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.     

Differential allo-response of murine thymocytes to H- 2 K region different recombinants and to H-2Kb mutants

Thymocytes used as responding cells in a mixed leukocyte culture with x-irradiated splenic stimulating cells generate highly significant proliferative and cytotoxic responses when responding and stimulating cells differ by the entire H-2 complex. On the other hand, when the genetic difference between responding and stimulating cells is only a K region, very little, if any, proliferative response is detectable and no cytotoxic response is found. In contrast, when responding and stimulating cell donors differ by a spontaneous mutation in the K region of the H-2 complex, as found in B6.C-H-2ba, B6-H-2bd and B6.C-H-2bf, highly significant proliferative and cytotoxic responses can be obtained. These results, thus, argue that the H-2 mutants cannot, with regard to their relationship to the parental strain, be readily equated with a K region difference as defined in the recombinant inbred strains.     

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.     

Biochemical evidence for expression of a semi-allogeneic, H-2 antigen by a murine adenocarcinoma

LT-85 is an alveolegenic adenocarcinoma induced in mutant C3HfB/HeN (C3Hf) mice. This tumor, however, grows preferentially in allogeneic, wild-type C3H/HeN (C3H) mice. The tumor-associated transplantation antigen has been mapped to the K end of the major histocompatibility complex. H-2K antigens were isolated from detergent extracts of LT-85 cells by immunoprecipitation with monoclonal antibody. The tryptic peptides of these antigens were compared, by using high-pressure liquid chromatography, with the tryptic peptides of H-2K antigens isolated from syngeneic mutant C3Hf and ancestral wild-type C3H spleen cells. We found that the H-2K antigens of the LT-85 tumor cells were very similar to, but distinct from, those present on syngeneic C3Hf lymphoid cells. We also found, however, that the H-2K antigens of LT-85 tumor cells were clearly different from the H-2K antigens of allogeneic C3H spleen cells. The H-2K antigens of LT-85 cells are therefore foreign to syngeneic C3Hf cells, but do not represent expression by the tumor cells of the allogeneic H-2K antigens expressed by normal C3H cells. Furthermore, the nature of the differences observed between the H-2K antigens of LT-85 cells and C3Hf and C3H spleen cells strongly suggests that the structure of the H-2K molecule of LT-85 cells is identical in some regions to the H-2K molecule of C3Hf cells, and in other regions to the H-2K molecule of C3H cells.     

Crystallization of murine major histocompatibility complex class I H-2Kb with single peptides.

X-ray quality crystals of a soluble murine class I H-2Kb molecule complexed with three different peptide antigens were grown in several forms by streak seeding and macroseeding methods. Co-crystals with VSV-8 (RGYVYGQL), OVA-8 (SIINFEKL) and SEV-9 (FAPGNYPAL) peptides were grown either from NaH2PO4/HPO4 or from polyethylene glycol 4000 within the pH range 5.0 to 7.5, with the use of 4-methyl-2-pentane diol (MPD) as an additive. The VSV-8 crystals grew in space groups P1, with cell dimensions a = 63.1 A, b = 69.1 A, c = 72.0 A, alpha = 89.9 degrees, beta = 77.1 degrees, gamma = 123.3 degrees and P2(1)2(1)2, with a = 138.1 A, b = 88.6 A, c = 45.7 A, and diffract to 2.9 and 2.3 A, respectively. Crystals of the SEV-9 complex grew from similar crystallization conditions to those of the orthorhombic VSV-8 complex with similar cell parameters and diffract to at least 2.5 A resolution. Crystals of the OVA-8 complex were obtained from either phosphate (space group C2, a = 118.7 A, b = 61.6 A, c = 85.3 A, beta = 108.4 degrees) or polyethylene glycol (space group P1, a = 64.5 A, b = 71.0 A, c = 66.3 A, alpha = 89.7 degrees, beta = 95.7 degrees, gamma = 123.3 degrees) and diffract to 3 A resolution. The crystallization procedures used here significantly increased the rate and production of X-ray quality crystals.     

Differential induction of H-2K vs H-2D class I major histocompatibility complex antigen expression by murine recombinant interferon-gamma

The results presented here indicate that recombinant murine interferon-gamma can cause a dramatic differential induction of two distinct class I MHC molecules. Thus, IFN-gamma treatment of the murine leukemia virus (MuLV)-induced AKR SL3 tumor, a cell line that normally expresses moderate levels of class I MHC antigens, resulted in a large increase in H-2Dk expression, but no change or a slight decrease in H-2Kk expression as measured by cytofluorography. Explanations of the selective enhancement of Dk expression based on increased Fc receptor display or differential kinetics of induction were ruled out. The phenomenon was observed over a wide range of doses of IFN-gamma and with two different monoclonal antibodies to Kk, the latter finding making it unlikely that an altered form of the Kk molecule was induced. The same differential induction of the Dk antigen was observed for the LBRM.5A4 tumor cell line. Because LBRM.5A4 is also MuLV+ but of congenic B10.BR (H-2k) origin, these results were consistent with the possibility that such differential induction was associated with the H-2k haplotype and/or MuLV. The implications of these results, as a possible mechanism of tumor cell escape from an immune surveillance system monitored by class I MHC-restricted T cells and as a useful model system to dissect the mechanism of IFN-gamma induction of class I MHC antigens, are discussed.     

Reduced tumorigenicity of murine hepatoma cells after treatment with antioxidants and melatonin

The tumor growth of murine hepatoma cells MH22a treated with N-acetylcysteine (NAC, 10 mM) and alpha-lipoic acid (ALA, 1.25 mM) antioxidants or hormone melatonin (1 μM) and transplanted into syngeneic (C3HA) mice has been studied. NAC, ALA, or melatonin treatment for 20 h reduced the tumor development and the number of dead mice. Melatonin produced the most pronounced effect. Tumors appeared in 10 days in 100% of control mice injected with untreated cells; the injection of cells pretreated by NAC or ALA generated tumors in 40 and 53% of mice, respectively. Cells pretreated with melatonin produced tumors 18–20 days after injection; 67% of control mice died in 36 days (the observation period). The mortality rate was 20 and 53% if the injected cells were treated with NAC or ALA, respectively. No mice died during this period with melatonin-pretreated cells. We found that treatment with antioxidants delayed (NAC) or completely inhibited (ALA) the progression of the cell cycle of murine hepatoma cells. After the antioxidant removal, the cell cycle was restored. Melatonin did not affect the cell cycle phase distribution. We conclude that there is no direct correlation between the loss of tumorigenic properties and the altered proliferative activity of hepatoma cells. Different mechanisms of antioxidants and melatonin action that underlie the transient normalization of the tumor phenotype are discussed.     

The effect of radiation with polychromatic visible and infrared light on the tumorigenicity of murine hepatoma 22A cells and their sensitivity to lysis by natural killers

The tumorigenicity of murine hepatoma cells (MH-22a) and their sensitivity to lysis by natural killers (NKs) have been studied after exposure to polychromatic visible and infrared light (VIS-IR, 480–3400 nm, 40 mW/cm²), similar to the terrestrial solar spectrum without its minor UV component, with the aim of clarifying the participation of this important environmental and physiotherapeutic factor in regulation of antitumor protective system. MH-22 cells were exposed in vitro to VIS-IR light and their sensitivity to lytic activity of NKs was evaluated. It was found that, after exposure to VIS-IR light at a dose of 4.8 J/cm², the sensitivity of MH-22a cells to lysis by NKs increased by 1.5–2 times, while after exposure at a dose of 9.6 J/cm² it did not change at all the ratios of the NKs-number (effectors) to that of hepatoma cells — targets (1 : 5–1 : 50). An increase of the hepatoma cell sensitivity to NKs was accompanied by structural changes of cell surface: the capability of supramembranous glycoproteins (glycocalyx) to sorb the vital dye alcian blue (AB) was significantly lower than in the case of unexposed cells of the control group. However, no changes in AB sorption was revealed in hepatoma cells exposed to light at a dose of 9.6 J/cm². The tumorigenicity of photoirradiated MH-22a cells has been studied in the experiments in vitro. For 25 days after transplantation of light-exposed hepatoma cells to C3HA syngene mice, the tumor volume proved to be smaller after exposure to light at both doses of 4.8 and 9.6 J/cm² than in the control group (by 4–4.5 times and 2.5–4 times, respectively), which correlated with an increase of sensitivity to lysis by NKs and with a decrease of AB sorption after light exposure only at a dose of 4.8 J/cm². Using the flow-cytometry method, we could show that VIS-IR light at the doses used did not interfere with the distribution of hepatoma cells over the cell-cycle phases and, thus, deceleration of the tumor growth was not associated with a cytostatic effect of the VIS-IR light. To evaluate the effect of polychromatic light on growth of the preformed tumors, a 5-day course of daily light exposure of C3HA tumor-bearing mice was performed on the 10th day after subcutaneous transplantation of 2 × 10⁵ cells of syngene hepatoma, when tumors developed in all (100%) animals. As in the case of transplantation of light-exposed cells, irradiation of tumor-bearing mice at doses 4.8–9.6 J/cm² resulted in a deceleration of tumor growth (by 2.1–2.9 and 2,2 times, respectively) for 4 weeks compared with unirradiated mice.     

Dominant reduced responsiveness controlled by H-2(Kb)Ab. a new pattern evoked by Thy-1 antigen and F liver antigen

In the most frequently used panel of H-2 recombinant strains, B10.A, B10.A(4R), B10.A(5R), and B10, inhibition of the immune response has hitherto mapped to H-2E. Inhibition of the responses to Thy-1 antigen and to F liver protein, as described here, maps in a novel pattern to H–2K ^b A ^b , and presumably to H–2A ^b .Enhancement of the adoptively transferred anti-Thy-1 response by treatment with CD8-specific antibody suggests, very provisionally, that T cells with suppressive activity mediate the inhibition. The evolution of this new pattern, and of dominant reduced responsiveness in general, is discussed and its relevance to immunological diseases assessed. An enzyme-linked immunosorbent assay (ELISA) for F-specific antibodies is introduced.     

H-2-restricted cytolysis of L cells doubly transformed with a cloned H-2Kb gene and cloned retroviral DNA

We report the construction of target cells by the double transformation of mouse L cells with a cloned H-2Kb gene and molecular clones of Akv or Gross murine leukemia virus (MuLV) or the cloned gag gene of Akv MuLV. Cytolytic T lymphocytes (CTL) generated in BALB.B mice specific for Gross MuLV (closely related to Akv MuLV) kill these doubly transformed cells. This is a direct indication that a CTL subpopulation recognizes H-2Kb antigen in association with a viral antigen encoded by the gag gene of Gross MuLV.     

Cytotoxic T lymphocyte response to minor H-43a alloantigen in H-43b mice. Privileged H-2Kb restriction to the response is not due to immunodominance or epistatic effect but due to Ir gene function of H-2Kb itself

Previous study demonstrated that anti-H-43a cytotoxic T lymphocyte (CTL) response of H-43b CWB (H-2b) stain carrying non-major histocompatability complex (MHC) genes of C3H and F1 strains raised by crossing CWB with various H-43b strains was restricted exclusively by self H-2Kb (Kb). In the present study, newly produced C3W strain (H-2k, H-43b), which is H-43-congenic to C3H/HeN (H-2k, H-43a), was used as H-43b mice, and possibility of immunodominance of Kb was examined. No anti-H-43a CTL response could be induced in C3W strain and F1 strains raised by crossing C3W with other H-43b strains not carrying Kb. Thus, the possibility of immunodominance of Kb over the other MHC class I alleles could not be supported. We also examined possibility of epistatic effect of I region genes and non-MHC genes on the Kb restriction. (C3W x C57BL/6)F1(I-Ak/b) and (C3W x B6.CH-2bm12)F1(I-Ak/bm12)mice showed equally anti-H-43a CTL response restricted exclusively by self Kb, and (C3W x B10.MBR)F1(Ik/k) mice also showed anti-H-43a CTL response restricted solely by self Kb. Cold target competition experiments demonstrated that H-43b C57BL/10 or A.BY mice, which do not have non-MHC genes of C3H mounted anti-H-43a CTL response restricted solely by self Kb. Thus, no relation of I region genes or non-MHC genes to the Kb restriction was shown. All the results indicate that H-43b mouse strains, including F1, can not achieve anti-H-43a CTL response unless they carry Kb allele. Notably, (C3W x C57BL/6)F1 mice mounted self Kb-restricted anti-H-43a CTL response, whereas (C3W x B6.CH-2bm1)F1 mice carrying mutated Kb could not mount anti-H-43a CTL response at all. These findings indicate strongly that Kb itself is classical Ir gene of anti-H-43a CTL response and directs self Kb restriction of the response.     

Cutting edge: the minor histocompatibility antigen H60 peptide interacts with both H-2Kb and NKG2D

Minor histocompatibility Ags elicit cell-mediated immune responses and graft rejection in individuals receiving MHC-matched tissues. H60 represents a dominant Ag that elicits a strong CTL response in C57BL/6 mice immunized against BALB.B. An 8-aa peptide in the H60 protein is presented by H-2K(b) and this is recognized by the TCR as an alloantigen. The intact H60 glycoprotein is a ligand for the costimulatory NKG2D receptor that is expressed by activated CD8(+) T cells. Thus, H60 may provide both an allogeneic peptide and its own costimulation. We show that mutation of an H-2K(b)-binding anchor residue in the H60 peptide completely abrogates binding of H60 glycoprotein to NKG2D and a synthetic H60 peptide partially blocks the binding of NKG2D to its ligand. Ligands of the human NKG2D receptor are remarkably polymorphic, suggesting that these may also serve as minor histocompatibility Ags.     

Influence of H-2 complex on susceptibility to infection by murine leukemia virus.

Titers of infectious ecotropic MuLV in mouse spleen were examined after deliberate infection. In congenic mice differing only in H-2 haplotype, a gene (or genes) within the H-2 complex determined either a high virus titer (H-2k, H-2d, H-2a) or a low titer (H-2b, H-2q). Susceptibility to high virus titers was inherited as a dominant trait. Kinetic studies revealed similar initial patterns of infection in both groups, with a fall in titer in the "resistant" strain occurring from week 6 through 10 after infection. Anti-VEA antibody titers differed significantly between the groups, but no mechanistic role for antibody in eliminating virus was apparent. Genes outside the H-2 complex were shown to influence MuLV titers after infection as well.     

Intraspecies identification of cultured murine cells by using H-2 antigen typing

17 mouse cell lines have been screened with specific sera against H-2 antigens. All the cell lines tested expressed H-2 antigens characteristic of the donor haplotype. The data obtained indicate that H-2 typing of cultured mouse cells can be used as an approach to control their intraspecies diversity.     

H-2 class I antigen expression on mouse teratocarcinoma cell lines

Immunity against PCC3 teratocarcinoma cells (129, H-2 ^b) was induced in allogeneic (C3H, H-2 ^k) mice by preimmunization with L cells (C3H, H-2 ^k) expressing cosmid-introduced K ^b or D ^b genes, but not with nontransfected L cells. In addition, the growth of PCC3 cells in sublethally irradiated (C3H × B6-H-2 ^bm1)F₁ and (C3H × B6-H-2 ^bm13 )F₁ mice bearing the K ^bm1 and D ^bm13 mutations, respectively, was either prevented, stopped, or delayed in comparison with the (C3H × B6)F₁ (k × b) mice, which failed to reject the PCC3 cells. The teratocarcinoma line OC15S was exceptional because it reacted specifically with K^b- and D^b-specific (but not I^b-specific) alloantisera, and because K^b- and D^b-specific antibodies could be absorbed by OC15S cells. The subpopulation of OC15S cells bearing the ECMA-7 antigen characteristic for embryonic carcinoma (EC) cells was isolated by the fluorescence-activated cell sorter and was shown to react specifically with K^b- and D^b-specific antisera. These experiments show that teratocarcinoma cells express antigens similar or identical to the K-and D-region products of differentiated cells. The lack of expression of class I antigens is thus neither a condition nor a consequence of the pluripotentiality of the EC cells. The exact nature of the major histocompatibility complex antigens on EC cells has yet to be established using the methods of molecular biology and biochemistry.     

Peripheral obestatin has no effect on feeding behavior and brain Fos expression in rodents

Obestatin is produced in the stomach from proghrelin by post-translational cleavage. The initial report claimed anorexigenic effects of obestatin in mice. Contrasting studies indicated no effect of obestatin on food intake (FI). We investigated influences of metabolic state (fed/fasted), environmental factors (dark/light phase) and brain Fos response to intraperitoneal (ip) obestatin in rats, and used the protocol from the original study assessing obestatin effects in mice. FI was determined in male rats injected ip before onset of dark or light phase, with obestatin (1 or 5 μmol/kg), CCK8S (3.5 nmol/kg) or 0.15 M NaCl, after fasting (16 h, n = 8/group) or ad libitum (n = 10–14/group) food intake. Fos expression in hypothalamic and brainstem nuclei was examined in freely fed rats 90 min after obestatin (5 μmol/kg), CCK8S (1.75 nmol/kg) or 0.15 M NaCl (n = 4/group). Additionally, fasted mice were injected ip with obestatin (1 μmol/kg) or urocortin 1 (2 nmol/kg) 15 min before food presentation. No effect on FI was observed after obestatin administration during the light and dark phase under both metabolic conditions while CCK8S reduced FI irrespectively of the conditions. The number of Fos positive neurons was not modified by obestatin while CCK8S increased Fos expression in selective brain nuclei. Obestatin did not influence the refeeding response to a fast in mice, while urocortin was effective. Therefore, peripheral obestatin has no effect on FI under various experimental conditions and did not induce Fos in relevant central neuronal circuitries modulating feeding in rodents.     

Overexpression of Hsp25 in K1735 murine melanoma cells enhances susceptibility to natural killer cytotoxicity

In the present study we used a murine melanoma model to investigate the effect of the 25-kDa heat shock protein (Hsp25) on natural killer (NK) cytotoxicity. The melanoma lines K1735-C123 (low metastatic potential) and K1735-M2 (high metastatic potential) were transfected with hsp25 and a control plasmid. Highly purified interleukin (IL)-2-stimulated DX-5+ NK cells showed enhanced lysis of Hsp25-overexpressing K1735-C123 targets in comparison with controls. In contrast, there was no difference in susceptibility to lysis by purified IL-2-stimulated DX-5+ NK cells between Hsp25-overexpressing and control-transfected K1735-M2 targets. Fluorescence-activated cell sorter analysis revealed that Hsp25 is displayed on the cell surface independently of Hsp25 overexpression and metastatic phenotype. Thus, surface localization of Hsp25 does not correlate with the target cell susceptibility to killing. To sum up, a cytoplasmic overexpression of Hsp25 is associated with an increased susceptibility to lysis by DX-5+ NK cells in the low-metastatic murine melanoma model investigated.     

Mapping the orientation of an antigenic peptide bound in the antigen binding groove of H-2Kb using a monoclonal antibody.

The major histocompatibility complex class I molecules are receptors for intracellular peptides, both of self and non-self origin. When non-self peptides (eg., pathogen derived) are bound to the class I molecules, they form ligands for T cell receptors resulting in antigen specific lysis of the infected cells by cytotoxic T lymphocytes. Therefore, an understanding of the process of antigen recognition requires the precise definition of the structural features of the bimolecular complex formed by a single well defined antigenic peptide bound to the class I molecule. A strategy using antibodies was developed to probe the structural features of the H-2Kb containing a defined peptide in the antigen cleft. We report that the binding surface area of a Kb specific monoclonal antibody (28-13-3s) includes residues in the alpha 1 (Gly56 and Glu58) and alpha 2 (Trp167) helices of Kb thus, binding across the antigen binding groove. When cells treated with the antigenic peptide of vesicular stomatitis virus, N52-59, and its alanine substituted analogs were tested for 28-13-3s binding, it was found that position 1 of the peptide also forms a part of the antibody binding site. This finding strongly supports the positioning of the N-terminus of N52-59 proximal to pocket A, thus, assuming an orientation parallel to the alpha 1 helix.