have target recognition receptors for autologous normal cells and express at least one self-reactive IRM in order to prevent auto-killing. Type II NK cells participate in killing those transformed cells which down-regulate their MHC class I expression in order to escape cytotoxic T-cell surveillance. It is also postulated that mechanism of inverse correlation of target cell MHC class I expression levels and their susceptibility to NK cells, involves interference model of missing self hypothesis for type I NK cells and inhibitory signal model of missing self hypothesis for type II NE cells. Finally, it is proposed that acid treatment of tumour cells enhances their lysis susceptibility by making them additionally susceptible to type II NK cells, rather than enhancing their killing by type I NK cells. This proposition would explain the lack of effect of acid treatment on the competing ability of tumour cells, when target cells are only lysed by type I NE cells.

Heat shock protein 72 (HSP72), hyperthermia-inducible immunogenic determinant on leukemic K562 and Ewing's Sarcoma cells

Following non-lethal heat stress (41.8°C) and a recovery period at 37°C, the inducible 72kDa HSP (HSP72) is detectable selectively on the cell surface of human Ewing's Sarcoma (ES) and of leukemic K562 cells but not on EBV transformed B cells (B-LCL) which were generated from PBL of healthy human volunteers. The HSP72 expression was measured by flowcytometric analysis using a monoclonal antibody (moAb) that specifically recognizes HSP72, the inducible form of the HSP70 group. The major histocompatibility complex (MHC) class I expression, detected with the moAb W6/32 was not affected by non-lethal heat exposure and a recovery period at 37°C for 12 h: ES cells express MHC class I molecules on about 80% of the cells; K562 cells exhibited no MHC class I expression neither before nor after heat shock. Inhibition of RNA- (actinomycin D) or protein-synthesis (cycloheximide) prior to heat treatment completely inhibits the expression of HSP72 on the cell surface of both tumour cells, thus indicating that de novo protein synthesis is required for HSP72 cell surface expression. Since, apart from HSP72, protein synthesis in general is down-modulated by heat shock we speculate that HSP72 molecules that are expressed on the cell surface of tumour cells might be recruited from newly synthesized proteins. The heat-inducible HSP72 cell surface expression on tumour cells could be correlated with an increased sensitivity of leukemic and sarcoma cells to lysis mediated by NK effector cells. The results of cold target inhibition assays revealed that histologically different tumour cells (sarcoma and leukemic cells) that were exposed to non-lethal temperatures have to share a similar if not identical HSP72 immunogenic determinant.

Protective effects of interferon‐γ on squamous‐cell carcinoma of head and neck targets in antibody‐dependent cellular cytotoxicity mediated by human natural killer cells

An in vitro model of antibody-dependent cellular cytotoxicity (ADCC) was established, using squamous-cell carcinoma of the head and neck (SCCHN) targets, human/mouse chimeric monoclonal antibodies (cMAbs) SF-25 and 323/A3 and human peripheral blood mononuclear cells (PBMC). We previously showed that natural killer (NK) cells are the main effector population mediating ADCC in the presence of the cMAbs. ADCC was significantly inhibited by the overnight pre-treatment of SCCHN targets with exogenous interferon-γ (IFN-γ). This inhibition was dose-dependent, reproducible and consistently observed with various SCCHN cell lines. SCCHN cells pre-treated with IFN-γ had a significantly higher expression of intercellular adhesion molecule-1 (ICAM-I) and major histocompatibility complex (MHC) class I antigens compared with untreated target cells. No differences in expression of the SCCHN-associated antigens on these targets or in the formation of NK-SCCHN conjugates were found, using flow cytometry. IFN-γ-pre-treated SCCHN cells were less effective in competing with untreated targets in cold target inhibition assays and in inducing cytokine production from NK cells in co-incubation experiments. Protective effects of IFN-γ on target cell sensitivity to lysis were blocked by pre-treatment of target cells with actinomycin-D or cycloheximide. The susceptibility of the target cells was restored by removal of MHC class I antigens from their surface by acid stripping before ADCC. Our results suggest that the decreased ADCC seen with SCCHN targets pre-treated with IFN-γ is related to post-binding events, possibly altered signaling from targets to effector cells, and requires protein synthesis in the target cells. © 1996 Wiley-Liss, Inc.

Protective effects of interferon-γ on squamous-cell carcinoma of head and neck targets in antibody-dependent cellular cytotoxicity mediated by human natural killer cells

An in vitro model of antibody-dependent cellular cytotoxicity (ADCC) was established, using squamous-cell carcinoma of the head and neck (SCCHN) targets,human/mouse chimeric monoclonal antibodies (cMAbs) SF-25 and 323/A3 and human peripheral blood mononuclear cells (PBMC). We previously showed that natural killer (NK) cells are the main effector population mediating ADCC in the presence of the cMAbs. ADCC was significantly inhibited by the overnight pre-treatment of SCCHN targets with exogenous interferon-gamma (IFN-gamma). This inhibition was dose-dependent, reproducible and consistently observed with various SCCHN cell lines. SCCHN cells pre-treated with IFN-gamma had a significantly higher expression of intercellular adhesion molecule-I (ICAM-I) and major histocompatibility complex (MHC) class I antigens compared with untreated target cells. No differences in expression of the SCCHN-associated antigens on these targets or in the formation of NK-SCCHN conjugates were found, using flow cytometry. IFN-gamma-pre-treated SCCHN cells were less effective in competing with untreated targets in cold target inhibition assays and in inducing cytokine production from NK cells in co-incubation experiments. Protective effects of IFN-gamma on target cell sensitivity to lysis were blocked by pre-treatment of target cells with actinomycin-D or cycloheximide. The susceptibility of the target cells was restored by removal of MHC class I antigens from their surface by acid stripping before ADCC. Our results suggest that the decreased ADCC seen with SCCHN targets pre-treated with IFN-gamma is related to post-binding events, possibly altered signaling from targets to effector cells, and requires protein synthesis in the target cells.

Genes in two MHC class I regions control recognition of a single rat NK cell allodeterminant.

We have previously presented evidence suggesting that the non-classical class I region of the rat MHC, RT1.C, encodes polymorphic molecules which induce the cytolytic activity of alloreactive NK cells. Those studies used target cells from a panel of MHC congenic rat strains possessing recombined portions of the RT1a, RT1l and RT1u MHC haplotypes. We have now examined in addition a set of rat strains bearing MHC haplotypes recombinant between RT1av1 and RT1c, and a more complex picture of the MHC control of rat NK alloreactivity has emerged. The expression of a major NK allodeterminant [the allogeneic lymphocyte cytotoxicity (ALC) determinant 2 or ALC-2, defined operationally using cold-target inhibition assays], appears to be under the control of both the RT1.C and the classical class I RT1.A region. Similarly, the alloreactive repertoires of NK cells from these recombinant strains are influenced by elements encoded within these two MHC class I regions. We present a model in which the classical class I autoantigen RT1.Ac exhibits dominant inhibition of NK cytotoxicity specific for the stimulatory determinant ALC-2 shared by the nonclassical class I molecules RT1.Cav1, RT1.Ca and RT1.Cc, and also prevents the deletion of NK cells of this specificity during their development.

Tumor-specific and HLA-A2-restricted cytolysis by tumor-associated lymphocytes in human metastatic breast cancer.

A tumor-specific cytotoxic T lymphocyte (CTL) immune response has been well documented in melanoma, renal cell carcinoma, and ovarian cancer. Conflicting evidence exists regarding the existence of tumor-specific CTL populations in breast cancer. Tumor cells and tumor-associated lymphocytes (TAL) were isolated from the pleural effusions of six consecutive patients with metastatic breast cancer. After solid-phase anti-CD3 stimulation, TAL cultures were expanded with weekly autologous tumor stimulation and low-dose IL-2 for 3 wk. T cell populations were characterized using flow cytometric analysis and ranged from 49 to 91% CD8+, > 98% CD3+, and < 3% CD16+. Functionally, tumor-stimulated TAL showed tumor-specific recognition of autologous tumor cells (241 +/- 142 LU20/10(7)) and no detectable lysis of autologous fibroblasts, Daudi or K562. Cytotoxicity of TAL against HLA-A2+ allogeneic targets was significantly higher when compared with HLA-A2- tumor cell lines (127 +/- 76 vs 6 +/- 18 LU, p = 0.0001). This cytotoxicity against autologous and allogeneic tumor cells was blocked by anti-HLA-A2 mAb and cold HLA-A2+ targets in cold-target inhibition assays. TAL from all HLA-A2+ patients recognized GP2, a known, HER2/neu-derived tumor-associated peptide Ag that is HLA-A2 restricted. We have shown that TAL obtained from metastatic effusions of breast cancer patients contain lymphocytes that can recognize and lyse autologous and allogeneic tumor cells in a tumor-specific, HLA-A2-restricted fashion. In addition, tumor-specific TAL derived from breast cancer patients can selectively lyse HLA-A2+ pancreatic and ovarian tumor cell targets, suggesting a common HLA-A2-restricted tumor-associated Ag between these distinct epithelial cancers. Further elucidation of the cell-mediated immune response to breast cancer and the identification of shared TAA could result in the development of broadly applicable vaccine therapies for many cancers.

Heat Treatment of Leukemic Cell Lines Can Increase Their Sensitivity to NK Lysis

The effect of stress on target cell susceptibility to human natural killer cell-mediated lysis was examined. Targets were incubated at 37, 42, or 45°C for 1 hr and then tested for NK sensitivity in chromium-release assays. The T cell target JURKAT displayed minor increases in susceptibility to NK lysis with 42°C pretreatment (20-50% increases) and dramatic increaser in lysability with 45°C pretreatment (100-300% increase) compared to control. In contrast, lysis of the NK prototypic target K562 is not increased after 42 or 45°C pretreatment. Kinetic studies indicated an optimal NK sensitivity enhancement time of 1 hr at 45°C for JURKAT. Inhibition of target cell protein synthesis by emetine pretreatment does not produce an increase in susceptibility to NK lysis. JURKAT cells pretreated with sodium arsenite exhibited a comparable increase in NK sensitivity to the heat treatments. Cold target inhibition assays suggest that the increase in sensitivity after heat treatment is at a postbinding stage. This was exemplified by the increased sensitivity of JURKAT, but not K562, to lysis mediated by isolated rat NK granules. These results indicate that a heat-sensitive, de novo protein synthesis-independent defense mechanism against lysis may exist in some tumors, altering their susceptibility to lysis by NK cells.