Tumor infiltrating lymphocytes of spontaneous versus transplanted mouse mammary tumors

Summary Tumor infiltrating lymphocytes (TIL) were isolated by centrifugal elutriation from C4 mouse mammary tumors and characterized with regard to phenotype and natural killer (NK) activity. Tumors that had arisen spontaneously in prenoplastic hyperplastic alevolar nodules and tumors that had been passaged one to two times in either naive or presensitized mice were studied. Mice were sensitized by limited s.c. tumor growth and subsequent surgical removal of the tumor. The total numbers of T or B cells in the infiltrates were similar in spontaneous tumors and in passaged tumors from either naive or sensitized mice. The ratio of L3T4-positive to lyt-2-positive cells was reduced, however, from 1.10±0.2 in spontaneous tumors to 0.53±0.28 or 0.48±0.04 in passaged tumors from untreated or sensitized mice. The site of tumor implantation, whether intramammary fat pad or s.c., did not affect the profiles of the infiltrates. The TIL from both spontaneous and passaged tumors demonstrated enhanced NK activity relative to peripheral lymphoid cells. The TIL of passaged tumors sensitized mice, however, had lower NK activity than those from naive mice.     

T cell locomotion in the tumor microenvironment. I. A collagen-matrix assay

Previous studies have shown that lymphocytic infiltrates of different mouse mammary tumors contain different proportions of the T cell subsets Lyt-1+ and Lyt-2+. These characteristic subset ratios may be established, at least in part, by differential locomotion of subsets in response to components of the tumor microenvironment, such as soluble chemotactic and chemokinetic factors, cell and stromal surfaces, oxygen tension, and pH. We describe a new in vitro assay for determining how such microenvironmental variables affect lymphocyte locomotion. Suspended lymph node cells, alone or mixed with other cell types, are sandwiched between two layers of type I collagen gel and bathed in culture medium. A halo of locomotory cells fans out around the flattened droplet. Locomotion requires energy and exogenous protein. After a 24-hr incubation at 37 degrees C, 10% CO2 in air, the cell density of the halo is analyzed optically and the subset ratios are characterized by immunofluorescence staining of the gel sandwich. Under standard culture conditions, the locomotory population is enriched 12% in Thy-1+ cells compared with the bulk population, and the ratio of Lyt-1+ to Lyt-2+ cells is significantly increased. Lymphocyte locomotion is inhibited by 1 microM PGE2, by decreased pH and oxygen tension, and by the presence of normal mammary cells or mammary adenocarcinoma cells. The Lyt-1+:2+ ratio in the locomotory population is not altered by PGE2 but is reduced by acidity and hypoxia. The ratio is also reduced by the presence of mammary cells and cells of one of the mammary adenocarcinoma cell lines tested (168) but not by two others (68H and 410). Our data support the hypothesis that the locomotion of T cell subsets is differentially responsive to the types of microenvironmental conditions that vary among tumors.     

Association between Inflammatory Infiltrates and Isolated Monosomy 22/del(22q) in Meningiomas

Meningiomas contain highly variable levels of infiltrating tissue macrophages (TiMa) and other immune cells. In this study we investigated the potential association between the number and immunophenotype of inflammatory and other immune cells infiltrating the tumor as evaluated by multiparameter flow cytometry, and the clinico-biological, cytogenetic and gene expression profile (GEP) of 75 meningioma patients. Overall, our results showed a close association between the amount and cellular composition of the inflammatory and other immune cell infiltrates and the cytogenetic profile of the tumors. Notably, tumors with isolated monosomy 22/del(22q) showed greater numbers of TiMa, NK cells and (recently)-activated CD69⁺ lymphocytes versus meningiomas with diploid and complex karyotypes. In addition, in the former cytogenetic subgroup of meningiomas, tumor-infiltrating TiMa also showed a more activated and functionally mature phenotype, as reflected by a greater fraction of CD69⁺, CD63⁺, CD16⁺ and CD33⁺ cells. GEP at the mRNA level showed a unique GEP among meningiomas with an isolated monosomy 22/del(22q) versus all other cases, which consisted of increased expression of genes involved in inflammatory/immune response, associated with an M1 TiMa phenotype. Altogether, these results suggest that loss of expression of specific genes coded in chromosome 22 (e.g. MIF) is closely associated with an increased homing and potentially also anti-tumoral effect of TiMa, which could contribute to explain the better outcome of this specific good-prognosis cytogenetic subgroup of meningiomas.     

Cellular tumorigenicity in nude mice. Role of susceptibility to natural killer cells

The athymic nude mouse is a useful animal model for assaying the neoplastic growth potential in vivo of animal cells transformed in vitro. Despite the demonstrated absence of thymus-dependent immunological functions, however, the nude mouse has now been shown to reject transplants of certain highly malignant heterologous tumors. In addition, a few transformed mammalian cell lines that exhibit all or most of the cellular phenotypes usually associated with malignancy fail to grow as tumors when injected into nude mice. In a continuing study to identify the in vitro phenotypes associated with tumor-forming ability in vivo, we investigated the role of cellular susceptibility to the naturally occurring, thymus-independent lymphocytes (natural killer or NK cells) in determining tumor induction by animal cells in nude mice. A representative collection of animal cells (ranging from normal human diploid cell strains to highly tumorigenic clonal cell lines, either transformed in vitro or derived from experimental tumors) was tested to see if the ability of cells to form tumors is consistently correlated with their susceptibility to NK cell-mediated lysis measured in vitro with splenic leukocytes from nude mice. If the physiological role of the NK cells in vivo were to recognize, and possibly to destroy, incipient tumor cells in situ, a direct association between cellular tumorigenicity and susceptibility to NK activity, might be expected. If, on the other hand, the formation of growing tumors by animal cells in nude mice depended on their ability to escape the cytolytic activity of NK cells, cellular tumorigenicity would be associated with cellular resistance to NK cells. Results obtained in this study failed to confirm either of these associations. Thus, cellular suscepbibility to NK cells, at least as determined by direct cytotoxicity assay in vitro, is not a useful predictive indicator of cellular tumorigenicity in nude mice.     

Macrophage regulation of tumor angiogenesis: implications for cancer therapy

This article reviews the evidence for macrophages playing an important role in the regulation of tumor angiogenesis. Findings in mouse models show that macrophages promote angiogenesis in tumors both by producing excessive amounts of proangiogenic factors and by physically assisting sprouting blood vessels to augment the complexity of the intra-tumoral vascular network. Recent studies however suggest that macrophages may be dispensable for the initiation of angiogenesis in tumors. Rather, these cells express proangiogenic programs that enhance the complexity of the tumor-associated vasculature, leading to aberrant, plethoric and dysfunctional angiogenesis. Gene expression and cell depletion studies further indicate that tumor-associated macrophages (TAMs) comprise phenotypically and functionally distinct subsets. This may reflect “education” of the macrophage phenotype by signals in some areas of the tumor microenvironment and/or TAM subsets derived from distinct macrophage precursors. Among the better characterized TAM subsets are the proangiogenic (TIE2⁺) and the angiostatic/inflammatory (CD11c⁺) macrophages, which coexist in tumors. Such antagonizing TAM subsets occupy distinct niches in the tumor microenvironment and are present at ratios that vary according to the tumor type and grade. Specifically targeting TAMs or reprogramming them from a proangiogenic to an angiostatic function may “normalize” the tumor vasculature and improve the efficacy of various anticancer therapies, including radiotherapy, chemotherapy and vascular-disrupting agents.     

Identification and quantitation of T lymphocyte subsets found in the spinal cord of the Lewis rat during acute experimental allergic encephalomyelitis

Monoclonal antibodies specific for different rat T cell subsets and Ia-positive cells were used in a quantitative morphologic study of the cellular infiltrates in the spinal cords of Lewis rats during acute, actively induced experimental allergic encephalomyelitis (EAE). The predominant cell types in the inflammatory spinal cord lesions are W3/25-positive and Ia-positive cells. The relative percentages represented by each cell type remain quite constant regardless of the degree of clinical illness exhibited by the rat. These data demonstrate a quantitative profile of the infiltrating cells in acute, active EAE, and suggest that the principal inflammatory cells in these lesions are T helper cells and Ia-bearing cells (macrophages, B cells, or activated T helper cells).     

Lysis of fresh murine mammary tumor cells by syngeneic natural killer cells and lymphokine-activated killer cells

Summary We have compared the ability of natural killer (NK) cells from two substrains of C3H mice that differ with respect to their susceptibility to the development of mammary adenocarcinomas to lyse fresh syngeneic mammary tumor cells. Single cell suspensions of mammary tumors from retired breeder females were used as targets in 22-h ⁵¹Cr-release cytotoxicity assays with syngeneic NK cells. Tumor cell suspensions were prepared by enzymatic digestion of finely minced tissue followed by centrifugation through a discontinuous Percoll gradient. Effector cells were prepared by passing spleen cells over nylon wool followed by centrifugation through Percoll fraction 7. Syngeneic NK cells had significant levels of lysis against 5/8 tumors studied. NK cells from low risk animals (C3Heb/FeJ) consistently demonstrated greater cytotoxicity against tumor cell preparations than did effectors from the high tumor substrain (C3H/OuJ). Study of cytocentrifuge preparations stained with Wright-Giemsa revealed that the two substrains were identical with respect to the number of azurophilic granules present in the cytoplasm of their NK cells. We have also shown that lymphokine-activated killer (LAK) cells can be generated from splenocytes in C3H mice. While LAK cells from both substrains were capable of lysing fresh syngeneic mammary tumor cells in vitro, LAK cells from the animals at high risk for the formation of mammary adenocarcinomas had greater cytotoxicity against tumor cell suspensions than LAK cells from the low tumor substrain.     

Changes in the host natural killer cell population in mice during tumor development. 1. Kinetics and in vivo significance

Our earlier studies revealed an increase in the level of null (surface IgM-negative, Thy 1-negative) lymphocytes in mice shortly after tumor transplantation and before the clinical appearance of spontaneous mammary tumors. The present study examined the splenic natural killer (NK) cell activity as well as the incidence of NK lineage cells in these hosts, since NK cells are considered to be a subset of null lymphocytes. Splenic NK activity against YAC-1 lymphoma targets was measured with a 4-hr 51Cr-release assay in CBA mice transplanted ip with 10(6) Ehrlich ascites tumor (EAT) cells, in elderly C3H mice prior to and during the growth of spontaneous mammary tumors (SMT) and in young C3H mice transplanted sc with 5 X 10(6) SMT cells or 10(6) cells from two syngeneic mammary tumor lines (T-58 and MT-2) of recent origin. In EAT-transplanted mice total NK activity in the spleen increased rapidly to a peak (11-fold) at 3 days, coincident with the null cell rise, but then declined to subnormal levels by Day 7 when the null cell level was still high. A similar pattern of activity was exhibited by intratumor lymphocytes isolated from the EAT. In SMT-transplanted mice splenic NK activity showed a small rise at Day 3, followed by a drop to below normal at Day 7, subnormal levels lasting for the tumor life span. Similar results were noted in T-58- or MT-2-transplanted mice. Null lymphocytes recovered during the peak NK activity from the spleen of 3-day EAT-bearing mice, when mixed with 10(6) EAT cells at 25:1 E:T ratio and adoptively transferred into fresh mice in a Winn type assay either ip or sc, completely prevented tumor development indicating a high enrichment of NK cells functionally effective in vivo. Elderly clinically tumor-free C3H mice showed measurable NK activity, which dropped after the appearance of spontaneous mammary tumors to very low levels, the magnitude of decline rising with increasing tumor age (1-11 weeks) or size. The incidence of NK lineage cells was measured from the tumor target (YAC-1 lymphoma)-binding ability of the splenic null cells, identified with a radioautographic technique. Null target-binding cells (TBC) were NK-1+ and included both active as well as inactive NK lineage cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Perforin is a major contributor to NK cell control of tumor metastasis.

We provide the first demonstration, using experimental and spontaneous models of metastasis in C57BL/6 (B6) (RM-1 prostate carcinoma) and BALB/c (DA3 mammary carcinoma) mice, that tumor metastasis is primarily controlled by perforin-dependent cytotoxicity mediated by NK1.1+ cells. MHC class Ilow RM-1 and DA3 tumor cells were sensitive in vitro to Fas-mediated lysis or spleen NK cells in a perforin-dependent fashion. Perforin-deficient NK cells did not lyse these tumors, and perforin-deficient mice were 10-100-fold less proficient than wild-type mice in rejecting the metastasis of tumor cells to the lung. Fas ligand mutant gld mice displayed uncompromised protection against tumor metastasis. Depletion of NK subsets resulted in greater numbers of metastases than observed in perforin-deficient mice, suggesting that perforin-independent effector functions of NK cells may also contribute to protection from tumor metastasis.     

Mouse Lung and Spleen Natural Killer Cells Have Phenotypic and Functional Differences,in Part Influenced by Macrophages

NK cells are lymphocytes of the innate immune system which are a first line of defense against infections and tumor cells, in bone marrow and peripheral organs like lung and spleen. The lung is an organ in contact with respiratory pathogens and the site of inflammatory disorders triggered by the respiratory environment. In contrast, spleen is a lymphatic organ connected to the blood system which regulates the systemic immune response. Here we compare NK cell maturation and expansion as well as expression of NK cell receptors in spleen and lung compartments. We show that spleen and lung NK cells differ in phenotypic and functional characteristics due to a difference of maturity and cellular microenvironment. Indeed we observe that spleen and lung macrophages have the capacity to influence the cytotoxicity of NK cells by cell-to-cell contact. This suggests that the differences of NK cell subsets are in part due to a modulation by the organ environment.     

Indomethacin therapy abrogates the prostaglandin-mediated suppression of natural killer activity in tumor-bearing mice and prevents tumor metastasis

We have shown earlier that a decline in splenic natural killer (NK) activity during the development of transplanted or spontaneous tumors in mice results from an inactivation of NK lineage cells, mediated by prostaglandins (primarily PGE2) secreted by NK suppressor cells of the monocyte-macrophage lineage. In the present study we have used a C3H mouse mammary carcinoma model to examine whether this mechanism of NK suppression is conducive to tumor metastasis in vivo and whether a reversal of this suppression by a chronic indomethacin therapy can prevent metastatic spread from the primary tumor site. Three mammary tumor lines, all derived in our laboratory from a spontaneous C3H mammary tumor were employed: T-58 (uncloned parental line, having weak lung metastasizing ability from the subcutaneous site), C3 (a clone of T-58, showing high metastatic ability), and C10 (a nonmetastatic clone of T-58). Although the degree of NK susceptibility of these lines varied inversely with their metastatic potential, none was NK resistant. A chronic administration of indomethacin in the drinking water (14 micrograms/ml) to mice beginning on Day 4 after subcutaneous transplantation of 10(6) tumor cells resulted in a significant reduction in the growth rate of primary tumors in all hosts and led to a complete or nearly complete abrogation of lung metastasis in T-58- or C3-transplanted hosts examined at 1 month after tumor transplantation; C10-transplanted mice showed no metastasis in the control or the treated group. Concomitantly, there was a substantial restoration of splenic NK activity in all indomethacin-treated hosts. Plastic-adherent cells (greater than 95% macrophages) isolated from tumors growing in control mice, when coincubated for 20 hr with normal splenic effector cells caused a suppression of NK activity, reversible in the presence of indomethacin (10(-5) M) in vitro. Similar cells recovered from the residual primary tumors in indomethacin-treated mice had no suppressor ability. Chemically pure PGE2 (at concentrations of 0.5 to 1 X 10(-6) M, but not 0.25 X 10(-6) to 10(-8) M) also caused a suppression of NK activity of normal splenic effector cells, when added during the 4-hr 51Cr-release assay or allowed to interact with effector cells alone for a 20-hr incubation period; a removal of the cell-free PGE2 in the latter case prior to the NK assay did not relieve the suppression.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of tumor-associated lymphocytes in a series of mouse mammary tumor lines with differing biological properties

Tumor-associated lymphocytes were isolated by isokinetic gradient separation from five related mouse mammary tumor lines with different immunological and growth characteristics. Although considerable variation in recovery rates was seen from experiment to experiment, the five tumor types were found to have reproducible and characteristic patterns of T lymphocyte subpopulations, as detected by cytotoxicity assay using monoclonal antisera to Thy-1, Lyt-1, and Lyt-2 antigens. Tumors of line 168, which are weakly immunogenic at best, had the lowest numbers of recovered ALS+, Thy1+ lymphocytes (12% and 9%, respectively), in contrast to immunogenic lines (mean 38% and 26%, respectively). Line 68H tumors, which grow after prolonged latency periods and also produce tumor cell variants in vivo, were unique in that the numbers of recovered Lyt 1+ lymphocytes exceeded the number of Lyt 2+ lymphocytes, whereas these two T cell subpopulations were either equal or Lyt 2+ cells predominated in the other faster growing, non-variant-producing tumors. No differences in T lymphocyte distribution were associated with the presence or absence of metastatic behavior. These results indicate that distinctive lymphocyte infiltrates may be characteristic of tumors with distinct biological differences.     

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.     

CD8 T cell help for innate antitumor immunity

Innate immunity is considered to initiate adaptive antitumor responses. We demonstrate that monoclonal CD8 T lymphocytes reactive to tumor Ag P1A on P815 mastocytoma cells provide essential "help" to NK cells for rejection of P1A-deficient tumors. RAG-deficient mice have normal NK cells but do not reject either tumor. Reconstitution of these mice with P1A-specific T cells conferred resistance to both P1A-expressing and -deficient tumor cells provided they were present at the same site. Elimination of Ag-negative tumor variants required both activated T and NK cells. Gene expression profiling of NK cells infiltrating P1A-positive tumors in mice with specific CD8 T cells demonstrated an activated effector phenotype. However, CD8 T cell help to NK cells appeared ineffective for P1A-negative variants separated from the P1A-positive tumor. Local tumor Ag-specific T cell-NK cell collaboration results in the elimination of tumor cells whether they express or not the T cell tumor Ag epitope, thus containing the emergence of tumor escape variants before metastasis.     

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.     

Murine mammary carcinoma exosomes promote tumor growth by suppression of NK cell function

Many tumor cells shed specialized membrane vesicles known as exosomes. In this study, we show that pretreatment of mice with exosomes produced by TS/A or 4T.1 murine mammary tumor cells resulted in accelerated growth of implanted tumor cells in both syngeneic BALB/c mice and nude mice. As implanted TS/A tumor cells grew more rapidly in mice that had been depleted of NK cells, we analyzed the effects of the tumor-derived exosomes on NK cells. The tumor-derived exosomes inhibit NK cell cytotoxic activity ex vivo and in vitro as demonstrated by chromium release assays. The treatment of mice with TS/A tumor exosomes also led to a reduction in the percentages of NK cells, as determined by FACS analysis, in the lungs and spleens. Key features of NK cell activity were inhibited, including release of perforin but not granzyme B, as well as the expression of cyclin D3 and activation of the Jak3-mediated pathways. Human tumor cell lines also were found to produce exosomes that were capable of inhibiting IL-2-stimulated NK cell proliferation. Exosomes produced by dendritic cells or B cells did not. The presentation of tumor Ags by exosomes is under consideration as a cancer vaccine strategy; however, we found that pretreatment of mice with tumor exosomes blunted the protective effect of syngeneic dendritic cells pulsed ex vivo with tumor exosomes. We propose that tumor exosomes contribute to the growth of tumors by blocking IL-2-mediated activation of NK cells and their cytotoxic response to tumor cells.     

Mechanisms involved in NK resistance induced by interferon-gamma.

Human tumor cell lines were treated with interferon-gamma (IFN-gamma) and then used as target cells in NK assays to measure their ability to form conjugates and stimulate the production of NK cytotoxic factors (NKCF) and to determine their susceptibility to NKCF lysis. K562 and cell lines RS1, RS3, RS7, CAC, and CAP2, obtained from solid brain tumors, were used as targets, and peripheral blood lymphocytes (PBL) from normal donors were used as effector cells. IFN-gamma-treated cell lines had a decreased susceptibility to NKCF lysis and a decreased ability to induce the release of these factors without affecting target-effector cell binding. These results were not due to changes in HLA class I antigen expression, given that the level of HLA class I antigens on the tumor cell lines was not affected, the only exception being K562. In an attempt to further clarify the possible influence of HLA class I expression on K562, IFN-gamma-pretreated K562 cells were separated into HLA class I positive and HLA class I negative subsets for the NK assays. The results showed that both populations behaved similarly upon target-effector conjugate formation, whereas the HLA class I positive population showed a reduced susceptibility to lysis by NK cells and NKCF. Thus, these results establish that NK resistance induced by IFN-gamma is mediated by blocking the target cell's ability to activate NK cell triggering and release of NKCF and by blocking its susceptibility to lysis by these factors. This analysis helps to clarify not only the NK process but also the controversial regulatory effect of IFN in NK lysis.     

Abrogation of junctional adhesion molecule-A expression induces cell apoptosis and reduces breast cancer progression

Intercellular junctions promote homotypic cell to cell adhesion and transfer intracellular signals which control cell growth and apoptosis. Junctional adhesion molecule-A (JAM-A) is a transmembrane immunoglobulin located at tight junctions of normal epithelial cells of mammary ducts and glands. In the present paper we show that JAM-A acts as a survival factor for mammary carcinoma cells. JAM-A null mice expressing Polyoma Middle T under MMTV promoter develop significantly smaller mammary tumors than JAM-A positive mice. Angiogenesis and inflammatory or immune infiltrate were not statistically modified in absence of JAM-A but tumor cell apoptosis was significantly increased. Tumor cells isolated from JAM-A null mice or 4T1 cells incubated with JAM-A blocking antibodies showed reduced growth and increased apoptosis which paralleled altered junctional architecture and adhesive function. In a breast cancer clinical data set, tissue microarray data show that JAM-A expression correlates with poor prognosis. Gene expression analysis of mouse tumor samples showed a correlation between genes enriched in human G3 tumors and genes over expressed in JAM-A +/+ mammary tumors. Conversely, genes enriched in G1 human tumors correlate with genes overexpressed in JAM-A-/- tumors. We conclude that down regulation of JAM-A reduces tumor aggressive behavior by increasing cell susceptibility to apoptosis. JAM-A may be considered a negative prognostic factor and a potential therapeutic target.     

Role of cytokines in the adoptive immunotherapy of an experimental model of human head and neck cancer by human IL-2-activated natural killer cells.

Peritumoral injection of human IL-2-activated natural killer cells into nude mice consistently induced regression of xenografts of human squamous cell carcinoma of the head and neck (SCCHN). To determine the mechanisms responsible for the tumor regression, the lymphoid cells infiltrating the tumor stroma at 24 to 48 h after adoptive immunotherapy were examined by in situ hybridization for the presence of mRNA for cytokines or IL-2R. Numerous lymphoid cells expressing cytokine or IL-2R genes were observed in these tumors, whereas the cultured IL-2-activated NK cells used for therapy were negative. Thus, it appeared that the transferred NK cells became activated in situ after coming into proximity with the tumor cells. To analyze this phenomenon, fresh or cultured human NK cells were coincubated in vitro with irradiated human SCCHN cell line, PCI-1, with or without the presence of IL-2. Expression of mRNA for IL-2R, perforin, and various cytokines was observed within 5 h. Contact with the tumor cells stimulated NK cells to proliferate, secrete IFN-gamma, TNF-alpha, and soluble IL-2R, up-regulate cell surface expression of IL2R p55 and p75 as well as CD16 Ag, and mediate higher levels of antitumor activity in 51Cr-release assays. In addition, supernatants of in vitro-activated NK cells significantly inhibited proliferation of SCCHN cell lines. By examining the effects of neutralizing mAb to various cytokines, this inhibitory activity was shown to be partially attributable to IFN-gamma. To determine the possible in vivo role of soluble factors produced by activated human NK cells, the supernatants (0.2 ml) or rIFN-gamma (10(5) U) were injected perilesionally each day for 2 wk into 3-day SCCHN established in immunosuppressed nude mice. These treatments caused significant (p less than 0.02) inhibition of tumor growth. The results of our studies indicate that human NK cells are strongly activated by SCCHN cells and that the consequent release of cytokines contribute to the regression of SCCHN growing in nude mice.     

Increase in tumor size following intratumoral injection of immunostimulatory CpG-containing oligonucleotides in a rat glioma model

The immunosuppressive environment of malignant gliomas is likely to suppress the anti-tumor activity of infiltrating microglial cells and lymphocytes. Macrophages and microglial cells may be activated by oligonucleotides containing unmethylated CpG-motifs, although their value in cancer immunotherapy has remained controversial. Following injection of CpG-containing oligonucleotides (ODN) into normal rat brain, we observed a local inflammatory response with CD8+ T cell infiltration, upregulation of MHC 2, and ED1 expression proving the immunogenic capacity of the CpG-ODN used. This was not observed with a control ODN mutated in the immunostimulatory sequence (m-CpG). To study their effect in a syngeneic tumor model, we implanted rat 9L gliosarcoma cells into the striatum of Fisher 344 rats. After 3 days, immunostimulatory CpG-ODN, control m-CpG-ODN, or saline was injected stereotactically into the tumors (day 3 group). In another group of animals (day 0 group), CpG-ODN were mixed with 9L cells prior to implantation without further treatment on day 3. After 3 weeks, the animals were killed and the brains and spleens were removed. Rather unexpectedly, the tumors in several of the animals treated with CpG-ODN (both day 0 and day 3 group) were larger than in saline or m-CpG-ODN treated control animals. The tumor size in CpG-ODN-treated animals was more variable than in both control groups. This was associated with inflammatory responses and necrosis which was observed in most tumors following CpG treatment. This, however, did not prevent excessive growth of solid tumor masses in the CpG-treated animals similar to the control-treated animals. Dense infiltration with microglial cells resembling ramified microglia was observed within the solid tumor masses of control- and CpG-treated animals. In necrotic areas (phagocytic), activation of microglial cells was suggested by ED1 expression and a more macrophage-like morphology. Dense lymphocytic infiltrates consisting predominantly of CD8+ T cells and fewer NK cells were detected in all tumors including the control-treated animals. Expression of perforin serving as a marker for T cell or NK cell activation was detected only on isolated cells in all treatment groups. Tumors of all treatment groups revealed CD25 expression indicating T cells presumed to maintain peripheral tolerance to self-antigens. Cytotoxic T cell assays with in vitro restimulated lymphocytes (⁵¹chromium release assay) as well as interferon-gamma production by fresh splenocytes (Elispot assay) revealed specific responses to 9L cells but not another syngeneic cell line (MADB 106 adenocarcinoma). Surprisingly, the lysis rates with lymphocytes from CpG-ODN-treated animals were lower compared to control-treated animals. The tumor size of individual animals did not correlate with the response in both immune assays. Taken together, our data support the immunostimulatory capacity of CpG-ODN in normal brain. However, intratumoral application proved ineffective in a rat glioma model. CpG-ODN treatment may not yield beneficial effects in glioma patients.