A group-specific inhibitor of lysosomal cysteine proteinases selectively inhibits both proteolytic degradation and presentation of the antigen dinitrophenyl-poly-L-lysine by guinea pig accessory cells to T cells

A limited intralysosomal proteolytic degradation is probably a key event in the accessory cell processing of large protein antigens before their presentation to T cells. With the aid of highly specific inhibitors of proteinases, we have examined the role of proteolysis in the presentation of antigens by guinea pig accessory cells. The proteinase inhibitor benzyloxycarbonyl-phenylalanylalanine-diazomethyl-ketone, which selectively inhibits cysteine proteinases, was used to block this set of enzymes in cultured cells. We demonstrate that the selective inhibition of the cysteine proteinases of antigen-presenting cells causes a profound inhibition of both the proteolytic degradation and the presentation of the synthetic antigen dinitrophenyl-poly-L-lysine. In contrast, the presentation of another synthetic antigen, the copolymer of L-glutamic acid and L-alanine, was enhanced by the same inhibitor. Another inhibitor, pepstatin A, which selectively blocks aspartic proteinases, did not block the presentation of dinitrophenyl-poly-L-lysine. The results identify cysteine proteinases, probably lysosomal, as one of the groups of enzymes involved in antigen processing.     

Cerulenin is a potent inhibitor of antigen processing by antigen-presenting cells

Cerulenin is an antibiotic that inhibits eukaryotic lipid and sterol synthesis and blocks lipid modification of proteins. The effect of cerulenin on the ability of accessory cells to present antigen to T cells was investigated. This antibiotic strongly inhibits the ability of accessory cells to present antigen to murine T-T hybrids. This effect is observed for multiple distinct antigens including L-glutamic acid60-L-alanine30-L-tyrosine10, bovine insulin, L-glutamic acid56-L-lysine35-L-phenylalanine9, and ovalbumen. Presentation by both macrophage and B lymphoblastoid cell lines is inhibited. The ability to effectively pulse these cells with antigen is inhibited but not the ability of these same cells to present antigen that they have previously processed. Furthermore, this inhibition is selective as it can occur without significant inhibition of the antigen-presenting cell protein or DNA synthesis. Cerulenin does not inhibit antigen uptake or catabolism as assessed with labeled antigen. By these criteria this drug is shown to interfere with an antigen-processing step. The ability of cerulenin to block processing was compared with other known inhibitors. Although cerulenin was effective with all antigens tested, at least one inhibitor was not. Taken together, these results suggest that the effect of cerulenin may define a distinct step in antigen processing and provides evidence that some other processing events are not universally required. The ability of cerulenin to interfere with antigen processing is discussed in the context of the known actions of this antibiotic and events of antigen processing and presentation.     

Immunologic function of endothelial cells: guinea pig aortic endothelial cells support mitogen-induced T lymphocyte activation, but do not function as antigen-presenting cells

The possibility that vascular endothelial cells (EC), like macrophages (M phi), can function as accessory cells necessary for mitogen- and antigen-induced T cell activation was examined. EC were enzymatically detached from the luminal surfaces of guinea pig aortas and then propagated in culture. Lymph node T lymphocytes were rigorously depleted of adherent cells, such that they completely lost the capacity to respond to mitogenic stimulation with phytohemagglutinin or concanavalin A. In this system, EC restored mitogen-induced T cell DNA synthesis as effectively as did M phi. This effect could not be explained by a facilitation of residual accessory cell activity within the responding T cell population, because EC restored mitogen responsiveness to T cells that had been treated with anti-Ia antibody and complement. Support of mitogen responsiveness could not be accounted for by secreted products of M phi or EC in the absence of intact accessory cells. In addition to the capacity to serve as fully sufficient accessory cells for the induction of mitogen-stimulated T cell proliferation, EC exerted a number of modulatory influences on T lymphocyte responses in cultures supported by M phi. When such cultures were supplemented with small numbers of EC, responses were dramatically augmented; larger numbers of EC resulted in marked suppression. At least part of these immunomodulatory effects could be accounted for by the activity of secreted products of EC. EC did not express detectable Ia antigens assayed either by indirect immunofluorescence, with the use of the fluorescence-activated cell sorter, or by complement-mediated cytotoxicity. Moreover, treating the EC population with anti-Ia antibody and complement had no effect on its capacity to support mitogen-induced T cell DNA synthesis. As would be expected from the lack of Ia antigen expression, EC were incapable of presenting antigen to primed T cells. They did, however, carry enough antigen into the cultures such that effective antigen presentation could occur when the cultures were supplemented with M phi that were syngeneic but not allogeneic to the responding T cells. Moreover, EC were capable of dramatically augmenting antigen-specific responses stimulated by antigen-pulsed M phi. There was no genetic restriction for this EC-mediated augmentation of antigen responsiveness. These results indicate that EC are capable of functioning as completely sufficient accessory cells for mitogen-induced T cell DNA synthesis and, in addition, are able to modulate ongoing M phi-supported T lymphocyte responses in both a positive and negative manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of glutamic acid uptake by bovine pulmonary arterial endothelial cells

L-Glutamic acid uptake by bovine pulmonary arterial endothelial cells in culture increased linearly with time up to 30 min and did not show saturation with increased substrate concentration up to 6 X 10(-3) M. The uptake per cell decreased as cell density increased and was lowest when the cells became fully confluent. Most of the uptake was sodium dependent, although the relative contribution of sodium-independent uptake increased with an increase in cell density. Cysteic and aspartic acid strongly inhibited L-glutamic acid uptake, but at higher cell densities this effect was less pronounced than at low densities. Other amino acids, including leucine, glutamine, and serine, exerted a modest inhibitory effect at both high and low cell densities. Thus pulmonary arterial endothelial cells contain similar membrane transport systems for L-glutamic acid as those previously described for fibroblasts, hepatocytes, and nerve cells. However, quantitative properties of the transport systems differ depending on the state of cellular density in monolayers.     

A subpopulation of adherent accessory cells bearing both I-A and I-E or C subregion antigens is required for antigen-specific murine T lymphocyte proliferation.

A murine T lymphocyte proliferation assay that used antigen-primed lymph node T cells, was antigen specific, and required exogenous accessory cells was used to characterize the accessory cells that supported proliferation. These cells were Thy 1.2 negative, radioresistant, glass-adherent, and were functional only if alive. The accessory cell function of spleen adherent cells was much greater than that of peritoneal cells. Also, the accessory cell function of spleen adherent cells was proportional to the length of time such cells were incubated with antigen and very small numbers of such cells provided accessory cell function. Cytotoxic studies with subregion-restricted anti-Ia antibodies and complement indicated that accessory cell function resided in a subpopulation of spleen adherent cells that bore both I-A and I-E or C subregion antigens. The function of such cells was not related to a selective ability (vs other spleen adherent cells) to take up antigen. These data indicate that antigen-specific stimulation of T lymphocyte proliferation requires at least one specific subpopulation of spleen adherent cells that can be phenotypically identified by its expression of Ia antigens and are consistent with the possibility that Ia antigens may be Ir gene products.     

Dissection of the functions of antigen-presenting cells in the induction of T cell activation

The functions of antigen-presenting cells (APC) in the initiation of T cell activation was examined by culturing antigen-bearing guinea pig macrophages (M phi) with T cells obtained from antigen-primed animals. Although such antigen-bearing M phi stimulated primed syngeneic T cell DNA synthesis, as assessed by tritiated thymidine incorporation, paraformaldehyde fixation (0.15% for 1 min at 37 degrees C) abolished this capacity. Analysis with acridine orange staining indicated that fixed antigen-bearing M phi could not trigger primed syngeneic T cells to progress from the G0 to the G1 phase of the cell cycle. The addition of control non-antigen-bearing syngeneic or allogeneic M phi but not interleukin 1 or 2 to cultures of T cells and fixed APC permitted a proliferative response. Although the interaction between fixed antigen-bearing M phi and responding T cells was genetically restricted, there was no similar restriction for the supplemental control M phi. In fact, completely Ia-negative endothelial cells (EC) and fibroblasts (FB) could restore antigen responsiveness to cultures of fixed antigen-bearing M phi and syngeneic responding T cells, although they could not directly present antigen. Moreover, metabolically intact accessory cells, including Ia-negative EC and FB, could take up and process antigen to an immunogenic moiety, which fixed Ia-positive M phi could present to primed T cells. These data indicate that recognition of the antigen-Ia complex on an APC is necessary but not sufficient to trigger proliferation of freshly obtained primed T cells. The results additionally support the conclusion that APC carry out at least two separate functions necessary for the initiation of antigen-induced T cell activation. Not only must the APC display the antigen-Ia complex, but it must also convey another required effect. This influence, which apparently involved the establishment of cell to cell contact, was neither Ia nor antigen dependent and could only be provided by a metabolically intact cell. By contrast, genetically restricted antigen presentation could be accomplished by a fixed Ia-positive cell. Only when both the antigen-Ia complex and the influence of an intact accessory cell were provided by the same or different accessory cell were T cells triggered to enter the cell cycle.     

Accessory cell function of thoracic duct nonlymphoid cells, dendritic cells, and splenic adherent cells in the Brown-Norway rat

Thoracic duct lymph of lymphadenectomized Brown-Norway (BN) rats is highly enriched for nonlymphoid cells (NLC) which share several characteristics with splenic dendritic cells (DC), e.g., the binding of monoclonal antibody OX2. The accessory cell activity of NLC was analyzed by comparing these cells with DC and splenic adherent cells (SAC). In concanavalin A (Con A)-induced T-cell proliferation NLC, like DC, were very effective accessory cells at low cell numbers, as a consequence of an efficient induction of interleukin 2 (IL-2) production and IL-2 responsiveness. Responses in the presence of SAC were poor, even after the addition of excess IL-2. A fourfold enhancement of accessory cell activity of SAC was achieved by the depletion of FcR-positive cells, which were responsible for suppression of the Con A response. Low responsiveness of BN rats with respect to Lewis rats can in part be explained by a higher suppressive activity of macrophages in the BN rat.     

Acquisition of syngeneic I-A determinants by T cells proliferating in response to poly (Glu60Ala30Tyr10)

The T cell proliferative response in mice to the synthetic polymer GAT is under Ir gene control, mapping to the I-A subregion of the H-2 major histocompatibility complex (MHC). Antigen-dependent proliferation in vitro of in vivo GAT-primed lymph node cells can be inhibited by a monoclonal antibody to Ia-17, an I-A public determinant. Using this antibody for direct immunofluorescent analysis, T cells in GAT-stimulated proliferative culture are identified that express syngeneic I-A during culture. This expression is strictly antigen dependent, requires restimulation in vitro, and requires the presence of I-A-positive adherent antigen-presenting cells. T cells bearing I-A can be enriched by a simple affinity procedure, and I-A-positive cells separated on a FACS are shown to retain antigen-specific reactivity. The acquisition of I-A determinants by T cells under these culture conditions is not nonspecific. The Ia determinants borne by T cell blasts appear to be dictated by the I subregion to which the relevant Ir gene maps, and which codes for the Ia molecule involved in presentation of the antigen. Thus, (B6A)F1 (H-2b X H-2a)F1 LNC express I-Ak antigens when proliferating to GAT but not when stimulated by GLPhe, the response to which is under I-E subregion control. The relation of Ir gene function to Ia-restricted antigen presentation and self-Ia recognition is discussed.     

The role of adherent accessory cells in the generation of effector suppressor T cells

The role of accessory cell populations in the generation of effector suppressor (Ts3) cells was studied. By using an in vitro culture system, it was previously determined that the induction of NP-specific effector suppressor activity requires T cells, antigen, and an anti-idiotypic B cell population. We now demonstrate that the generation of Ts3 cells in this system also requires accessory cells. The accessory population appears to play a role in the processing and presentation of antigen. These antigen-presenting accessory cells are required early in the induction phase of Ts3 generation. These accessory cells can present NP coupled to immunogenic or non-immunogenic polypeptide carriers, including polymers of L-amino acids. However, NP coupled to polymers of poorly metabolized D-amino acids fail to induce suppressor T cell generation. Furthermore, the data demonstrate that an H-2 homology must exist between the Ts3 precursors and the antigen-presenting cell population if suppressor activity is to be generated. We also characterize the differential genetic restrictions that govern the induction of Ts3 cells that control suppression of either T cell or B cell responses. The data suggest that although I-J region encoded gene products control the induction and effector phases of suppressor cell activity as measured on T cell responses, the suppression of B cell responses appear to be controlled by I-A gene products. Possible cellular mechanisms that might explain these findings are discussed.     

L-glutamic acid--a modulator of the physiological status of myeloid series blood cells

The effects of L-glutamic acid on the differentiation of HL-60 and K-562 cell lines and on the expression level of mRNAs encoding IL-1 beta, IL-6, and TNF alpha were studied. It was shown that TNF alpha actively induces the differentiation of these cell lines, whereas L-glutamic acid enhances its effect. Our results indicated that L-glutamic acid modulates the physiological state of the myeloid cell line in blood, in particular, by affecting both the reception and expression of cytokines functionally important for these cells.     

Mutations of the Corynebacterium glutamicum NCgl1221 gene, encoding a mechanosensitive channel homolog, induce L-glutamic acid production

Corynebacterium glutamicum is a biotin auxotroph that secretes L-glutamic acid in response to biotin limitation; this process is employed in industrial L-glutamic acid production. Fatty acid ester surfactants and penicillin also induce L-glutamic acid secretion, even in the presence of biotin. However, the mechanism of L-glutamic acid secretion remains unclear. It was recently reported that disruption of odhA, encoding a subunit of the 2-oxoglutarate dehydrogenase complex, resulted in L-glutamic acid secretion without induction. In this study, we analyzed odhA disruptants and found that those which exhibited constitutive L-glutamic acid secretion carried additional mutations in the NCgl1221 gene, which encodes a mechanosensitive channel homolog. These NCgl1221 gene mutations lead to constitutive L-glutamic acid secretion even in the absence of odhA disruption and also render cells resistant to an L-glutamic acid analog, 4-fluoroglutamic acid. Disruption of the NCgl1221 gene essentially abolishes L-glutamic acid secretion, causing an increase in the intracellular L-glutamic acid pool under biotin-limiting conditions, while amplification of the wild-type NCgl1221 gene increased L-glutamate secretion, although only in response to induction. These results suggest that the NCgl1221 gene encodes an L-glutamic acid exporter. We propose that treatments that induce L-glutamic acid secretion alter membrane tension and trigger a structural transformation of the NCgl1221 protein, enabling it to export L-glutamic acid.     

Dose-response effects of phytoestrogens on the activity and expression of 3beta-hydroxysteroid dehydrogenase and aromatase in human granulosa-luteal cells

There is evidence that certain phytoestrogens can inhibit key steroidogenic enzymes although most studies have been carried out on microsomal or purified enzyme preparations, some using cell lines. This study was designed to test the hypothesis that low doses of phytoestrogens, at concentrations that would be attained through the diet, could inhibit 3beta-hydroxysteroid dehydrogenase (HSD) and/or aromatase in primary cultures of human granulosa-luteal (GL) cells and that this effect was due to a decrease in the expression of these proteins. Based on published evidence, eight compounds were selected for investigation and these included the flavones apigenin and quercetin, the isoflavones genistein, biochanin A and daidzein, the lignans, enterodiol and enterolactone, and the mycotoxin zearalenone. Human GL cells were cultured for 48 h in the presence of these phytoestrogens at concentrations ranging from 0.01 to 100 microM and after addition of fresh media the conversion of pregnenolone to progesterone or androstenedione to oestradiol over a 4h period was measured. Biochanin A was the only phytoestrogen that displayed any dose-dependent inhibition of 3beta-HSD, others showing inhibition at doses >/=10 microM. Apigenin and quercetin only inhibited aromatase/17beta-HSD at high doses as did genistein, biochanin A and daidzein. The lignans had weak inhibitory effects on aromatase/17beta-HSD, whilst zearalenone showed potent inhibition at 0.1 microM. Phytoestrogens did not exert any significant effects on protein expression of 3beta-HSD or aromatase as determined by Western blots. It is concluded that steroidogenic enzymes are inhibited by phytoestrogens in primary cultures of human GL cells but these cells are less sensitive to the effects of phytoestrogens than cell-free systems. This may be due to poor lipid solubility or cellular metabolism. We have also shown for the first time that phytoestrogens do not act by inhibiting the cellular concentration of 3beta-HSD and aromatase even though exposure time would have allowed for changes in gene expression.     

H2 gene control and biological activities of a T-cell mitogen derived from Mycoplasma arthritidis: a review

Mycoplasma arthritidis generates a soluble, non-dialysable, polyclonal mitogen which is active for murine and human T lymphocytes in the presence of an adherent, radio-resistant, Ia-bearing accessory cell population. Genetic analysis has established that the I-E sub-region of the murine H2 gene complex controls responses to the mitogen and that this control is exercised at the level of the Ia-bearing accessory cell. Lymphocyte proliferation, induction of cytotoxic lymphocytes, and interferon induction are all under Ir gene control and appear to be dependent upon binding of the mitogen to a specific Ia antigen present on a subset of splenic cells. This mycoplasma mitogen provides a new model system to define the mechanisms of Ir gene control of lymphocyte activation.     

Identification of suppressor T cells in virgin non-responder spleen cells responsible for primary unresponsiveness to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)

T cell subsets that regulate antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in mice that are Ir gene non-responders have been further characterized. We previously defined several T cell subsets in GAT-primed non-responder mice. The Lyt-2+ suppressor-effector T cells suppress responses to GAT and GAT complexed to methylated BSA (GAT-MBSA). The Lyt-1+ cell population is complex and can be separated into I-J- Th cells, which support responses to GAT and GAT-MBSA. After priming, the Lyt-1+, I-J+ cell population contains suppressor-inducer cells that activate precursors of suppressor-effector cells to suppress responses to GAT and GAT-MBSA as well as Ts cells that directly inhibit responses to GAT but not GAT-MBSA. By contrast, the Lyt-1+ cells from virgin mice contain only cells that directly suppress responses to GAT but not GAT-MBSA. The major question addressed in the present studies was whether the Lyt-1+, I-J+ Ts cells in virgin and primed mice and the suppressor-inducer cells in GAT-primed mice were functionally and serologically distinct subsets. The studies used mAb and panning procedures to separate cell populations and inhibition of PFC cell responses to functionally define the activity of the cell populations. We used the following two mAb that were raised by immunizing rats with GAT-specific suppressor factors: 1248A4.10 (known to react with suppressor-inducer cells) and 1248A4.3, another reagent from the same fusion. Lyt-1+ cells from virgin spleens contained Ts cells that were A4.10-, A4.3+ and no suppressor-inducer T cells, whereas Lyt-1+ cells from GAT-primed spleens contained Ts cells that were A4.10-, A4.3+ as well as A4.10+, A4.3- suppressor-inducer cells. Thus, the Lyt1+, I-J+ cell subset can be divided into two functionally and serologically distinct subsets, direct Ts cells (1248A4.3+), which suppress responses to GAT but not GAT-MBSA, and GAT-primed suppressor-inducer T cells (1248A4.10+).     

Qualitative and quantitative studies of antigen-presenting cell function by using I-A-expressing L cells

I-A-expressing transfected murine L cells were analyzed as model antigen-presenting cells. Four features of accessory cell function were explored: antigen processing, interaction with accessory molecules (LFA-1, L3T4), influence of Ia density, and ability to stimulate resting, unprimed T lymphocytes. I-A+ L cells could present complex protein antigens to a variety of T cell hybridomas and clones. Paraformaldehyde fixation before but not subsequent to antigen exposure rendered I-A+ L cells unable to present intact antigen. These results are consistent with earlier studies that made use of these methods to inhibit "processing" by conventional antigen-presenting cells. The ability of anti-L3T4 antibody to inhibit T cell activation was the same for either B lymphoma or L cell antigen-presenting cells. In striking contrast, anti-LFA-1 antibody, which totally blocked B lymphoma-induced responses, had no effect on L cell antigen presentation, measured as interleukin 2 (IL 2) release by T hybridomas, proliferation, IL 2 release, or IL 2 receptor upregulation by a T cell clone. I-A+ L cell transfectants were found to have a stable level of membrane I-A and I-A mRNA, even after exposure to interferon-gamma-containing T cell supernatants. In agreement with earlier reports, a proportional relationship between the (Ia) X (Ag) product and T cell response was found for medium or bright I-A+ cells. However, dull I-A+ cells had a disproportionately low stimulatory capacity, suggesting that there may be a threshold density of Ia per antigen-presenting cell necessary for effective T cell stimulation. Finally, I-A-bearing L cells were shown to trigger low, but reproducible primary allogeneic mixed lymphocyte responses with the use of purified responder T cells, indicating that they are capable of triggering even resting T cells. These studies confirm the importance of antigen processing and I-A density in antigen-presenting cell function, but raise questions about the postulated role of the LFA-1 accessory molecule in T cell-antigen-presenting cell interaction. They also illustrate the utility of the L cell transfection model for analysis and dissection of antigen-presenting cell function.     

Plasma lipoproteins can suppress accessory cell function and consequently suppress lymphocyte activation

The low density classes of plasma lipoproteins (d less than or equal to 1.063 g/ml) suppress mitogenic activation and proliferation of peripheral blood T lymphocytes. Here we demonstrate that lipoprotein suppression can be directed against the accessory cells (greater than 85% monocytes) required for optimal activation of lymphocytes by polyclonal mitogens. Preincubation of accessory cells for 24 h with very low (VLDL) and low (LDL) density lipoproteins suppressed their ability to enhance lymphocyte activation, whereas preincubation of T lymphocytes with lipoproteins did not alter their responsiveness to mitogens. The phenotypic distribution of the accessory cell population was not specifically altered by the lipoproteins, nor did loss of viability account for the suppressive effect of the lipoproteins. Furthermore, the lipoprotein-preincubated accessory cells did not secrete stable inhibitory substances, nor was their ability to produce interleukin 1 diminished. The results of mixing experiments indicate that VLDL-incubated accessory cells had not differentiated into suppressor cells. The lipoprotein-incubated accessory cells appeared to induce the interleukin 2-responsive state in the mitogen-activated lymphocytes, but could not deliver a signal or signals required for the further progression of the activated lymphocytes through the cell cycle. These important findings define at least two types of accessory cell function in the in vitro activation of T lymphocytes.     

Combined RAR alpha- and RXR-specific ligands overcome N-myc-associated retinoid resistance in neuroblastoma cells

Retinoids induce human neuroblastoma cells to undergo growth inhibition and neuritic differentiation in vitro, through interactions with nuclear retinoid receptor proteins. In this study, we found that three different neuroblastoma cell lines exhibited wide variation in their responsiveness to the growth inhibitory effects of the retinoic acid receptor (RAR) agonist, all-trans-retinoic acid (aRA). Resistance to the growth inhibitory effect of aRA correlated with the presence of N-myc gene amplification and not aRA-induced RAR beta levels. Over-expression of N-myc in a neuroblastoma cell line with no endogenous N-myc expression caused a marked reduction in retinoid-induced growth inhibition. Combination of receptor-specific retinoid agonists for RXR and RAR alpha significantly enhanced the sensitivity of N-myc-amplified neuroblastoma cells to the growth inhibitory effects of aRA. Our results indicate that combination receptor-specific retinoid therapy can overcome N-myc-mediated retinoid resistance and may be a more effective chemo-preventive strategy in the disease.     

A single monoclonal anti-Ia antibody inhibits antigen-specific T cell proliferation controlled by distinct Ir genes mapping in different H-2 I subregions

A xenogeneic rat anti-mouse Ia monoclonal antibody, M5/114 (gamma 2b, kappa), was studied for its effects in vitro on T cell proliferative responses. Strain distribution studies revealed that M5/114 could inhibit I-A subregion-restricted T cell responses of the H-2b,d,q,u but not the H-2f,k,s haplotypes, indicating that this xenoantibody recognizes a polymorphic determinant on mouse Ia molecules. This same monoclonal antibody was found to inhibit BALB/c (H-2d) T cell proliferation to both G60A30T10 and G58L38 phi 4. The Ir genes regulating responses to these antigens map to either the I-A subregion (GAT), or the I-A and I-E subregions (GL phi), raising the possibility that M5/114 recognizes both I-A and I-E subregion-encoded Ia glycoproteins. It could be shown, using appropriate F1 responding cells, that M5/114 does in fact affect GAT and GL phi responses by interaction with both the I-A and the I-E subregion products, and not by any nonspecific effect resulting from binding to the I-A subregion product alone. These results are consistent with genetic and biochemical studies directly demonstrating that M5/114 recognizes A alpha A beta and E alpha E beta molecular complexes. The existence of a shared epitope on I-A and I-E subregion products suggests the possibility that these molecules arose by gene duplication. Finally, the precise correlation between the Ia molecules recognized by M5/114 and the ability of this antibody to block T cell responses under Ir gene control strengthens the hypothesis that Ia antigens are Ir gene products.     

Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. III. Ir gene control of lymphocyte transformation correlates with binding of the mitogen to specific Ia-bearing cells

The supernatant from Mycoplasma arthritidis broth cultures (MAS) contains a T cell mitogen that is under Ir gene control. Responsiveness to this mitogen is dictated by the I-E/I-C subregion of the major histocompatibility complex and is dependent upon adherent radioresistant Ia+ accessory cells from responding haplotype animals. In this study, we established that MAS could be removed from culture supernatants by absorption with spleen cells from mice that themselves are responsive to the mitogen (k and d haplotypes), but activity is not removed by spleen cells from mouse strains that are nonresponsive to the mitogen (b, q, and s haplotypes). Absorption studies with lymphoid cells from congenic and recombinant strain mice established that absorption of the mitogen was itself linked to the I-E/I-C subregion of the major histocompatibility complex. Thymocytes from responding haplotype strains were incapable of removing MAS activity, and spleen cells devoid of Thy-1-positive cells retained their full absorbing capacity. The ability to effectively absorb MAS activity was abrogated by the pretreatment of spleen cells with anti-Ia antiserum and complement. Furthermore, the ability of spleen cells from responding haplotype strains to respond to MAS was blocked by the addition of anti-Ia serum to the cell cultures. Whereas the latter treatment resulted in an almost complete elimination of MAS responsiveness, the ability of similarly treated spleen cells to respond to the mitogens PHA and Con A was only minimally depressed. These results are consistent with our hypothesis that the mitogenic moiety of MAS actually binds to I-E/I-C-coded Ia antigens.     

Effects of staphylococcin 1580 on cells and membrane vesicles of Bacillus subtilis W23.

1. Uptake of L-glutamic acid is inhibited, and preaccumulated L-glutamic acid is released from Bacillus subtilis cells treated with staphylococcin 1580. Uptake of alpha-methylglycoside is enhanced at low bacteriocin concentrations and inhibited by excess bacteriocin. 2. Inhibition of amino acid uptake into membrane vesicles is somewhat less sensitive to staphylococcin 1580 than uptake into whole cells under similar conditions, when the bacteriocin concentration is expressed per weight unit of membrane protein. Inhibition of uptake into vesicles is independent of the electron donor system used, but varies with different substrates. 3. Influx of L-glutamic acid into vesicles under anaerobic conditions is severely hampered by staphylococcin 1580. The L-glutamic acid carrier functions are slightly affected only. 4. Staphylococcin 1580 abolished the membrane potential induced by respiration or by a potassium diffusion potential in the presence of valinomycin, as measured with the fluorescent dye 3,3'-dipropylthiadicarbocyanine. 5. The effects of staphylococcin 1580 on cells and membrane vesicles allowed the classification into three groups with different sensitivity to the staphylococcin concentration.