The rapid induction of HLA-E is essential for the survival of antigen-activated naive CD4 T cells from attack by NK cells

Increasing evidence shows that NK cells regulate adaptive immunity, but the underlying mechanisms are not well understood. In this study, we show that activated human NK cells suppress autologous naive CD4 T cell proliferation in response to allogeneic dendritic cells (DCs) by selectively killing Ag-activated T cells. Naive CD4 T cells, which were initially resistant to NK cell-mediated cytotoxicity, became substantially susceptible to NK cells within a day after priming with DCs. Ag-activated T cells showed various degrees of susceptibility to NK cells. After 1 d of priming with LPS-matured DCs, T cells were less susceptible to NK cells than were T cells primed with TNF-α-matured DCs. Subsequently at day 3, Ag-activated T cells regained resistance to NK cells. The level of HLA-E expression on Ag-activated T cells was closely correlated with resistance to NK cells. HLA-E was highly expressed at day 1 by T cells primed with LPS-matured DCs but not by T cells primed with TNF-α-matured DCs. An Ab blockade revealed a critical role for the HLA-E-NKG2A interaction in the protection of Ag-activated T cells from NK cells. Collectively, this study demonstrates that NK cells impact adaptive immunity through the finely controlled kinetics of HLA-E expression on T cells. Thus, HLA-E may be a new target for immunoregulation.     

Cellular interaction between fixed and living cells. Transfer of radioactive materials from living cells to fixed cells

Transfer of radioactive materials to fixed cells from an overlying layer of living cells has been examined to determine whether fixed cells can act as acceptors of glycosyltransferases of living cells. After the incubation of living cells were removed by EDTA treatment, and the radioactivity associated with the fixed cells was determined. Lipids, proteins and carbohydrates were found to be transfered from the living cells to the fixed cells. The amount of radioactivity transferred to the fixed cells was dependent on the number of both fixed and living cells and increased with the time of incubation. When fixed cells were treated with chloroform-methanol before the addition of living cells, the transfer of both lipids and proteins to the fixed cells decreased drastically, but only a slight decrease incarbohydrate transfer was observed. Most of the radioactive materials transferred from living cells labeled with glucosamine or fucose to chloroform-methanol-treated fixed cells were solubilized by trypsin but not by the detergents tested. Approximately 55% of the materials transferred from the cells labeled with glucosamine could be solubilized by hyaluronidase and chondroitinase, and the rest was solubilized by neuraminidase and a glycosidase mixture. The treatment of chloroform-methanol-extracted fixed cells with trypsin caused a significant decrease in the transfer from cells labeled with glucosamine. When nucleotide sugars were used as the radioactive precursor, no significant amount of radioactivity was transferred to the fixed cells.     

Eight types of endocrine cells in the abomasum of sheep

In the ovine abomasum, 8 types of endocrine cells were classified at ultrastructural level. The gastric-type EC cells contained oval and pleomorphic granules with high electron density. The intestinal-type EC cells were filled with oval, irregular and highly dense granules. ECL cells contained irregular granules with high density and wide clear spaces. D cells were filled with round granules showing low to moderate density and finely granular matrix D1 cells contained round or oval granules with variable, low to moderate density and finely granular content. G cells showed round and oval granules with moderate density, densely packed or flocculent content. F cells were filled with oval or elliptic granules showing low density with a finely granular and flocculent matrix. X cells contained round granules with high density and homogeneous material. Gastric-type EC cells, intestinal-type EC cells, D cells, and D1 cells were represented in the cardiac, fundic and pyloric gland areas of the ovine abomasum. ECL cells and F cells were confined to the fundic glands, G cells and X cells to the pyloric glands.     

Generation of suppressor cells by concanavalin A: a new perspective

Significantly lower mitogenic responses of fresh cells co-cultured with Con A-stimulated cells were found when compared with the responses of fresh cells co-cultured with preincubated control cells. We do not agree with the interpretation that this effect represents the generation of suppressor cells by Con A, since the responses of fresh cells cultured alone were also significantly less than when co-cultured with control cells and the same as when co-cultured with the Con A-stimulated cells. Treatment with mitomycin C was sufficient to prevent the preincubated cells from contributing to the mitogenic response of the fresh cells. The increased responses of fresh cells when co-cultured with preincubated cells seems analagous to the increased mitogenic responses of cells aged in vitro by preincubation without mitogen. This effect seems to be transferable to fresh cells in the absence of cell division. Although preincubation in the presence of Con A abrogates this effect, we do not interpret this as the generation of suppressor cells.     

Effect of "immune" RNA on various types of cells interacting in the immune response]

Simultaneous injection of marrow cells previously incubated with "immune" RNA, thymus cells and sheep red cells to X-ray treated mice resulted in a greater accumulation of antibody-forming cells in the spleen, as compared to mice inoculated with intact cells or with those incubated with normal RNA. Preliminary incubation of thymus cells with "immune" RNA did not affect the accumulation of antibody-forming cells. Incubation of peritoneal exudate cells with "immune" or normal RNA failed to influence the mode of accumulation of the antibody-forming cells either.     

Interferon gamma modulates the ability of autologous non-T cells to stimulate T cells to produce and respond to interleukin 2

The interactions of T-cell receptor with self-Ia antigen on non-T cells induced IL-2 production and IL-2 receptors on the cell surface and thus responsiveness to IL-2 of T cells in autologous mixed-lymphocyte reaction (AMLR). Four-day-cultured autologous non-T cells lost their ability to stimulate T cells to produce and respond to IL-2 with concurrent decrease of HLA-DR and HLA-DQ antigen expressed on the cell surface. Culturing of non-T cells with 500 U/ml of recombinant interferon gamma (IFN-gamma) maintained their stimulating ability which was otherwise lost. Treatment of non-T cells with monoclonal anti-HLA-DR or anti-HLA-DQ antibody before mixture with T cells abrogated their ability to induce IL-2 production and IL-2 responsiveness of T cells. The combined data suggested that Ia antigen expressed on non-T cells is modulated by IFN-gamma, which increases the ability of non-T cells to stimulate autologous T cells to produce and respond to IL-2.     

Stem cells for the replacement of inner ear neurons and hair cells

Stem cells in the nervous system have some capacity to restore damaged tissue. Proliferation of stem cells endows them with self-renewal ability and accounts for in vitro formation of neurospheres, clonally derived colonies of floating cells. However, damage to the nervous system is not readily repaired, suggesting that the stem cells do not provide an easily recruited source of cells for regeneration. The vestibular and auditory organs, despite their limited ability to replace damaged cells, appear to contain cells with stem cell properties. These inner ear stem cells, identified by neurosphere formation and by their expression of markers of inner ear progenitors, can differentiate to hair cells and neurons. Differentiated cells obtained from inner ear stem cells expressed sensory neuron markers and, after co-culture with the organ of Corti, grew processes that extended to hair cells. The neurons expressed synaptic vesicle markers at points of contact with hair cells. Exogenous stem cells have also been used for hair cell and neuron replacement. Embryonic stem cells are one potential source of both hair cells and sensory neurons. Neural progenitors made from embryonic stem cells, transplanted into the inner ear of gerbils that had been de-afferented by treatment with a toxin, differentiated into cells that expressed neuronal markers and grew processes both peripherally into the organ of Corti and centrally. The regrowth of these neurons suggests that it may be possible to replace auditory neurons that have degenerated with neurons that restore auditory function by regenerating connections to hair cells.     

Analysis of the murine lymphokine-activated killer (LAK) cell phenomenon: dissection of effectors and progenitors into NK- and T-like cells

Murine as well as human lymphokine-activated killer (LAK) cells have been reported to have several characteristics of T lymphocytes and to be clearly distinct from natural killer (NK) cells. The present study of murine LAK cells showed that cytotoxic cells generated in the presence of interleukin 2 IL 2 were heterogeneous with respect to cell surface markers of progenitor as well as effector cells. Negative selection of cells with antibodies and complement or positive selection by fluorescence-activated cell sorting unequivocally showed that LAK effector cells consisted of at least two clearly distinct populations, the relative contribution of which was dependent on donor organ and target cells studied. Approximately 40% of the cytotoxic activity of spleen-derived effector cells active against the NK-resistant targets EL-4 or MCA-5 was eliminated by treatment with antibodies to the NK-markers asialo-GM1 and NK 1 (NK-LAK). Approximately 60% of cytotoxic activity was associated with cells expressing the T cell marker Lyt-2, lacked NK 1, and was lacking or expressed only small amounts asialo-GM1 (T-LAK). The NK-LAK cells were of greater importance for the cytotoxic activity against the standard NK target YAC-1, although T-LAK cells also excerted significant cytotoxicity against this cell line. Limiting dilution analysis estimated that the minimal frequency of precursors developing into cells with cytotoxic activity against EL-4 was 1/6700 in spleen and 1/4200 in peripheral blood. The frequency of cells developing into cytotoxic effectors against YAC-1 cells was 1/3700 and 1/1450 in spleen and peripheral blood, respectively. Depletion of progenitor cells from spleen or peripheral blood expressing NK 1 or Lyt-2 by treating the cells with antibodies to these structures and complement indicated that NK-1-expressing cells were the dominating progenitor of the LAK cells irrespective of target cells used. Culture of murine lymphoid cells from spleen or peripheral blood with high concentrations of IL 2 results in the emergence of two different killer cell populations with phenotypic similarities to NK and T cells, respectively, both being able to kill targets resistant to resting NK cells. In contrast to numerous earlier reports, we concluded that LAK cells are heterogeneous with respect to surface markers, with a major population of LAK cells apparently representing IL 2-activated cells expressing cell surface markers associated with NK cells.     

Ultrastructural identification of round-granule EC cells in rabbit fundic mucosa

In endocrine (EC) cells of rabbit fundic mucosa, it is practically impossible to obtain unequivocal ultrastructural identification of all cells found in order to perform morphometric analysis. In addition to classic EC cells with pleomorphic granules, a cell type with entirely round granules is encountered which can be confused with non-EC cells. To solve this problem, all EC cells in our study were first identified by their 5-HT (immunocytochemistry) and argentaffinity. Examination of the fine structures of reactive cells then revealed that the round granules of EC cells were differentiated from those of non-EC cells by the existence of a dense core surrounded by a less dense halo, a feature providing unequivocal ultrastructural identification. EC cells with round granules showed less argentaffinity and less immunoreactivity to 5-HT as compared with classic EC cells. After labelling with [3H]L-dopa, EC cells with round-granules displayed an overall staining index higher than that of classic EC cells and comparable with that of D cells; however, the nuclear staining index was higher than that of D cells.     

Selective adhesion of embryonal carcinoma cells and differentiated cells by Ca2+-dependent sites

The specificity of adhesion between embryonal carcinoma cells and fibroblastic cells of various origins was studied. Embryonal carcinoma cells have intercellular adhesion sites requiring Ca²⁺ (CDS). These sites were found to be sensitive to proteases but resistant to them in the presence of Ca²⁺. CDS with a similar protease sensitivity is present in fibroblastic cells. When embryonal carcinoma cells of different lines were mixed, they adhered to each other nonselectively by CDS. Nonselective adhesion by CDS occurred also between fibroblastic cells of various lines. When embryonal carcinoma and fibroblastic cells were mixed, they preferentially adhered to homotypic cells. Fab fragments of antibodies raised against F9 cells (a nullipotent line of embryonal carcinoma) inhibited the adhesion between embryonal carcinoma cells but not between fibroblastic cells. This inhibitory activity of Fab was absorbed with embryonal carcinoma cells with CDS, but not with fibroblastic cells with CDS or embryonal carcinoma cells from which CDS was experimentally removed. SDS-polyacrylamide gel electrophoresis of radioiodinated cell surface proteins showed that the presence of a 140K-dalton component correlated with the presence of CDS in embryonal carcinoma cells, while the presence of a 150K-dalton component correlated with the presence of CDS in fibroblastic cells. These results suggest that CDS in embryonal carcinoma and fibroblastic cells comprise distinct molecules.     

The influence of 2-mercaptoethanol (2-ME) treatment of IgG antibody on its ability to induce cytotoxicity in nonsensitized lymphocytes

Administering dextran 2 hr before giving antigen (sheep red blood cells) to X-irradiated mice repopulated with B and T cells caused alterations in antibody synthesis. Besides a slight increase in the number of cells making IgM antibodies, cells producing IgG antibodies were detected at a time when none are usually present in untreated control animals.Repopulating X-irradiated mice with B cells treated with dextran either in vivo or in vitro and immunizing with sheep red blood cells resulted in twice the background number of sheep red blood cell-specific IgM-plaque-forming cells at 4.5 days of immunization as well as a small number of IgG-plaque-forming cells at 8.5 days. At these times, control animals given only B cells and sheep red blood cells possessed a background number of IgM-plaque forming cells and no IgG plaque-forming cells.Incubating B cells with θ-specific antiserum and complement to remove residual T cells prior to transplantation obliterated dextran's stimulus. Dextran's alterations of immunological responses towards unrelated antigens therefore appears to be manifested through T cells. The latter must be in company with B cells at the time of exposure to dextran. Thus, T cells upon contact with dextran apparently release B cell stimulatory factor (s) responsible for increasing the number of IgM-forming cells and for triggering IgG-forming cells.     

A role of HLA-DQ molecules of stimulator-adherent cells in the regulation of human autologous mixed lymphocyte reaction

In this study, we have found that treatment of stimulator autologous adherent cells with anti-HLA-DQ mAb resulted in markedly enhanced proliferative response of T cells in human autologous mixed lymphocyte reaction system wherein T cells were cultured with autologous adherent cells at near ratio of adherent cells to T cells in peripheral blood, in which T cells minimally proliferate. However, treatment of stimulator-adherent cells with anti-HLA class I, anti-DR and anti-DP mAb had no effect on the proliferative response of T cells under the condition. It was further observed that CD4-enriched cells could significantly proliferate in the presence of autologous adherent cells either untreated or treated with anti-DQ mAb, although treatment of adherent cells with anti-DR mAb blocked proliferative response of CD4-enriched cells. Moreover, the proliferative response of CD4-enriched cells was suppressed by addition of CD8-enriched cells in the presence of untreated adherent cells, whereas the proliferative response of CD4-enriched cells could not be suppressed by CD8-enriched cells when the adherent cells in the culture were treated with anti-DQ mAb. These observations suggest that CD8 T cells are required for suppression of proliferative response of CD4 T cells to HLA-DR molecules on autologous stimulator-adherent cells, and that HLA-DQ molecules on the surface of adherent cells play an important role in the induction of suppression by CD8 T cells.     

Prolactin and growth hormone cells in the human hypophysis: A study with immunoenzyme histochemistry and differential staining

Growth hormone and prolactin cells were immunostained in human hypophyses with antibody against rat growth hormone or prolactin and the peroxidase-antiperoxidase complex. Growth hormone cells were round and, in normal pituitaries, arranged in sizable groups. Prolactin cells occurred singly and were less numerous; they were often extensively branched. Only a few prolactin cells stained with carmoisine. Incubation of the antibody with an excess of the appropriate antigen greatly diminished or abolished immunostaining; absorption of anti-prolactin with growth hormone often enhanced it. Prolactin cells were somewhat hypertrophied and hyperplastic in a neonate. Many of them stained with carmoisine. An even greater hypertrophy and hyperplasia of these cells (which pushed apart the growth hormone cells) was found in a lactating woman. Immunostained giant prolactin cells were also observed. Staining of the prolactin cells with carmoisine was extensive. Upon prolonged exposure to anti-growth hormone antibody, ACTH/MSH cells also showed immunostaining which was abolished by absorption of the antiserum with growth hormone but not with synthetic 1-24ACTH. Growth hormone cells evidently correspond to the alpha acidophils of Romeis, prolactin cells in lactation to his eta cells; the relation of his epsilon cells to the pleomorphic "resting" prolactin cells is not clear.     

SV40 T antigen and telomerase are required to obtain immortalized human adult bone cells without loss of the differentiated phenotype.

In most human primary bone cells, SV40 T-antigen expression was able to expand life span for a few passages before cells undergo growth arrest, described as crisis. In this study, telomerase activity was reconstituted in human osteoblast precursors (hPOB cells) and marrow stromal cells (Saka cells) transformed with the SV40 T antigen. Bone cells with telomerase activity were able to bypass crisis and show unlimited life span. Despite chromosomal aberrations observed in hPOB-tert cells, these immortalized precursors were able to differentiate into osteoblasts like precrisis hPOB cells. Saka-tert cells enhanced the formation of human osteoclast-like cells in a similar manner as Saka cells. These results demonstrate that reconstitution of telomerase activity in transformed SV40 T-antigen human osteoblast precursors or marrow stromal cells leads to the generation of immortalized cells with a preserved phenotype.     

Tumor-specific immunity induced by somatic hybrids. II. Elicitation of enhanced immunity against the parent plasmacytoma.

Hybrid cells derived from fusion of a BALB/c plasmacytoma (TEPC-15) and L cells (C3H origin) were used to stimulate tumor-specific immunity against the parental plasmacytoma cells. Live hybrid cells induced tumor-specific immunity against TEPC-15 more effectively than mitomycin-treated hybrid or TEPC-15 tumor cells. Adoptive transfer of immunity with spleen cells of mice immunized with hybrid cells was also more effective than that with mitomycin-treated tumor cells. The immunity induced by the hybrid cells was specific to the TEPC-15 tumor because the mice that received immune spleen cells were not protected against challenge with either HOPC-8 or McPC-603 plasmacytomas. T cell populations were primarily responsible for the transfer of specific immunity based on the sensitivity of immune cells to anti-Thy 1.2 and complement. Mice that had established solid tumors were treated with 5 x 10(7) spleen cells to evaluate the therapeutic value of the hybrid-induced immune cells. Tumors in the mice that received immune cells gradually regressed over a 40-day period, whereas tumors on the control mice continued to grow. These results suggest that a rearrangement of tumor-specific antigens on allogeneic hybrid cells can enhance their immunogenicity.     

体外人表皮细胞对胰蛋白酶耐受的探讨

目的:观察人表皮细胞对胰酶消化的耐受能力,通过不同细胞恢复贴壁时间不同来探索分离和纯化表皮细胞的新方法,并探讨胰酶耐受细胞的干性表达,及其与muse细胞的可能相关性。方法:中性蛋白酶及胰酶消化获取表皮细胞,用0.25%的胰酶悬浮表皮细胞,以3.0×105/m L的细胞密度种植于12孔板,每间隔半小时或1小时终止胰酶1孔,其中胰酶作用时间最长达46小时。记录不同细胞贴壁时间、生长状态,并在接种后第7天,对贴壁细胞进行Nestin、Sox10抗体免疫细胞化学染色。结果:随着胰酶作用时间的延长,贴壁细胞数目递减,细胞贴壁所用时间也延长。所有孔中最早出现的贴壁细胞为树突状细胞,这些细胞开始生长缓慢,大约4天后生长迅速,10天后部分孔出现鱼群样细胞团。部分孔经Nestin、SOX10抗体的免疫细胞化学染色结果均为阳性,其中以Nestin抗体较明显。结论:人表皮细胞对胰酶消化的耐受能达46小时,从形态学观察判断,黑素细胞贴壁早于角质形成细胞,大多数贴壁后细胞增殖力强,干细胞表面标记显示部分阳性。     

二硝基氟苯修饰的黑色素瘤细胞激活树突状细胞诱导特异性T细胞反应

目的:探索半抗原二硝基氟苯(DNP)修饰的恶性黑色素瘤细胞(恶黑)激活树突状细胞(DC)后,在体外诱导特异性T细胞反应的抗肿瘤效应。方法:采用DNP修饰恶黑细胞M3(H-2d),然后在体外激活BALB/c小鼠(H-2d)外周血来源的DC,用于激发自体的T细胞,观察对T细胞的增殖和特异性T细胞的杀伤功能。结果:经DNP修饰的M3细胞激活的DC,其诱发的T细胞增殖能力和对M3细胞的特异性杀伤效应均明显高于未修饰的M3细胞组和DC组。结论:DNP修饰M3所激活的DC可以诱导更强的恶黑特异性T细胞效应。     

Induction of mammotroph development by a combination of epidermal growth factor,insulin, and estradiol-17beta in rat pituitary tumor GH3 cells

Several reports have indicated that prolactin-secreting cells (PRL cells) are generated from growth hormone-secreting cells (GH cells). We have shown that treatment with a combination of epidermal growth factor (EGF), insulin, and estradiol-17beta (E (2)) induces the appearance of PRL cells in pituitary tumor GH3 cells. The aim of the present study was to clarify the involvement of mitosis in the cytogenesis of PRL cells in rat pituitary and GH3 cells. The effects of the treatment with EGF, insulin and E(2) on DNA-replication were studied by detecting the uptake of bromodeoxyuridine (BrdU) into the nucleus. In cultured rat pituitary cells, BrdU-labeled PRL cells were observed irrespective of the hormone treatment. In GH3 cells, BrdU-labeled GH cells and mammosomatotrophs (MS cells) were detected; BrdU-labeled PRL cells were not detected, however, when GH3 cells were treated with BrdU for 3 hr and then immediately examined for BrdU-labeling. BrdU-labeled PRL cells were found only when GH3 cells treated with BrdU were allowed to grow for another 3 days. This finding suggests that during the additional 3-day culture, BrdU-labeled PRL cells were generated from BrdU-labeled cells other than PRL cells. These results indicate that PRL cells are transdifferentiated from GH cells or MS cells in GH3 cells by a combined treatment with EGF, insulin and E(2), while PRL cells in rat pituitaries are able to proliferate in response to the hormone treatment. Thus, there may be two pathways for cytogenesis of PRL cells: the transdifferentiation of GH cells or MS cells, and a self-duplication of PRL cells.     

Rapid response of marginal zone B cells to viral particles

Marginal zone (MZ) B cells are thought to be responsible for the first wave of Abs against bacterial Ags. In this study, we assessed the in vivo response of MZ B cells in mice immunized with viral particles derived from the RNA phage Qbeta. We found that both follicular (FO) and MZ B cells responded to immunization with viral particles. MZ B cells responded with slightly faster kinetics, but numerically, FO B cells dominated the response. B1 B cells responded similarly to MZ B cells. Both MZ and FO B cells underwent isotype switching, with MZ B cells again exhibiting faster kinetics. In fact, almost all Qbeta-specific MZ B cells expressed surface IgG by day 5. Histological analysis demonstrated that a population of activated B cells remain associated with the MZ, probably due to the elevated integrin levels expressed by these cells. Thus, both MZ and FO B cells respond with rapid proliferation to viral infection and both populations undergo isotype switching, but MZ B cells remain in the MZ and may be responsible for local Ab production, opsonizing pathogens entering the spleen.     

Pre-clinical assessment of autologous DC-based therapy in ovarian cancer patients with progressive disease

Dendritic cell–based vaccines offer promise for therapy of ovarian cancer. Previous studies have demonstrated that oxidation of several antigens, including ovarian cancer cells, using hypochlorous acid strongly enhances their immunogenicity and their uptake and presentation by dendritic cells. The response of T cells and dendritic cells to autologous tumour from patients with active disease has not previously been investigated. Monocyte-derived dendritic cells were generated from patients with active disease and activated by co-culture with oxidised tumour cells and the TLR agonist poly I:C. The dendritic cells showed an activated phenotype, but secreted high levels of TGFβ. Co-culture of the antigen-loaded dendritic cells with autologous T cells generated a population of effector T cells that showed a low level of specific lytic activity against autologous tumour, as compared to autologous mesothelium. The addition of neutralising antibody to TGFβ in DC/T cell co-cultures increased the levels of subsequent tumour killing in three samples tested. Co-culture of monocytes from healthy volunteers with the ovarian cell line SKOV-3 prior to differentiation into dendritic cells reduced the ability of dendritic cells to stimulate cytotoxic effector cells. The study suggests that co-culture of dendritic cells with oxidised tumour cells can generate effector cells able to kill autologous tumour, but that the high tumour burden in patients with active disease may compromise dendritic cell and/or T cell function.