A secreted form of the major histocompatibility complex class II-associated invariant chain inhibiting T cell activation.

Major histocompatibility complex (MHC) class II molecules function at the cell surface to present antigenic peptides to T helper cells. Intracellularly, MHC class II molecules are associated with the invariant chain (Ii). Ii can modulate MHC class II-dependent T cell activation through (i) assistance in the export of MHC class II molecules from the endoplasmic reticulum, (ii) providing a targeting signal for endosomal/lysosomal compartments, and (iii) preventing peptides from associating prematurely with MHC class II molecules. Here we describe the generation and subsequent secretion of a lumenal form of Ii, IiP25. IiP25 lacked the targeting sequences for transport to MHC class II compartments but contained part of the CLIP region that is known to compete with antigenic peptides for binding to MHC class II molecules. When added to an antigenic peptide presentation model system, IiP25 inhibited T cell activation by competing for the CLIP binding site at the plasma membrane. Secretion of a lumenal Ii fragment may represent an additional mechanism to modulate T cell activation by MHC class II molecules.

Walking through the forest of transgenic models of human disease.

In the investigation of human disease, molecular biology has provided immunologists with several enormously powerful tools. Transgenic and knockout mice provide animal models to investigate mechanisms, as well as aid in the design of therapies for these diseases. These mice have been useful in several different ways. First, as direct models of disease they provide direct tools for the study of the disease. Second, expression of individual molecules can be altered in the context of established disease models. We describe here some of the models in use as well as the limitations and promise of this research.

Distinct effects of Jak3 signaling on alphabeta and gammadelta thymocyte development.

Janus kinase 3 (Jak3) plays a central role in the transduction of signals mediated by the IL-2 family of cytokine receptors. Targeted deletion of the murine Jak3 gene results in severe reduction of alphabeta and complete elimination of gammadelta lineage thymocytes and NK cells. The developmental blockade appears to be imposed on early thymocyte differentiation and/or expansion. In this study, we show that bcl-2 expression and in vivo survival of immature thymocytes are greatly compromised in Jak3-/- mice. There is no gross deficiency in rearrangements of the TCRdelta and certain gamma loci in pre-T cells, and a functional gammadelta TCR transgene cannot rescue gammadelta lineage differentiation in Jak3-/- mice. In contrast, a TCRbeta transgene is partially able to restore alphabeta thymocyte development. These data suggest that the signals mediated by Jak3 are critical for survival of all thymocyte precursors particularly during TCRbeta-chain gene rearrangement, and are continuously required in the gammadelta lineage. The results also emphasize the fundamentally different requirements for differentiation of the alphabeta and gammadelta T cell lineages.

Local expression of TNFalpha in neonatal NOD mice promotes diabetes by enhancing presentation of islet antigens.

The relationship of inflammation to autoimmunity has been long observed, but the underlying mechanisms are unclear. Here, we demonstrate that islet-specific expression of TNFalpha in neonatal nonobese diabetic mice accelerated diabetes. In neonatal transgenic mice, disease was preceded by apoptosis of some beta cells, upregulation of MHC class I molecules on residual islet cells, and influx and activation of both antigen-presenting cells bearing MHC-islet peptide complexes and T cells. Infiltrating dendritic cells/macrophages, but not B cells, from neonatal islets activated islet-specific T cells in vitro. Thus, inflammation can trigger autoimmunity by recruiting and activating dendritic cells/macrophages to present self-antigens to autoreactive T cells.

Autoimmunity without diabetes in transgenic mice expressing beta cell-specific CD86, but not CD80: parameters that trigger progression to diabetes.

To define more clearly the roles of CD80 (RIP-CD80) and CD86 (RIP-CD86) in the activation of autoreactive T cells in vivo, we generated transgenic mice expressing either or both costimulatory molecules on the beta cells of the pancreas. While RIP-CD80 mice do not show any sign of autoimmunity, at the age of 7 mo RIP-CD86 transgenic mice develop a lymphoid infiltrate with both IFN-gamma- and IL-4-positive cells in the vicinity of the islets; these mice, however, never progress to diabetes. This fundamental difference in the ability of CD80 and CD86 to activate self-reactive T cells in vivo is, however, obliterated when the level of TCR signaling is increased by either TNF-alpha or transgenic MHC class II expression. These results support the suggestion that CD80 and CD86 mainly differ at the level of the intensity of the signals they deliver.

Activation and re-activation potential of T cells responding to staphylococcal enterotoxin B.

To elucidate the parameters that lead to superantigen induced non-responsiveness, an in vitro model for studying primary and secondary responses to the bacterial superantigen staphylococcal enterotoxin B (SEB) was established. Upon re-activation with SEB, in vitro SEB primed T cells show an early proliferative response that 'quenches' in time and is severely impaired 3 days after re-stimulation. Despite their overall impaired proliferative capacity and IL-2 production, these T cells are able to produce IFN-gamma and to up-regulate activation markers CD69 and IL-2R alpha upon re-stimulation with SEB, demonstrating that SEB non-responsiveness is not absolute. Rather, it reflects the inability to mount an ongoing proliferative response upon re-stimulation with SEB. Our results also demonstrate that SEB-induced non-responsiveness is not simply the result of presentation in the absence of co-stimulation, since presentation of SEB on highly purified dendritic cells during the primary response did not prevent the induction of non-responsiveness. As previously shown, SEB induces a Th1 phenotype in responding CD4+ T cells. Skewing towards a Th2 phenotype by adding IL-4 and antibodies to IFN-gamma did not prevent the induction of non-responsiveness by SEB. Interestingly, T cells pretreated with plate-bound anti-CD3 epsilon and anti-V beta 8 were also non-responsive to SEB re-stimulation. Thus, non-responsiveness to SEB (defined here as inability to produce IL-2 and proliferate) seems to reflect an intrinsic inability of previously activated T cells to respond to SEB, probably reflecting differences in signal transduction pathways used by naive versus previously activated T cells.

A molecule expressed on accessory cells, activated T cells, and thymic epithelium is a marker and promoter of T cell activation.

T cell maturation results in part from direct cell-cell interactions between developing thymocytes and thymic stromal cells. Identification of the cell surface molecules involved in these interactions has been approached by production of mAbs reactive to thymic stromal cell surface Ags. A mAb against one such Ag, mouse thymic stroma (MTS) mAb MTS 23, stains a subset of thymic medullary epithelium by immunohistology. In addition, it was found to detect, by flow cytometry, an Ag constitutively expressed on peripheral B cells and macrophages as well as thymic and splenic dendritic cells. This Ag was also up-regulated on T cells and thymocytes within 24 to 48 h after activation. We then investigated whether the Ag identified by MTS 23 may represent a functional accessory molecule. MTS 23 was able to block up to 75% of T cell proliferation in soluble anti-CD3 and Ag-induced responses in a dose-dependent manner, but not under conditions in which no APCs were required. The molecule detected by this mAb has an apparent molecular mass of 120 kDa under reducing and nonreducing conditions. On the basis of these molecular properties and expression pattern, it is therefore postulated that MTS 23 may detect a novel accessory molecule important for T cell activation. Its expression on thymic epithelium is consistent with the notion that T cell development is not solely a consequence of unique molecular interactions, but also of signals arising from combinations of interactions involving molecules also expressed extrathymically.

Parameters of tolerance induction by antigen targeted to B lymphocytes.

We have shown that a foreign protein Ag (the F(ab) fragment of rabbit IgG) becomes a particularly effective tolerogen when it is targeted to B lymphocytes in vivo. This is done by injecting the Ag intravenously into mice in the form of the F(ab) fragment of rabbit anti-mouse IgD antibody (F(ab)) rabbit anti-delta). Our hypothesis is that resting B cells are tolerogenic APC for CD4+ T cells in unprimed animals, and induce Ag-specific nonresponsiveness in the Th cell compartment by presenting Ag without appropriate costimulatory signals. In this report, we find that Ag-activated T cells appear to be resistant to tolerance induction, because F(ab) rabbit anti-delta given 5 days after challenge with rabbit F(ab) in alum adjuvant has little or no effect of the subsequent antibody response. Tolerance also fails in this model when B cells are activated independently of Ag by simultaneous injection of activating concentrations of divalent, IgG mouse anti-delta at the same time as F(ab) rabbit anti-delta. Finally, nonresponsiveness in the T cell compartment is not limited to a lesion in T cell help for the antibody response, because T cells from mice treated with F(ab) rabbit anti-delta and then primed with rabbit F(ab) fragments in CFA show reduced T cell proliferation and IL-2 production when restimulated with Ag in vitro.

Predictability of alloreactivity among unrelated individuals.

Extended haplotypes are specific HLA-B, BF, C2, C4A, C4B and DR allelic combinations that occur at high frequencies and show positive linkage disequilibrium among these highly polymorphic MHC markers. About 30% of all normal caucasian haplotypes are extended, and the matching of two extended haplotypes in unrelated individuals has been shown to match for the determinants of primary mixed lymphocyte reactivity (MLR-I). In this work we report that the matching of one extended haplotype and a serologically defined HLA-DR generic type on the second chromosome in unrelated individuals is associated with the absence of mixed lymphocyte reactivity in 15 to 30% of the cases studied. Our results suggest that, for those individuals who carry either one or two extended haplotypes, it is relatively easy to identify an unrelated MLR-I-matched subject. However, for individuals lacking at least one extended haplotype, it should be difficult to find an MLR-I-matched unrelated subject.

Small B cells as antigen-presenting cells in the induction of tolerance to soluble protein antigens.

We have investigated the ability of resting B cells, acting as antigen-presenting cells, to induce tolerance to soluble protein antigens in mice, using an antigen targeted specifically to B cells. We inject mice intravenously with ultracentrifuged Fab fragments of rabbit anti-mouse immunoglobulin D (IgD) (Fab anti-delta). Treatment with Fab anti-delta results in profound tolerance to challenge with 100 micrograms Fab nonimmune rabbit Ig (Fab NRG), precipitated in alum, as measured by antibody production. Tolerance to rabbit Fab is antigen specific, since the treated mice make normal antibody responses to a control antigen, chicken Ig. Tolerance is dependent on antigen presentation by B cells, since intravenous injection of soluble Fab NRG, which is not targeted to B cells, results in a much lower frequency and degree of tolerance, especially at lower doses. T cell help in this system is affected, since T cells from Fab anti-delta-treated mice fail to provide help for an adoptive primary antibody response to Fab NRG when transferred together with normal B cells into severe combined immunodeficient (SCID) mice. The antigen-specific B cell compartment is also affected during tolerance induction, since B cells from treated animals make less antibody than normal B cells when transferred into SCID mice with normal T cells. Although the mechanism of nonresponsiveness in the helper T cell compartment remains to be determined, we think it is likely that the precursors of helper T cells are inactivated or deleted by encountering antigen presented by small, resting B cells, which lack accessory signals necessary to induce helper T cell proliferation and differentiation to effector function.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen presentation in acquired immunological tolerance.

In acquired tolerance, previous exposure to antigen under certain conditions induces specific unresponsiveness instead of specific immunological memory. It has been studied as an approach to the mechanisms of self-tolerance that operate on immunocompetent T and B lymphocytes once they leave their sites of origin in the thymus and the bone marrow. Possible mechanisms involve induction of specific suppressor cells or inactivation of antigen-specific lymphocytes (clonal anergy) as a consequence of abortive antigen presentation, in which the antigen receptor is effectively engaged but certain poorly defined accessory signals the T lymphocytes require are lacking. We propose that small, resting B lymphocytes, which lack these accessory signals, are the inactivating antigen-presenting cells in acquired tolerance to proteins and to the class II transplantation antigens. B lymphocytes, which can use their antigen receptors to gather and process antigens that are present at very low concentrations, may play a role in self-tolerance. In addition, B lymphocytes and T lymphocytes rendered anergic by encounter with self antigens could persist as self-specific suppressor cells to block an autoimmune response of autoreactive clones that had escaped deletion or anergy.

Demonstration of the antiviral role of natural killer cells in vivo with a natural killer cell-specific monoclonal antibody (NK 1.1).

A monoclonal antibody (NK 1.1) to mouse natural killer (NK) cells selectively depleted NK cell activity in virus-infected mice without significantly depressing other immune functions, including the development of virus-specific cytotoxic T cells. NK cell depletion with this antibody resulted in markedly enhanced plaque-forming unit titers of some (murine cytomegalo, Pichinde) but not other (mouse hepatitis, lymphocytic choriomeningitis) viruses. This confirms that NK cells do play a role in regulating certain infections and shows that this antibody provides a convenient tool for examining the role of NK cells in viral infections.

Identification of HLA-B44 subtypes associated with extended MHC haplotypes.

The HLA class I antigen B44 is found in each of two different extended major histocompatibility haplotypes (allele combinations of HLA-B, HLA-DR, and complement genes BF, C2, C4A, and C4B in linkage disequilibrium). Using isoelectric focusing, two variants of HLA-B44 were identified. The basic variant was found in all cell lines with the extended haplotype HLA-B44, DR7, FC31, and the acidic variant in all cell lines with the extended haplotype HLA-B44, DR4, SC30. The occurrence of each antigen variant with a unique extended haplotype explains previous observations concerning the nonrandom association of B44 variants with DR antigens.

Characterization of HLA-Bw73 by serology and one-dimensional isoelectric focusing patterns.

The HLA-Bw73 antigen has been characterized by antisera in the Ninth International Histocompatibility Workshop. The International Workshop antibodies 9w245, 9w246, and 9w247 detected HLA-B7 and one or more antigens of this group (HLA-B40, Bw22, Bw42, or Bw48) in addition to HLA-Bw73. We have serologically characterized three additional antibodies, in two family studies, which contain anti-Bw73 (two of the antisera also contain anti-B7 activity). We have performed absorption studies with the three antisera, which indicate that anti-Bw73 activity is removed by HLA-B7 positive lymphocytes in two of the antisera and that, in one case, anti-B7 activity is removed by HLA-Bw73 positive HLA-B7 negative lymphocytes. The third antiserum is cytotoxicity negative absorption positive for HLA-B7. Neither HLA-B27 positive nor HLA-B8 positive lymphocytes removed any antibody activity. Using one-dimensional isoelectric focusing, unique bands have been characterized for over 30 Caucasian allotypes, including HLA-B7 and HLA-B27. Lymphocytes from two families carrying the HLA-Bw73 antigen were analyzed by isoelectric focusing. These two families show that HLA-Bw73 has a band migrating in the gel very close to HLA-B35 but distant from the cross-reactive group HLA-B7. These studies indicate that HLA antigens which share common epitopes (including those recently characterized, such as HLA-Bw73 and HLA-B7), can be distinguished serologically and by isoelectric focusing.

Differential expression of T cell differentiation antigens and major histocompatibility antigens on activated T cells during the cell cycle.

In this report we have analyzed cell cycle-related fluctuations of both quantity and density of the T cell differentiation antigens, CD3 (T3), CD4 (T4) and CD8 (T8), as well as the major histocompatibility complex (MHC) antigens on the cell surface of activated T cells. Phytohemagglutinin-activated T cells cultured for 3 days with or without conditioned medium or for 10 days with conditioned medium and mixed lymphocyte culture-derived T cell clones were used for the analysis. Correlated measurements of the surface antigen quantity (immunofluorescence), DNA content (dye Hoechst 33342), and cell size (light scatter), not influenced by synchrony induction methods and cell fixation, were performed by dual-beam flow cytometry. Our results demonstrate that the T cell differentiation antigens, CD3, CD4 and CD8, and class I MHC antigens are increased in density in the G1 phase for all activated T cells tested. In contrast, class II MHC antigens are increased in density in the G2 phase of activated T cells maintained with conditioned medium. Since it is known that the T cell differentiation antigens and class I MHC antigens on activated T cells are necessary for proliferation of T cells, our study suggests that this effect is more significant in the G1 phase. The cell cycle changes in expression of class I and class II MHC antigens, but not of the T cell differentiation antigens, appear to be mediated by soluble factors, probably including interferon-gamma, which could produce a differential increase of class I and class II MHC antigens on G2 phase cells.

Unrelated individuals matched for MHC extended haplotypes and HLA-identical siblings show comparable responses in mixed lymphocyte culture.

Extended haplotypes are specific HLA B, HLA DR, BF, C2, C4A, and C4B combinations in significant linkage disequilibrium in chromosomes of unrelated individuals. The possibility that matching unrelated individuals for extended haplotypes may match for the genes that cause mixed lymphocyte reactivity was tested. 22 of 26 unrelated extended-haplotype-matched subjects had similar mixed lymphocyte reactivity to HLA-identical siblings.

Mechanism of the pressor response to tetradecapeptide renin substrate in the rat.

The synthetic tetradecapeptide renin substrate (TDP; Asp-arg-val-tyr-ile-his-pro-phe-his-leu-leu-val-tyr-ser) has been employed frequently to elucidate the enzymatic action of renin in vitro and, to a lesser extent, in vivo. We assessed the utility of TDP as a renin substrate in vivo using conscious spontaneously hypertensive rats. Intravenous injection of TDP (1 and 3 micrograms/kg) increased diastolic pressure by 45 +r2 and 67 +/- 2 mmHg, respectively. The pressor response to TDP was significantly inhibited by captopril (3 mg/kg, po), indicating its dependence on conversion by ACE to some active molecule. Pressor responses to TDP also were less in animals subjected to bilateral nephrectomy 18-24 hr before study. However, responses to angiotensin I and II also were reduced, implying a non-specific effect of nephrectomy. Intravenous infusion of the renin inhibitor pepstatin (200 micrograms/min) inhibited pressor responses to hog renin by approximately 60%, but did not affect those to TDP. Intravenous infusion of the water soluble renin inhibitor, pepstatinyl-arginine-o-methyl ester (500 micrograms/min), also inhibited pressor responses to renin (approx. 80%) and did not affect those of TDP. Incubation to TDP (5 microM) with rabbit lung ACE resulted in generation of AI that was blocked by captopril (1 microM). These data suggest that TDP is a substrate for ACE and that the increase in blood pressure produced by TDP is due to its sequential cleavage by ACE to AII and can be independent of renin.

Antihypertensive activity of SCH 31846, a non-sulfhydryl angiotensin-converting enzyme inhibitor.

The antihypertensive, hemodynamic, and autonomic actions of SCH 31846, a new, potent and long-acting non-sulfhydryl angiotensin-converting enzyme (ACE) inhibitor, were evaluated in several experimental preparations. Oral administration of 0.3-3 mg/kg caused dose-related decreases in blood pressure in spontaneously hypertensive rats (SHRs). Pretreatment with a diuretic augmented the maximum hypotensive response attainable. Single doses (3 mg/kg) of SCH 31846 reduced pressure for over 24 h. Five-day treatment lowered pressure progressively. Single oral doses of 3.2 and 10 mg/kg reduced blood pressure of conscious normotensive dogs. Diuretic pretreatment also enhanced the response. The antihypertensive action of SCH 31846 in SHRs was eliminated by nephrectomy, but not attenuated by indomethacin, indicating its dependency on renal renin but not on prostaglandin synthesis. Other studies using SHRs pointed to an absence of a central effect. SCH 31846 (1 mg/kg i.v.) decreased blood pressure and peripheral resistance of anesthetized dogs but did not alter cardiac output. Autonomic interactions were examined in normal and diuretic-pretreated SHRs and anesthetized dogs. SCH 31846 affected the response to sympathetic nerve stimulation and cardiovascular reflexes only minimally. It is concluded that SCH 31846 is a potent and long-lasting antihypertensive agent, the action of which is mediated, in all probability, by ACE inhibition.