Ek Molecules Research Articles

Ligation of Retinoic Acid Receptor α Regulates Negative Selection of Thymocytes by Inhibiting Both DNA Binding of nur77 and Synthesis of Bim

Negative selection refers to the selective deletion of autoreactive thymocytes. Its molecular mechanisms have not been well defined. Previous studies in our laboratory have demonstrated that retinoic acids, physiological ligands for the nuclear retinoid receptors, selectively inhibit TCR-mediated death under in vitro conditions, and the inhibition is mediated via the retinoic acid receptor (RAR) alpha. The present studies were undertaken to investigate whether ligation of RARalpha leads to inhibition of TCR-mediated death in vivo and to identify the molecular mechanisms involved. Three models of TCR-mediated death were studied: anti-CD3-mediated death of thymocytes in wild-type mice, and Ag- and bacterial superantigen-driven thymocyte death in TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c in the context of the E(k) (class II MHC) molecule. Our data demonstrate that the molecular program of both anti-CD3- and Ag-driven, but not that of superantigen-mediated apoptosis involves up-regulation of nur77, an orphan nuclear receptor, and bim, a BH3-only member of the proapoptotic bcl-2 protein family, proteins previously implicated to participate in the negative selection. Ligation of RARalpha by the synthetic agonist CD336 inhibited apoptosis, DNA binding of nur77, and synthesis of bim induced by anti-CD3 or the specific Ag, but had no effect on the superantigen-driven cell death. Our data imply that retinoids are able to inhibit negative selection in vivo as well, and they interfere with multiple steps of the T cell selection signal pathway.

Novel sequential solid-phase synthesis of N-linked glycopeptides from natural sources

In the present report a practical and versatile procedure for the solid-phase synthesis of N-linked glycopeptides from natural sources has been demonstrated. The approach is based on the mild hydrazinolysis procedure to release N-linked oligosaccharides in their intact unreduced form from the natural glycoproteins, e.g. fetuin and ribonuclease B and subsequent formation of the corresponding glycosylamines. Treatment of the reducing sugars 1–7 with a saturated solution of ammonium hydrogen carbonate in either water or dimethyl sulfoxide (DMSO) gives in almost quantitative yields the glycosylamines 8–14. Coupling of the unprotected glycosylamines 8–14 to the side-chain-activated aspartic acid derivative Fmoc-Asp(ODhbt)-OBut 16 affords the N-glycosylated asparagine derivatives 17–23. Subsequent acetylation of the carbohydrate hydroxy groups and cleavage of the tert-Bu ester by trifluoroacetic acid (TFA) treatment yields the glycosylated N-linked building blocks 31–37. The building blocks 31–37 are then incorporated into the multiple-column peptide-synthesis protocol of the glycopeptide T-cell epitope analogues 40–46 of the mouse haemoglobin-derived decapeptide Hb (67–76), VITAFNEGLK. The decapeptide sequence VITAFNEGLK binds well to the MHC Class II Ek molecule and is non-immunogenic in CBA/J mice. Syntheses of several natural and unnatural glycosylations, e.g. N-acetylglucosamine, N,N′-diacetylchitobiose, glucose, maltotriose, maltoheptose and di- and tri-antennary complex oligosaccharides on the decapeptide Hb (67–76) affording the N-linked glycopeptides 40–46 are described. The N-linked glycopeptides 40–46 have been fully characterised by 1D- and 2D-1H and 13C NMR spectroscopy and by ES-MS.

Versatile solid-phase thiolytic reduction of azido and N-Dts groups in the synthesis of haemoglobin (67–76) O-glycopeptides and photoaffinity labelled analogues to study glycan T-cell specificity

E. Meinjohanns, M. Meldal, T. Jensen, O. Werdelin, L. Galli-Stampino, S. Mouritsen and K. Bock, J. Chem. Soc., Perkin Trans. 1, 1997, 871 DOI: 10.1039/A606725E

CD40‐CD40 ligand interactions stimulate B cell antigen processing

The interactions between B cell CD40 and T cell CD40 ligand (CD40L) have been shown recently to play an important role in T cell-dependent activation of B cells. Here, we show that the ligation of CD40 stimulates the processing of antigen by B cells. The activation of an antigen-specific T cell hybrid by B cells co-cultured with insect cells expressing recombinant CD40L or with a CD40-specific monoclonal antibody requires less antigen and fewer B cells compared to control cells. The augmentation was observed both for processing initiated by antigen binding to and cross-linking the surface immunoglobulin, and processing of antigen taken up by fluid-phase pinocytosis. CD40 appears to affect a step in the intracellular processing of antigen, as CD40 has no effect on the presentation of an antigenic peptide which does not require processing. In addition, the CD40-induced augmentation of processing is not attributable to the effect of CD40 ligation on the cell surface expression of B7, LFA-1 or CD23. CD40 ligation does not affect the biosynthesis of the class II EK molecules, and although ligation of CD40 induces B cell proliferation, the augmentation of processing does not require proliferation. The ability of CD40 to stimulate B cell antigen processing has the potential to influence significantly the outcome of antigen-dependent T cell-B cell interactions.

Cross-linking cell surface class II molecules stimulates Ig-mediated B cell antigen processing.

Th cells bind to peptide-class II complexes presented on B cell surfaces. Recent evidence indicates that upon cross-linking, class II molecules transduce signals that modulate a variety of B cell functions. One possible function of class II signaling is to regulate the assembly of processed Ag-class II complexes. Here we show that cross-linking B cell surface class II molecules augments the processing and presentation of an Ek-restricted Ag to a specific T cell hybrid. Significantly, class II cross-linking only affects processing initiated by Ag binding to the surface Ig. The processing of Ag taken up by fluid phase pinocytosis is not affected by class II cross-linking, nor is the presentation of an antigenic peptide that does not require processing. Augmentation of Ag processing is enhanced by treatment of B cells with dibutyryl cAMP, a second messenger in the class II signaling pathway. The cross-linking of class II molecules does not alter the rate or number of Ig molecules internalized or the biosynthesis or expression of Ek molecules. Moreover, changes in the expression of the B7 family of costimulatory molecules or the adhesion molecule LFA-1 (CD11a/CD18) induced by class II cross-linking do not appear to account for the augmentation of processing observed here. Thus, the cross-linking of class II molecules on B cell surfaces selectively stimulates Ig-mediated Ag processing, indicating that a step in this pathway is a target of class II-mediated signaling events.

Requirements of class II-mediated B cell differentiation for class II cross-linking and cyclic AMP.

Cells of the mouse B cell clone, CH12.LX, receive Ag-dependent differentiative signals through their surface membrane class II molecules. The present study was performed to determine the role of class II cross-linking and cAMP in the successful delivery of these signals. Delivery of differentiative signals by anti-Ek mAb was increased by further cross-linking with a secondary anti-isotype antibody. Intact or (Fab')2, but not Fab forms of anti-Ek successfully delivered the Ag-dependent differentiative signal. Inability of monovalent Fab fragments to deliver the signal could not be attributed to an inability to adequately bind Ek molecules. The requirement for cAMP for class II-mediated signaling was also examined, because previous studies have implicated elevated cAMP levels as necessary for class II signaling. Both Ag-dependent, Ek-mediated differentiation and the Ek-mediated inhibition of Ag-independent LPS-induced differentiation were inhibited by the adenyl cyclase inhibitor 2'5'ddA, although elevation of cAMP was not in itself sufficient to deliver the differentiative signal. Inhibition of LPS-induced differentiation could be mediated by mAb binding to either Ek, Abk, or Abb on CH12.LX or an Ab-bearing transfectant, CH12.ABB1. This inhibition was abrogated by 2'5'ddA in the case of Ek or Abb, both of which deliver Ag-dependent differentiative signals to CH12.LX cells. In the case of Abk, which does not deliver such signals to CH12.LX, 2'5'ddA did not abrogate anti-Abk-mediated inhibition of the LPS response. The effects of 2'5'ddA were reversed by the cAMP analog, dibutyryl cAMP, and Ag-dependent-induced differentiation of CH12.LX or CH12.ABB1 was accompanied by an increase in cAMP levels.

Expression of a class II major histocompatibility complex (MHC) heterodimer in a lipid-linked form with enhanced peptide/soluble MHC complex formation at low pH.

A murine class II major histocompatibility complex (MHC) heterodimer, Ek, expressed as a glycan-phosphatidyl inositol-anchored chimera on Chinese Hamster Ovary cells, can present peptides, but not processed antigen to T cells. This chimeric MHC requires a 100-times higher peptide concentration to achieve a two- to four-times lower level of T cell stimulation. Cleavage with phosphatidylinositol-specific phospholipase C and purification result in large quantities of heterodimer in a water-soluble form. Plates coated with this material and then incubated with peptide can efficiently stimulate the appropriate T cell hybridomas. This stimulation is significantly enhanced when peptides are preincubated with the plate-bound MHC molecules in a pH range (5.0-5.5) similar to that of late endosomes. More than half of the soluble Ek molecules can form a specific complex with cytochrome c peptides in this pH range. This suggests that class II MHC molecules undergo distinct conformational changes in endosomal compartments that render them more capable of forming functional complexes with peptide antigens, irrespective of other cell components.

Antibodies specific for Ig idiotype, but not isotype, can substitute for antigen to induce IgM secretion by a B cell clone

Cells of the mouse B cell clone, CH12.LX, secrete IgM when cultured with nominal antigen (sheep erythrocytes, SRBC) and mAbs which bind their membrane Ek molecules. To determine whether anti-Ig antibodies can substitute for antigen in the induction of IgM secretion by CH12.LX, the B cells were cultured with anti-Ek mAbs and anti-IgM or anti-idiotype antibodies. Anti-IgM antibodies were capable of cross-linking the membrane IgM of CH12.LX, and inhibited mitogen-induced differentiation of the B cells. However, anti-IgM could not substitute for SRBC in delivering a major histocompatibility complex-restricted differentiative signal. In contrast, either polyclonal or monoclonal antibodies specific for the CH12.LX Ig idiotype were fully capable of substituting for antigen in the induction of IgM secretion by CH12.LX. The binding of anti-IgM antibodies did not prevent anti-idiotype antibodies from delivering a differentiative signal. Thus, binding of ligand to different parts of the membrane Ig molecule can result in the delivery of different biological signals to the B cell.

Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor

T lymphocytes are predisposed to recognition of foreign protein fragments bound to cell-surface molecules encoded by the major histocompatibility complex (MHC). There is now compelling evidence that this specificity is a consequence of a selection process operating on developing T lymphocytes in the thymus. As a result of this positive selection, thymocytes that express antigen receptors with a threshold affinity for self MHC-encoded glycoproteins preferentially emigrate from the thymus and seed peripheral lymphoid organs. The specificity for both foreign antigen and MHC molecules is imparted by the alpha and beta chains of the T-cell antigen receptor (TCR). Two other T-cell surface proteins, CD4 and CD8, which bind non-polymorphic regions of class II and class I MHC molecules respectively, are also involved in these recognition events and play an integral role in thymic selection. In order to elucidate the developmental pathways of class II MHC-restricted T cells in relation to these essential accessory molecules, we have produced TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c and the Ek (class II MHC) molecule. The transgenic TCR is expressed on virtually all T cells in mice expressing Ek. The thymuses of these mice contain an abnormally high percentage of mature CD4+CD8- cells. In addition, the peripheral T-cell population is almost exclusively CD4+, demonstrating that the MHC specificity of the TCR determines the phenotype of T cells during selection in the thymus.

Haplotype-specific differences in signaling by transfected class II molecules to a Ly-1+ B-cell clone.

CH12.LX B cells are responsive to antigen-dependent differentiative signals transmitted through their surface Ek molecules. Although CH12.LX cells express surface Ak molecules with normal protein sequence, the Ak molecules do not deliver differentiative signal to these B cells. To determine whether introduction of a new A molecule into CH12.LX cells would correct this deficiency, CH12.LX cells were transfected with the genes encoding new Aalpha and/or Abeta molecules. It was found that transfected cells responded to antigen-specific signals delivered via the Ealphak Ebetak, Aalphab Abetab, or Aalphak Abetab molecule. However, the B cells did not respond to signals generated by the molecule Aalpha kAbetak, Aalphab Abetak, Aalphak Abetad, or Aalpha kAbetau. Comparison of these sequences suggests that two Abeta residues, His-47 and Trp-197, are important to the transmission of differentiative signals to B cells.

Signaling to a B-cell clone by Ek, but not Ak, does not reflect alteration of A k genes

The mouse B-cell clone, CH12.LX (Iak, Ly-1+, mu+, delta+), can be induced to differentiate and secrete antibody in an antigen-specific, H-2-restricted manner. Induction requires two signals. One must be provided by the binding of specific antigen to the membrane IgM; the other is delivered by the binding of Ek-specific T-cell hybridomas to the Ek molecules of CH12.LX (Bishop and Haughton 1986). Previous studies demonstrated that Ek-specific monoclonal antibodies (mAbs) could substitute for T cells in delivering the second differentiative signal (Bishop and Haughton 1986). Although CH12.LX cells present Ak to Ak-restricted or alloreactive T-helper cells, neither T cells nor mAbs specific for Ak induce differentiation (Bishop and Haughton 1986). However, since the Akspecific mAbs tested previously were beta-chain-specific and the Ia epitope specificity of the T cells used was unknown, it is possible that the differentiative signal delivered to the CH12.LX class II molecule is chain-specific. Here we report the effects of ten additional Iak-specific mAbs upon the differentiation of CH12.LX. In addition, a cDNA library was prepared from CH12.LX cells, clones corresponding to the alpha and beta chains of the Ak molecule were isolated, and their nucleotide sequences were determined. Finally, the Ak and Ek molecules of CH12.LX and H-2k spleen cells were compared by two-dimensional gel electrophoresis to examine possible post-translational differences in the Iak molecules of CH12.LX.

Antigen presentation by liposomes: inhibition with antibodies.

The involvement of both antigen and class II major histocompatibility complex (MHC) molecules in T cell activation by liposome-bound antigens was investigated. We used a pigeon cytochrome c (PCC)-specific Ek-restricted T cell hybridoma that can be activated to produce interleukin 2 by liposomes carrying either PCC and Ek molecules, or a high concentration of PCC alone. We demonstrated that the MHC-restricted response of this hybridoma to liposomes is specifically blocked by both anti-MHC and anti-PCC monoclonal antibodies, whereas unrestricted activation is only inhibited by PCC-specific antibody.

Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). IV. Distinct H-2-linked Ir genes control antibody responses to different HBsAg determinants on the same molecule and map to the I-A and I-C subregions.

We have previously demonstrated that the murine humoral immune responses to the group-specific a and subtype-specific d/y determinants of hepatitis B surface antigen (HBsAg) are controlled by H-2-linked immune response (Ir) genes. High responder (H-2d,q), intermediate responder (H-2a greater than b greater than k) and nonresponder (H-2f,s) haplotypes have been identified (8, 9). The kinetics and specificity of in vivo antibody production after HBsAg immunization in congeneic, H-2-recombinant strains was analyzed to further define relevant Ir genes and their influence on the immune response to distinct antigenic determinants. These studies indicate that the humoral anti-HBs response is regulated by at least two Ir genes, one in the I-A subregion (Ir-HBs-1) and one in the I-C subregion (Ir-HBs-2) of the murine H-2 complex. Ir-HBs-1 regulates the primary responses to all HBsAg determinants, whereas the influence of Ir-HBs-2 is determinant specific, affecting the responses to the d or y determinants. The anti-a response is regulated exclusively by Ir-HBs-1. Strains possessing only the Ir-HBs-2 gene [B10.S(9R) and B10.HTT] produce no anti-a response and a subtype-specific antibody response is detected only after secondary or tertiary immunization. In contrast, the influence of Ir-HBs-2 in the presence of Ir-HBs-1 is detected upon primary immunization and is additive rather than exclusive. There is also suggestive evidence that the presence of the Ek molecule, at least in the context of I-Ak, may have a suppressive influence on the anti-HBs response. Additionally, HBsAg-specific, T cell proliferative responses were H-2 restricted and the kinetics and specificity of T cell proliferative responses paralleled in vivo antibody production. These data indicate that, although the I-A subregion exerts a dominant influence, distinct Ir-HBs genes, mapping in separate I subregions, control immune responses to alternate HBsAg determinants on the same protein molecule.

Cross-reactivity of suppressor T cells specific for lactate dehydrogenase B and IgG2a myeloma protein.

The major histocompatibility complex-controlled immune responsiveness of mice to two unrelated antigens, lactate dehydrogenase B (LDHB) and IgG2a myeloma protein is remarkably similar (1, 2). The similarity is explained by our recent work, demonstrating that both antigens, when presented in the context of cell surface Ek (Ek alpha Ek beta) molecules, generate strong suppression that causes nonresponsiveness to all mouse strains expressing this molecule. The suppression is mediated by antigen-specific, Ek-restricted Lyt-1+2+ suppressor T (Ts) cells, which act by inhibiting the proliferation of A(A alpha A beta)-restricted, Lyt-1+2- (possibly T helper [Th]) cells specific for the same antigen (3-5). However, the question of why the Ek molecule, in combination with two structurally unrelated proteins, preferentially induces Ts cells has remained unanswered. We demonstrate here that the anti-LDHB and anti-IgG2a Ts cells are fully cross-reactive, which indicates that the LDHB + Ek and IgG2a + Ek determinants recognized by these cells are very similar or identical.

Haplotype-specific suppression of T cell response to lactate dehydrogenase B in (responder x nonresponder)F1 mice.

Mouse strains that express the Ek (Ek beta E-1k alpha) molecule are nonresponders (NR) to the enzyme lactate dehydrogenase B (LDHB) in terms of T cell proliferation. Nonresponsiveness is caused by T suppressor (Ts) cells recognizing LDHB in the context of Ek molecules on the antigen-presenting cells. The data presented here demonstrate that the Ek-restricted Ts cells function in (R x NR)F1 mice in a remarkable haplotype-specific fashion: they selectively interfere with the Ak (ANR)-restricted response, and do not affect the response channeled through the A molecules of the responder parent. This haplotype-specificity of suppression provides an explanation of the dominance of responsiveness in (R x NR)F1 mice.

H-2-controlled suppression of T cell response to lactate dehydrogenase B. Characterization of the lactate dehydrogenase B suppressor pathway.

We characterized the cell types involved in the H-2-controlled suppression of T cell response to lactate dehydrogenase B (LDHB). The suppressor effector (Tse) was found to be an Lyt-1+2+, J+ cell that recognizes antigen together with Ek molecules of antigen-presenting cells (APC). To become functional, the Tse cell requires a second signal from a nonspecific, Lyt-1+2-, J+ suppressor-inducer (Tsi) cell. The Tsi-Tse interaction is not subject to any genetic restriction. The target cell of suppression is an Lyt-1+2-, J- (most likely T helper [Th]) cell that recognizes LDHB in the context of A molecules on APC. The suppression is manifested in inhibition of the antigen-specific, A-restricted proliferation of Th cells. The interaction between Tse and Th is restricted by the A region of the H-2 complex. Because this restriction is determined by the receptor of Th cells, the mechanism of Th-Tse interaction most likely involves a concomitant recognition of LDHB and A region-controlled molecules by Th cells on the surface of Tse cells.

Role of the Ek molecule in the generation of suppressor T cells in response to LDHB.

The role of the Ek (E alpha kE beta k) molecule in the generation of suppressor T (Ts) cells specific for lactate dehydrogenase B (LDHB) was studied using different approaches. First, lymph node cells from LDHB-primed B10.A(2R) (AkEk) nonresponder mice were shown to suppress the LDHB-specific and Ak-restricted proliferative response of T cells from the congenic responder strain B10.A(4R), which does not express E molecules (AkEo). Similarly, lymph node cells from primed CBA (AkEk) mice suppressed the anti-LDHB response of Lyt-1+Lyt-2-T cells (depleted of Lyt-2-bearing Ts cells) from the same mice. Second, in vitro priming of 2R (AkEk) T cells with LDHB-pulsed 4R (AkEo) antigen-presenting cells (APC) generated T-cell proliferation but not suppression. Third, nonresponder 2R mice were turned into responders by injecting them with LDHB-pulsed 4R APC or monoclonal Ia.m7 antibody that blocks the Ek molecule. The data demonstrate that expression of Ek molecules by the APC is necessary to generate LDHB-specific Ts cells, which in turn prevent the proliferation of Lyt-1+Lyt-2- (probably helper) cells recognizing the same antigen in the context of the Ak molecule.

Cell-specific drug transfer from liposomes bearing monoclonal antibodies.

Liposome-encapsulated drugs and fluorescent markers have been proposed as useful tools in studies of cell biology and in chemotherapy1–3. Until recently, this approach has been limited by the lack of means to fix protein ligands, such as antibodies, to liposome surfaces where they can mediate specific interactions with cells. Our newly developed technique4 for the covalent coupling of monoclonal antibodies to small, sonicated liposomes was used to study the specific delivery of methotrexate (MTX) to cellular targets in vitro. Liposomes containing MTX and carboxyfluorescein (CF)5–7 were coupled to monoclonal antibodies against determinants of molecules encoded by the major histocompatibility complex of the murine H–2k haplotype. These were incubated with lipopolysaccharide (LPS)-induced blast cells from CBA (H–2k), or C57BL/6 (B6) (H–2b) spleens. Only spleen cells from the CBA strain showed evidence of drug effect, as indicated by inhibition of radiolabelled deoxyuridine incorporation, demonstrating that the interaction of the liposomes with the cells was specific. With regard to the different determinants, the effect of the liposome-encapsulated MTX was not correlated with the number of liposomes bound to cells. More lipsomes became bound to the H–2Kk molecule than to the H–2 I–Ek molecule, but liposomes bound to the latter were more effective for drug delivery. An endocytic pathway for the liposome-encapsulated drug is suggested by the fact that the effect of the drug in liposomes, but not in free form, was inhibited by the lysosomotropic amine, NH4CI. The techniques described here provide a simple quantitative assay for the ‘endocytic potential’ of any cell-surface determinant for which a ligand, such as an antibody or hormone, is available.