Robert G. Ulrich

HLA-DR peptide inhibits HIV-induced syncytia.

The infectivity of the human immunodeficiency virus (HIV) is related to the structure of its envelope protein, gp160, which is responsible for viral entry. We considered the possibility that a structural homology between gp160 and major histocompatibility complex (MHC) molecules might be associated with the extraordinary affinity that gp120 has for its receptor, CD4. Amino acid sequence comparisons revealed five regions of structural similarity between the HLA-DR beta molecule and gp160. The DR2 beta synthetic peptides containing these regions were examined for their ability to block HIV-induced syncytia formation using a 51Cr release assay. The peptide beta 141-155 inhibited the formation of syncytia whereas the other four DR beta peptides with gp160 similarity did not. Our results indicate that this region in gp120, which is similar to an HLA-DR region, is crucial to T cell-gp120 interactions, and should be considered in the design of future vaccines.

Incorporation of fatty acids into phospholipids in L cells stimulated by antibody

Binding antibodies to surface membranes stimulated incorporation of fatty acids (FA) into phospholipids of L cells. Antibodies stimulated at least a 3.4-fold greater incorporation of arachidonic acid into phosphatidylinositol than into any other class of phospholipid when compared on a molar basis (p<0.003). This enhanced incorpoation was selective, depending on the character of the FA, because antibodies stimulated the incorporation of arachidonic acid at least 2.4-fold more than oleic acid, palmitic acid or stearic acid (p<0.001). Surprisingly, an antibody-stimulated incorporation of palmitic acid into sphingomyelin (SM) was at least 2.2-fold greater than that into any other class of phospholipid (p<0.001) and the antibody-stimulated incorporation of palmitic acid into SM was at least 60-fold greater than that of arachidonic acid, stearic or oleic acid (p<0.001). Nontoxic doses of ethylenediamine tetraacetic acid (EDTA), dexamethasone, 4-bromophenacylbromide and indomethacin inhibited the antibody-stimulated incorporation of arachidonic acid into cellular phospholipids, principally phosphatidylinositol (PI), and similarly inhibited the antibody stimulation of DNA synthesis. We conclude that when antibody binds to surface antigens on L cells, a rapid and selective incorporation of fatty acids into certain cellular phospholipids occurs, possibly mediated by calcium-dependent phospholipases. Degradation products of arachidonic acid, i.e., prostaglandins, may be important in these antibody stimulation events, as well. These early changes in phospholipid metabolism may serve as an important signal or mechanism for the subsequent stimulation of DNA synthesis in L cells.

Antibody to immunoselected L-cell antigens mimics stimulating activity of antibody to whole L cells.

A heterogeneous IgG antibody raised in rabbits in response to injections of whole L cells was used to identify and select relevant antigens in a nonionic detergent extract of L cells prelabeled with [35S]methionine by means of immunoprecipitation and immunoaffinity chromatography. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the immunoprecipitate and immunoeluate contained far fewer protein bands than the whole cell extract but selectively retained a 42,000-MW protein species. In response to injections of the immunoprecipitate, rabbits produced a new antiserum which reacted predominantly with the 42,000-MW protein when reacted with L-cell proteins separated by sodium dodecyl sulfate-gel electrophoresis and transferred to nitrocellulose paper by the Western blot technique. The new antiserum (raised to the immunoprecipitate) and the original antiserum (raised to whole cells) were equipotent in stimulating calcium transport, phospholipid metabolism, and DNA synthesis in L cells. Binding of the IgG fractions of the two antisera displayed identical high affinity binding to L-cell surface antigens, with the same average association constant of 1.5 X 10(6) M-1. These studies have shown that an antiserum raised to whole L cells has a much narrower reactive spectrum with L-cell membrane antigens than might be imagined and has identified a 42,000-MW membrane protein as an important immunogen which itself elicits a potent immune response resulting in an antibody capable of mimicking the cell stimulatory properties of the original antiserum.

Enhanced N-acylation of palmitic acid in sphingomyelin of antibody-stimulated L cells

Metabolism of sphingomyelin was stimulated in a fibroblast-like transformed cell line, L-929, when the cells were incubated with antibodies of a specific rabbit antiserum. The cells responded with an increased incorporation of [3H] palmitic acid into sphingomyelin. The stimulated uptake of palmitic acid into sphingomyelin can not be explained by simple mass increases in cellular phospholipids but probably represents a selective N-acyl group turnover. Palmitic acid composed only 7.5 percent of the acyl substituents, as assessed by gas-liquid chromatographic analysis, but [3H] palmitic acid was incorporated at a two-fold higher level into the acyl position than into the long chain base precursor (sphingosine). Since it is known that palmitic acid is the predominant fatty acid forming sphingosine, this represents a considerable selection for N-acyl group turnover. Another saturated fatty acid, stearic acid, which was over twice as abundant constituently was incorporated at a much lower rate when the cells were stimulated. Thus palmitic acid was observed to be selectively turning over in a manner suggestive of acylation-reacylation cycles observed with other classes of phospholipids.

Alloreactive T cell recognition of the HLA-DRβ N-terminal polymorphic region

Abstract T cell alloreactivity, originally discovered as a tissue transplantation effect, is believed to be a manifestation of the normal major histocompatibility complex (MHC) restriction of antigen presentation by accessory cells to T cells. The molecular features of the human class II-MHC proteins (HLA) which are recognized by alloreative T cells are not at present understood, although they are clearly related to the polymorphic nature of the MHC proteins. Human CD4 4 T cell clones were selected by response to the HLA-DR2 peptides β1–15 or β51–65, in an MHC-restricted manner. In addition, these clones respond to cell lines expressing the DR2 haplotype, without the requirement for accessory antigen presenting cells. DR2β peptide 1–15 blocks the T cell alloresponse and polymorphic residues are shown to stimulate the peptide-specific response of these clones. Thus, the polymorphic residues contained within the DRβ sequence 1–15 are demonstrated to be directly recognized by alloreactive T cells.

Cyclic AMP and theophylline enhance DNA synthesis in L cells stimulated with anti-actin IgG and [(IgG)2protein A]2 complex by recruiting cells into S-phase

Surface binding of anti-actin IgG alone or in a Mr = 716 000 [(IgG)2Protein A]2 complex results in a stimulation of DNA synthesis and cell growth in L cells. Cyclic-AMP (0.01–1.0 mM) added to such cell cultures augmented DNA synthesis as measured by incorporation of [3H]thymidine into DNA. Theophylline (0.1–1.0 mM), a phosphodiesterase inhibitor which prevents enzymatic breakdown of cAMP, had similar effects, but cGMP (0.01–1.0 µM) reversed the effects of cAMP and theophylline upon DNA synthesis. Analysis of the cell cycle by flow cytometry revealed that antibody produced a shift (7%) of cells from the G1-phase to the S-phase (DNA-synthetic) of the cell cycle at 72 hr of incubation. Addition of cAMP (0.5 mM) to cell cultures, however, produced significant shifts of antibody stimulated cells from G1-phase to S-phase at all time points measured, i.e., 24 (12%),48 (22%),72 hr (23%). Thus, antibody recruited cells into S-phase of the cell cycle and cAMP greatly augmented the effect. These observations suggest that the mechanism of activation of L cell growth by antibody to surface antigens involves a recruitment of cells into the DNA-synthetic phase and that the effect may be mediated by cAMP.

DRβ peptides block the antigen-specific response but not the alloresponse of a dual-reactive T-cell clone

Abstract It is generally assumed that alloreactivity and the response to foreign antigens are equivalent T-cell recognition events. We have addressed this issue by examining the antigen presentation of beef insulin by two DR1-restricted human T-clones. One of the clones was dual-reactive and exhibited alloreactivity. By employing 20 synthetic consecutive overlapping peptides representing the entire extracellular (residues 1–198) and intracellular (residues 222–273) parts of the DR2β molecule, the effects of each of these peptides on antigen presentation and alloreactivity were examined. The DR1-restricted response to beef insulin by both clones was inhibited by peptides from DR2β region 1–25. Other DR2β (residues 21–198 en 222–237) or control peptides had no effect. The DR3-associated alloresponse of the dual-reactive clone was not inhibited by any peptide. These observations suggest that recognition of antigen is fundamentally different from allorecognition.

Immune Recognition of Human MHC DR2 Antigen

The human class II major histocompatibility complex (MHC) antigens are homologues of the murine Ia antigens. Class II HLA glycoproteins have a limited tissue distribution, being found primarily on the macrophage, dendrite cells, B cells and activated T cells, but they may also be induced on other cell types by interferon-γ (Pober et al, 1983). Class II HLA antigens are composed of a dimer of a heavy (33–35 kDa) a chain and a light (27–29 kDa) B chain (Shackelford et al., 1981). Both the a and B subunits are composed of two extracellular domains, each containing a disulfide loop, with the exception of the N-terminal domain of the a chain. Allelic polymorphism is a characteristic of the B chains of DP and DR (Goyert et al., 1982; van Rood et al., 1976; Giles and Capra, 1985) and of both chains of the DQ molecules (Giles and Capra, 1985; Giles et al., 1985).