Monocyte-independent interleukin-2 production and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody: interleukin-1 is not required for T-cell proliferation

We report a new, monocyte-independent system for the induction of activation and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody (OKT3 hybridomas). Incubation of nylon-wool-nonadherent (NA) lymphocytes or purified T cells with OKT3 hybridomas resulted in interleukin-2 (IL-2) production, expression of IL-2 receptor, modulation of the CD3 antigen, and proliferation. In contrast, murine hybridomas (OKT4, OKT8, anti-HLA-DR, and others) expressing monoclonal antibodies (mAb) other than OKT3 did not induce T-cell activation and proliferation. T cells did not respond to OKT3 mAb alone. OKT3 hybridomas alone did not produce interleukin-1 (IL-1) or other soluble factors that might be involved in the induction of IL-2 production by T cells, and they did not contain membrane-bound IL-1. In addition, IL-1 activity was not detected in cultures of NA-lymphocytes and OKT3 hybridomas, clearly demonstrating that IL-1 was not required, at least in this system, for T-cell activation and proliferation. Direct cell-cell contact between T cells and OKT3 hybridomas was required for IL-2 production. Thirty to fifty percent of T cells formed conjugates with the OKT3 hybridomas but not with the OKT4 or OKT8 hybridomas. Both conjugate formation and IL-2 production were significantly inhibited by the OKT3 mAb and by the anti-LFA-1 mAb. The cells responsible for IL-2 production were found to be of the T3+ T4+ T8- Leu 7- Leu 11- phenotype. IL-2 activity produced by NA-lymphocytes in response to OKT3 hybridomas became detectable as early as 1 hr and reached a maximum by 8 hr, preceding IL-2 receptor expression, modulation of the CD3 antigen, and [3H]thymidine incorporation of T cells. T cells produced higher concentrations of IL-2 in response to OKT3 hybridomas than in response to equal numbers of monocytes and OKT3 mAb. Addition of monocytes to cultures of T cells and OKT3 hybridomas resulted in suppression of IL-2 production in a concentration-dependent manner, suggesting that monocytes regulate the levels of IL-2 production. This monocyte-independent system may be useful for further dissection of T-cell activation and proliferation and its regulation by monocytes.     

ATP-coupled transport of vesicular stomatitis virus G protein between the endoplasmic reticulum and the Golgi

The temperature and ATP dependence of transport of the vesicular stomatitis virus strain ts045 G protein from the endoplasmic reticulum (ER) to an early Golgi compartment containing mannosidase I was studied in the mutant Chinese hamster ovary cell clone 15B. Appearance of G protein containing the Man5GlcNAc2 oligosaccharide species occurred after a shift to the permissive temperature with a lag period of 5 min and without detectable formation of the intermediate Man7GlcNAc2 and Man6GlcNAc2 species. Two biochemically distinct transport steps were detected during transport from the ER to the Golgi. An initial step is temperature sensitive, thermoreversible, and requires a high threshold of cellular ATP for maximal rate of transport (80% of the normal cellular ATP pool). Export from the ER is inhibited at 65% of the normal cellular ATP pool. Prolonged incubation at reduced levels of cellular ATP or at the restrictive temperature resulted in the accumulation of G protein in either the Man8GlcNAc2 species or the Man7GlcNAc2 and Man6GlcNAc2 species, respectively. Reversal of the temperature-sensitive block is ATP coupled. A second step is insensitive to incubation at the restrictive temperature and proceeds efficiently when the cellular ATP pool is reduced to 20% of the control. G protein accumulates at this intermediate step during prolonged incubation at 15 degrees C. The data suggest a functional division of processes required for transport of protein between the ER and Golgi compartments. The two steps may reflect the export (budding) and delivery (fusion) of proteins through vesicular trafficking between the ER and Golgi.     

Suppressor cell function of human granular lymphocytes identified by the HNK-1 (Leu 7) monoclonal antibody

The HNK-1 (Leu 7) differentiation antigen defines a subpopulation of human granular lymphocytes with natural killer (NK) and K cell function. In this study, we investigated whether HNK-1+ cells, identified with the monoclonal antibody and purified with a fluorescence-activated cell sorter (FACS), could function as suppressor cells. The results demonstrated that purified HNK-1+ cells efficiently suppressed both PWM-induced IgG production by B cells and T cell proliferation in mixed lymphocyte reactions (MLR). Manifestation of this suppressor cell activity required immune complex activation and was partially sensitive to 2000 rad irradiation. This suppressor cell activity was predominantly mediated by a subset of HNK-1+ cells that have previously been shown to have maximum NK function and lack expression of the E rosette (ER) receptor and T cell antigens (e.g., T3 and T8). Thus, HNK-1+ER- cells suppressed a MLR by an average 52%; HNK-1+ER+ were one-half as efficient, causing an average 23% suppression. For comparison, we also examined the characteristics of Leu 2a+ suppressor T lymphocytes. In contrast to HNK-1+ cells, unactivated Leu 2a+ cells suppressed both B and T cell responses. This suppressor activity was not augmented by immune complex activation and was absolutely radio-sensitive in PWM assays. HNK-1+ cells, especially the HNK+ER- subset, can therefore mediate suppressor cell function in addition to their spontaneous cytotoxic function. Furthermore, some of their suppressor cell properties are distinct from those attributed to other types of suppressor lymphocytes.     

Altered biodistribution of indium-111-labeled monoclonal antibody 96.5 to tumors and normal tissues of nude mice bearing human melanoma xenografts in visceral organs

Summary Human melanoma xenografts were produced in the subcutis, kidney, cecum and liver of different nude mice. An¹¹¹In-labeled anti-(human melanoma) monoclonal antibody (96.5) or an¹¹¹In-labeled nonspecific control monoclonal antibody (ZCE-025) was injected intravenously in separate groups of mice. Radioactive antibody accumulation was measured in tumor, blood, viscera, and carcasses. mAb 96.5 targeted specifically to tumor tissue regardless of site of growth. Tumors in the liver exhibited significantly (P <0.05) higher tumor-to-blood ratios (45±6, mean ±SEM) than xenografts at other visceral organs, the lowest value being found for subcutaneous melanoma (2.6±0.5). The differences in tumor-to-blood ratio were due to significant alterations of antibody biodistribution, since the actual antibody concentration in the different tumor sites was similar. The percentage of recovered anti-melanoma antibody per milliliter of blood in mice with visceral lesions (4.6±1.1%/ml) was significantly lower than that found in mice with subcutaneous tumors (9.5±1.4%/ml,P <0.05). Moreover, significantly higher levels (18.2±3.2%/g, 31.0±5.1%/g, respectively) of the melanoma mAb 96.5 were found in normal liver and spleen tissue recovered from mice with visceral tumors as compared to tissue from mice with subcutaneous tumors (9.2±0.9%/g, 13.5±1.9%/g, respectively;P <0.05). These results demonstrate that the presence of visceral tumor can significantly affect tumor-to-blood ratios, blood levels, and biodistribution of¹¹¹In-labeled mAb 96.5.This work was supported in part by funds from the National Institutes of Health, National Cancer Institute, Grant R35-CA42107 and Core Grant CA16672     

Use of Synthetic Peptides to Study the Function of GTPases Regulating Vesicular Traffic

We describe the application of synthetic peptides to explore the function of GTPases regulating vesicular traffic through the constitutive and regulated exocytic pathways. The utility of the approach is discussed in the context of the parameters that limit or enhance the effectiveness of these reagents in eliciting a biologically relevant response.     

Role of uncultured human melanoma cells in the proliferation of autologous tumor-specific cytotoxic T lymphocytes.

The role of uncultured melanoma cells in the proliferation of autologous tumor-specific cytotoxic T lymphocytes (CTLs) was investigated. Uncultured autologous tumor cells by themselves induced modest, but significant, proliferation in 10 of 13 (77%) CTL clones and in only two of nine non-CTL clones. Uncultured allogenic melanoma cells mostly failed to induce CTL proliferation. Autologous tumor-induced CTL proliferation declined with increasing age of the culture. It did not correlate with IL-2 receptor-alpha expression or was not inhibited by addition of anti-IL-2 antibody to the culture. It was inhibited by pretreatment of tumor cells with anti-MHC class II, but not -MHC class I mAb. IL-2 alone was sufficient for the potent proliferation of five of nine CTL clones. In all these five CTL clones, autologous tumor cells suppressed IL-2-induced proliferation. The remaining four CTL clones, however, required both uncultured autologous melanoma cells and IL-2 for the proliferation. IL-4 or IL-6, in particular IL-6, facilitated IL-2-induced CTL proliferation, but not their cytotoxicity. In summary, uncultured melanoma cells by themselves induced modest levels of CTL proliferation in the context of MHC class II antigens, whereas they suppressed IL-2-induced CTL proliferation in more than half of the clones.     

Spectral properties of Noctiluca miliaris Suriray, a heterotrophic dinoflagellate

This note examines the role of Noctiluca miliaris Suriray, aheterotrophic dinoflagellate, as a spectral light absorber andcompares its light absorption to that of other dinoflagellates.The discussion emphasizes that carotenoids are responsible formuch of Noctiluca's orange coloration and that an algal heterotrophcan significantly alter ocean color. A bloom of Noctiluca couldbe detected by the Coastal Zone Color Scanner thus causing errorin the estimate of autotrophic pigments as well as estimatesof primary production. ¹This paper is dedicated to Martin W.Johnson on the occasionof his 90th birthday.     

Phytanyl-glycerol ethers and squalenes in the archaebacteriumMethanobacterium thermoautotrophicum

Summary The lipids of a thermophilic chemolithotroph,Metbanobacterium thermoautotropbicum, have been analyzed by chromatographic techniques and identified by infrared spectrometry and combined gas chromatography-mass spectrometry. Of the total chloroform soluble lipids 79% and 21% are polar and non-polar lipids, respectively. The major components of the polar lipids are dialkyl ethers of glycerol or its derivatives. The nature of the glycerol ether alkyl groups was found to be that of the saturated tetraisoprenoid hydrocarbon phytane. The non-polar lipids of the chloroform soluble fraction consist principally of three series of C₂₀, C₂₅ and C₃₀ acyclic isoprenoid hydrocarbons, the major components being squalene and a continuous range of hydrosqualene derivatives, from dihydrosqualene up to and including decahydrosqualene. These data establish thatM. tbermoautotropbicum contains predominantly non-sapo-nifiable lipids as doHalobacterium, Halococcus, Sulfolobus andTbermoplasma. In particular, the composition of the chloroform soluble lipids ofM. tbermoautotropbicum is quite similar to that ofHalobacterium cutirubrum. The results strongly support the recent proposal, based on 16S rRNA sequence homologies, that the extreme halophiles and methanogens share a common ancestor. In addition, it is pointed out that the occurrence of phytane and related polyisoprenoid compounds in ancient sediments can no longer be considered unequivocally as indicative of past photosynthetic activity. Finally, speculations are made concerning the possible role of and evolutionary significance of the presence of squalene and hydrosqualenes in these organisms. To our knowledge this is the first report of squalene and hydrosqualenes in a strictly anaerobic microorganism.To either of whom reprint requests should be sent.