A mouse plasma cell surface antigenic determinant (PLKB) recognized by xenoantiserum. Its relationship to the PC.1 antigenic determinant.

The specificities of the xenoantisera made against mouse myeloma cells have been compared to those recognized by alloantiserum by studying patterns of cytotoxicity on both normal and malignant plasma cells. Goat antiserum obtained by immunization with Balb/c mouse myeloma ADJ-PC-22A cells and purified by in vivo absorption could detect cell surface antigenic determinants present on plasma cells and on cells of liver, kidney, and brain (PLKB antigen), as we had previously reported for a similarly prepared rabbit antiserum. In spite of an apparent similarity between the tissue representation of the PLKB determinant and that of PC.1 antigenic determinants which were detected by DBA/2 anti-ADJ-PC-22A cell alloantiserum, the PLKB antigenic determinant is not identical with the PC.1 antigenic determinant, since the former is found on the tissues of PC.1-negative as well as PC.1-positive strains of mice. However, it was deduced that the PLKB antigenic determinant and the PC.1 antigenic determinant reside in close proximity on the cell surface or maybe even on the same molecule, since Fab fragments of antiserum against either PLKB or PC.1 blocked the cytotoxicity against both antigens. On the other hand, these Fab fragments did not inhibit the cytotoxicity of anti-H-2 antiserum, indicating that neither PLKB nor PC.1 antigenic determinants are in close proximity to H-2 antigens. Association of PLKB and PC.1 determinants was further supported by the finding that the loss of the PLKB determinant in a variant of myeloma MOPC-70A corresponds to the loss of PC.1 determinant on the same cells.     

A surface membrane determinant shared by subpopulations of thymocytes and B lymphocytes.

Utilizing a quantitative fluorescence assay with the fluorescence-activated cell sorter (FACS), we have demonstrated that a rabbit antiserum obtained by immunization with cells of a mouse IgM-producing plasma cell tumor (MOPC104E) is reactive with at least two surface determinants, designated Th-B and ML2, on subpopulations of normal murine lymphocytes. The ML2 determinant is restricted to B lymphocytes. The Th-B determinant is shared by splenic B lymphocytes and a large subpopulation of thymocytes, the latter of which express a 3-fold higher density of Th-B on their surface than do the B lymphocytes. Neither Th-B nor ML2 were found on peripheral T cells or on brain, liver, or kidney cells. The available evidence suggesting that Th-B may be a stem cell determinant that is lost upon maturation is discussed.     

Further studies on the specificity of the N- and C-terminal antigenic determinant of hen egg-white lysozyme.

The specificity of the N- and C-terminal antigenic determinant (P17: sequence Lys1-Cys6-Asn27, Trp123-Cys127-Leu129) of hen egg-white lysozyme (HL) was studied in more detail. In a Scatchard plot of the binding of 14C-acetyl HL with guinea pig purified anti-P17 antibody experimental values bent sharply near r=1. This suggests the presence of two antibody populations with different affinities for HL or possible steric hindrance in the binding of a second HL molecule to the second binding site of the antibody molecule. The antigenic activities of various peptides were tested by measuring their inhibition of the binding of 14C-acetyl-P17 with the anti-P17 antibody. Only P17 and P17t (sequence Lys1-Cys6-Homoser12, Trp123-Cys127-Leu129) were inhibitory, with KI values of 2.0 times 10(4) and 8.1 times 10(3), respectively. These results indicate that the direct binding site of P17 to anti-P17 antibody may be located in the terminal portion of P17 (sequence Lys1-Cys6-Homoser12, Trp123-Cys127-Leu129) while the rest of P17 may be important in maintaining the conformation of this determinant. The single disulphide blood involved in this determinant is essential for manifestation of immunological activity.     

Detection of a cell surface antigen common to a mouse myeloma and a population of mouse thymocytes.

A new antigen termed PTA (plasmocytoma thymus antigen) is described which occurs on 70--80% of mouse thymocytes of all mouse strains tested and on an IgG 2b producing BALB/c plasma cell tumor. PTA is detectable on mouse spleen and lymph node cells. It is absent from liver, brain, bone marrow, and antibody plaque forming cells. PTA is not related to theta antigen and is not detectable on rat thymocytes.     

Functional HLA-DM on the surface of B cells and immature dendritic cells

HLA-DM (DM) plays a critical role in antigen presentation through major histocompatibility complex (MHC) class II molecules. DM functions as a molecular chaperone by keeping class II molecules competent for antigenic peptide loading and serves as an editor by favoring presentation of high-stability peptides. Until now, DM has been thought to exert these activities only in late endosomal/lysosomal compartments of antigen-presenting cells. Here we show that a subset of DM resides at the cell surface of B cells and immature dendritic cells. Surface DM engages in complexes with putatively empty class II molecules and controls presentation of those antigens that rely on loading on the cell surface or in early endosomal recycling compartments. For example, epitopes derived from myelin basic protein that are implicated in the autoimmune disease multiple sclerosis are down-modulated by DM, but are presented in the absence of DM. Thus, this novel concept of functional DM on the surface may be relevant to both protective immune responses and autoimmunity.     

Reduced responsiveness of immature B cells to the B cell mitogen, lipoprotein.

When pre-B cells are isolated from bone marrow and incubated in vitro for several days, they spontaneously differentiate into LPS-reactive B cells. These newly produced B cells do not respond to another B cell mitogen, lipoprotein (Lp). The lack of Lp reactivity of newly produced B cells represents a qualitative difference between these cells and B cells in peripheral tissues that respond equally well to both LPS and Lp. Thus, responsiveness to different mitogens provides unique markers to identify B cells at early stages of development.     

Developmentally regulated expression of the beta 4 integrin on immature mouse thymocytes.

Integrins are a superfamily of alpha beta heterodimers, most of which serve as cell surface receptors for extracellular matrix proteins. In this report, we demonstrate that the recently described alpha 6 beta 4 integrin, previously thought to be limited to epithelial cells and Schwann cells, is expressed on immature mouse thymocytes. The presence of alpha 6 beta 4 is controlled by regulation of beta 4 expression, because alpha 6 was expressed by virtually all cells examined, paired with the beta 1 integrin chain to form VLA-6. During fetal ontogeny, beta 4 was highly expressed by 35% of day-13 thymocytes, 75% of day-14 to -15 thymocytes, then rapidly declined to low levels by birth. In neonates and adults, beta 4 expression was highest on CD4- CD8- CD3- and TCR(+)-gamma delta subsets. Correlation of IL-2R, CD44 and beta 4 on CD4- CD8- thymocytes revealed maximal levels on the intermediate CD44- IL-2R+ subset. Most CD4- CD8+ TCR- thymocytes and a significant fraction of CD4+ CD8+ thymocytes were beta 4lo, whereas the most mature J11d- single positive thymocytes were beta-4. Overall, down-regulation of beta 4 was associated with up-regulation of CD4, CD8, and CD3 in the thymus. alpha 6 beta 4 was undetectable on fetal liver or bone marrow cells, lymphocytes from lymph node, spleen, or blood, and mitogen-activated splenic T cells cultured up to 10 wk with IL-2. The data suggest that alpha 6 beta 4 is up-regulated after pro-T cells enter the thymus and may have a thymus-specific function for T cells. The developmentally regulated pattern of expression and the prominence of alpha 6 beta 4 on day-13 to -16 fetal and adult CD4- CD8- CD3- thymocytes further suggest this unusual integrin may play a role in early T cell development, including stages before acquisition of the TCR.     

Expression of TASK-2 and its upregulation by B cell receptor stimulation in WEHI-231 mouse immature B cells

Stimulation of B cell receptors (BCR ligation) induces apoptosis of immature B cells, which is critical to the elimination of self-reactive clones. In the mouse immature B cell line WEHI-231, the authors previously reported two types of background K+ channels with large (~300 pS, LK(bg)) and medium (~100 pS, MK(bg)) conductance in divalent cation-free conditions. While the authors have recently identified LK(bg) as TREK-2, the molecular nature of MK(bg) is unknown yet. In the present study, the authors found that BCR ligation markedly increased the background K+ conductance of WEHI-231. A single-channel study revealed that MK(bg) activity is increased by BCR ligation and that the biophysical properties (unitary conductance and pH sensitivity) of MK(bg) are consistent with those of TWIK-related acid-sensitive K+ channel 2 (TASK-2). The expression of TASK-2 and its upregulation by BCR ligation were confirmed by RT-PCR and immunoblot assays in WEHI-231. The BCR ligation-induced increase of K+ current was prevented by calcineurin inhibitors (cyclosporine A or FK506), and also by TASK-2-specific small interfering RNA (siRNA) transfection (si-TASK-2). Furthermore, si-TASK-2 attenuated the apoptosis of WEHI-231 caused by BCR ligation. TASK-2 activity and its mRNA were also confirmed in the primary splenic B cells of mouse. Summarizing, the authors report for the first time the expression of TASK-2 in B cells and surmise that the upregulation of TASK-2 by BCR ligation is associated with the apoptosis of immature B cells.     

T cell surface markers expression by immature human thymocytes in in vitro culture: role of Ia+ accessory cells

Previous studies have indicated that the human thymus is composed of several discrete compartments. Cortical thymocytes are reactive with the monoclonal antibody anti-T6, whereas most medullary cells, unreactive with anti-T6, stain brightly with anti-T3 antibody, which defines mature T cell populations. By using an indirect immune rosette method, we isolated the minor thymocyte population (1 to 2% of all thymocytes) lacking both T3 and T6 but expressing T11 antigens. These cells could be maintained in culture supplemented with recombinant IL 2 (Rec-IL 2) for several days. Under these conditions, T3-T6- cells were shown to undergo phenotypic changes. In the absence of thymic macrophage (Mo), T3+ and T8+ thymocytes appeared in culture, whereas the development of T4+ cells strictly required the presence of Mo. The expression of T4 antigen could be largely prevented by the addition of anti-HLA-DR antibody, further indicating that Ia+ accessory cells had the ability to promote in vitro development of T4+ thymocytes. In the presence of Mo, not only T4+ but also T8+ cells were obtained. Double fluorescence staining with anti-T8-FITC and anti-T4-biotin demonstrated that after 12 days of culture, T4 and T8 antigens were mutually exclusive. Furthermore, during the course of these studies, we observed that under the culture conditions utilized (e.g., presence or absence of Mo), T3-T6-thymocytes failed to express the T6 antigen. Thus, the in vitro development of T cells bearing a mature phenotype could be obtained in the absence of intermediate expression of cortical (T6+) thymocytes.     

Further studies on the rabbit erythroid cell plasma membrane transferrin receptor

Studies reproted here indicate that a major cell surface glycoprotein of the rabbit bone marrow erythroid cell may be involved in binding transferrin. The glycoprotein has an apparent molecular weight of 18,000. It is suggested that bone marrow erythroid cells may provide an invaluable source of the red cell membrane transferrin receptor for future studies.     

Functional studies on B cell hybridomas with B cell surface antigens. IV. Direct effects of cytochalasin B on differentiation

We previously established B cell hybridomas between M12.4.1 B lymphoma of BALB/c mice and normal B cell of C57BL/6 (B6) mice. These hybridomas express Iab, Iad, and IgM molecules on the cell membrane, and can induce the generation of IgM secretion when treated with purified goat anti-mouse-mu antibody (anti-mu) without T cell factors. In this study, TH2.54, a subclone of a B cell hybridoma, was treated with cytochalasin B (CB), a fungal product that disrupts microfilaments, and the direct effect of CB on the proliferation and differentiation of TH2.54 was examined. CB considerably suppressed the spontaneous proliferation of hybrid cells. This product, however, did not inhibit the generation of IgM secretion by TH2.54 treated with anti-mu. Surprisingly, CB could directly induce the development of IgM-secreting cells by TH2.54 at a relatively high frequency. Among cytochalasins, dihydrocytochalasin B (H2CB), cytochalasin C (CC), and cytochalasin D (CD) showed marked effects on the induction of IgM secretion as well as CB. In addition, the differentiative effect of CB was greatly inhibited by N6, O2-dibutyryladenosine 3':5'-cyclic monophosphate (dbc-AMP), but not by N2, O2-dibutyrylguanosine 3':5'-cyclic monophosphate (dbc-GMP). Analysis by flow microfluorometry (FMF), cytotoxicity assays, and quantitative absorption tests demonstrated that CB treatment of TH2.54 resulted in a significant decrease in the expression of Iab, Iad, and IgM molecules on the cell membrane. In contrast, parental M12.4.1 neither generated any IgM secretion nor changed Iad expression on the cell membrane under the same conditions. The present study suggests very strongly that microfilament-microtubule systems are not involved in the differentiative process of TH2.54 induced by anti-mu. The results also indicate that CB can provide the initiative signal for differentiation of TH2.54 into the maturation lineage; this is followed by a significant change in the expression of Ia and IgM molecules on the cell membrane.     

An antigenic determinant on human centromere protein B (CENP-B) available for production of human-specific anticentromere antibodies in mouse.

Centromere protein B (CENP-B) is one of the centromere DNA binding proteins constituting centromere heterochromatin throughout the cell cycles. Some components of mammalian centromeres including CENP-B are target antigens for autoimmune disease patients, often those with scleroderma. Recent isolations of CENP-B genes from human and mouse suggested that CENP-B was highly conserved among mammals. From the previous analysis of the reactivity of patient anticentromere sera, two autoepitopes have been located on the DNA binding domain at the amino-terminal region. The amino acid sequences for both the epitopes are perfectly conserved in the two species, human and mouse. In this study, to identify a human-specific antigenic determinant, the remaining two epitopes were further located in separate carboxyl-terminal regions of human CENP-B. Although the amino acid sequence of one epitope is identical to that of the corresponding region in mouse CENP-B, the other has a less homologous sequence. To confirm that the latter epitope was available for production of human-specific anticentromere antibodies, mice were immunized with the recombinant human CENP-B product. One serum that exclusively stained human centromere structure, but not that of other mammals, was identified in the immunofluorescence microscopic observation. The epitope analysis showed that the less conserved one was recognized by this serum. These results suggested that the corresponding region defines the antigenic determinants for the species specificity.     

Preferential infection of immature dendritic cells and B cells by mouse mammary tumor virus

Until now it was thought that the retrovirus mouse mammary tumor virus preferentially infects B cells, which thereafter proliferate and differentiate due to superantigen-mediated T cell help. We describe in this study that dendritic cells are infectable at levels comparable to B cells in the first days after virus injection. Moreover, IgM knockout mice have chronically deleted superantigen-reactive T cells after MMTV injection, indicating that superantigen presentation by dendritic cells is sufficient for T cell deletion. In both subsets initially only few cells were infected, but there was an exponential increase in numbers of infected B cells due to superantigen-mediated T cell help, explaining that at the peak of the response infection is almost exclusively found in B cells. The level of infection in vivo was below 1 in 1000 dendritic cells or B cells. Infection levels in freshly isolated dendritic cells from spleen, Langerhans cells from skin, or bone marrow-derived dendritic cells were compared in an in vitro infection assay. Immature dendritic cells such as Langerhans cells or bone marrow-derived dendritic cells were infected 10- to 30-fold more efficiently than mature splenic dendritic cells. Bone marrow-derived dendritic cells carrying an endogenous mouse mammary tumor virus superantigen were highly efficient at inducing a superantigen response in vivo. These results highlight the importance of professional APC and efficient T cell priming for the establishment of a persistent infection by mouse mammary tumor virus.     

Further studies on amplification of cell-associated immunological memory by secondary antigenic stimulus.

Using the hapten-carrier system in which the dinitrophenyl group (DNP) served as a B cell reactive hapten and bovine serum albumin (BSA) or human gammaglobulin (HGG) as a T cell reactive carrier, changes in the hapten-specific memory (B cell-associated memory) and the carrier-specific memory (T cell-associated memory) after a secondary antigenic stimulus were analyzed in mice. Since an immunological adjuvant was indispensable in the induction of the primary increase in memory, antigen used for the primary antigenic stimulus was injected together with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) which has already been shown to exhibit a potent adjuvant effect. With the cell-transfer technique, it was found that the cell-associated hapten-specific memory for anti-DNP antibody response to DNP-BSA was truly amplified by the secondary injection of DNP-HGG into mice primed with DNP-HGG, and that the cell-associated carrier-specific memory as judged by the helper effect on anti-DNP response to DNP-BSA was also truly amplified by the secondary injection of BSA into mice primed with BSA. However, when memory was assessed in actively immunized mice, the secondary injection of BSA into mice primed with DNP-BSA and HGG decreased anti-DNP responsiveness to the tertiary injection of DNP-BSA, whereas the secondary injection of DNP-HGG secondarily increased anti-DNP responsiveness. In mice primed with DNP-BSA the titers of serum antibodies to BSA increased after the secondary injection of DNP-BSA or BSA. From these results it has been concluded that, like B cell-associated memory, T cell-associated memory is also amplified by a secondary antigenic stimulus, although its expression is inhibited in actively immunized mice through negative control by their antibodies.     

Functional studies on B cell hybridomas with B cell surface antigens. I. Effects of anti-immunoglobulin antibodies on proliferation and differentiation

B cell hybridomas with Ia and IgM molecules on the cell membrane were treated with either purified goat anti-mouse mu antibody (anti-mu) or monoclonal rat anti-mouse IgM antibody (anti-IgM). The spontaneous uptake of [3H] thymidine by these cells was markedly inhibited by both reagents. These hybrid cells could be induced to differentiate into IgM-secreting cells in the presence of these reagents at high frequency. Furthermore, the induction of IgM secretion by B cell hybridomas treated with these antibodies was completely T cell independent, and cell division was not required for the differentiative response to anti-mu. In addition, F(ab')2 fragments of anti-mu showed more effects on proliferation and differentiation of these cells than intact anti-mu. Interestingly, TH2.54, a subline of B cell hybridomas, could generate IgG2a production as well as IgM when incubated with anti-mu. These findings suggest very strongly that the interaction of either goat anti-mu or monoclonal rat anti-IgM with surface IgM molecules on the cell membrane of the B cell hybridomas inhibits in vitro spontaneous proliferation, and results in providing signals for differentiation into Ig-secreting cells without T cell factors.