Obligatory role of gamma interferon in T cell-replacing factor-dependent, antigen-specific murine B cell responses.

The role of gamma interferon (IFN-gamma) in T cell-replacing factor (TRF) activity for antigen-specific plaque-forming cell (PFC) responses in vitro was studied using antibodies to murine IFN-gamma (Mu IFN-gamma). TRF activity was present in supernatants (Sn) of Con A- or mixed leukocyte reaction-stimulated murine spleen cells as well as in an IL-2-rich fraction of phytohemagglutinin-stimulated human peripheral blood lymphocyte Sn and in the Sn of the Gibbon T lymphoma MLA-144. The human TRF was highly active with cells from nu/nu mice and normal mice but not with cells from animals with the xid immunologic defect, similar to the activity of murine TRF. Antibodies to IFN-gamma consisted of hyper-immune rabbit antisera, IFN-gamma affinity-purified rabbit immunoglobulin and an interspecies hybridoma specific for Mu IFN-gamma. The results show that the activities of all preparations of TRF are markedly diminished or abrogated by antibody to Mu IFN-gamma but not by antibodies to human IFN-gamma (Hu IFN-gamma), nor by normal rabbit sera or purified rabbit Ig. The degree of inhibition was dose dependent and was quantitatively reversed by the addition to the cultures of recombinant-derived Mu IFN-gamma (Mu rIFN-gamma) but not Hu rIFN-gamma. This reversal was fully antigen specific and thus not attributable to polyclonal B cell activation by IFN-gamma, which is inactive alone in the TRF assay. Kinetic analysis shows that IFN-gamma must act by 24-48 h to produce PFC responses at 4 d. Together, the data demonstrate that IFN-gamma is a necessary mediator for TRF effects and that IFN-gamma is induced by TRF from T-depleted murine spleen cells in sufficient quantity to support large antibody responses. The source of this IFN-gamma may be the potent natural killer cells that are induced in cultures stimulated with TRF.

Persistent calcium elevation correlates with the induction of surface immunoglobulin-mediated B cell DNA synthesis.

Surface immunoglobulin (sIg)-mediated stimulation of B lymphocytes induces a tyrosine kinase-dependent sequence of events leading to rapid and large elevations in intracellular ionized calcium ([Ca2+]i). These early biochemical events do not necessarily lead to proliferation of B cells, however, and conversely, the absence of or inhibition of these events does not necessarily prevent cellular proliferation. We now show by digital image analysis of single B cells that conditions which lead to B cell proliferation are associated with low-level but persistent sustained or cyclic elevations in [Ca2+]i. In marked contrast, early and nonsustained elevations in [Ca2+]i are induced in B cells by stimuli that lead to G1 transition but fail to progress to DNA synthesis. Thus, when B cells were stimulated with mitogenic and nonmitogenic anti-IgD antibodies, both of which induce entry of cells into G1 and early calcium transients of comparable magnitude, persistent low-level calcium elevations were only detected in cells stimulated with the mitogenic antibody. Furthermore, persistent calcium elevations were also seen when B cells were stimulated with a multivalent dextran-anti-Ig conjugate which induced very high levels of B cell proliferation in the absence of detectable phosphatidylinositol 4,5-biphosphate hydrolysis or elevations in [Ca2+]i as detected by flow cytometry. Finally, B cells from X-linked B cell-defective mice, which do not proliferate in response to anti-Ig antibody, show marked and early increases in [Ca2+]i, but do not show persistent calcium elevations. These data suggest that the rapid and large increases of [Ca2+]i seen in lymphocytes within seconds after antigen receptor ligation may be associated with entry in G1, whereas low-level but persistent elevations may be the hallmark of a cell destined to synthesize DNA.

The isolated major histocompatibility complex class I alpha3 domain binds beta2m and CD8alphaalpha dimers.

The MHC class I molecule plays a crucial role in cytotoxic lymphocyte function. The heavy chain of the MHC class I molecule can form many non-covalent interactions with other molecules on multiple domains and surfaces. We have generated an isolated alpha3 domain of a murine MHC class I molecule and evaluated the contribution of this domain to binding with the MHC class I light chain, beta2m, and CD8. The alpha3 domain binds beta2m at a thousand-fold higher concentration than the whole MHC, and binds CD8alphaalpha with a dependence on the alpha3 CD loop. Our results are relevant for models of MHC folding and CD8-MHC function. The study of individual domains of complex molecules is an important strategy for understanding their dynamic structure and function.

Functional interactions of human and murine lymphoid cells. I. Analysis of primary and secondary responses.

In this study human T-cell responses against murine alloantigens were analyzed. The results show that optimal primary responses are obtained from peripheral blood mononuclear cells only when murine splenic adherent cells (SAC) were used as antigen. Further analysis revealed that human T cells were able to respond directly to murine cells without the need for antigen reprocessing; however, human interleukin 1 (IL-1) was required for optimal stimulation. In contrast, secondary proliferative responses to murine cellular antigens could be induced from primed T cells even in the absence of SAC and/or IL-1. These proliferative responses, and in addition, cytotoxic T-cell responses, were specific for the priming antigen. Long-term human T-cell lines specific for murine alloantigens were found to replace the need for murine T cells in antigen-specific murine B-cell responses to sheep red blood cells. The mechanism of help delivered by the human T cells appeared to be by the release of nonspecific helper-T-cell factors. The evidence presented for this is the inability of these cells to stimulate cells from mice that express the X chromosome B-cell defect xid.

T-cell replacing factor activity of recombinant-derived interleukin-2 requires gamma-interferon.

In this study we show that recombinant-derived human interleukin-2 (rIL-2) has potent T-cell replacing activity in antigen-driven murine antibody responses of T-depleted spleen cells. This T-cell replacing factor (TRF) effect of rIL-2 is antigen specific. By the use of a variety of antibodies to gamma-interferon (IFN-gamma), including antibody from an interspecies hybridoma, we demonstrate that this TRF activity depends upon the production of IFN-gamma in the responding cell cultures, i.e., antibody to IFN-gamma blocks TRF activity. In addition, we show that the cell-to-cell allogeneic helper effect of T cells for antibody responses is similarly inhibited by antibodies to IFN-gamma. The inhibition of response produced by the antibodies is reversed by the addition of excess rIFN-gamma to the cultures. The results demonstrate that IFN-gamma is a necessary intermediate mediator for TRF activity mediated by IL-2 and also by T-cell-mediated help for B cells. Thus, even recombinant-derived lymphokines have complex secondary levels of action on responding cells. Antibodies to individual lymphokines are clearly a powerful resource to analyze the role of soluble factors in cellular responses.

In vitro antibody production.

This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. A generalized system for inducing in vitro antibody production is presented along with a procedure for quantifying the number of antibody-producing cells by plaque-forming cell (PFC) assays: the Cunningham-Szenberg technique and the Jerne-Nordin technique. The assay can be modified as described to measure all classes of antibodies or to enumerate total immunoglobulin-secreting B cells. A protocol for preparing the resting B cells by Percoll gradient centrifugation is also described.

A role for IFN-gamma and NK cells in immune responses to T cell-regulated antigens types 1 and 2.

Responses to antigens that have been previously recognized as T cell-independent are now known to be dependent on some form of T-cell (or T cell-derived) help. This T-cell dependency, however, can be most easily noted when responses to small resting B cells are examined, since responses of larger, size-separated B cells to TNP-Ficoll require little if any T-cell help. The type of ancillary "help" that is required for the responses to type 1 and type 2 antigens exhibits certain similarities as well as differences. Thus, responses to both of these antigens can be stimulated in the presence of L3T4- or L3T4+ cells as well as in the presence of IL2. However, while the presence of IFN-gamma may be significantly inhibitory for the responses to the type 2 antigen TNP-Ficoll, they do not appear to influence the response to the type 1 antigen TNP-BA. Furthermore, while vigorous elimination of NK cell activity from purified B-cell populations enhances the response to TNP-Ficoll, it has no discernible effect on the response to TNP-BA. These results suggest that determination of type 1 and type 2 antigens may be made not only based on the characteristics of the carrier molecule but also on the nature of ancillary help that needs to be provided for these antigens to stimulate optimal responses. Type 1 antigens have polyclonal B-cell activating properties and stimulate responses which are not influenced by IFN-gamma, type 2 antigens have no polyclonal B-cell activating properties and stimulate responses which can be influenced by IFN-gamma.

Surface immunoglobulin crosslinking activates a tyrosine kinase pathway in B cells that is independent of protein kinase C.

It has been found that the principal biochemical pathway activated in B cells stimulated by antigen- or anti-immunoglobulin-mediated crosslinking of surface immunoglobulin is that resulting in hydrolysis of phosphatidylinositol bisphosphate with generation of diacylglycerol and inositol trisphosphate. Recent evidence suggests that surface immunoglobulin-mediated B-cell activation can proceed without detectable increases in the concentration of either diacylglycerol or intracellular Ca2+ concentration, implicating involvement of other non-protein-kinase-C/Ca2(+)-dependent signal-transduction pathways. Therefore, we sought evidence for activation of a signaling pathway that is associated with growth regulation in other cell types--i.e., the protein-tyrosine kinases. We now show that crosslinking of membrane immunoglobulin by mitogenic antibodies leads to rapid tyrosine phosphorylation of several cellular substrates, consistent with the induction of a tyrosine kinase activity. This increase in tyrosine phosphorylation is weakly (if at all) stimulated by other B-cell mitogens, including phorbol esters and ionophores, and does not require the presence of detectable protein kinase C. Furthermore, inhibition of anti-immunoglobulin-stimulated phosphatidylinositol bisphosphate hydrolysis does not inhibit activation of this tyrosine kinase-dependent pathway. These findings suggest that occupancy of the membrane immunoglobulin receptor may induce multiple pathways of activation.

B lymphocyte immunoglobulin receptor desensitization is downstream of tyrosine kinase activation.

Resting mature B cells have two classes of immunoglobulin receptors on their surface, IgD and IgM. Activation of a cell by crosslinking one of these receptors leads to homologous and heterologous receptor anergy as judged by the inability to induce a second calcium signal 2 hr after the initial activation step. The mechanism for this energy is not known. In this report we show that this receptor anergy is downstream of tyrosine kinase activation in that cells pretreated with anti-IgM, when stimulated with anti-IgD, showed tyrosine phosphorylation comparable to that of naive cells, but had no calcium response.

Viral antigens act as helper determinants for antibody responses to cell surface antigens.

Thy-1 alloantigens on murine thymus cells are weak immunogens in vivo for PFC responses in the absence of other antigenic disparities between the donor and recipient. Our previous work showed that non-H-2 alloantigens acted as helper determinants to augment anti-Thy-1 PFC responses. In this report we demonstrate that strong helper antigens are also produced by infection of donor thymus cells with viruses such as HSV-1, NDV, or vaccinia. This helper effect (as much as 30-fold) for a cellular antigen, requires linked recognition (expression of Thy-1 and virus in the same cell membrane), is T-dependent, antigen- (virus) specific, and is Thy-1-specific. The recognition of the viral helper sites is not restricted by the MHC genotype of the thymus cell donor, indicating that host reprocessing of antigen occurs. These are the first results that show that adventitious antigens may function as helper determinants for antibody responses to native membrane antigens and may be the mechanism that initiates several forms of acute post-viral autoimmune disease.

Anti-immunoglobulin stimulation of B lymphocytes activates src-related protein-tyrosine kinases.

Stimulation of resting B lymphocytes with antibodies to surface immunoglobulin (sIgD or sIgM) induces protein tyrosine phosphorylation, implicating one or more B-cell protein-tyrosine kinases (PTKs) in sIg signal transduction. We have evaluated whether members of the src family of PTKs are involved in this process. Our results show that addition of antibodies to IgD or to IgM can stimulate the PTK activity of the blk, fyn, and lyn gene products. Additionally, all three PTKs were found to coimmunoprecipitate with sIg in digitonin lysates from resting B cells. In all stimulatory conditions, whether initiated through sIgD or sIgM, the blk gene product p56blk displayed the strongest activation index. The kinetics of activation of these kinases, particularly that of p56blk, paralleled the early appearance of newly tyrosine-phosphorylated B-cell proteins, suggesting that this group of kinases may account for some portion of the tyrosine kinase activity in sIg-activated B cells. These observations demonstrate a functional and possible physical association between the members of the src family of PTKs and the B-cell antigen receptors.

Naive alloreactive CD8 T cells are activated by purified major histocompatibility complex class I and antigenic peptide.

In this report, we demonstrate stimulation of T cell receptor (TCR) transgenic CD8 T cells by isolated major histocompatibility complex (MHC) class I H-2Ld complexes and antigenic peptide. This is the first demonstration of CD8 T cells activated by MHC and antigenic peptide in the absence of antigen priming. Furthermore, isolated MHC and a potent peptide antigen can stimulate phenotypically naive CD44- T cells to become CTL effectors and to produce interleukin-2 in nanogram per milliliter amounts. These results demonstrate that particular TCR antigen pairs may overcome the need for specialized antigen-presenting cells and have implications for mechanisms of autoimmunity and tolerance induction.

Comparison of tyrosine kinase activation by mitogenic and nonmitogenic anti-IgD antibodies.

Low concentrations of anti-Ig dextran conjugates that stimulate very high levels of B cell proliferation and Ig secretion stimulate no detectable increases in tyrosine phosphorylation. To study this point further, we compared tyrosine phosphorylation patterns induced by mitogenic and nonmitogenic anti-Ig antibodies. Whereas the mitogenic, strongly cross-linking, antibody H delta a/1 induced greater levels of tyrosine phosphorylation than did the nonmitogenic antibody FF1-4D5, the pattern of substrate phosphorylation was equivalent. At lower concentrations of H delta a/1, which were still mitogenic, the degree of phosphorylation that was induced was similar to that induced by high concentrations of FF1-4D5. Both antibodies stimulated comparable increases in the kinase activity of the three src-related kinases present in normal B cells and linked to the IgR, i.e., Blk, Fyn, and Lyn. These results suggest that the extent of tyrosine kinase activation is proportional to mIg cross-linking, that induction of B cell DNA synthesis may require little tyrosine kinase activation, and that activation of tyrosine kinase per se does not necessarily lead to B cell DNA synthesis.

Purified MHC class I and peptide complexes activate naive CD8+ T cells independently of the CD28/B7 and LFA-1/ICAM-1 costimulatory interactions.

T cells play a central role in the initiation, maintenance, and regulation of the immune response. Effector responses of T cells are controlled by complex combinations of lymphokines and adhesion/costimulatory molecule signals. To isolate the effects of specific adhesion/costimulatory molecules and to define the minimal molecular requirements of naive CD8+ T cell activation, we have developed an APC-free system for stimulation of naive CD8+ T cells. In this report, we demonstrate that immobilized MHC class I-peptide complexes can activate naive CD8+ T cells from TCR transgenic mice at low cell densities. The CD8+ T cells were stimulated to proliferate and secrete IL-2 independently of the molecular interactions between CD28/B7.1-B7.2 or LFA-1/ICAM-1 surface receptors. Previous reports have shown that CD28 ligation is necessary for late T cell survival of APC-stimulated naive CD8+ T cells. Our data suggest that under certain specific conditions of high intensity T cell signaling, early activation and late cell proliferation can occur independently of APC-derived costimulatory signals.

Rural telemedicine: a national snapshot.

OBJECTIVES: To estimate the use of telemedicine in rural hospitals in the U.S. and to identify and describe those rural hospitals that are active in telemedicine. MATERIALS AND METHODS: Nationwide mailed survey, with telephone follow-up, to all hospitals not located in a Metropolitan Statistical Area. RESULTS: The overall response rate was 95% of all rural hospitals. Of these, 416 (17.55%) reported having telemedicine, and more than 530 more have plans to begin telemedicine programs during the next few years. Rural hospitals of all sizes and in all regions of the country are initiating telemedicine programs, but there is significant variation by region. Specifically, hospitals located in more populous rural counties near metropolitan areas are less likely to have telemedicine than are hospitals located in less populous rural counties in more remote areas. Conservatively, more than 4000 teleconsults per month are estimated among rural hospitals nationwide in 1995, including all forms of telemedicine. CONCLUSIONS: Telemedicine is becoming an important means of providing specialty medical services in rural areas. This screening survey generated information about the extent of telemedicine use in rural communities, but it also raised many new questions. These questions are being pursued through a detailed follow-up survey.

I-J restriction of T cells that suppress in vivo antibody responses to Thy-1.

The contact-sensitizing haptens dinitrophenyl (DNP) and oxazalone (Ox) act as helper determinants for antibody responses to Thy-1 when conjugated to donor thymus cells. The helper effect is transferrable from primed to naive mice with spleen cells, producing specific augmentation of in vivo PFC responses to Thy-1. The helper cells are hapten-specific and require associative recognition of hapten and Thy-1, excluding a role for nonspecific B cell activation. The phenotype of the helper cells is Thy-1+ and Lyt-1+2-. Antigen-specific suppression could be readily generated by using an inoculum of DNP-modified syngeneic RBC. T cells from these suppressed donors (Ts) were shown to abolish the helper effects of TH in adoptive transfer experiments in vivo. These Ts were characterized as Thy-1+ and Lyt-1-2+. A requirement for MHC compatibility at the I-J subregion was necessary between the Ts and the recipient to obtain a transfer of suppression.

IL4 is not required for proliferative responses in B cells stimulated by anti-IgD-dextran conjugates even under limiting conditions of cell-cell interaction.

The experiments in this manuscript confirm and extend our previous finding that IL4 has minimal enhancing activity on B cell activation stimulated by anti-Ig-dextran conjugates. The absence of significant IL4-mediated enhancement is seen also under conditions which are limiting for optimal B cell proliferation. Thus, even when B cells are cultured at low cell densities where cell to cell contact is minimized and are stimulated with picogram per milliliter concentrations of anti-Ig-dextran, IL4 mediates low levels of enhanced proliferation, if at all. The low level of IL4-induced enhancement does not reflect the anti-Ig-dextran-mediated downregulation of IL4 receptors on B cells, since anti-Ig-dextran stimulates an increase in IL4 receptors similar in magnitude to that stimulated by IL4 by itself. To exclude the possibility that anti-Ig-dextran was stimulating IL4 secretion by B cells and thus masking an effect of added IL4, we added inhibiting concentrations of monoclonal anti-IL4 antibody, with the B cells and found that it was without effect on anti-Ig-dextran-stimulated proliferation. Our results suggest that IL4 may not have a prominent role in influencing B cell growth that is stimulated by multivalent T cell-independent antigens.

Absence of demonstrable phospholipid turnover in B cells stimulated by low mitogenic concentrations of dextran-anti-immunoglobulin conjugates.

Previously we have demonstrated that when anti-immunoglobulin (Ig) is conjugated to high molecular weight dextran (Dex) it stimulates B cell activation at pg/ml concentrations in the absence of detectable phosphoinositide hydrolysis or increases in intracellular ionized calcium. To study carefully whether anti-Ig-Dex recruited a phosphoinositide-dependent pathway of activation, we stimulated B cells that were labeled with 32P and [3H]glycerol with anti-Ig-Dex conjugates at concentrations ranging from 1-1 x 10(-4) micrograms/ml. Thirty seconds to thirty minutes after stimulation lipids were extracted and analyzed by thin layer chromatography and spots correlating with known lipid standards were isolated and counted. There was a four- and tenfold increase in the ratio of 32P/3H incorporated into phosphatidic acid (a metabolite of diacylglycerol) and phosphatidylinositol, respectively, when cells were stimulated with 0.1-1.0 microgram/ml of anti-Ig-Dex for 30 min. Below 1 ng/ml there was no detectable increase in the turnover of these metabolites despite the fact that in parallel cultures B cells were stimulated to proliferate by this concentration of anti-Ig-Dex. To determine whether a cAMP-dependent pathway was recruited by low concentrations of conjugates, we evaluated cAMP levels from B cells that were stimulated with anti-Ig-Dex for 5-60 min using a radioimmunoassay. While cholera toxin stimulated a 50-100-fold increase in the levels of cAMP, we observed no alteration in cAMP in anti-Ig-stimulated cells. These results support and extend our previous findings by demonstrating that B cell activation that is induced by cross-linking of surface Ig may not stimulate phosphoinositide-dependent or cAMP-dependent pathways of activation. Possible alternative mechanisms of activation will be discussed.

Different patterns of inositol polyphosphate production are seen in B lymphocytes after cross-linking of sIg by anti-Ig antibody or by a multivalent anti-Ig antibody dextran conjugate.

Anti-delta antibody conjugated to 2 x 10(6) m.w. dextran (dex) stimulates B lymphocyte proliferation at 10,000-fold lower concentrations than that required by the unconjugated antibody. Dex conjugated antibody also stimulates a greater and more sustained increase in intracellular ionized calcium [( Ca2+]i) than does the unconjugated anti-Ig antibody. Inasmuch as inositol phosphate metabolites have been linked to rises in [Ca2+]i, we analyzed by FPLC the relative amounts of the inositol polyphosphates (IP) in these cells. Anti-Ig-dextran induced a threefold greater increase in total IP than did the unconjugated anti-Ig. Furthermore, in cells stimulated by unconjugated anti-Ig there was a transient induction of I(1,4,5)P3 followed by a rapid accumulation of the I(1,3,4)P3 isomer with little accumulation of I(1,4)P2, whereas in anti-Ig-dex-stimulated cells there was prolonged elevation of I(1,4,5)P3 with more accumulation of I(1,4)P2. In addition, levels of I(1,3,4,5)P4 were maintained over a longer period of time in B cells stimulated by anti-Ig-dex than in those stimulated by unconjugated anti-Ig. The enhanced ratio of I(1,4,5)P3/I(1,3,4)P3 was also seen when suboptimal concentrations of anti-Ig-dex were used which stimulated a level of total inositol phosphate that was similar to that stimulated by the unconjugated anti-Ig. The possibility that the greater stimulation of increased [Ca2+] by anti-Ig-dex than by unconjugated anti-Ig was a predominant factor in influencing the metabolic pathway of I(1,4,5)P3 was excluded. These results show that 1) stimulation of increases in the various IP isomers occurs in anti-Ig stimulated normal B cells as has been shown in B cell lines and 2) that signal transduction and consequent PIP2 hydrolysis that is stimulated by Ag-mediated cross-linking of sIg is strongly influenced by the extent and type of cross-linking that is induced.

Bimodal effect of phorbol ester on B cell activation. Implication for the role of protein kinase C.

The role of protein kinase C PKC in B cell activation is controversial. These studies were undertaken to determine whether protein kinase C has a stimulatory or inhibitory role in B cell activation. We found that treatment of B cells for a short period of time (30 min) with the PKC activator phorbol 12,13-dibutyrate (PDBU) primed the cells for enhanced proliferative responses to anti-immunoglobulin (anti-Ig) antibody whereas treatment for a longer period of time (3 h or more) resulted in suppression of proliferation. The enhanced proliferative response to treatment of B cells with PDBU for short periods of time was associated with inhibition of anti-Ig-stimulated increases in phosphatidyl 4,5-bisphosphate (PIP2) hydrolysis and inhibition of increases in [Ca2+]i, indicating that activation of PKC per se might be sufficient for enhancing B cell activation. The time-dependent effect of phorbol esters on the inhibition of B cell proliferation was found to be closely correlated with the kinetics of disappearance of PKC as measured by Western blot and by enzymatic activity but not with inhibition of [Ca2+]i and PIP2. These data demonstrate a bimodal time-dependent effect of PDBU on B cell activation and suggest that (a) the inhibitory effect of phorbol ester on anti-Ig-induced proliferation may be due to the disappearance of PKC rather than to the inhibition of PIP2 and Ca2+; and (b) the early activation of PKC is a stimulatory rather than an inhibitory signal in the induction of B lymphocyte proliferation by anti-Ig.