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IL-23 has been implicated in the pathogenesis of several tissue-specific autoimmune diseases. Currently, celiac disease (CD) is the only autoimmune disease in which both the major genetic (95% HLA-DQ2(+)) and etiologic factors (dietary glutens) for susceptibility are known. We demonstrate that wheat gliadin induces significantly greater production of IL-23, IL-1beta, and TNF-alpha in PBMC from CD patients compared with HLA-DQ2(+) healthy controls, strongly advocating a role for IL-23 in the pathogenesis of CD. Moreover, IL-1beta alone triggered IL-23 secretion and the IL-1R antagonist inhibited this response in PBMC and purified monocytes. This sequence of events was replicated by beta-glucan, another substance known to induce IL-23 production. Our results suggest that gliadin and beta-glucan stimulate IL-23 secretion through induction of the IL-1 signaling pathway and reveal for the first time that the IL-1 system regulates IL-23 production. These findings may provide therapeutic targets for this disease and other inflammatory conditions mediated by IL-23.

Negative selection is the process whereby immature thymocytes expressing TCRs with high affinity for self-peptide:MHC complexes are induced to undergo apoptosis. The transcriptional events that occur as a result of TCR signaling during negative selection are not well-characterized. Using oligonucleotide arrays, we have identified 33 genes that exhibit changes in RNA levels in CD4(+)CD8(+) thymocytes during negative selection in vivo. Of 18 genes that have been further characterized, 13 are regulated in response to stimulation with Ag or anti-CD3 and anti-CD28 Abs ex vivo, indicating that these genes are regulated independently of activation of the peripheral immune system. These data also support the idea that anti-CD3/CD28-mediated thymocyte apoptosis is a valid model for negative selection in vivo. A detailed examination of the regulation of many of the identified genes in response to treatment with dexamethasone or gamma-radiation or in response to anti-CD3/anti-CD28 stimulation in the presence of pharmacological inhibitors of mitogen-activated protein kinase kinase kinase 1, p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase, calcineurin, and cyclin-dependent kinase 2 has facilitated the elucidation of a map of the transcriptional events that occur downstream of the TCR. These studies support a model whereby similar signal transduction pathways are activated by stimuli that induce positive and negative selection and are consistent with the idea that the balance between opposing proapoptotic and antiapoptotic pathways determines cell fate. The data presented in this study also suggest that calcineurin functions to amplify TCR signals by promoting sustained increases in the levels of specific transcripts.

It has been postulated that production of TNF-alpha is central to the pathogenesis of septic shock induced by group B Streptococcus (GBS). In vitro studies using human cord blood monocytes have demonstrated that GBS induces TNF-alpha secretion, but little is known about the intracellular signaling pathways of TNF-alpha induction. In this report we show that heat-killed serotype III GBS induces host cell signal transduction pathways that lead to activation of the transcription factors NF-kappaB and AP-1. Using adenoviral transfer of IkappaBalpha (IkappaBalpha overexpression), the production of TNF-alpha induced by whole GBS was inhibited by only 20%. We also show that the p38 mitogen-activated protein kinase (MAPK) pathway is involved in GBS-induced TNF-alpha secretion, because TNF-alpha protein and mRNA levels in the presence of a specific inhibitor of p38 MAPK, SB 202190, were dramatically diminished. EMSAs showed that SB 202190 inhibited GBS-induced AP-1 activation, but had no effect on NF-kappaB-DNA binding activity. These results indicate that both NF-kappaB and AP-1 (via p38 MAPK) are involved in the regulation of TNF-alpha production in GBS-stimulated neonatal monocytes. Therefore, disrupting the signal transduction pathways induced by GBS has the potential to attenuate the production of immune response mediators, thereby halting or possibly reversing the course of this potentially fatal disease.

HIV-1 envelope-specific CD4+ T cell lines were established simultaneously from PBMC and lymph node mononuclear cells of two HIV-1-infected patients. Three recombinant envelope proteins were used to establish the CD4+ T cell lines: gp160NL4-3, gp120IIIB, and gp120MN. Six T cell lines were established from the first patient, one for each Ag from each compartment, and four T cell lines, two per compartment, were established from the second patient. Each line was challenged with a panel of overlapping peptides spanning the entire gp120 sequence to define its T cell epitope specificity. The pattern of recognition for all the lines from any given patient was similar between compartments. Each patient had a different pattern of peptide recognition. TCR analysis showed a heterogeneous usage of Vbeta between lines with same peptide specificity and established from different compartments. These data suggest that the cellular immune response does not phenotypically vary between the peripheral blood and lymph node compartments, but demonstrates genotypic heterogeneity, showing possible redundancy of the immune response to HIV-1 gp160.

Monocyte/macrophages (MM) were isolated from HIV-1 seronegative individuals, infected with HIV-1 and examined for their ability to infect autologous T lymphocytes with and without concomitant presentation of exogenous Ag. HIV-1-infected MM presented tetanus toxin (TT) and streptokinase to T cells (as measured by [3H]thymidine incorporation) comparable to presentation by uninfected MM. In these studies, it was observed that HIV-1-infected MM without additional exogenous Ag stimulated autologous T lymphocytes, however, to a lesser degree than with TT and streptokinase. Virus production in T cells appeared to be relative to the degree of stimulation with the highest levels of stimulation and infection observed when T cells were exposed to HIV-1-infected TT-presenting MM. Studies were carried out to examine some of the restricting elements in MM-mediated infection of T lymphocytes with and without TT presentation. Antibodies to CD4, as well as soluble immunopurified gp120, blocked cell-mediated infection indicating that infection of T cells was through the CD4 molecule as has been demonstrated with cell-free virus. In addition, soluble gp120 inhibited Ag presentation by HIV-1-infected and uninfected MM. mAb to MHC class II Ag HLA-DR and -DP blocked T cell infection by HIV-1-infected MM with and without presentation of TT. No effect was observed with mAb to MHC class I Ag. These results indicate that virus transmission to T lymphocytes can be mediated by HIV-1-infected MM and that these cells maintain their function as APC. Activation of T cells appears to be important in the process of T cell infection in this system inasmuch as antibodies that block Ag presentation and thus a T cell proliferative signal inhibit infection.

Human peripheral blood lymphocyte subpopulations were analyzed for the presence of B cell alloantigens with a microcytotoxicity assay. B cell alloantigens were found exclusively on sIg-positive lymphocytes and were not present on sIg-negative, Fc receptor-bearing lymphocytes or sIg-negative, Fc receptor-negative T lymphocytes.

Antisera detecting HLA-DRw antigens (human Ia-like) react in cytotoxicity assays with peripheral blood B cells and monocytes but not T lymphocytes. Antisera detecting DRw specificities were used in flow microfluorometry studies to determine whether T lymphocytes express these antigens in quantities not detected by cytotoxicity. Peripheral blood lymphocytes depleted of monocytes were incubated with DRw antisera, reacted with fluoresceinated goat anti-human IgG, and analyzed on the fluorescence-activated cell sorter (FACS). Anti-DRw sera were found to be reactive on all of the lymphocytes as evidenced by a positive fluorescence signal. The reactivity was specific in that only those sera that detected DRw antigens on B cells and monocytes were reactive with the total lymphocyte population from the same donor. Reactivity of the antisera was removed by absorbing the antiserum with monocyte/B cells and T cells from a donor with the same DRw antigen as that found on the target cell of the donor tested. The results demonstrate the presence of DRw antigens on resting peripheral blood T lymphocytes. The implications of quantitative differences in DRw antigen expression on lymphoid cells are discussed.

Human T cell lymphocyte lines, established from lymphoid tissues from patients with adult T cell malignancies infected with the human T cell lymphoma virus (HTLV), were used in co-culture experiments to infect newborn cord blood lymphocytes (CBL). The infected and non-infected CBL cell lines were typed for HLA alloantigen determinants and tested for cell surface antigens using selected monoclonal antibodies. Infected cord blood lymphocytes showed inappropriate expression of alloantigenic determinants of the HLA-A and -B alleles. The monoclonal antibody 4D12, detecting an antigen common to the HLA-B5 cross-reactive group, was reactive with all infected cultures; this determinant appeared de novo in CBL cells lacking B5 cross-reactive group antigens in the uninfected state, and it increased in density in the infected cultures where the HLA-B5 cross-reactive alloantigens were present in uninfected CBL. HLA-DR was expressed in low levels or not detected on non-infected cultured cord blood lymphocytes and was present on all infected cells. OKT4 positive cells predominated in infected cultures, whereas OKT8 positive cells decreased in number or were absent. The TCGF receptor also increased both in density and in percent positive cells in all infected cell lines. The results suggest that HTLV may be tropic for a subset of T cells expressing mature T cell markers and that viral infections directly affect expression of cell surface antigens controlled by the human major histocompatibility complex (MHC).

The human class II, HLA-linked, B cell alloantigens include the HLA-DR, MB, MT, and Te determinants. Interest in the molecular relationships of these antigens has recently intensified because of their homology to the murine Ia antigens and their possible importance in disease predisposition and transplantation. We have used alloantisera with carefully defined immunochemical as well as serologic specificity, and two immunochemical techniques, sequential immunoprecipitation with analysis by SDS-PAGE and two-dimensional gel electrophoresis, to explore the molecular relationships of the MT2, MB3, MT4, and HLA-DR5 antigenic determinants. The data presented here indicate that 1) all class II molecules that bear the DR5 antigenic determinant also bear the MT2 antigenic determinant; (2) the homozygous DR5 cell line, Swei, expresses at least two structurally distinct class II molecules, both of which bear MT2: one bears the MT2, MB3, and MT4 antigenic determinants, and the second bears the MT2, but not the MB3 or MT4 antigenic determinant; and (3) the DR5 determinant is located on at least one and possibly both of these distinct class II molecules.

The addition of inactivated preparations of purified human T cell lymphotropic virus (HTLV-III) was found to inhibit normal human lymphocyte phytohemagglutinin (PHA)-induced blastogenesis but had no effect on concanavalin A (Con A), pokeweek mitogen, or allogeneic stimulation. The inhibition was concentration-dependent and also dependent on adding the virus preparation before or at the same time as PHA. The CD4 molecule is the receptor for HTLV-III binding. Immunopurified large envelope protein (gp120) from HTLV-III was found to bind to the CD4 molecule and also inhibited PHA-induced lymphocyte blastogenesis. These results suggest that the gp120 viral protein may alter immune function by binding to the CD4 molecule, which in turn serves as an "off" signal to lymphocyte response to PHA stimulation. Alternatively, by binding to the CD4 molecule, gp120 may interfere with the interaction of this molecule with class II histocompatibility antigens on accessory cells, thus selectively suppressing PHA response.

Alloantigens specific for human B lymphocytes can be identified with selected antisera. These antigens have similarities to murine Ia antigens in that they are found on human B lymphocytes and are controlled by genes linked to genes controlling HLA. Chronic lymphocytic leukemia cells bearing B cell antigens were labeled with 3H leucine and the membrane components reacting with the B cell antisera isolated by immunoprecipitation. These membrane components had m.w. of 33,000 and 24,000 daltons similar to the murine Ia antigens. The results complete the homology of murine Ia and human B cell alloantigens.

Heterologous antisera directed against human B cell antigens were tested for their ability to inhibit the function of Fc receptors on human lymphocytes. These antisera coated on the attacking cells in a lymphocyte-dependent antibody-mediated cytotoxic assay inhibited lysis of sensitized target cells. However, the F(ab)2 fragments of the IgG fraction of an anti-human Ia antiserum failed to inhibit the killing of sensitized targets by attacking cells. Antisera against other lymphocyte membrane antigens also inhibited target cell lysis when coated on attacking cells. These results suggest that there is not identity between human B cell antigens and Fc receptors of antibody-dependent cytotoxic lymphocytes.

The primary interaction of HIV-1 with the target cell involves the viral large envelope protein (gp120) and the cellular CD4 molecule. mAb reacting with portions of CD4 have been shown to block HIV-1 attachment and infection. In one of the early reports describing HIV-1 cell interaction, some mAb reacting with MHC class II Ag were also found to block infection. To investigate further a possible role for MHC class II in HIV-1 binding, a cultured T lymphocyte cell line (H-9) that expresses MHC class II molecules and PHA-stimulated PBL was exposed for various time periods to concentrated viral particles and individual HIV-1 proteins. A decrease in the ability to detect the CD4a epitope and HLA-DR was observed after the cells were exposed to virus for 15, 30, and 60 min whereas HLA-DP and HLA-DQ Ag increased or remained unchanged. After 120 min of virus exposure, the CD4a epitope remained diminished whereas HLA-DR was detected at levels found on cells not exposed to virus. mAb detecting the CD4a epitope and HLA-DR, as well as alloantisera detecting the specific HLA-DR Ag on the target cell, blocked HIV-1 binding. When immunopurified gp120 was added to PHA-stimulated and unstimulated PBL, the CD4a epitope decreased in the same manner as was observed with whole virus preparations. In contrast to exposure to the intact virus, HLA-DR expression appeared to increase. Other viral proteins, p17, p24, and a portion of the small envelope protein, gp41, had no effect on the ability to detect cell surface Ag. Thus, although CD4 is the primary receptor for HIV-1 binding, HLA-DR appears to be involved in the binding site, probably by virtue of its close proximity to the CD4 molecule on the cell surface.

Mono and oligosaccharides were used to inhibit the cytotoxicity of antisera detecting HLA-DRw antigens on monocytes isolated from peripheral blood. Certain sugars preferentially inhibited antisera detecting some DRw antigens and not others. Sera detecting HLA-A and B antigens were not inhibited with the sugars used. Anti-DRw sera blocked the attachment of radiolabeled sugars to monocytes with the same patterns as the reciprocal blocking of cytotoxicity by the sugars. The results were not consistent with the intrepretation that carbohydrates are an antigenic "component" of DRw specificity, but rather that the surface structures, possibly those bearing the DRw antigens, are receptors for mono and oligo saccharides.

This study describes the monoclonal antibody 5E9 and the cell surface antigen it defines. The hybridoma cell line T3-5E9 was derived from fusion of P3 X 63/Ag8 myeloma cells and spleen cells from BALB/c mice immunized with HSB-2 cells, a human T cell line. Although binding to only 1 to 5% of peripheral blood (PB) and spleen mononuclear cells, 5E9 antibody bound to 40 to 80% of Con A-, PHA-, or PWM-activated PB cells. Moreover, 5E9 antibody bound to variable numbers of Sezary, acute myelogenous leukemia, and ALL PB leukemia cells. 5E9 antibody bound to all hematopoietic and nonhematopoietic cell lines tested, to 11 +/- 1% of thymocytes, and 40% of nucleated bone marrow cells. Under reducing conditions, immunoprecipitation studies using 5E9 antibody demonstrated 5E9 antigen to be an 90,000 m.w. glycoprotein. Under nonreducing conditions, antigen 5E9 is a disulfide-linked dimer of approximately 190,000 daltons. Sequential precipitation experiments using antibody 5E9, alpha OD heteroantiserum (raised against T ALL cells), and monoclonal antibody OKT9 demonstrated that the 3 antibody preparations recognized the same 90,000 m.w. glycoprotein. Thus, antibody 5E9 defines an 90,000 m.w. human cell surface antigen that is absent on the majority of PB mononuclear cells and is expressed on rapidly dividing normal and malignant human cells. This monoclonal antibody should be a useful marker of human cell activation.

It is widely recognized that antisera that interact with determinants encoded by the Ia region of the mouse and its counterpart, the DR locus of man, are capable of interfering with the ability of monocytes and lymphocytes to respond in vitro to an antigenic stimulus. Using an in vitro assay that measures antigen-specific proliferation of human lymphocytes, we found that both a heteroantiserum raised in rabbits (anti-P29,34) and alloantisera which recognize determinants encoded for by the human DR locus dramatically block in vitro antigen-specific proliferation. These anti-DR antisera appear to act at the level of the monocyte; monocytes pulsed and washed free of excess antisera fail to promote proliferation in the presence of soluble antigen and untreated T cells whereas identically pulsed and washed T cells respond normally with untreated monocytes. Furthermore, the addition of unpulsed monocytes fails to restore in vitro antigen-specific reactivity. Our data suggest that membrane-bound anti-DR-specific antisera on monocytes is profoundly suppressive, and also suggests simple steric hindrance may not account for all of the observed effects such antisera have on monocyte-T cell interactions.

Antibodies which react with products encoded by the human DR locus precipitate a biomolecular membrane glycoprotein complex with m.w. of 29,000 and 34,000 daltons. Such antibodies are directed against HLA-DR Ia-like antigens found on human B cells and human monocytes and have been shown previously to inhibit a variety of in vitro cellular and humoral immune reactions. We have compared the in vitro effects of such antibodies on two assays of human immunity: antigen-specific proliferation and polyclonal immunoglobulin production. Intact IgG fractions of a rabbit heteroantiserum (anti-P29,34), a human MT-2 locus alloantiserum (Ia 172), and a mouse monoclonal antibody (LB 3.1) markedly inhibited in vitro immune reactivities of human mononuclear cells. Interestingly, F(ab')2 preparations of these antibodies failed to inhibit these in vitro immune responses at the concentrations tested. These data suggest that under certain conditions, Fc domains play a critical role in the inhibitory activity of antibodies to Ia-like molecules.

Treatment of lymphoid cell preparations with ammonium chloride buffers to remove contaminating erythroid cells markedly reduced the ability of these cells to function as attacking cells in antibody dependent cell mediated cytotoxicity tests (ADCC). The lymphoid cells, however, retained their ability to adhere to antibody-coated target cells. Recovery of function 20 hr after exposure to ammonium chloride suggests a metabolic rather than receptor effect of these salts on the ADCC function of lymphocytes.