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Although self peptides bound to HLA-DQ and, especially, HLA-DR allotypes have been described in some detail, few ligands that bind to HLA-DP have been identified. Toward this aim, naturally processed peptides were isolated from immunoaffinity-purified HLA-DP2 molecules expressed in cultured B lymphocytes. The size distribution of the peptide repertoire is generally similar to those reported for self peptides bound to HLA-DR and HLA-DQ molecules. Twelve peptides representing individual sequences including two nested sets were sequenced by mass spectrometry and/or N-terminal Edman analysis. Source proteins included MHC molecules and other integral membrane proteins as well as secretory and serum proteins. No dominant amino acid markers suggestive of particular enzymatic processing events were detected. Peptide specificity and affinity were examined in binding assays using synthetic peptides and purified HLA-DP and HLA-DR molecules. Anchor residues were tentatively assigned using alanine-substituted analogues of two self peptides. Some structural features of HLA-DP2 that may relate to peptide binding are considered.

The proximal promoters of all MHC class II genes contain a sequence element, the 19-bp X box, that is conserved in both sequence and position. Extensive analysis using a wide variety of approaches has demonstrated that the integrity of the X box is essential for transcription initiation from all class II genes studied. However, the X box is now recognized to contain two subregions, termed X1 and X2. Radiolabeled oligonucleotides corresponding to the X2 box of the MHC class II genes DPA and DQB were used to screen B cell and T cell expression libraries. A novel cDNA, termed XBR (X box repressor), encoding a putative zinc finger protein that binds specifically to the DPA X2 box was isolated from a human T cell line. The XBR gene encodes a 7-kb message that is ubiquitously transcribed, although at higher levels in tissues of the lymphocytic compartment. Southern blots indicate that this gene is single copy in primates and contains regions that are highly divergent in other species. Overexpression of XBR in a B cell line resulted in a dramatic reduction of transcription from a reporter gene construct driven by the DPA promoter, but not from similar constructs with mutations in the X2 box. Similarly, overexpression of XBR reduced induction of reporter gene activity driven from the DPA promoter in HeLa cells treated with IFN-gamma. XBR may, therefore, mediate transcriptional repression, thus preventing inappropriate MHC class II expression. XBR function may in part explain the dominant trans-acting repression of MHC class II expression reported in cell fusion experiments.

Mutational studies indicate that the superantigen staphylococcal enterotoxin A (SEA) has two separate binding sites for major histocompatibility complex (MHC) class II molecules. Direct evidence is provided here for the formation of SEA-MHC class II trimers in solution. Isoelectric focusing separated SEA-HLA-DR1 complexes into both dimers and HLA-DR1.SEA2 trimers. The molar ratio of components was determined by dual isotope labeling. The SEA mutant SEA-F47S, L48S, Y92A, which is deficient in MHC class II alpha-chain binding, formed only dimers with HLA-DR1, whereas a second SEA mutant, SEA-H225A, which lacks high-affinity MHC class II beta-chain binding was incapable of forming any complexes. Thus SEA binding to its MHC receptor is a two-step process involving initial beta-chain binding followed by cooperative binding of a second SEA molecule to the class II alpha chain.

A previously unrecognized element, located downstream of the start site of transcription in the first exon of the DR alpha gene, has been defined that enhances promoter activity up to eightfold in a position-dependent manner. Mutations in this DNA-binding site abolished binding of a nuclear factor in human B cell nuclear extract and decreased the activity of the DR alpha promoter to a basal level. Significant sequence homology of this element was found in the DNA of the DR beta, DP alpha and -beta, and DQ alpha genes, always located downstream of the transcriptional start site. The nuclear factor binds to the DR alpha and DP alpha element but not to the element in the DQ alpha gene. It was identified as NF-E1 (YY1). This protein, previously identified by its binding to the Ig kappa 3' enhancer and the Ig heavy chain mu E1 site, thus also appears to be quite important in the regulation of MHC class II gene expression.

The immune response to viruses partially depends on the biochemical interaction between viral peptides and histocompatibility molecules. In this study, the refolding of recombinant HLA-A*0201 heavy chain and beta 2-microglobulin (beta 2-m) in the presence of peptides from influenza B nucleoprotein (BNP), influenza A matrix protein, and HIV gp120 and their analogues was examined. The plateau value for the amount of refolded complex with three peptides, a 10-mer BNP 85-94 (A86) with alanine substituted for leucine at the P2 anchor residue and two BNP 8-mers, was significantly lower than the native peptide epitope BNP 85-94 or with other peptides tested. To attempt to understand the basis for the lower yield of complex, equilibrium dissociation constants (KdS) for the two 10-mers, BNP 85-94 (A86) and BNP 85-94, were determined from association and dissociation rates and from Scatchard plots, all measured at 10 degrees C. In addition, dissociation rates were measured at 0 degrees, 26 degrees, and 37 degrees C. Although the kinetics were similar at 0 degrees and 10 degrees, at 37 degrees these two complexes had distinct rates of dissociation, resulting in relatively stable or unstable complexes. The behavior of the unstable complexes paralleled the behavior of empty complexes described in vivo; they are unstable at physiologic temperature, produced in low yield, and stabilized by low temperature. Comparison of all of the kinetic data suggests that the equilibrium amounts of the two HLA/peptide complexes (plateau values) result from distinct reaction pathways, i.e., that the molecules that form stable complexes may undergo an additional reaction to those that form unstable complexes.

The superantigen staphylococcal enterotoxin A (SEA) binds to major histocompatibility complex (MHC) class II molecules at two sites on either side of the peptide groove. Two separate but cooperative interactions to the human class II molecule HLA-DR1 were detected. The first high affinity interaction to the DR1 beta chain is mediated by a zinc atom coordinated by H187, H225, and D227 in SEA and H81 in the polymorphic DR1 beta chain. The second low affinity site is to the DR1 alpha chain analogous to SEB binding and is mediated by residue F47 in SEA. Binding of one SEA to the DR1 beta chain enhances the binding of a second SEA molecule to the DR1 alpha chain. The zinc site is on the opposite side of the SEA molecule from residue F47 so that one SEA molecule can readily bind two class II molecules. Both binding sites on SEA are required for maximal activity. Thus, unlike, SEB, SEA requires two separate binding sites for optimal activity, which may allow it to stabilize SEA interaction with T cell receptors, as well as to activate the antigen-presenting cell by cross-linking MHC class II.

CD43, CD44, CD16, and L-selectin have been previously shown to be enzymatically cleaved from stimulated leukocytes. However, little is known about the enzymes involved in these processes. Here, metalloprotease(s) inhibitable by 1,10-phenanthroline together with serine protease(s) inhibitable by N alpha-p-tosyl-L-lysine chloromethyl ketone and 3,4-dichloroisocoumarin are shown to be involved in the cleavage of CD43, CD44, and CD16 but not in the cleavage of L-selectin on granulocytes. In addition, mAbs that recognize these individual receptors and induce their specific cleavage did not initiate cleavage of the others. In one case only, L-selectin, cleavage was also triggered by mAbs interacting with CD16 (the low affinity Fc gamma R). Thus, this mechanism represents a novel pathway of L-selectin cleavage induction.

By fluorescence resonance energy transfer, we have previously demonstrated that upon anti-CD3 mAb-mediated activation of a murine T cell hybridoma expressing human CD4, CD4 moves into close association with the TCR/CD3 complex. It was shown that this association between CD4 and the TCR/CD3 complex was dependent upon the presence of an intact CD4 cytoplasmic domain. We have now expressed, in a murine T cell hybridoma, mutated forms of CD4 containing cysteine to serine point mutations at positions 420, 422, or 430. The mutations at positions 420 and 422, but not 430, abolish association with p56lck. By using fluorescence resonance energy transfer, we demonstrate that mutations of CD4 which fail to interact with p56lck are unable to associate with the TCR/CD3 complex under conditions in which wild-type CD4 and the 430 mutant CD4 do associate with the TCR/CD3 complex. In addition, these mutants have a diminished response to CD4-dependent stimuli. We conclude that the association between CD4 and the TCR/CD3 complex during T cell activation plays an important role in CD4-dependent responsiveness and this association requires the interaction of CD4 with p56lck. These results also suggest that a substrate for p56lck may be expressed in the TCR/CD3 complex.

A highly malignant human T-cell leukemia was identified by cell surface analysis as a member of the T-cell receptor (TCR) gamma delta lineage. Cytogenetic and molecular analysis showed a novel t(8;14)(q24;q11) rearrangement involving the J delta 1 gene segment on chromosome 14 and the distal end of chromosome 8 near the c-myc proto-oncogene locus. The gamma delta TCR of the leukemia blasts was functionally intact and could be activated to generate intracellular calcium flux and to target Fc receptor-mediated redirected tumor cell lysis. In addition, non- major histocompatibility complex restricted lysis of a limited target cell panel was shown by fresh leukemic blasts and by the in vitro- maintained leukemia cells that was comparable to known T-cell lines with natural killer-like activity. These data suggest that the T-cell leukemia potentially had in vivo functional cytolytic activity. However, whether this activity did contribute to the patient's clinical condition could not be determined.

CD14, expressed on the surface of monocytes as a phospholipid-linked protein, is a receptor for serum LPS binding protein/LPS complex. It was specifically down-modulated after stimulation of monocytes by physiologic activating/differentiating agents such as bacterial LPS and IFN-gamma, by the pharmacologic agents PMA and calcium ionophore A23187, and by anti-CD14 antibodies. The down-modulation was almost totally blocked at 4 degrees C or at pH 4.5 and markedly inhibited by the protease inhibitors diisopropylfluorophosphate and PMSF. A soluble labeled CD14 was isolated from culture supernatant of surface iodinated monocytes after their activation, indicating that CD14 is shed from the cell surface rather than internalized. The size of the soluble CD14 shed from the monocytes in vitro was smaller than that of either the membrane-bound form or a soluble CD14 cleaved from the cell surface by phosphatidyl inositol-specific phospholipase C, but identical to the size of one of the two major soluble CD14 forms normally found in human serum. These data suggest that CD14 shedding induced by monocyte stimulation may play an important role in the regulation of surface CD14 expression.

The germline repertoire of variable genes for the TCR-gamma/delta is limited. This, together with the availability of several V delta-specific and a C delta-specific mAbs, has made it possible to assess differences in the TCR-gamma/delta repertoire in man. TCR-gamma/delta cells expressing particular V gene segments have been previously shown to be localized in different anatomical sites. In this study, analysis of TCR-gamma/delta V gene segment usage performed on subjects from the time of birth through adulthood revealed striking age-related changes in the TCR-gamma/delta repertoire in peripheral blood. V delta 1+ gamma/delta T cells predominated in thymus as well as in peripheral blood at birth and then persisted as a relatively constant proportion of CD3+ PBL. However, V delta 2+ gamma/delta T cells that constitute a small proportion of the CD3+ cells in thymus and in peripheral blood at birth, then expand and account for the major population of gamma/delta T cells in PBL in adults. No parallel postnatal expansion of V delta 2+ cells in the thymus was observed, even when paired thymus-peripheral blood specimens were obtained on subjects between the ages of 3 d and 8 yr. The subset of V delta 2+ lymphocytes that was expanded in peripheral blood expressed high levels of CD45RO suggesting prior activation of these cells, consistent with the possibility that their expansion might have resulted from exposure to foreign antigens or superantigens. In contrast, V delta 1+ T cells in PBL showed no comparable increase in relative numbers and were either negative or expressed only low levels of CD45RO. Consistent with evidence for extrathymic peripheral expansion of selective TCR-gamma/delta subsets, no link between MHC haplotype and differences in the TCR-gamma/delta V gene usage between individuals was apparent, and identical twins displayed TCR-gamma/delta variable gene segment phenotypes that were strikingly different from one another. The elements that determine the TCR-gamma/delta repertoire in individuals are not known. It is possible that both thymic selection and extrathymic factors may influence the peripheral repertoire. Recently, TCR-gamma/delta+ lymphocytes have been shown to expand markedly in peripheral lymphoid tissues and infectious lesions in response to mycobacterial antigens, and a correlation between mycobacterial responses and TCR-gamma/delta V gene usage has been shown in mice. The data presented here demonstrated peripheral age-related changes in the gamma/delta repertoire and point to the importance of extrathymic expansion of specific gamma/delta subsets in generating the human TCR-gamma/delta repertoire.

Polypeptide constructs containing up to four copies of the T cell epitope 306-318 of influenza virus haemagglutinin have been synthesized on solid phase. Between the copies, a non-natural PEG-based spacer amino acid has been introduced. The oligomeric epitopes were analysed by RP-HPLC and ES-MS. The arrangement of the epitopes within the peptide constructs was either linear or comb-like. The proliferative response in a T helper cell assay induced by these oligomerized epitopes has been tested, showing that the linearly arranged epitopes are more effective than the comb-like oligomers.

In a previous study we reported that oligomerized T cell epitopes "superactivated" CD4+ T cells. These oligomers, consisting of 12-16 copies of a peptide epitope derived from the hemagglutinin protein of influenza virus (HA306-318), induced a specific T cell response in amounts as little as 5 pg/ml. We now show that the improved antigenicity of these multimerized epitopes can also be utilized to induce "high zone tolerance". Tolerization, similar to activation, occurred at about 3 logs lower concentration of oligomer than of peptide. HA306-318-specific T cell cultures became nonresponsive to stimulation with peptide after incubation with 0.5-5 microg/ml HA306-318 12-mer. The nonresponsiveness was accompanied by a drastic down-regulation of the TCR and by T cell elimination by apoptotic cell death. In contrast, stimulation with peptide even at 50 microg/ml led to temporary induction of anergy. Consequently, induction of tolerance with the oligomer was permanent and no recovery of the cultures was seen in recall experiments 12-14 days after high zone exposure to the 12-mer.

Killer cell immunoglobulin-like receptors (KIR) bind HLA class I proteins in an allele- and locus-specific manner. This report describes the use of transfectants expressing recombinant chimeric proteins, comprising the extracellular portions of KIR molecules and the transmembrane and cytoplasmic tails of CD3-zeta, to create an in vitro system in which signaling is readily measured and that preserves the specificity of the KIR / HLA-C interaction. The identity of the amino acid residues on the KIR molecule important for binding to the HLA protein is not well understood; although some KIR2D residues involved in HLA-C recognition have been identified, their relative importance and whether other amino acids contribute to binding was unclear. This novel system was used to study, by site-directed mutagenesis, the role of various amino acids in KIR binding to HLA-C ligand. The data presented here show that while multiple polymorphic residues contribute to the HLA-C binding site on KIR proteins, two clusters of polymorphic residues define the group allotype specificity of HLA-C binding to a KIR2D molecule.

NK T cells are a T cell subset in the human that express an invariant alpha-chain (V alpha 24invt T cells). Because of the well-described immunomodulation by glucocorticoids on activation-induced cell death (AICD), the effects of dexamethasone and anti-CD3 stimulation on V alpha 24invt T cell clones and CD4+ T cell clones were investigated. Dexamethasone significantly enhanced anti-CD3-mediated proliferation of V alpha 24invt T cells, whereas CD4+ T cells were inhibited. Addition of neutralizing IL-2 Ab partially abrogated dexamethasone-induced potentiation of V alpha 24invt T cell proliferation, indicating a role for autocrine IL-2 production in corticosteroid-mediated proliferative augmentation. Dexamethasone treatment of anti-CD3-stimulated V alpha 24invt T cells did not synergize with anti-Fas blockade in enhancing proliferation or preventing AICD. The V alpha 24invt T cell response to dexamethasone was dependent on the TCR signal strength. In the presence of dexamethasone, lower doses of anti-CD3 inhibited proliferation of V alpha 24invt T cells and CD4+ T cells; at higher doses of anti-CD3, which caused inhibition of CD4+ T cells, the V alpha 24invt T cell clones proliferated and were rescued from AICD. These results demonstrate significant differences in TCR signal strength required between V alpha 24invt T cells and CD4+ cells, and suggest important immunomodulatory consequences for endogenous and exogenous corticosteroids in immune responses.

W6/32 is one of the most common monoclonal antibodies (mAb) used to characterize human class I major histocompatibility complex (MHC) molecules. It recognizes a conformational epitope on the intact MHC molecule containing both beta2-microglobulin (beta2-m) and the heavy chain. Labelling proteins by biotinylation is a very useful technique of for their detection, purification and analysis. A common method for biotinylating proteins is through the use of N-hydroxysuccinimide (NHS) biotin or Sulfo-NHS-biotin where the free amino groups on the protein are used for coupling the biotin moiety. However, W6/32 was unable to effectively immunoprecipitate biotinylated human class I MHC molecules including the human non-classical HLA-G molecule. FACScan analysis confirmed that biotinylating human class I MHC and HLA-G molecules prevents the recognition of these molecule by W6/32. In contrast, the recognition by another conformation-dependent monoclonal antibody, ME1, specific to HLA-B27 molecules, remained totally unaffected.

Copolymer 1 (Cop 1, poly (Y, E, A, K)) is a random synthetic amino acid copolymer effective in the treatment of relapsing forms of multiple sclerosis (MS). Cop 1 binds promiscuously, with high affinity and in a peptide-specific manner to purified MS-associated HLA-DR2 (DRB1*1501) and rheumatoid arthritis-associated HLA-DR1 (DRB1*0101) or HLA-DR4 (DRB1*0401) molecules. In the present work at least 95% of added Cop 1 could be bound to recombinant "empty" HLA-DR1 and -DR4, and 80% could be bound to HLA-DR2 proteins. Amino acid composition, HPLC profiles, and sequencing patterns of Cop 1 eluted by acid extraction from HLA-DR molecules were similar to those of the unseparated Cop 1. Protruding N-terminal ends of Cop 1 bound to HLA-DR1, -DR2, or -DR4 molecules were then treated with aminopeptidase I, followed by elution, HPLC, and pool sequencing. In contrast to untreated or unbound Cop 1, this material exhibited distinct motifs at some positions with increases in levels of E at the first and second cycles, of K at the second and third cycles, and of Y (presumably at P1 of the bound peptide) at the third to fifth cycles, regardless of the HLA-DR molecule employed. No preference was seen at the following cycles that were mainly A. These first pooled HLA-DR binding epitopes provide clues to the components of Cop 1 that are biologically active in suppressing MS and possibly rheumatoid arthritis.

The TAL1 gene is disrupted by translocation or deletion (tal(d)) in up to 30% of T-cell acute lymphoblastic leukaemia (T-ALL), leading to aberrant transcriptional activation, as a SIL-TAL1 fused transcript in tal(d). It has been suggested that TAL1 transcription occurs in approximately 50% of a T-ALLs without apparent rearrangement. SIL-TAL1 was positive in 15/60 (25%) of T-ALL, whereas wild-type TAL1 transcripts were detected in all 13 SIL-TAL1 and in 19/43 (44%) T-ALL without SIL-TAL1. To investigate the cellular origin of TAL1 we exploited the fact that GATA1 and TAL1 are co-ordinately expressed in non-lymphoid haemopoietic cells, whereas only the latter is found in T-ALL. GATA1 was detected in 10/23 (43%) TAL1-negative T-ALLs but in 17/19 (89%) 'unexplained' TAL1-positive cases, suggesting a common non-lymphoid cellular origin. Immunocytochemical analysis with a TAL1-specific monoclonal antibody showed nuclear expression in the blasts of 10/34 (29%) cases, including 8/10 SIL-TAL1+ and two RT-PCR TAL1+, SIL-TAL1- cases. In the remaining cases TAL1 expression was restricted to a minor population (5%) of larger, strongly TAL1-positive cells which comprised erythroid cells, CD34+ CD3- precursors and an unidentified TAL1+ CD45- population which morphologically resembled monocytes/macrophages. We therefore suggest that appropriate diagnostic evaluation of T-ALL should include molecular detection of SIL-TAL1 transcripts and in situ immunocytochemical detection of TAL1 protein expression by leukaemic blasts. This approach will enable accurate analysis of the prognostic significance of TAL1 deregulation in T-ALL.

Copolymer 1 (Cop 1) is a random synthetic amino acid copolymer of L-alanine, L-glutamic acid, L-lysine, and L-tyrosine, effective both in suppression of experimental allergic encephalomyelitis and in the treatment of relapsing forms of multiple sclerosis. Cop 1 binds promiscuously and very efficiently to living APCs of various HLA haplotypes. In the present study, a substantial part of the whole mixture of random polypeptides that compose Cop 1 was shown to bind to purified human HLA-DR1, DR2, and DR4 with high affinity in a temperature- and time (and, in the case of DR4, pH)-dependent manner, and was competitively inhibited by DR-restricted peptides, but not by peptide derivatives that bind with low affinity. Bacterial superantigens inhibited Cop 1 binding only at very high concentrations. The formation of the Cop 1-DR1 complex was also shown by SDS-PAGE. These findings represent the first direct evidence for interactions of Cop 1 with purified DR molecules, and suggest that its effectiveness in experimental allergic encephalomyelitis and multiple sclerosis may be directly related to its binding in the groove of HLA-DR proteins.

Genetic immunization has been widely applied in efforts to find novel and efficient mechanisms of stimulating the immune response. An effective attack against viral pathogens or tumors often requires activation of T cell-mediated immunity and the generation of cytotoxic T cells. Intramuscular immunization with plasmid DNA containing cDNAs that encode proteins results in expression and secretion of the foreign antigen by muscle cells. T cell activation occurs when peptide fragments of the exogenous protein are presented by major histocompatibility complex class I molecules on the surface of professional antigen-presenting cells. Identification of specific peptide epitopes from a protein antigen presented to T cells during an infectious process or tumor situation would provide all of the antigenic information needed to stimulate effective T cell-mediated immunity. Such peptides represent the naturally processed epitopes selected by the processing machinery of antigen presenting cells. Delivery of this information to the appropriate cells in vivo might be sufficient to stimulate T cell immunity and overcome the difficulties associated with overexpression of large protein antigens or those with potentially toxic side effects. This report describes the use of naturally processed T cell epitopes, administered in plasmid DNA vaccines, to stimulate cytotoxic T cell responses to two viral antigens effectively.

Type 1 diabetes (insulin-dependent diabetes mellitus, IDDM) is a disease controlled by the major histocompatibility complex (MHC) which results from T-cell-mediated destruction of pancreatic beta-cells. The incomplete concordance in identical twins and the presence of autoreactive T cells and autoantibodies in individuals who do not develop diabetes suggest that other abnormalities must occur in the immune system for disease to result. We therefore investigated a series of at-risk non-progressors and type 1 diabetic patients (including five identical twin/triplet sets discordant for disease). The diabetic siblings had lower frequencies of CD4-CD8- Valpha24JalphaQ+ T cells compared with their non-diabetic sibling. All 56 Valpha24JalphaQ+ clones isolated from the diabetic twins/triplets secreted only interferon (IFN)-gamma upon stimulation; in contrast, 76 of 79 clones from the at-risk non-progressors and normals secreted both interleukin (IL)-4 and IFN-gamma. Half of the at-risk non-progressors had high serum levels of IL-4 and IFN-gamma. These results support a model for IDDM in which Thl-cell-mediated tissue damage is initially regulated by Valpha24JalphaQ+ T cells producing both cytokines; the loss of their capacity to secrete IL-4 is correlated with IDDM.

Although self peptides bound to HLA-DQ and, especially, HLA-DR allotypes have been described in some detail, few ligands that bind to HLA-DP have been identified. Toward this aim, naturally processed peptides were isolated from immunoaffinity-purified HLA-DP2 molecules expressed in cultured B lymphocytes. The size distribution of the peptide repertoire is generally similar to those reported for self peptides bound to HLA-DR and HLA-DQ molecules. Twelve peptides representing individual sequences including two nested sets were sequenced by mass spectrometry and/or N-terminal Edman analysis. Source proteins included MHC molecules and other integral membrane proteins as well as secretory and serum proteins. No dominant amino acid markers suggestive of particular enzymatic processing events were detected. Peptide specificity and affinity were examined in binding assays using synthetic peptides and purified HLA-DP and HLA-DR molecules. Anchor residues were tentatively assigned using alanine-substituted analogues of two self peptides. Some structural features of HLA-DP2 that may relate to peptide binding are considered.

During T cell activation, CD4 is intimately involved in colocalizing the T cell receptor (TCR) with its specific peptide ligand bound to class II molecules of the major histocompatibility complex (MHC). Previously, the COOH-terminal residues, Trp62/63, which flank the immunodominant epitope of hen egg lysozyme (HEL 52-61), were shown to have a profound effect on TCR recognition. CD4 maintains the fidelity of this interaction when short peptides are used. To determine which portion of CD4 was responsible for this effect, a series of CD4 mutants were made and transfected into CD4 loss variants of two HEL 52-61-specific T cell hybridomas. Surprisingly, some CD4 mutants that failed to interact with MHC class II molecules (D2 domain mutant) or with p56kk (cytoplasmic-tailless mutant) restored responsiveness. Nevertheless, a significant reduction in association between cytoplasmic-tailless CD4 and the TCR, as determined by fluorescence resonance energy transfer, was observed. Thus, neither colocalization of CD4 and the TCR nor signal transduction via CD4 was solely responsible for the functional restoration of these T cell hybridomas by wild-type CD4. However, substitution of the two membrane proximal domains of murine CD4 (D3 and D4) with domains from human CD4 or intercellular adhesion molecule 1 not only abrogated its ability to restore function, but also substantially reduced its ability to associate with the TCR. Furthermore, the mouse/human CD4 chimera had a potent dominant negative effect on T cell function in the presence of equimolar concentrations of wild-type CD4. These data suggest that the D3/D4 domains of CD4 may interact directly or indirectly with the TCR-CD3 complex and influence the signal transduction processes. Given the striking structural differences between CD4 and CD8 in this region, these data define a novel and unique function for CD4.

The proximal promoters of all MHC class II genes contain a sequence element, the 19-bp X box, that is conserved in both sequence and position. Extensive analysis using a wide variety of approaches has demonstrated that the integrity of the X box is essential for transcription initiation from all class II genes studied. However, the X box is now recognized to contain two subregions, termed X1 and X2. Radiolabeled oligonucleotides corresponding to the X2 box of the MHC class II genes DPA and DQB were used to screen B cell and T cell expression libraries. A novel cDNA, termed XBR (X box repressor), encoding a putative zinc finger protein that binds specifically to the DPA X2 box was isolated from a human T cell line. The XBR gene encodes a 7-kb message that is ubiquitously transcribed, although at higher levels in tissues of the lymphocytic compartment. Southern blots indicate that this gene is single copy in primates and contains regions that are highly divergent in other species. Overexpression of XBR in a B cell line resulted in a dramatic reduction of transcription from a reporter gene construct driven by the DPA promoter, but not from similar constructs with mutations in the X2 box. Similarly, overexpression of XBR reduced induction of reporter gene activity driven from the DPA promoter in HeLa cells treated with IFN-gamma. XBR may, therefore, mediate transcriptional repression, thus preventing inappropriate MHC class II expression. XBR function may in part explain the dominant trans-acting repression of MHC class II expression reported in cell fusion experiments.

A previously unrecognized element, located downstream of the start site of transcription in the first exon of the DR alpha gene, has been defined that enhances promoter activity up to eightfold in a position-dependent manner. Mutations in this DNA-binding site abolished binding of a nuclear factor in human B cell nuclear extract and decreased the activity of the DR alpha promoter to a basal level. Significant sequence homology of this element was found in the DNA of the DR beta, DP alpha and -beta, and DQ alpha genes, always located downstream of the transcriptional start site. The nuclear factor binds to the DR alpha and DP alpha element but not to the element in the DQ alpha gene. It was identified as NF-E1 (YY1). This protein, previously identified by its binding to the Ig kappa 3' enhancer and the Ig heavy chain mu E1 site, thus also appears to be quite important in the regulation of MHC class II gene expression.

The immune response to viruses partially depends on the biochemical interaction between viral peptides and histocompatibility molecules. In this study, the refolding of recombinant HLA-A*0201 heavy chain and beta 2-microglobulin (beta 2-m) in the presence of peptides from influenza B nucleoprotein (BNP), influenza A matrix protein, and HIV gp120 and their analogues was examined. The plateau value for the amount of refolded complex with three peptides, a 10-mer BNP 85-94 (A86) with alanine substituted for leucine at the P2 anchor residue and two BNP 8-mers, was significantly lower than the native peptide epitope BNP 85-94 or with other peptides tested. To attempt to understand the basis for the lower yield of complex, equilibrium dissociation constants (KdS) for the two 10-mers, BNP 85-94 (A86) and BNP 85-94, were determined from association and dissociation rates and from Scatchard plots, all measured at 10 degrees C. In addition, dissociation rates were measured at 0 degrees, 26 degrees, and 37 degrees C. Although the kinetics were similar at 0 degrees and 10 degrees, at 37 degrees these two complexes had distinct rates of dissociation, resulting in relatively stable or unstable complexes. The behavior of the unstable complexes paralleled the behavior of empty complexes described in vivo; they are unstable at physiologic temperature, produced in low yield, and stabilized by low temperature. Comparison of all of the kinetic data suggests that the equilibrium amounts of the two HLA/peptide complexes (plateau values) result from distinct reaction pathways, i.e., that the molecules that form stable complexes may undergo an additional reaction to those that form unstable complexes.

The chromosomal breakpoints of follicular lymphomas carrying the t(14;18)(q32;q21) are known to be clustered within a 150-bp region in the major breakpoint region (mbr) of the Bcl-2 oncogene. We have demonstrated that nuclear proteins specifically bind to a novel target sequence within this 150-bp region and a region of Dxp genes, members of the immunoglobulin (Ig) diversity (DH) gene family. One protein, designated BCLF-1, appears to be specifically expressed in lymphoid lineage cells. Two other proteins, BCLF-2 and -3, bind only to the complementary single strand of the target sequence. The manner in which these proteins interact with the target sequence is similar to the interaction of the ReHF-1 and -2 proteins to the signal-like sequence at the chromosomal breakpoint junctions in patients with the t(8;14)(q24;q11) and t(1;14)(p32;q11) translocations. It was further suggested that the BCLF-1 is quite similar to or identical to the ReHF-1. It is therefore hypothesized that these conserved target sequences found in recombination hotspot regions may define novel sequence motifs recognized by two classes of DNA binding proteins. One class of DNA binding proteins is specifically expressed in lymphoid cells while the other class binds to the complementary single strand DNA. These binding activities may play a crucial role in chromosomal translocation in lymphoid neoplasms.

CD43, CD44, CD16, and L-selectin have been previously shown to be enzymatically cleaved from stimulated leukocytes. However, little is known about the enzymes involved in these processes. Here, metalloprotease(s) inhibitable by 1,10-phenanthroline together with serine protease(s) inhibitable by N alpha-p-tosyl-L-lysine chloromethyl ketone and 3,4-dichloroisocoumarin are shown to be involved in the cleavage of CD43, CD44, and CD16 but not in the cleavage of L-selectin on granulocytes. In addition, mAbs that recognize these individual receptors and induce their specific cleavage did not initiate cleavage of the others. In one case only, L-selectin, cleavage was also triggered by mAbs interacting with CD16 (the low affinity Fc gamma R). Thus, this mechanism represents a novel pathway of L-selectin cleavage induction.

Human CD4 is an accessory molecule found on the cell surface of a subset of T lymphocytes. We have previously shown by infection of simian fibroblasts with an SV40-CD4 recombinant virus that CD4 acts as an adhesion molecule by binding to human MHC class II Ag expressed on the surface of human B lymphocytes. This report confirms that human B lymphoblastoid cell lines expressing class II molecules at the cell surface can bind to Chinese hamster ovary cells that have been stably transfected with human CD4. This cellular adhesion is a late event, which is first detected after 2 h, but remains stable for up to 16 h. The association between CD4 and class II is energy dependent, as it is detected at 37 degrees C, but not at 4 degrees C or if either cell type is fixed with paraformaldehyde. ATP is required for the establishment and maintenance of stable CD4/class II-mediated cell conjugates. Cytoskeletal interactions also regulate CD4/class II adhesion as treatment with the microtubule and microfilament inhibitors colchicine, cytochalasin-D, and nocodazole rapidly dissociates even preformed cell conjugates. Our observations also indicate that the Ag-independent engagement of class II by CD4 induces homotypic clustering of human B cells. This effect is blocked by reagents directed against lymphocyte function-associated Ag-1 and may result from the induction of a high affinity phenotype of lymphocyte function-associated Ag-1 induced by class II signaling. Finally, we discuss the implications of CD4/class II-mediated adhesion and the role of CD4 in the regulation of T cell adhesion.

hXBP-1 is a transcription factor of the leucine zipper (b-zip) family important in the expression of the class II major histocompatibility complex gene, DRA. Studies with mouse-human hybrids have mapped hXBP-1 hybridizing fragments to human chromosomes 5 and 22 and the frequent detection of two mRNA transcripts suggested that hXBP-1 may be a member of a small gene family. To analyze the structure and regulation of hXBP-1 further, cosmid clones from both chromosomes were isolated. Mapping and sequence analyses reveal that chromosome 22 contains the functional hXBP-1 gene while chromosome 5 contains a processed pseudogene. hXBP-1 promoter analysis has revealed that cis-active elements within the 5'-untranslated region of hXBP-1 are essential for full promoter activity. One such element, hX2, is identical to the hXBP-1 target sequence in the DRA promoter. Mutagenesis of the hX2 site substantially decreases promoter activity. This element interacts with four distinct protein complexes in mature B cells and cross-competition experiments show that two of these complexes (complex 1 and complex 4) also interact with the hXBP-1 target sequence (X2) from the DRA promoter. The similarities of the hXBP-1 promoter and of the DRA promoter (the gene that the hXBP-1 protein regulates) are further emphasized by the fact that a Y box element is located 3' of both hX2 and X2.

Staphylococcal enterotoxins bind with high affinity to class II major histocompatibility complex proteins and subsequently stimulate large numbers of T cells via the V beta portion of the T-cell receptor. Binding of enterotoxin A and enterotoxin E to HLA-DR was completely abolished by low levels of EDTA, whereas binding of toxic shock toxin was unaffected. Addition of Zn2+ to as little as 2 microM excess over EDTA completely reconstituted binding, but Ca2+, Mg2+, Cu2+, Fe2+, and Mn2+ had no effect. The dissociation constant (Kd) of 65Zn2+ binding to a single site on purified enterotoxin A was 2 microM, and addition of purified HLA-DR1 did not alter the Kd, indicating that the binding site was exclusive to enterotoxin A. In the presence of saturating levels of zinc the Kd for enterotoxin A binding to purified HLA-DR1 was 25 nM. Thus, zinc binding is an essential first step in the formation of the major histocompatibility complex binding domain of at least two bacterial superantigens. Given the measured Kd of zinc binding to enterotoxin A, serum levels of free zinc (0.2-1.0 microM) may well regulate the toxic sequelae by these two superantigens.

By fluorescence resonance energy transfer, we have previously demonstrated that upon anti-CD3 mAb-mediated activation of a murine T cell hybridoma expressing human CD4, CD4 moves into close association with the TCR/CD3 complex. It was shown that this association between CD4 and the TCR/CD3 complex was dependent upon the presence of an intact CD4 cytoplasmic domain. We have now expressed, in a murine T cell hybridoma, mutated forms of CD4 containing cysteine to serine point mutations at positions 420, 422, or 430. The mutations at positions 420 and 422, but not 430, abolish association with p56lck. By using fluorescence resonance energy transfer, we demonstrate that mutations of CD4 which fail to interact with p56lck are unable to associate with the TCR/CD3 complex under conditions in which wild-type CD4 and the 430 mutant CD4 do associate with the TCR/CD3 complex. In addition, these mutants have a diminished response to CD4-dependent stimuli. We conclude that the association between CD4 and the TCR/CD3 complex during T cell activation plays an important role in CD4-dependent responsiveness and this association requires the interaction of CD4 with p56lck. These results also suggest that a substrate for p56lck may be expressed in the TCR/CD3 complex.

CD14, expressed on the surface of monocytes as a phospholipid-linked protein, is a receptor for serum LPS binding protein/LPS complex. It was specifically down-modulated after stimulation of monocytes by physiologic activating/differentiating agents such as bacterial LPS and IFN-gamma, by the pharmacologic agents PMA and calcium ionophore A23187, and by anti-CD14 antibodies. The down-modulation was almost totally blocked at 4 degrees C or at pH 4.5 and markedly inhibited by the protease inhibitors diisopropylfluorophosphate and PMSF. A soluble labeled CD14 was isolated from culture supernatant of surface iodinated monocytes after their activation, indicating that CD14 is shed from the cell surface rather than internalized. The size of the soluble CD14 shed from the monocytes in vitro was smaller than that of either the membrane-bound form or a soluble CD14 cleaved from the cell surface by phosphatidyl inositol-specific phospholipase C, but identical to the size of one of the two major soluble CD14 forms normally found in human serum. These data suggest that CD14 shedding induced by monocyte stimulation may play an important role in the regulation of surface CD14 expression.

A long-term CD4-CD8- TCR alpha beta human T-cell line, as well as similar CD4-CD8- TCR gamma delta T-cell lines for comparison, were generated from various tissues by negative selection using anti-CD4 and anti-CD8 monoclonal antibodies (MAbs) followed by positive selection with specific anti-TCR MAb and then repeated in vitro stimulation with interleukin-enriched media and lectin. These cell lines all demonstrated non-MHC-restricted cytolysis on a variety of human tumor cell lines. However, removal of lymphokines from the culture media for 24 hr abrogated most of the non-MHC-restricted target-cell lysis without affecting TCR alpha beta or TCR gamma delta cell viability or TCR function as determined by antibody-triggered redirected target-cell lysis. Subsequent re-exposure to lymphokines reconstituted non-MHC-restricted cytolysis by these cell lines. Thus, much of the non-specific, non-MHC-restricted cytolytic activity generated by CD4-CD8- TCR alpha beta or TCR gamma delta cells is secondary to lymphokine-activated killing (LAK) activity. These cells have potent LAK activity and may be prominent in LAK-cell populations. In addition, after lymphokine deprivation, both CD4-CD8- TCR alpha beta and TCR gamma delta cells showed residual activity against some tumor-cell targets, the nature of which remains to be defined.

Human lymphocyte function-associated antigen (LFA)-1, a heterodimeric lymphocyte surface glycoprotein of 177,000 and 95,000 relative molecular weight has been implicated to function in the cytotoxic T lymphocyte (CTL) effector mechanism. Seven mouse hybridoma lines producing monoclonal antibodies (MAb) reactive with this structure were studied. Three unique and 3 partially over-lapping epitopes on human LFA-1 were defined by competitive cross inhibition binding assays using biosynthetically labeled anti-LFA-1 MAb. In contrast, of five rat antimouse LFA-1 MAb, all five recognized a common or shared epitope. An HLA-B7 specific human CTL line expressed 1.1 X 10(5) LFA-1 sites per cell with a direct saturation binding assay. Human CTL expressed two to four times more LFA-1 than peripheral blood lymphocytes or B and T lymphoblastoid cell lines. Titration of each of the anti-LFA-1 MAb in a 51chromium release cytolytic assay revealed quantitative differences in the ability of the different anti-LFA-1 MAb to block cytolysis indicating distinct functional and antigenic epitopes exist on the human LFA-1 molecule. Anti-LFA-1 MAb reversibly inhibited the CTL reaction by slowing the initial rate of cytolysis. These results suggest anti-LFA-1 MAb inhibit CTL function by specific blockade of a functionally relevant molecule.

The HLA-DR antigen expressed on the surface of the human melanoma cell line SK-MEL-37 was characterized and compared with the HLA-DR antigen from the MU B lymphoblastoid cell line originating from the same individual. The HLA-DR heavy chain from SK-MEL-37 cells had an apparent mobility on SDS-PAGE slightly slower than that isolated from MU cells. In contrast, the HLA-DR light chains and the HLA-A,-B heavy chains from the two cell lines had identical mobilities. Double-labeled tryptic peptide mapping and limited N-terminal sequencing showed that the SK-MEL-37 HLA-DR antigen, like all previously examined B lymphoblastoid cell HLA-DR antigens, was homologous to the murine I-E/C subregion antigens and that the mobility difference of the SK-MEL-37 HLA-DR heavy chain was not attributable to differences in the primary structure of the polypeptide. Treatment of the cells with tunicamycin abolished the m.w. difference, suggesting that it was due to a change in glycosylation in SK-MEL-37. This was confirmed by analysis of the glycopeptides from pronase-digested HLA-DR light and heavy chains and HLA-A,B heavy chains purified from the two cell types. The results suggest 1) there is a difference in asparagine-linked oligosaccharide processing in the two cell types, with more of the larger complex glycans synthesized in the melanoma cells than in the B lymphoblastoid cells, 2) the effect is more pronounced with HLA-DR heavy chains than with HLA-DR light chains or HLA-A,B heavy chains, and 3) the oligosaccharide size difference is not solely due to sialic acid content.

Penicillin-binding proteins 5 and 6 have been purified to homogeneity from the dacA mutant strain of Escherichia coli (JE 11191). Protein 6 from the mutant strain appears to be identical to that from the wild type, but protein 5 is a mutant protein which has no D-alanine carboxypeptidase activity. Moreover, the mutant protein 5 binds, but does not release, [14C]penicillin G. Correspondingly, an acyl-enzyme intermediate derived from a synthetic substrate is accumulated by the mutant protein. A comparison of the acylation site for the synthetic substrate and for penicillin G by limited proteolysis and some other properties of the mutant protein are described.