Major histocompatibility complex class I-restricted presentation of protein antigens without prior intracellular processing.

Proteins in their native form are incapable of stimulating antigen (Ag)-specific T cells, which can only recognize major histocompatibility complex (MHC)-bound peptides that have been generated by intracellular processing within antigen-presenting cells (APCs). Here, we show that APCs can trigger MHC class I-restricted T-cell responses after presenting proteins without conventional intracellular processing, provided the immunostimulatory MHC class I-binding peptide sequence is incorporated at the carboxy-terminal position. Such MHC-bound proteins do not stimulate T cells directly, because the contact between MHC/peptide complex and its cognate ligand is sterically hindered by the amino-terminal bulk of the protein. Removal of the latter via an extracellular Ag proteolysis by the T-cell- and/or APC-derived enzymes is required for effective T-cell stimulation. Our data challenge the established concept that only small peptides can bind to the MHC class I molecules.

Selective pharmacologic inhibition of murine and human IL-12-dependent Th1 differentiation and IL-12 signaling.

We have previously shown that lisofylline (LSF) inhibits murine Th1-mediated disease in vivo by blocking IL-12-induced differentiation of Th1 cells. The cellular and molecular mechanisms underlying this inhibition were further explored by testing LSF in several IL-12-responsive model systems in vitro. IL-12-dependent Th1 differentiation was abrogated by LSF and yielded effector T cells that were deficient in proinflammatory cytokine secretion, including IFN-gamma, IL-2, and TNF-alpha. The diminished Th1 phenotype resulted from both a lower frequency of IL-12-derived Th1 clones and a reduced capacity of individual clones to secrete IFN-gamma due to lower levels of IFN-gamma mRNA. The arrest in Th1 development resulted from a blockade of IL-12 signaling that preceded the Th0 to Th1 transition. Thus, LSF blocked IL-12-enhanced IFN-gamma production in anti-CD3-stimulated T cells and prevented IL-12-mediated repression of the transcription factor GATA-3. Lisofylline also inhibited IL-12-induced increases in STAT4 tyrosine phosphorylation, but did not block TCR signaling or inhibit acquisition of IL-12 responsiveness. These findings were extended to show that LSF also inhibits IL-12-dependent responses in human T cells. LSF, which has one asymmetric chiral center, was selectively inhibitory for IL-12 signaling compared with its S-enantiomer (1501-S) and the oxidized side chain analog, pentoxifylline. The results suggest that LSF may be useful as a modulator of Th1-mediated disease in humans.

Co-ligation of the antigen and Fc receptors gives rise to the selective modulation of intracellular signaling in B cells. Regulation of the association of phosphatidylinositol 3-kinase and inositol 5'-phosphatase with the antigen receptor complex.

Cross-linking of the Fc receptor (FcR) to surface immunoglobulin (sIg) on B cells inhibits the influx of extracellular calcium and abrogates the proliferative signal. The mechanism by which this occurs is not well understood. In this report we show that co-cross-linking the FcR to the antigen receptor gives rise to very selective modulation of signal transduction in B cells. Co-cross-linking sIg and the FcR enhanced the phosphorylation of the FcR, the adapter protein, Shc, and the inositol 5'-phosphatase Ship. Furthermore, phosphorylation of the FcR induced its association with Ship. Cross-linking of the FcR and sIg decreased the tyrosine phosphorylation of CD19, which led to a reduction in the association of phosphatidylinositol 3-kinase. In addition, the phosphorylation of several other proteins of 73, 39, and 34 kDa was reduced. Activation of the cells with either F(ab')2 or intact anti-IgG induced very similar changes in levels of tyrosine phosphorylation of most other proteins, and no differences in the activation of several protein kinases were observed. These results indicate that the inhibitory signal that is transmitted through the FcR is not mediated by a global shutdown of tyrosine phosphorylation but is, rather, a selective mechanism involving localized changes in the interactions of adapter proteins and the enzymes Ship and phosphatidylinositol 3-kinase with the antigen receptor complex.

Cross-linking of Fc gamma receptor to surface immunoglobulin on B cells provides an inhibitory signal that closes the plasma membrane calcium channel.

Stimulation of B lymphocytes by the cross-linking of surface Ig (sIg) with an F(ab')2 antibody fragment leads to the rapid activation of several tyrosine kinases. This gives rise to the activation of phospholipase C gamma (PLC gamma) and the generation of inositol phosphates. These, in turn, lead to a prolonged elevation of intracellular Ca2+ ([Ca2+]i) consisting of a rapid release of Ca2+ from intracellular stores and a sustained influx of extracellular Ca2+. In contrast, co-cross-linking sIg to Fc gamma receptor (Fc gamma RII) with intact anti-sIg induces a much more transient increase in [Ca2+]i. Stimulation of the murine B cell lymphoma, A20, with F(ab')2 anti-sIgG leads to the production of high levels of IL-2, while co-cross-linking of sIgG with Fc gamma RII blocks this response. In studies reported here, we show that co-cross-linking of Fc gamma RII with sIg prevents the influx of extracellular Ca2+ without significantly affecting the tyrosine phosphorylation of substrates including PLC gamma 1, PLC gamma 2, and Syk or the mobilization of Ca2+ from intracellular stores. In cells that had been previously activated with F(ab')2 anti-IgG, co-cross-linking of sIg to Fc gamma RII rapidly abrogated the influx of extracellular Ca2+ by closing the plasma membrane Ca2+ channel. Additionally, even 2-3 h after stimulation of the cells with F(ab')2 fragment, addition of intact anti-IgG to the cells, or removal of extracellular Ca2+, markedly inhibited (> 90%) IL-2 production. These results indicate that co-cross-linking sIg with Fc gamma RII both prevented the opening of and actively closed the Ca2+ channel, and, through this mechanism, Fc gamma RII was able to control production of IL-2. Overall, since influx of extracellular Ca2+ has been found to be necessary for the proliferation and differentiation of B cells, Fc gamma RII may play a critical role in controlling these responses by regulating the opening of the Ca2+ channel.

Regulation of HIV production by blood mononuclear cells from HIV-infected donors: I. Lack of correlation between HIV-1 production and T cell activation.

The relationship between production of HIV-1 by peripheral blood mononuclear cells (PBMCs) from HIV-1-infected donors and the level of T cell activation by various stimuli was examined. Stimulation of PBMCs with soluble anti-CD3 antibody or staphylococcal enterotoxin/superantigen (SAg) was found to be 100-1000 times more effective at inducing production of HIV-1 than was stimulation with immobilized anti-CD3 or various other T cell activating agents. However, proliferation of CD4+ T cells and lymphokine production following stimulation with soluble anti-CD3 were less than with immobilized anti-CD3. To determine whether immobilized anti-CD3 stimulated cells may produce a factor(s) that suppresses HIV production, dual-chamber coculture experiments were performed in which soluble and immobilized anti-CD3-stimulated CD8-depleted PBMCs were separated by porous membranes. Stimulation of cells by immobilized anti-CD3 suppressed HIV-1 production by soluble anti-CD3-stimulated cells in the inner chamber, suggesting that diffusible factor(s) are involved in suppressing HIV-1 production. Experiments in which exogenous cytokines were added to cells stimulated with soluble anti-CD3 did not reveal the suppressive factor(s) produced; however, IL-7 was found to markedly increase HIV-1 production. Both T cells and monocytes were found to be required for soluble anti-CD3 to induce high levels of HIV-1 production, suggesting a role for adhesion molecules. Our results thus show that (1) soluble anti-CD3 is a powerful stimulus for HIV production, (2) there is not an absolute correlation between the level of HIV-1 production and T cell activation following stimulation of PBMCs with T cell activating agents, (3) immobilized anti-CD3 stimulation produces a factor that decreases HIV replication, and (4) T cell monocyte interactions are important for production of HIV-1 following stimulation with soluble anti-CD3.

Regulation of HIV production by blood mononuclear cells from HIV-infected donors: II. HIV-1 production depends on T cell-monocyte interaction.

Cell-cell interactions induced between T cells and monocytes by certain soluble anti-CD3 monoclonal antibodies (MAbs) were previously shown to be required for high-level production of HIV-1 by peripheral blood mononuclear cells (PBMCs) from infected donors. Staphylococcal enterotoxin or superantigen (SAg) is another mitogen inducing monocytes-T cell interactions that exhibit potent induction of HIV-1 production. Antibodies to several adhesion molecules were used to test the requirements for T cell- and monocyte-associated adhesion molecules in HIV-1 production following activation with anti-CD3 or SAg. Blocking of either CD2-LFA-3, or CD18-ICAM-1, inhibited anti-CD3- or SAg-induced HIV-1 production by more than 90% without inhibiting CD4+ T cell proliferation. Inhibition of HIV production was observed when either the T cell or monocyte coreceptor was bound by MAbs to these adhesion molecules. Blocking of CD28-B7 interactions by soluble CTLA-4 fusion protein, a CD28 homolog, inhibited both HIV-1 production and CD4+ T cell proliferation. Fc binding was not required for HIV-1 inhibition by MAbs to CD2 and CD18, because Fab or F(ab')2 fragments of these MAbs inhibited HIV-1 production by more than 80%. A chimeric single-chain MAb to CD2 was produced, containing heavy and light chain variable regions from MAb 35.1 to CD2 linked to the constant regions of human IgG1 (CD2 SFv-Ig). This humanized CD2 SFv-Ig inhibited HIV-1 production by 30% to > 98%. These results thus indicate that simultaneous engagement of multiple adhesion pathways between T cells and monocytes are required for HIV production by patients PBMCs and may have implications for therapy of HIV infections.

Inhibition of virus production in peripheral blood mononuclear cells from human immunodeficiency virus (HIV) type 1-seropositive donors by treatment with recombinant HIV-like particles.

We have previously reported on the assembly of recombinant human immunodeficiency virus (HIV)-like particles that contain gag structural proteins and present env glycoproteins gp120 and gp41 on their surfaces (O. Haffar,. J. Garriques, B. Travis, P. Moran, J. Zarling, and S.-L. Hu, J. Virol. 64:2653-2659, 1990). On the basis of their structures, we hypothesized that the recombinant particles would interfere with virus infection and tested our hypothesis in vitro by using peripheral blood mononuclear cells (PBMC) from HIV type 1-seropositive donors. Addition of the recombinant particles to PBMC concomitant with stimulation by anti-CD3 inhibited virus production, as determined by reduced levels of p24 in the culture supernatants. This inhibition of p24 production correlated with lower levels of cell-associated viral DNA. Several lines of evidence suggested that the recombinant particles exerted their antiviral effects primarily by inhibiting virus production from latently infected cells and not by inhibiting subsequent virus spread. Importantly, CD4+ T-cell stimulation by specific antigen or by anti-CD3 was not inhibited by treatment with the recombinant particles. This apparent selective inhibition of virus replication in infected PBMC represents a novel property of the recombinant HIV-like particles.

Dihydrotestosterone exerts a depressive influence on the production of interleukin-4 (IL-4), IL-5, and gamma-interferon, but not IL-2 by activated murine T cells.

The present study examined the effects of the androgen steroid, dihydrotestosterone (DHT), on murine T-cell production of a number of lymphokines. Direct exposure of murine T cells to DHT in vitro was found to reduce the amount of interleukin-4 (IL-4), IL-5, and gamma-interferon (gamma IFN) produced after activation with anti-CD3 without affecting the production of IL-2. Exposure of T cells to either androstenedione or testosterone (the metabolic precursors of DHT) affected no change in the biosynthesis of either of these lymphokines. We have determined that macrophages possess 5 alpha-reductase, and are thus competent to metabolize testosterone to DHT. This physicochemical information is complemented by a functional analysis of macrophage metabolism of testosterone. By incubating bone marrow macrophages with testosterone, before their use as accessory cells, the IL-4 and IL-5 producing potential of the activated T cells cocultured with them was depressed. That the observed effect was mediated by the conversion of testosterone to DHT was further corroborated by illustrating that the inhibition of IL-4 production was abrogated if 4MA, a specific 5 alpha-reductase inhibitor, was added to macrophage cultures containing testosterone. The biologic role of DHT in lymphokine and immune response regulation in vivo was addressed using several lines of investigation. First, transdermal delivery of DHT to groups of mice altered the capacity of T cells residing in the draining lymph nodes, only, to produce lymphokines. Second, treatment of either aged mice or the T cells isolated from them with a combination of dehydroepiandrosterone and DHT restored the capacity of their T cells to produce IL-2, IL-4, and gamma IFN to levels equivalent to that of younger mice. Finally, we observed a difference between males and females of a given age to produce IL-2, IL-4, and gamma IFN, with both IL-4 and gamma IFN production being elevated in females. Collectively, our findings indicate that DHT, similar to other steroid hormones, may play an important role in lymphokine regulation in vivo.

Inhibition of HIV-1 replication in seropositive patients' CD4+ T-cells by pokeweed antiviral protein-monoclonal antibody conjugates.

Pokeweed antiviral protein (PAP) inhibits HIV-1 replication in HIV-1 infected CD4+ cells and PAP targeted to CD4+T-cells by conjugation with monoclonal antibodies (mAb) against CD4 is approximately 1000 times more potent than non-conjugated PAP. Furthermore, PAP-antiCD4 inhibits HIV-1 production in seropositive patients' CD4+ T-cells activated with mAb to CD3 which was found to be the most potent means to activate HIV-1 production. These findings, together with previous observations that PAP-mAb conjugates have an in vivo plasma half-life of about 30 times that of non-conjugated PAP, suggest that PAP-antiCD4 may be a useful therapy in HIV-infected humans. Additionally, because PAP is known to have antiviral activity against several other human viruses, PAP-mAb conjugates may also have clinical potential for treating other viral diseases.