Effects of in vivo morphine treatment on antibody responses in C57BL/6 bgJ/bgJ (beige) mice

C57BL/6J bg^J/bg^J (beige) mice are less sensitive than other strains to the analgesic effects of morphine, although they have normal numbers of μ receptors. In the present study, beige mice and their normal littermates (beige⁺) were treated in vivo with morphine or the opioid antagonist, naltrexone and their primary in vitro antibody responses were assessed. Morphine treatment caused splenic atrophy and suppressed the primary in vitro antibody response in beige and beige⁺ mice. However, these effects were not blocked by naltrexone co-treatment. In these mouse strains, naltrexone decreased spleen size and antibody responses by itself, which may mask its ability to antagonize morphine. In beige mice, placebo pellet implantation suppressed the primary in vitro antibody response. Morphine did not cause a further suppression of the antibody response in beige mice compared to placebo. Because of this anomalous response to placebo treatment, the immunosuppressive effects of morphine on the antibody response/10⁷ cells can not be attributed to a specific drug effect in this strain. However, when antibody responses were expressed on a per spleen basis, the overall capacity to respond to antigenic challenge was suppressed by morphine treatment.     

Host-mediated antiviral activity ofLactobacillus casei against cytomegalovirus infection in mice

The protective effect of heat-killedLactobacillus casei (LC) against murine cytomegalovirus (MCMV) infection was examined. ICR mice treated once with LC 1 day or 2 days before challenge survived lethal infection, but untreated orLactobacillus fermentum (LF)-treated mice did not. The protective effect was evidenced by an increase in plaque-forming units (PFU) per 50% lethal dose (LD₅₀) and a decrease in titers of infectious viruses replicated in the target organs. This was further confirmed by severity of histopathological damage to the target organs, especially the liver. LC neither inactivated MCMV nor inhibited its replication in mouse embryonic fibroblasts (MEF). The spleen cells from LC-treated mice inhibited its replication in MEF on co-cultivation. Augmentation by LC of splenic natural killer (NK) cell activity correlated with survival of mice from otherwise lethal MCMV infection. Cytotoxic activity of peritoneal cells and level of serum interferon (IFN) were elevated after MCMV infection, but they were not associated with survival of mice nor with treatment of LC. The protective effect of LC was not clear in NK-deficient beige mutant (bg^J/bg^J) mice, when compared with that in their littermate (bg^J/+) mice. Poor protection of bg^J/bg^J mice by LC treatment correlated with failure to induce NK cell activity by LC treatment in the mutant mice. Thus, it is likely that LC protects mice from MCMV infection by augmentation of NK cell activity.     

Regulation of natural killer cell activity by anti-I-region monoclonal antibodies

The effects of a monoclonal antibody directed against immune response gene products on mouse NK activity were examined. In vivo administration of an anti-I-A^k antibody to C3H/He (H-2^k) mice modulated their peritoneal cell (PC) and spleen cell (SC) natural killer (NK) activity against YAC-1 lymphoma target cells in vitro. No such effect was observed when BALB/c (H-2^d) mice were treated with this antibody. Administration of anti-I-A^k antibody to mice before and after infection with Toxoplasma or treatment with poly(I:C) leads to suppression of NK activity in comparison to NK activity of mice infected with Toxoplasma or injected with poly(I:C) alone. A similar treatment regimen with M5/114 antibody which reacts with I-A^b, I-A^d, I-E^d, and I-E^k molecules resulted in decreased NK activity in B10.D2 (H-2^d) but not in B10.BR (H-2^k) mice. Serum and cell culture supernatant interferon (IFN) concentrations were not altered as a result of anti-I-A^k treatment. Removal of adherent cells did not restore NK activity of anti-I-A^k-treated Toxoplasma-infected mice to levels obtained with mice infected with Toxoplasma. In contrast, depletion of Ly 2.1⁺ cells from nylon-wool nonadherent SC of mice treated with anti-I-A^k antibody, before and after infection with Toxoplasma, resulted in restoration of NK activity to the same level as that observed in Toxoptasma-infected mice.     

Resistance to moloney murine sarcoma virus-induced tumorigenesis in NK-deficient beige mice

C57BL/6J-bg^J$\text{bg}\text{bg}$ mice are reported to be less susceptible to tumor induction by threshold doses of Moloney murine sarcoma virus than their +/bg littermates, and there are no significant differences between $\text{bg}\text{bg}$ and +/bg mice in which tumors were induced with respect to tumor latency, size, and regression rate. The difference in tumor frequency cannot be accounted for by M-MSV boosting of activity in $\text{bg}\text{bg}$ mice or by depression of activity in +/bg animals.     

Brain and ganglion development from two genotypic classes of cells in allophenic mice.

Several strain-specific markers were found to be histochemically visualizable in parts of the central nervous system in allophenic mice. These markers therefore provide a new basis for mapping the normal developmental lineages of major parts of the nervous system, and for identifying the focus of mutant gene action in some neurological mutations. Cell strains in mosaic animals were visualized on the basis of a quantitative difference in β-galactosidase activity (Bgs-locus), in the Purkinje zone of the cerebellum, and in the hippocampal pyramidal zone of the cerebrum. The differential between strains was increased if the beige ($\text{bg}{}^{\mathrm{J}}\text{bg}{}^{\mathrm{J}}$) mutation was included in the high-activity strain. (β-galactosidase is lysosomal, and enhanced visualization in beige results from its enlarged and aggregated lysosomes.) Purkinje cell-strain visualization was also obtained by an indirect fluorescent antibody technique, in sections treated with antisera containing antibodies against strain-type histocompatibility alloantigens, including H-2. The above markers reveal considerable interspersion of cells from separate lineages in short sequences of each genotype. Purkinje and pyramidal cells of the same brain sometimes differ appreciably in genotypic composition. The enzyme glucosephosphate isomerase was found histochemically to be localized in nerve fibers rather than cell bodies in the brain. However, it was prominent in the cell bodies of the spinal ganglia, so that biochemical determination of ganglion strain-types is possible by means of strain-specific isozymes (Gpi-1-locus). Individual ganglia contained both cell strains and thus are not individually derived as clones from the neural crest.     

The Rab Protein Family: Genetic Mapping of Six Rab Genes in the Mouse

Rab proteins constitute a family of GTP-binding proteins that are located in distinct intracellular compartments and play a role in the regulation of vesicular trafficking. Yeast mutations in Rab gene homologs cause defects in vesicular transport similar to those observed in beige (bg) mice. To investigate Rab genes as candidates for mouse mutations characterized by defects in vesicular trafficking, we utilized an intersubspecific backcross [C57BL/6J-bg^J × (C57BL/6J-bg^J × CAST/Ei)F₁] segregating for the bg locus. Restriction fragment length polymorphisms (RFLPs) were obtained through Southern hybridization of F₁ and C57BL/6J chromosomal DNA with the coding sequences of Rab genes. These RFLPs and 12 polymorphic microsatellites were used to determine the segregation of the Rab genes in 93 backcross mice. Rab4a, Rab4b, Rab7, Rab10, Rab22, and Rab24 were localized on mouse chromosomes 8, 7, 9, 12, 2, and 13, respectively. Although the results exclude these loci as candidates for bg , they demonstrate a wide dispersion of Rab genes throughout the mouse genome and reveal that Rab4b and Rab24 are possible candidates for the mouse mutations reduced pigmentation (rp) and purkinje cell degeneration (pcd), respectively.     

Enhanced natural killer (NK) cell activity and NK-sensitive thymic cells in murine muscular dystrophy

Studies have shown that there is an abnormality in the thymus of dystrophic mice with respect to age-dependent thymus weight changes and altered morphology (T. DeKretser and B. Livett, Nature (London), 263, 682, 1976). Recently, others have shown that natural killer (NK) cells can lyse cells of a large, immature, rapidly dividing cell subpopulation within the thymus of normal young (3 weeks of age) mice (M. Hansson, K. Karre, R. Kiessling, J. Roder, B. Anderson, and P. Hayry, J. Immunol., 123, 765, 1979). The NK susceptibility of dystrophic mouse thymocytes as targets was therefore studied. Spleen cells from normal (+/+) and dystrophic ($\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$) male C57BL/6J mice 8–10 weeks old were passed over nylon wool and the nonadherent cells were incubated with ⁵¹Cr-labeled YAC-1 lymphoma target cells or thymocytes in a ⁵¹Cr-release assay. Spleen cells from dystrophic mice killed twofold more YAC-1 target cells than did spleen cells from normal mice. Thymocytes from 3- to 4-week-old dystrophic mice were three to four times more susceptible to NK lysis by dystrophic mouse spleen cells as compared with normal mouse spleen cells. Spleen cells from dystrophic mice had the same NK activity against dystrophic and normal mouse thymocytes as targets. Normal mouse spleen cells killed three- to fourfold more dystrophic mouse thymocytes than that of normal mouse thymocytes as targets. Target cellbinding studies revealed that conjugate-forming cells from nylon nonadherent dystrophic mouse spleen cells were found to be two- to fourfold greater than for normal mouse spleen cells using YAC-1 tumor cells as targets. The number of lymphocytes bound per YAC-1 target cell ranged from 2 to 5 for dystrophic mouse spleen cells as compared with 1 to 2 for the normal control group. Using both normal and dystrophic mouse thymocytes as targets, the conjugate-forming cells from dystrophic mouse spleen cells were also found to be twofold greater than in the normal control group. Cold target inhibition studies revealed that the natural killing of dystrophic mouse thymocytes was due to a YAC-1-reactive NK cell. Effector cell depletion studies using monoclonal anti-Thy-1.2 plus complement treatment and plastic petri dish adherence also revealed that the natural killing of dystrophic mouse thymocytes was not due to either T lymphocytes or macrophages. Taken together, these results show an increase in NK-sensitive thymocyte targets in dystrophic mice, in combination with an increase in splenic NK activity.     

The accumulation and metabolism of glycosphingolipids in primary kidney cell cultures from beige mice

In the normal C57BL/6J male mouse a specific subset of the kidney glycosphingolipids which is associated with multilamellar bodies of lysosomal origin and represents about 10% of the total kidney glycolipids, is excreted into the urine each day. This excretion is blocked and glycosphingolipids accumulate in the kidney of bg ^J/bg^J mutants of this strain. To examine this process in vitro, glycosphingolipid metabolism and excretion were studied in beige mouse kidney cell cultures. Primary kidney cell cultures from male C57BL/6J control and bg ^J/bg ^J beige mutants were grown in D-valine medium and glycosphingolipids labeled with [³H]palmitate. As we have shown previously, the giant lysosomes of altered morphology were maintained in cultures of the beige kidney cells. Beige-J and control cells synthesized the same types of glycosphingolipids, but the mutant cells had quantitatively higher levels of these compounds than control cells, as determined by high performance liquid chromatography. Beige-J cells incorporated more [³H]palmitate into glycospingholipids than control cells on a cpm/mg protein basis and the specific activity (cpm/pmole glycosphingolipid) was lower in beige cells. Medium from beige-J cells accumulated more glycosphingolipids than that from control cells in a 24 h period. The glycosphingolipids released into the medium as determined by HPLC were primarily non-lysosomal types and both control and mutant cells retained the glycosphingolipids associated with lysosomal multilamellar bodies excreted in vivo. The elevated levels of lysosomal glycosphingolipids and the dysmorphic lysosomes in primary cultures of beige cells, then, are not caused by a mutant block in secretion of lysosomes. (Mol Cell Biochem 118: 61–66, 1992)     

Biochemical and morphological characterization of primary kidney cell cultures from beige mutant mice

Summary Primary kidney cultures from adult beige-J (bg ^J/ bg ^J) mice were selected for epithelial cell growth using D-valine medium. After 2 weeks of attachment and proliferation in vitro, the cells form a confluent or nearly confluent monolayer that retains several phenotypic characteristics of the beige-J mutant. These include large, multilamellar inclusion bodies that are apparently dysmorphic lysosomes, and higher concentrations of neutral glycosphingolipids and dolichols than control cells. -Glucuronidase activity, used as a lysosomal enzyme marker, is not elevated in beige-J-cultured kidney cells compared with controls, as it is in the intact kidney. The high levels of -glucuronidase activity in both control and mutant cells may mask expression of this difference in vitro. The action of the beige-J mutation in kidney cells is thought to be due to a block in exocytosis that results in the accumulation of abnormal lysosomes and their components. The maintenance of the beige phenotype in vitro indicates that the mutation is not suppressed in primary kidney cell cultures. The expression of the beige phenotype in vitro should be useful for studies concerning the primary lesion of this mutation.     

Interleukin 2 enhances natural killing of normal lymphocytes

Purified Interleukin 2 (IL-2), free of interferon (IFN), significantly enhanced NK activity of normal human peripheral blood mononuclear cells (PBMC). This enhancing activity was absorbed by IL-2 receptor-bearing cells but was not blocked by antibody to α-IFN. IFN in the culture supernatants was greatly increased after stimulation with poly(I:C) plus IL-2. There was less IFN produced by either modulator acting alone. Stimulation of PBMC with IL-2 and/or poly(I:C) increased the proportion of OKM1⁺ cells and anti-Leu-7⁺ cells. When cells expressing either surface antigen were specifically lysed to deplete NK, cytotoxic activity could be restored by overnight incubation in IL-2. This result suggests that IL-2 stimulates the development of NK cells from precursors that lack cell surface OKM1 or Leu-7. IL-2 acted directly on large granular lymphocytes and did not require the presence of adherent cells. These results suggest that IL-2 may act synergistically with other IFN inducers and may play an important role in the regulation of NK cells.     

Mutations in mice that influence natural killer (NK) cell activity

A series of mutations in mice was tested for splenic NK-cell activity against YAC-1 target cells. Mutations at six loci that reduce NK-cell activity in the homozygous state were identified, including beige (bg), hairless (hr), motheaten (me), obese (ob), steel (Sl) and, to a lesser extent, dominant spotting (W). Motheaten mice displayed the most profound NK-cell deficiency, with NK-cell activity virtually absent. Two mutations, nude (nu) and lymphoproliferation (Ipr), produced elevated NK-cell-mediated lysis. The double homozygous recessivenu/nu bg/bg nude-beige mouse was viable and NK-cell-deficient, with activity slightly higher than that of +/?bg/bg beige littermate controls. Pigmentation mutants related to beige, including pale ears (ep), pearl (pe), and ruby eyes (ru ^2J ) did not dramatically influence NK-cell levels. Unlike the obese gene, other mutations leading to obesity, diabetes (db) and yellow (Asuy), did not impair NK-cell function. The possible site of gene action of these mutants in the NK-cell pathway is discussed.     

Augmentation of mouse natural killer cell activity by muramyl dipeptide and its analogs

The ability of muramyl dipeptide (MDP) and its structural analogs (des-MDP, abu-MDP, and des-abu-MDP) to influence mouse natural killer (NK) cells in two different strains of mice was examined. In CBA/J mice, administration of MDP by both intraperitoneal (ip) and intravenous (iv) routes enhanced splenic NK cell activity. Maximum augmentation of NK cell activity was observed 3 days after MDP treatment. NK cell activity was also stimulated upon in vitro culture of CBA/J mouse spleen cells with MDP. Only iv inoculation of MDP to C57BL/6 mice 7 days previously enhanced NK cell activity of spleen cells. Peritoneal NK cell activity was not affected in either strain of mice, regardless of the route of inoculation of MDP. Two structural analogs of MDP, abu-MDP and des-abu-MDP, enhanced peritoneal NK cell activity, whereas des-MDP had no effect when tested 3 days after ip treatment of CBA/J mice with these compounds. Peritoneal NK cell activity of C57BL/6 mice was not modulated by des-MDP, abu-MDP, or des-abu-MDP. A synergistic effect on peritoneal NK cell activity was observed in both CBA/J and C57BL/6 mice treated first with MDP and then with lipopolysaccharide (LPS) or Bacillus Calmette-Guerin (BCG).     

Precursor of mast cells fixed in the skin of mice

The presence and origin of mast-cell precursors fixed in the skin tissue of mice were investigated. Giant granules of beige (C57BL/6-bg^j/bg^j, Chediak-Higashi syndrome) mice were used to distinguish different populations of mast cells. Pieces of the skin were grafted from the intact WBB6F¹ (WB × C57BL/6)F¹?+/+ mice onto the back of the WBB6F¹?+/+ mice which had been irradiated and injected with bone marrow cells of C57BL/6-bg^j/bj^j mice (bg^j/bg^j ?+/+ chimeras). Although the number of mast cells in the skin grafts decreased after the transplantation, the mast-cell precursors circulating in the bloodstream of bg^j/bg^j ?+/+ chimeras (bg^j/bg^j type) did not seem to enter into the skin grafts, because most of mast cells were of +/+ type after the recovery of mast-cell number to pregrafting levels. As a considerable proportion of +/+-type mast cells was labeled with ³H-thymidine, the recovery of mast-cell number in the grafts was attributed to the proliferation and differentiation of +/+-type precursor cells fixed in the skin tissue of the donor. On the other hand, the skin of WBB6F¹-W/W^v mice seemed to be depleted of fixed precursors, because most of mast cells were of bg^j/bg^j type in skin grafted from WBB6F¹-W/W^v mice to bg^j/bg^j?+/+ chimeras. Since the fixed precursor cells which proliferate and differentiate into mast cells after skin grafting may be transferred to WBB6F¹-W/W^v mice by bone marrow transplantation, such precursor cells seem to have been derived from the bone marrow.     

Enhancement by interferon of natural killer cell activity in mice.

Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for ⁵¹chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as 10³ to 10⁴ units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nu/nu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity of these cells.     

Natural killer cell activity in murine muscular dystrophy. III. NK-sensitive myoblast cells and lack of NK activity in beige/dystrophic hybrid mice

The NK-susceptibility of dystrophic mouse myoblast cells was investigated. Spleen cells from 8- to 10-week-old normal (+/+) and dystrophic (dy2J/dy2J) male C57BL/6J mice were fractionated on Percoll density gradients and the cells at each density interface were incubated with either 51Cr-labeled YAC-1 or myoblast cells in a 6 hr 51Cr-release assay. Myoblast target cells were obtained from either heterozygous (+/dy2J) or homozygous (dy2J/dy2J) muscle cultures or a transformed tetraploid myoblast line (M14D2). The data indicate that the interface between the 50 and 60% (1.060-1.075 g/ml) Percoll density fractions of spleen cells from either normal or dystrophic mice contains the largest proportion of asialo GM-1 positive and NK-1 positive cells displaying NK activity. Myoblast cells from either heterozygous (phenotypically normal) or homozygous dystrophic mice were not significantly different in susceptibility to NK-mediated lysis by Percoll enriched normal or dystrophic mouse NK cells. However, dystrophic mouse spleen cells had the highest NK activity against both myoblast targets as compared with normal mouse spleen cells. The transformed myoblast cell line, M14D2, was significantly less susceptible to NK-mediated lysis by dystrophic mouse spleen cells when compared with freshly cultured myoblast target cells. Target cell binding studies revealed that conjugate forming cells from the 50% Percoll density interface of dystrophic mouse spleen cells were approximately twofold greater than that of normal mouse spleen cells against either heterozygous or homozygous dystrophic mouse myoblast targets. Cold target inhibition studies revealed that the natural killing of dystrophic mouse myoblast cells was due to a YAC-1 reactive NK cell. Breeding experiments between C57BL/6J homozygous "beige" (bgJ/bgJ) mutant mice and dystrophic (dy2J/dy2J) mice produced beige/dystrophic hybrid mice which displayed clinical symptoms of the dystrophy process by 3 to 4 weeks of age. Spleen cells from these hybrid mice showed no significant differences in NK activity against YAC-1 target cells when compared with homozygous beige mice. Taken together, these results demonstrate the first reported evidence that murine myoblasts are susceptible to NK-mediated lysis. In addition, the data indicate that although dystrophic mouse NK cells recognize myoblast cells as targets, the NK cell studies with the beige/dystrophic hybrid mice do not indicate a direct in vivo role for NK cells in the dystrophy process.     

Echinococcus multilocularis: susceptibility and responses to infection in inbred mice

Kroeze W. K. and Tanner C. E. 1987. Echinococcus multilocularis: susceptibility and responses to infection in inbred mice. International Journal for Parasitology17: 873–883. Of six strains of mice examined, C57L/J mice were the most susceptible to intraperitoneal infections with Echinococcus multilocularis. Five of the six strains developed splenomegaly during the infection. Changes in leukocyte levels in infected mice were most pronounced in the C57L/J and BALB/cJ strains. Two of the six strains of mice, C57BL/6J and C57BL/6J (bg^J), showed low specific IgG responses to E. multilocularis when measured using an ELISA. Many of the responses observed were directly correlated with the parasite burden in infected animals. It is concluded that susceptibility or resistance to E. multilocularis in mice is probably controlled by non-H-2 gene(s); additionally, hematological and immunological responses to infection, although correlated to parasite burden, varied among strains of mice, suggesting some degree of host genetic control of these responses.     

Induction of natural killer cell activity of thoracic duct lymphocytes by polyinosinic-polycytidylic acid (poly(I:C)) or interferon

Natural killer (NK) cell activity of thoracic duct lymphocytes (TDL) was examined in normal mice and in mice treated with polyinosinic-polycytidylic acid (poly(I:C) and interferon (IFN). TDL from mice treated with phosphate-buffered saline (PBS) expressed little or no NK cell activity against YAC-1 target cells at effector-to-target ratios of up to 200:1, even after in vitro treatment with murine L-cell IFN. In contrast, TDL from poly(I:C)- or IFN-treated mice expressed significant NK activity, which correlated with the significantly higher NK activity of splenocytes from these mice compared to the NK activity of splenocytes from PBS-treated mice. These data indicate that although TDL from normal mice express no detectable NK cell activity, NK cell activity can be induced in TDL by in vivo treatment with poly(I:C) or IFN.     

Dysregulated TLR3-dependent signaling and innate immune activation in superoxide-deficient macrophages from nonobese diabetic mice

In type 1 diabetes (T1D), reactive oxygen species (ROS) and proinflammatory cytokines produced by macrophages and other innate immune cells destroy pancreatic β cells while promoting autoreactive T cell maturation. Superoxide-deficient nonobese diabetic mice (NOD.Ncf1^m1J) are resistant to spontaneous diabetes, revealing the integral role of ROS signaling in T1D. Here, we evaluate the innate immune activation state of bone marrow-derived macrophages (BM-M?) from NOD and NOD.Ncf1^m1J mice after poly(I:C)-induced Toll-like receptor 3 (TLR3) signaling. We show that ROS synthesis is required for efficient activation of the NF-κB signaling pathway and concomitant expression of TLR3 and the cognate adaptor molecule, TRIF. Poly(I:C)-stimulated NOD.Ncf1^m1J BM-M? exhibited a 2- and 10-fold decrease in TNF-α and IFN-β proinflammatory cytokine synthesis, respectively, in contrast to NOD BM-M?. Optimal expression of IFN-α/β is not solely dependent on superoxide synthesis, but requires p47^phox to function in a NOX-independent manner to mediate type I interferon synthesis. Interestingly, MHC-II I-A^g7 expression necessary for CD4 T cell activation is increased 2-fold relative to NOD, implicating a role for superoxide in I-A^g7 downregulation. These findings suggest that defective innate immune-pattern-recognition receptor activation and subsequent decrease in TNF-α and IFN-β proinflammatory cytokine synthesis necessary for autoreactive T cell maturation may contribute to the T1D protection observed in NOD.Ncf1^m1J mice.     

Live cell imaging of highly activated natural killer cells against human hepatocellular carcinoma in vivo

Background aimsTracking administered natural killer (NK) cells in vivo is critical for developing an effective NK cell-based immunotherapy against human hepatocellular carcinoma (HCC). Here the authors established a new molecular imaging using ex vivo-activated NK cells and investigated real-time biodistribution of administered NK cells during HCC progression.MethodsEx vivo-expanded NK cells from healthy donors were labeled with a near-infrared lipophilic cytoplasmic dye, and their proliferation, surface receptor expression and cytotoxicity activity were evaluated. Human HCC HepG2 cells were implanted into the livers of NOD.Cg-Prkdc^scid IL2rg^tm1Wjl/SzJ (NSG) mice. The authors administered 1,1’-dioctadecyltetramethyl indotricarbocyanine iodide (DiR)-labeled NK cells intravenously to non-tumor-bearing and intrahepatic HCC tumor-bearing NSG mice. Fluorescent imaging was performed using a fluorescence-labeled organism bioimaging instrument. Single cell suspensions from the resected organs were analyzed using flow cytometry.ResultsThe fluorescent DiR dye was nontoxic and did not affect the proliferation or surface receptor expression levels of the NK cells, even at high doses. The administered DiR-labeled NK cells immediately migrated to the lungs of the non-tumor-bearing NSG mice, with increased NK cell signals evident in the liver and spleen after 4 h. NK cells migrated to the intrahepatic tumor-bearing livers of both early- and late-stage HCC mice within 1 h of injection. In early-stage intrahepatic tumor-bearing mice, the fluorescence signal increased in the liver until 48 h post-injection and decreased 7 days after NK injection. In late-stage HCC, the NK cell fluorescence signal was the highest in the liver for 7 days after NK injection and persisted for 14 days. The purity of long-term persistent CD45⁺CD56⁺CD3⁻ NK cells was highest in early- and late-stage HepG2-bearing liver compared with normal liver 2 weeks after NK injection, whereas highest purity was still observed in the lungs of non-tumor-bearing mice. In addition, Ki-67 expression was detected in migrated human NK cells in the liver and lung up to 72 h after administration. With HepG2 tumor progression, NK cells reduced the expression of NKp30 and NKG2D.ConclusionsAdministered NK cells were successfully tracked in vivo by labeling the NK cells with near-infrared DiR dye. Highly expanded, activated NK cells migrated rapidly to the tumor-bearing liver, where they persisted for 14 days after administration, with high purity of CD45⁺CD56⁺CD3⁻ NK cells. Liver biodistribution and persistence of administered NK cells showed significantly different accumulation patterns during HCC progression.