Rejection of B16 melanoma induced by expression of a transfected major histocompatibility complex class I gene.

Transfection of a functional major histocompatibility complex class I gene into certain tumor cells, induced by oncogenic viruses or chemical carcinogens, can effectively abrogate their tumorigenic activity. Since experimentally induced tumors possess strong tumor-specific transplantation antigens, expression of cell surface class I antigens may present the tumor cells to appropriate immune effector cells. Most spontaneously arising tumors do not possess tumor-specific transplantation antigens, and their tumorigenicity may not be affected by the expression of a transfected class I gene. We demonstrate that the poorly immunogenic B16-BL6 melanoma can be rendered nontumorigenic in syngeneic mice by the expression of the class I H-2K antigen but not the class II I-A antigen. Furthermore, the poorly tumorigenic, class I-expressing B16-BL6-transfected cells can effectively immunize syngeneic C57BL/6 mice against the highly tumorigenic, class I-deficient B16-BL6 parental cells. Our success in experimentally manipulating the tumorigenicity of a spontaneously derived neoplasm offers hope for a potential modality for the effective treatment of human cancer.     

H-2 antigen expression and sensitivity of BL6 melanoma cells to natural killer cell cytotoxicity

The sensitivity of H-2b-high and H-2b-low variants of BL6 melanoma to the cytotoxic action of NK and lymphokine-activated killer cells was investigated. BL6 mouse melanoma cells lack detectable H-2Kb and had low levels of expression of H-2Db Ag. The BL6T2 variant cells, obtained after treatment of BL6 cells with mutagen N-methyl-N-nitro-N'-nitro-soguanidine, had relatively high levels of expression of class I H-2b Ag. Poly(I:C)-stimulated spleen cells of nude mice were highly cytotoxic for BL6T2, whereas H-2b-low BL6 cells were less sensitive to NK activity in an 18-h 51Cr-release assay. Similar results were obtained after 4-h incubation of radio-labeled tumor cells with IL-2-activated effector cells. In contrast, both lines were equally sensitive to lysis by purified granules derived from rat large granular lymphocytes (LGL) or by macrophages. By using various clones selected from BL6 or BL6T2 cells, it was found that BL6 or BL6T2 clones with low H-2b Ag expression were less sensitive to lysis by NK cells than H-2b-high clones. After IFN treatment of either BL6 or BL6T2, the target cells became more resistant to lysis by either NK cells or by purified LGL granules. IFN-treated BL6 cells had substantially increased expression of H-2b Ag and in this respect became similar to untreated BL6T2. However, IFN-treated BL6 cells were more resistant than BL6T2 cells to lysis by NK cells and LGL granules, suggesting that augmentation of H-2b Ag expression and NK resistance could be two independent IFN-induced effects. With a cold target inhibition assay, it was found that BL6T2 or its H-2 positive clones were highly competitive and inhibited the cytotoxic activity of NK and lymphokine-activated killer cells against radiolabeled YAC-1 and BL6T2, whereas BL6 cells or H-2-negative clones of BL6T2 and BL6 lines showed poor competitive ability. Thus, our data indicate that the NK resistance of H-2-low BL6 cells may be due to a paucity of NK recognizable determinants. N-Methyl-N-nitro-N'-nitroguanidine treatment of BL6 melanoma cells was associated with an increase in class I H-2b Ag expression and NK sensitivity, suggesting the involvement of class I MHC Ag in the sensitivity of tumor cells to NK cell-mediated cytotoxicity.     

Increased sensitivity to MHC-nonrestricted lysis of BL6 melanoma cells by transfection with class I H-2Kb gene

An H-2Kb- negative clone of BL6 melanoma (BL6-8) was transfected with neor, H-2Kb, or H-2IAk genes. In an 18-h cytotoxicity assay clones with high levels of H-2Kb Ag expression were found more sensitive to lysis by spleen cells of syngenic and allogeneic mice than H-2Kb low clones. NK cells were involved in the lysis of H-2Kb+ BL6 melanoma clones, with spleen cell cytotoxicity of mice increased after poly I:C stimulation or decreased after pretreatment with anti-asialo GM1 serum or NK1.1 mAb. Anti-TNF Ab were also able to reduce the cytotoxicity of normal spleen cells and completely abolished the cytotoxicity of the NK-depleted spleen cells suggesting involvement of NC cells in lysis of H-2Kb+ BL6 melanoma clones. Increase in sensitivity of H-2Kb+ BL6 cells to natural cell-mediated cytotoxicity was associated with the appearance of NK recognizable determinants as assessed by the cold target inhibition assay. All BL6 clones, irrespective of sensitivity to natural cell-mediated cytotoxicity, showed high sensitivity to lysis by LGL-derived granules. In contrast, all H-2Kb low BL6 clones were resistant and all H-2Kb highly positive clones were sensitive to lysis by TNF-alpha. When an H-2Kb highly positive clone was selected in vitro for resistance to TNF, it concomitantly showed increased resistance to cytotoxicity by spleen cells, confirming the importance of TNF in spleen cell cytotoxicity against H-2Kb+ melanoma cells. Taken together, the data indicate that class I H-2Kb but not class II H-2IAk gene product could increase the sensitivity of BL6 cells to lysis by NK and natural cytotoxic cells as well as TNF. We hypothesize that these effects could be due to pleiotropic effects of H-2Kb gene products on various biologic properties of BL6 melanoma cells some of which may be more directly involved in regulation of tumor cell sensitivity to lysis by NK and/or natural cytotoxic cells.     

PGE2 involvement in experimental infection with Trypanosoma cruzi subpopulations

PGE₂ involvement in experimental Trypanosoma cruzi infection depends on the lethal capacity of the parasite subpopulation used. Mice acutely infected with non-lethal K98 displayed an enhancement in PGE₂ serum levels during the acute period, while those infected with lethal T. cruzi subpopulations (RA or K98-2) showed levels not different from normal mice. The enhancement detected in K98 group could be related both to an increased number of CD8+ T cell number and to enhanced PGE₂ release per cell by CD8+; values of PGE₂ release by adherent cells were not altered in this group. Treatment with cyclooxygenase inhibitors enhanced mortality rates of mice infected with K98, and administration of 16,16-dimethyl PGE₂ (dPGE) reversed this effect. However, mice infected with RA did not reduce their mortality rates by administration of diverse doses of dPGE. These findings suggest that PGE₂ could play a role in resistance in mice infected with K98.     

Low-dose melphalan-induced shift in the production of a Th2-type cytokine to a Th1-type cytokine in mice bearing a large MOPC-315 tumor

The current studies demonstrate that MOPC-315 tumor cells secrete large amounts of interleukin-10 (IL-10), which contributes to the inhibitory activity of MOPC-315 culture supernatants for the in vitro generation of antitumor cytotoxicity by MOPC-315-immune spleen cells. Moreover, addition of neutralizing monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of cells from the tumor infiltrated spleens of mice bearing a large MOPC-315 tumor resulted in the generation of enhanced anti-MOPC-315 cytotoxicity. In contrast, addition of monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of splenic cells from mice that are in the final stages of immune-mediated tumor eradication as a consequence of low-dose melphalan (l-phenylalanine mustard; L-PAM) therapy (and whose spleens no longer contain metastatic tumor cells) did not lead to enhancement in the in vitro generation of antitumor cytotoxicity. The cessation of IL-10 secretion as a consequence of low-dose L-PAM therapy of MOPC-315 tumor bearers was found to be accompanied by the acquisition of the ability to secrete interferon (IFN) by the splenic cells. In addition, by day 2 after low-dose L-PAM therapy a drastic decrease in the amount of IL-10 secreted by the s.c. tumor nodules was noted, which preceded the accumulation of tumor-infiltrating lymphocytes capable of secreting IFN. Thus, low-dose L-PAM therapy of mice bearing a large MOPC-315 tumor leads to a shift in cytokine production from a Th2-type cytokine to a Th1-type cytokine, and it is conceivable that this shift in cytokine production plays an important role in the low-dose L-PAM-induced acquisition of antitumor immunity by hitherto immunosuppressed mice bearing a large MOPC-315 tumor.Supported by research grant IM-435 from the American Cancer Society and CA54413 from the National Cancer InstituteIn partial fulfillment of the requirements for the Doctor of Philosophy DegreeRecipient of career development award CA-01350 from the National Cancer Institute     

Lectin-induced apoptosis of tumour cells

The mechanisms of cytotoxic activity of Griffonia simplicifolia1-B₄ (GS1B₄) and wheat germ agglutinin (WGA) lectins againstvarious murine tumour cell lines were studied. Tumour cellsthat lack lectin-binding carbohydrates were resistant to lysisby these lectins. However, YAC-1 cells that expressed GS1B⁴lectin-binding sites showed low sensitivity to lysis. To furtheranalyse the relative importance of cell surface carbohydratesin lectin cytotoxicity, BL6–8 melanoma cells, which donot express the     

Assessment of in vivo natural antitumor resistance and lymphocyte

Clearance of IV-injected tumor cells has been correlated with levels of natural killer (NK) cell activity in recipient animals. Studies of in vivo tumor cell clearance strongly suggest a relationship between levels of NK cell activity and antitumor or antimetastatic effector function. This study outlines the applicability of three radiolabels, [125I]iododeoxyuridine, ( [125I]dUrd), indium-111-oxine chelate ( [111In]Ox), and chromium-51 (51Cr), to studies of tumor cell clearance in vivo. The suitability of these labels for analysis of the in vivo migration patterns of normal lymphocytes or thymus-derived T cells cultivated in vitro (CTC) is also discussed. The results indicate that [111In]Ox and 51Cr compare favorably with the more widely used [125]dUrd as radiolabels for the assessment of IV-injected tumor cell clearance from the lungs of mice. The rates of clearance of both [111In]Ox and 51Cr, like that for [125I]dUrd, correlate closely with levels of NK-cell activity of the host. Further studies with [111In]Ox reveal that treatment of recipients with anti-asialo GM1 serum, a regimen known to suppress NK-cell activity, demonstrates the appropriate reduction in isotope clearance from the lungs after NK suppression. However, clearance data obtained by monitoring levels of radioactivity in the liver after IV injection must be viewed cautiously, since the same cells labeled with [111In]Ox and [125I]dUrd had a different pattern of clearance from the liver. The same inconsistencies in clearance were observed when [111In]Ox and [125I]dUrd were injected intrafootpad (i.f.p.). Similar effects were observed when [111In]Ox or 51Cr was applied to studies of CTC migration. Levels of [111In]Ox and 51Cr remained high in the liver after IV injection, while [125I]dUrd was rapidly cleared. Normal spleen or thymic lymphocytes exhibited the expected homing to the spleen after labeling with [111In]Ox, indicating a suitability of this label for migration studies, except possibly in the liver. These results with CTC and normal lymphocytes should be considered during the formulation of immunotherapy protocols based on cell migration data, since the choice of radiolabel can result in widely divergent levels of radioactivity accumulated in some organs, and may not provide an accurate representation of the presence of viable, intact, or functional cells.     

Preferential ATP-binding cassette transporter A1-mediated cholesterol efflux from late endosomes/lysosomes

Recently, ATP-binding cassette transporter A1 (ABCA1), the defective molecule in Tangier disease, has been shown to stimulate phospholipid and cholesterol efflux to apolipoprotein A-I (apoA-I); however, little is known concerning the cellular cholesterol pools that act as the source of cholesterol for ABCA1-mediated efflux. We observed a higher level of isotopic and mass cholesterol efflux from mouse peritoneal macrophages labeled with [(3)H]cholesterol/acetyl low density lipoprotein (where cholesterol accumulates in late endosomes and lysosomes) compared with cells labeled with [(3)H]cholesterol with 10% fetal bovine serum, suggesting that late endosomes/lysosomes act as a preferential source of cholesterol for ABCA1-mediated efflux. Consistent with this idea, macrophages from Niemann-Pick C1 mice that have an inability to exit cholesterol from late endosomes/lysosomes showed a profound defect in cholesterol efflux to apoA-I. In contrast, phospholipid efflux to apoA-I was normal in Niemann-Pick C1 macrophages, as was cholesterol efflux following plasma membrane cholesterol labeling. These results suggest that cholesterol deposited in late endosomes/lysosomes preferentially acts as a source of cholesterol for ABCA1-mediated cholesterol efflux.     

Chronic stress influences the immune system through the thyroid axis

The aim of the present work was to analyze the effect of chronic stress on thyroid axis and its influence on the immune response. For this purpose a murine model of chronic stress was developed to evaluate and to correlate thyroid hormone levels with humoral alloimmune response. Results show a reduction in serum levels of thyroid hormones, specially a significant decrease in serum levels of triiodotyronine (T3) in stressed animals. On the other hand, alloimmunization was not able to induce an early increment in T3 and thyroxine (T4) levels as it was previously reported in normal animals. In addition, lower titers of alloantibodies were obtained in animals under stress conditions as compared to normal mice. The sustitutive T4 treatment in stressed animals increased significantly alloantibody production as well as the early increment in thyroid hormones after antigenic challenge. These findings suggest that chronic stress induces an alteration of the function of thyroid axis that alters the immune response.     

Effects of extracellular matrix elements on pseudopodial activity of rat keratinocytes

Effects of extracellular matrix elements on the migration activity of keratinocytes have been studied in the primary culture obtained from newborn rats. Collagen of type I, matrigel, its fractions and the matrix produced by fibroblasts were used as substrata for cultivation. A method of migration activity estimation using latex spheres 0.8 mkm in diameter was first used. We have revealed that keratinocytes from the primary culture do not migrate on matrigel and fibroblast matrix, though displaying some pseudopodial activity. This activity dramatically increases on type I collagen, and a weak migration ability appears correlating with a particular structure of the actin cytoskeleton, i.e. with the appearance of special lamellopodia-connected filopodia.