Functional analysis of cloned macrophage hybridomas. VII. Modulation of suppressor T cell-inducing activity

We have previously characterized a macrophage hybridoma clone, termed clone 59, which induced immunity but consistently failed to induce Ts responses. Macrophage 59 cells were cultured with supernatants from several activated T cell clones to determine if lymphokines could modulate the activity of this macrophage hybridoma to generate effector Ts. Culture supernatants from Th1 clones and from one atypical IL-4 and IFN-gamma-producing T cell clone successfully modulated clone 59 cells to induce effector Ts cells. In contrast, supernatants from activated Th2 cells failed to generate Ts-inducing activity in macrophage 59 cells. Culture with recombinant derived IFN-gamma was sufficient to cause modulation of Ts-inducing activity in macrophage 59 cells. The data imply that the differential functional activities ascribed to various macrophage hybridoma clones reflect macrophage heterogeneity instead of independent macrophage lineages. The suppression induced by clone 59 macrophages was genetically restricted to the putative I-J region. The ability of IFN-gamma containing supernatants to endow macrophage 59 with the capacity to induce effector suppressor cells was specifically abrogated by addition of an anti-IJk-idiotype antibody, which also reacts with IJ-interaction molecules, indicating that the mechanism of modulation most likely involves expression of IJ-interaction molecule determinants on antigen presenting cells.     

Functional analysis of cloned macrophage hybridomas. IV. Induction and inhibition of mixed lymphocyte responses

A series of macrophage (M phi) hybridomas were generated by fusion of drug-marked P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells. The ability of this panel of cloned M phi hybridomas expressing various levels of surface Ia antigens to induce allogeneic mixed lymphocytes responses (MLR) was examined. All MLR stimulatory M phi hybridomas expressed surface Ia antigens. However, some Ia+ and all Ia- M phi hybridomas were unable to induce vigorous MLR responses. Furthermore, even after induction of surface Ia antigen expression with Con A supernatants (Con A Sn) or purified interferon-gamma, the nonstimulatory M phi hybridomas remained ineffective at inducing strong MLR proliferative responses. Furthermore, addition of the latter M phi hybridoma clones (both with and without Con A Sn treatment) to conventional MLR cultures resulted in inhibition of MLR responses. The series of inhibitory M phi hybridomas secreted normal levels of IL 1 upon stimulation with lipopolysaccharide. After surface Ia induction with Con A Sn, the inhibitory M phi hybridomas could stimulate secretion of IL 2 and expression of IL 2 receptors. Moreover, although they inhibited conventional MLR responses, IL 2 production and IL 2 receptor expression were not significantly inhibited. Addition of these M phi hybridomas 24 to 48 hr after initiation of MLR response also inhibited MLR proliferation. The results indicated that the group of inhibitory M phi hybridomas can inhibit MLR responses after IL 2 secretion and acquisition of IL 2 receptors. Finally, this inhibitory activity has been maintained during 1 yr of continuous in vitro culture, and the hybridomas represent a stable "homogeneous" subpopulation of inhibitory macrophages. Thus, the inhibitory phenotype appears to reflect arrest at a distinct differentiation stage.     

Functional analysis of cloned macrophage hybridomas. VI. Differential ability to induce immunity or suppression

We previously screened a series of macrophage hybridomas derived from fusion of P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells for their ability to induce I-J restricted Ts cell responses. One Ia+ macrophage clone (63) consistently induced Ag-specific, I-J-restricted Ts. To evaluate whether macrophage hybridoma 63 also induced delayed-type hypersensitivity (DTH) immunity, mice were immunized with hapten-coupled macrophage hybridoma cells. Hapten-coupled splenic adherent cells and control macrophage hybridomas induced significant primary DTH responses, whereas hapten-coupled macrophage 63 induced little or no immunity when injected into H-2 compatible hosts. However, macrophage hybridoma 63 specifically activated I-Ak, I-Ad, or I-Ed restricted T cell hybridomas/clones, in vitro in the presence of appropriate Ag. Three different strategies designed to eliminate suppressor cell activity were successfully used to demonstrate that hapten-coupled macrophage 63 could also induce in vivo immunity. First, after immunization with hapten-coupled macrophages, mice were treated with cyclophosphamide. Second, macrophage 63 was treated with anti-IJ idiotype antibody before 4-hydroxy-3-nitrophenyl acetyl hapten (NP) coupling. Finally, haptenated macrophages were injected into I-A compatible but I-J incompatible recipients. These protocols are known to inhibit the induction of Ts activity, thus these results indirectly suggest that there is stimultaneous generation of Ts activity in vivo. The latter hypothesis was tested in adoptive transfer experiments. Transfer of lymph node cells from NP-63 primed B10.BR (H-2k) mice induced immunity in naive 4R animals, whereas the same number of immune cells suppressed NP-induced DTH responses in 5R mice. The combined results indicate that a cloned macrophage line can activate both Th and Ts cells. Macrophages which induce Ts activity may be responsible for maintaining the balance of immunity vs suppression. The data support the hypothesis that IJ interacting molecules (IJ-IM) expressed on macrophages are critical for induction of suppressor cell activity.     

Production of human suppressor T cell hybridomas

To study human T cell suppression of immunoglobulin (Ig) synthesis with homogeneous populations of immunoregulatory cells, human suppressor T cell hybridomas were prepared by somatic cell fusion of concanavalin A-activated peripheral blood T cells with hypoxanthine-guanine phosphoribosyltransferase-(HGPRT, EC 2.4.2.8) deficient human leukemic CEM T cells. After selection in hypoxanthine-aminopterin-thymidine (HAT) medium and cloning by limiting cell dilution, two human T cell hybridomas were identified that produced 60 to 80% suppression of in vitro polyclonal immunoglobulin production when cocultured with pokeweed mitogen- (PWM) stimulated peripheral blood lymphocytes. Further, one of the suppressor T cell hybridomas constitutively secreted a soluble suppressor factor(s) (TsF) of m.w. 70,000 to 85,000 daltons, which produced reversible noncytotoxic inhibition of lectin-activated B cell Ig production. In contrast, this TsF did not inhibit lectin- or antigen-induced T cell proliferation, nor did it interfere with the generation or effector function of cytotoxic T cells. Additional studies indicated that this Tsf acts directly on B cells or monocytes rather than indirectly modulating the activity of immunoregulatory T cells. In summary, these studies suggest that techniques of somatic cell fusion may provide a valuable approach to further study human immunoregulatory cell-cell interactions as well as provide a source of sufficient quantities of important lymphokines for further purification and characterization.     

Functional analysis of macrophage hybridomas. I. Production and initial characterization

A series of macrophage hybridomas were generated by fusion of splenic adherent cells with P388D1 tumor cells. Forty-two cell lines were established, and each was cloned by limiting dilution. Six clones that exemplified the spectrum of macrophage heterogeneity were selected for further analysis. Qualitative and quantitative differences in phenotype and functional activity were noted. Some clones constitutively expressed Ia antigens, whereas others only expressed detectable levels of Ia after lymphokine activation. The level of antigen-presenting activity generally correlated with the level of Ia expression. Furthermore, interclonal differences were noted in the levels of receptor-mediated phagocytosis and IL 1 secretion. Generally, the hybridoma clones maintained stable phenotypic and functional properties during approximately 1 yr of continuous in vitro culture. These cloned hybridoma cell lines represent a useful resource to analyze macrophage biology and to dissect structure and function relationships.     

Prostaglandin E2 inhibits the activation of cloned T cell hybridomas

To minimize complicating interactions inherent in heterogeneous cell populations, we used a panel of cloned murine autoreactive (E8.A1) and antigen-specific (HEL.C10, HEL.B14) T cell hybridomas to examine the effect of prostaglandin E2 (PGE2) on T cell activation. These T cells secrete interleukin 2 (IL 2) when co-cultured with a cloned population of I region-matched stimulator cells (TA3), or with mitogenic signals in the absence of TA3 stimulator cells. Physiologic concentrations of PGE2 inhibited the induction of IL 2 secretion by the T cell hybridomas tested, when they were activated either by TA3 cells or by mitogenic signals. IL 2 production was inhibited in a dose-dependent manner by concentrations of PGE2 between 10(-7) and 10(-11) M, with 50% inhibition occurring at 10(-10) M. Pretreatment of the T hybridoma cells with 10(-7) M PGE2 for 1 hr before culture also resulted in marked inhibition of IL 2 secretion. Similar pretreatment of the TA3 cells did not affect their ability to activate the T cell hybridomas. PGE2 at 10(-8) M induced a 30-fold increase in cAMP levels within 25 min of addition to culture of the E8.A1 T cell hybridoma, but caused no significant elevation of cAMP levels in TA3 cells. The direct addition of dibutyryl cAMP (dcAMP) to cultures of E8.A1 cells resulted in marked inhibition of IL 2 secretion when stimulated by TA3 or by mitogenic signals, with an average of 80% inhibition occurring at 10(-4) M dcAMP. PGE2 and dcAMP also inhibited the growth of E8.A1 cells. Initially, cell growth was virtually halted, but began to recover between 24 and 48 hr after the addition of either PGE2 or dcAMP. Neither PGE2 nor dcAMP inhibited the division of TA3 cells. High affinity binding sites for PGE2 were detected in the E8.A1 T cell hybridomas with an apparent Kd of 7.6 X 10(-10) M, which is consistent with the functional data. No specific binding was detected in the TA3 stimulator cells. These findings suggest that the immunosuppressive effects of PGE2 are localized to the T cell, are receptor regulated, and may be mediated by the associated increase of cAMP levels in the T cell hybridomas.     

Relationships between antigen-specific helper and inducer suppressor T cell hybridomas

Ts1, or inducer suppressor T cells, share many phenotypic and functional characteristics with helper/inducer subset of T cells. In order to evaluate the relationship between these cell types, we made a series of new Ts1 hybridomas by the fusion of Ts1 cells with the functionally TCR alpha/beta-negative BW thymoma (BW 1100). Three Ts1 hybridomas (CKB-Ts1-38, CKB-Ts1-53, and CKB-Ts1-81) were established that express TCR and produce Ag-specific suppressor factors constitutively, thus making it possible to study the nature and specificity of Ag receptors, MHC restriction, and lymphokine production by the Ts1 hybridomas. Results presented in this report demonstrate that all the Ts1 hybridomas described here express CD3-associated TCR-alpha beta. These three Ts1 hybridomas recognize Ag (NP-KLH) specifically in a growth inhibition assay and this recognition is restricted by IE molecules. Two of the hybridomas also produce IL-2 or IL-2 and IL-4 upon Ag-specific activation. Thus, by these three criteria the Ts1 hybridomas appear indistinguishable from Th cells. These three Ts1 hybridomas, however, release suppressor factors (TsF1) in the supernatant that suppress both in vivo DTH and in vitro PFC responses in an Ag-specific manner. Like the TsF1 factors characterized previously, the suppression mediated by these factors are Igh restricted and lack H-2 restriction. These factors mediate suppression when given in the induction phase but not during the effector phase of the immune response. The TsF1 factors are absorbed by Ag (NP-BSA), and anti-TCR affinity columns and the suppressor activity can be recovered by elution. The data are consistent with the interpretation that Ts1 inducer-suppressor T cells are related to Th cells; the feature that distinguishes these cells is the ability to produce Ag-binding factors that specifically suppress immune responses.     

Analysis of cloned T cell function. I. Dissection of cloned T cell proliferative responses using cyclosporin A

CsA interferes in a specific manner with the expansion of T cell clones in that it inhibits the antigen-driven component of the proliferative responses made by cloned helper T cells, cloned conventional cytolytic T cells, and cloned helper-independent cytolytic T cells. Cloned helper T cells and helper-independent cytolytic T cells, which share the ability to proliferate when cultured with specific alloantigen, fail to proliferate when cultured with specific alloantigen, fail to proliferate in response to this stimulus in the presence of CsA (10 to 100 ng/ml). In contrast, the proliferation observed when these cells are cultured with exogenous growth factors (but not alloantigen) is little influenced by as much as 1000 ng/ml CsA. When cloned helper T cells or helper-independent cytotoxic T cells are cultured with alloantigen plus exogenous growth factor, additive or synergistic proliferation occurs. However, CsA (10 to 1000 ng/ml) blocks only the component of proliferation induced by alloantigen, and leaves the lymphokine-driven component intact. CsA has similar effects on the proliferation of cloned conventional cytolytic T cells. Thus, CsA separates cloned T cell proliferation into two components: one driven by contact with alloantigens, the other driven by contact with mitogenic lymphokines.     

Functional analysis of macrophage hybridomas. III. Inhibition of lymphocyte blastogenesis and tumor proliferation

Four cloned macrophage hybridoma cells prepared by fusion of splenic adherent cells with a P388D1 macrophage tumor markedly inhibited the growth of lymphocytes and tumor cells. Macrophage clones 5, 8, 63, and 64 are strong inhibitors of B-cell blastogenesis, T-cell blastogenesis, and tumor proliferation, while the P388D1, tumor line and clones 13, 59, and 67 demonstrated little inhibitory activity in all three systems. The inhibitory effect of macrophage hybridomas can be detected within 8 hr, although greater inhibition was noted following longer incubation. The correlations among these three assay systems suggest that similar mechanisms may be involved. The data indicate that the inhibition of cell proliferation was not due to cell lysis. Furthermore, the inhibition of lymphocyte proliferation in Con A-stimulated cultures was not accompanied by inhibition of lymphokine production in the same cultures. Neither prostaglandins nor hydrogen peroxide appear to be primarily responsible for growth inhibition. The inhibitory properties of these macrophage hybridoma lines represent a stable phenotype and provide a homogeneous source of cells for further analysis of the phenomenon.     

Expression of cell surface antigens by suppressor T cell hybridomas. I. Comparison of phenotype and function

The phenotypic expression of cell surface markers by T cell hybridomas that elaborate suppressor factors specific for the polymers L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) or L-glutamic acid50-L-tyrosine50 (GT) has been analyzed. We found that determinants encoded by the I-J subregion of the H-2 complex were borne on the surface of these hybrid cells and on the factors they secrete, whereas I-J determinants were not expressed by the AKR thymoma fusion parent, BW5147. The level of expression of I-J determinants fluctuated widely depending upon culture conditions, but I-J products and other cell surface markers of normal T cells could be quantitatively increased, or induced to appear, by treatment of the hybridomas with chemical agents, such as dimethyl sulfoxide (DMSO) or phorbol myristate acetate (PMA). In contrast, the surface expression of the viral product gp70 was decreased by the same treatment. Using chemical induction, we typed BW5147, a group of antigen-specific suppressor T cell hybridomas, and two control hybridomas for expression of I-J, Thy-1, Lyt, and H-2K alloantigens. Also, a haplotype-specific hybridoma that produces an antigen-nonspecific factor was analyzed. The results demonstrated that BW5147 failed to express I-J or Lyt alloantigens but expressed Thy-1.1 and H-2Kk gene products. The pattern of expression of these antigens by T cell hybridomas was very complex, but three conclusions could be drawn: 1) Good correlation exists between the expression of certain I-J determinants and the ability of T cell hybridomas to produce suppressor factor. 2) The expression of Thy-1, Lyt, or H-2Kk determinants is variable, and no correlation was found between expression of these antigens and the ability to produce active suppressor factors. 3) I-Jk products contributed by the AKR thymoma fusion partner are expressed by T cell hybridomas.     

Association of glycosylation-inhibiting factor with plasma membranes of T suppressor cell hybridomas

The glycosylation inhibiting factor (GIF) was detected in EGTA extracts of the OVA-specific Ts cell hybridoma, 231F1 cells and 71B4 cells, which constitutively secrete GIF. The lymphokine in both culture supernatants and EGTA extracts failed to bind to OVA-Sepharose. Association of GIF with the plasma membrane was confirmed by surface labeling of the 231F1 cells with 125I. The major species of GIF in the extract was 14.4-kDa peptide as determined by SDS-PAGE, and was identical to that detected in culture supernatants. Pretreatment of the cells with monoclonal anti-GIF switched the cells from the formation of unglycosylated IgE-BF to the formation of glycosylated IgE-BF, indicating that the membrane-associated GIF is involved in the determination of the nature of IgE-binding factor during their biosynthesis. When the hybridoma was stimulated with OVA-pulsed APC, EGTA extracts of the cells contained GIF having affinity for OVA. The binding of the OVA-binding GIF in the EGTA extracts to OVA-Sepharose was inhibited by a synthetic peptide, which corresponds to amino acid residues 307-317 in the OVA molecule and represents the epitope recognized by TCR on the cells. The OVA-binding GIF in the extracts bound to the monoclonal anti-TCR-alpha chain, H-28-710 and the mAb 14-12, which is specific for the Ag-binding chain of effector type suppressor factor, and suppressed the in vivo antibody response of BDF1 mice to DNP-OVA in a carrier-specific manner. Evidence was obtained that indicated that the Ag-binding chain was associated with nonspecific GIF chain on the cell surface of the Ag-stimulated cells.     

Structure and expression of Fc gamma receptors on mouse suppressor T cell hybridomas

Antigen-specific and idiotype-specific mouse suppressor T cell hybridomas were analyzed for the presence and specificity of Fc gamma receptors (Fc gamma R) by EA rosetting and by flow microfluorometry with the use of monoclonal antibodies. We found that four hybridomas expressed Fc gamma R specific for IgG1 and IgG2b, one of which became Fc gamma R- during prolonged culture. Four other hybridomas and the fusion parent, BW5147, consistently lacked Fc gamma R. The 125I-labeled Fc gamma R were isolated from surface radioiodinated hybridoma cells solubilized with 1% Nonidet P-40, were purified by using single or repetitive chromatography on mouse IgG-Sepharose columns, and were analyzed by SDS-PAGE. An 125I-labeled 56,000 to 61,000 Mr macromolecule was isolated from each of the Fc gamma R+ hybridomas, but from none of the Fc gamma R- hybridomas nor from BW5147 cells. This macromolecule rebound to insolubilized mouse IgG1, IgG2b, and human Fc fragments, but not to insolubilized mouse IgG2a, IgG3, or IgA or human F(ab')2 fragments, consistent with the specificity observed for Fc gamma R on intact hybridoma cells. The mouse suppressor T cell Fc gamma R differs in size and specificity from mouse B cell Fc gamma R. A 70,000 Mr protein expressed on all hybridomas and on BW5147 cells was radiolabeled and, despite preclearing with ovalbumin-Sepharose, bound to the mouse IgG-Sepharose columns, presumably due to mouse antibodies to gp-70. This macromolecule was completely and specifically removed by using goat antiserum to gp-70.     

Cell cycle-dependent expression of antigen-binding and I-J-bearing molecules on suppressor T cell hybridomas

The I-J and antigen-binding chains with constant region determinant (Ct) that compose an antigen-specific suppressor T cell factor were found on the surface of suppressor T cell hybridomas, serologically and morphologically demonstrated by a fluorescence-activated cell sorter (FACS) and immunoelectron microscopic analyses. Moreover, the surface expression of the I-J and Ct-bearing chains fluctuating with the same kinetics depended entirely upon the cell cycle. The maximum expression of these two chains was observed in the early stage of the M phase, and the minimum in the S phase. Similarly, the magnitude of the suppressor activity was maximal in the late stage of the M phase, and was minimal in the S phase. The results therefore demonstrated that there exists good correlation between the cell surface expression of the I-J and Ct-bearing chains and the magnitude of the suppressor activity produced. The antigen recognition units on suppressor T cell hybridomas have serologically and morphologically been characterized by using radiolabeled antigens or monoclonal antibodies against the I-J or Ct on the antigen-binding molecule. Cell-binding assay and radioautographic analysis demonstrated that the suppressor T cell hybridoma possesses the capacity to bind native antigen in an antigen-specific fashion as does the hybridoma-derived, antigen-specific suppressor factor composed of the I-J and the Ct-bearing chains, indicating that the recognition unit on the cell surface is composed of a structure similar to the factor.     

Antigen presentation to T cell hybridomas by a macrophage cell line: an inducible function

We have investigated the ability of an Ia-, nonantigen-presenting macrophage tumor cell line, P388D, (H-2d), to present antigen to T cell hybridomas after incubation in a lymphokine-containing preparation. P388D, cells were incubated in microtiter wells with various concentrations of Con A-stimulated spleen cell supernatants. Antigen-specific stimulation of H-2d-restricted, KLH-specific T cell hybridomas was observed by P388D1 incubated with SUP.P388D1 cells incubated for 3 days in medium or control SUP did not present antigen. In addition, no stimulation of T hybridomas was seen by P388D1 in the inhibited by the appropriate monoclonal anti-Ia reagents. These results demonstrate that a macrophage tumor cell line can be induced to present antigen and provides for large numbers of readily available, homogeneous macrophages for studying the cellular biochemical requirements for antigen processing and presentation.     

Expression of functional alpha beta T cell receptor gene rearrangements in suppressor T cell hybridomas correlates with antigen binding, but not with suppressor cell function

We have examined the expression of TCR genes in 4-hydroxy-3-nitrophenyl-acetyl (NP)-specific Ts cell hybridomas. Each of three independently isolated hybridomas expressed in-frame TCR alpha-chain rearrangements derived from the original suppressor Ts cell. Different V alpha and J alpha gene segments were rearranged and expressed in each Ts cell line. The only TCR beta-chain expressed in these cells was derived from the BW5147 fusion partner. Expression of the BW5147 beta-chain was found to correlate with cell surface Ag binding, inasmuch as subclones derived from one of the original Ts lines expressed greatly reduced levels of beta-chain mRNA and no longer bound to NP-coupled RBC. Subclones that continued to express beta-chain mRNA did bind to NP-coupled RBC. This suggests that the Ag receptor on Ts hybridomas is a TCR-alpha beta dimer composed of a unique alpha-chain and the BW5147 beta-chain. Ag binding could be modulated by preincubation of Ts hybridoma cells with anti-TCR-alpha beta antibody, thereby supporting this conclusion. Suppressor factor activity was measured in the conditioned media of Ts subclones that differed by 250-fold in levels of beta-chain mRNA expression. No difference in suppressor factor activity was found; conditioned media from these subclones suppressed both plaque-forming cell responses and delayed-type hypersensitivity responses at approximately equivalent dilutions. Suppressor factor activity in the conditioned media of both a beta-chain negative subclone and a beta-chain positive subclone could be absorbed with an antibody that recognizes the TCR alpha-chain, but not with an antibody that recognizes the TCR beta-chain. We conclude that suppressor factor activity in the conditioned media of these Ts hybridomas is not derived from surface TCR-alpha beta receptors, although it does share TCR alpha-chain determinants.     

Specific in vivo suppression of lymph node cell proliferation and humoral immune responses by cloned antigen-specific T suppressor cells

Two BSA-specific Ts cell clones have been isolated from CBA/J mice tolerized by low doses of BSA. Together with one Ts cell clone isolated from an immune animal, they have recently been characterized with regard to phenotypes and in vitro functions. In the present report the in vivo effector functions of two of them (Ts cell clones BVI/5 and HF1.MS) are described. BSA-primed lymph node cells from CBA/J mice, which had received BVI/5 or HF1.MS Ts cells at the time of immunization, do not respond to a subsequent in vitro antigenic challenge. A human gamma-globulin (HGG)-specific lymph node cell proliferation is not influenced. BVI/5 Ts cells injected into mice at the time of priming with fluorescein (Flu)-conjugated BSA or Flu-HGG inhibit the humoral anti-Flu-response in Flu-BSA-primed animals. The anti-Flu-response in Flu-HGG-primed animals is only marginally affected by BVI/5 Ts cells. The data show that it is possible to induce immunologic unresponsiveness at the humoral level by reexposing in vitro propagated Ts cell clones to their syngenic in vivo environment.     

Inhibition of IgE production in B hybridomas by IgE class-specific suppressor factor from T hybridomas

The function of IgE class-specific suppressor factor (IgE-TsF) from T hybridomas was studied by employing IgE-producing B hybridomas. IgE-TsF was obtained from IgE class-specific T hybridomas, which had been established by the fusion of a phosphorylcholine-conjugated Mycobacterium-primed T cell population with the T lymphoma cell line BW5147. The absorption experiments showed that IgE-TsF from T hybridomas was composed of the binding site(s) for IgE and I region gene products as observed in conventional IgE-TsF. Incubation of IgE-producing B hybridomas with IgE-TsF for 1 hr at 37 degrees C resulted in the reduction of the number of IgE-secreting cells when assessed by a reverse plaque assay. The proportions of surface IgE-positive cells were concomitantly reduced. After 24 hr incubation with IgE-TsF, the number of cytoplasmic IgE-positive cells was reduced, showing that IgE synthesis was inhibited by IgE-TsF. Antigen-specific TsF from phosphorylcholine-specific T hybridomas did not show any inhibitory effect, and IgE-TsF did not block the antibody production of IgM-producing B hybridomas. Precapping of IgE receptors by anti-epsilon antibody or the simultaneous addition of soluble IgE with IgE-TsF abrogated the suppressive function, suggesting that IgE-TsF acted directly on B epsilon cells through binding with IgE receptors.     

Myeloid suppressor lines inhibit T cell responses by an NO-dependent mechanism.

CD11b(+)Gr-1(+) myeloid suppressor cells (MSC) accumulate in lymphoid organs under conditions of intense immune stress where they inhibit T and B cell function. We recently described the generation of immortalized MSC lines that provide a homogeneous source of suppressor cells for dissecting the mechanism of suppression. In this study we show that the MSC lines potently block in vitro proliferation of T cells stimulated with either mitogen or antigenic peptide, with as few as 3% of MSC cells causing complete suppression. Inhibition of mitogenic and peptide-specific responses is not associated with a loss in IL-2 production or inability to up-modulate the early activation markers, CD69 and CD25, but results in direct impairment of the three IL-2R signaling pathways, as demonstrated by the lack of Janus kinase 3, STAT5, extracellular signal-regulated kinase, and Akt phosphorylation in response to IL-2. Suppression is mediated by and requires NO, which is secreted by MSC in response to signals from activated T cells, including IFN-gamma and a contact-dependent stimulus. Experiments with inducible NO synthase knockout mice demonstrated that the inhibition of T cell proliferation by CD11b(+)Gr-1(+) cells in the spleens of immunosuppressed mice is also dependent upon NO, indicating that the MSC lines accurately represent their normal counterparts. The distinctive capacity of MSC to generate suppressive signals when encountering activated T cells defines a specialized subset of myeloid cells that most likely serve a regulatory function during times of heightened immune activity.     

Functional proteomic analysis for regulatory T cell surveillance of the HIV-1-infected macrophage

Regulatory T cells (Treg) induce robust neuroprotection in murine models of neuroAIDS, in part, through eliciting anti-inflammatory responses for HIV-1-infected brain mononuclear phagocytes (MP; macrophage and microglia). Herein, using both murine and human primary cell cultures in proteomic and cell biologic tests, we report that Treg promotes such neuroprotection by an even broader range of mechanisms than previously seen including inhibition of virus release, killing infected MP, and inducing phenotypic cell switches. Changes in individual Treg-induced macrophage proteins were quantified by iTRAQ labeling followed by mass spectrometry identifications. Reduction in virus release paralleled the upregulation of interferon-stimulated gene 15, an ubiquitin-like protein involved in interferon-mediated antiviral immunity. Treg killed virus-infected macrophages through caspase-3 and granzyme and perforin pathways. Independently, Treg transformed virus-infected macrophages from an M1 to an M2 phenotype by down- and up- regulation of inducible nitric oxide synthase and arginase 1, respectively. Taken together, Treg affects a range of virus-infected MP functions. The observations made serve to challenge the dogma of solitary Treg immune suppressor functions and provides novel insights into how Treg affects adaptive immunosurveillance for control of end organ diseases, notably neurocognitive disorders associated with advanced viral infection.     

Inhibition of specific immune responses by feeding protein antigens. V. Induction of the tolerant state in the absence of specific suppressor T cells

The effect of CY pretreatment on the ability of OVA feeding to induce both tolerance and active suppression was examined in mice. CY-pretreated, OVA fed mice were fully unresponsive in both OVA-specific DTH and antibody responses, but, in contrast to untreated OVA-fed mice, did not transfer suppression to normal recipients via splenic lymphocytes. Restoration of Ts activity in CY-pretreated mice was accomplished by reconstitution with normal T cells before antigen feeding, indicating that the CY effect was at the Ts precursor level. In addition, it was found that certain OVA-specific immune parameters (DTH and splenic PFC responses) in recipient mice were susceptible to suppression by transfer of spleen cells from OVA-fed donors, whereas other measures (antigen-induced T cell proliferation and serum antibody titers) were not. The data suggest that CY-sensitive Ts are not necessary for either induction or maintenance of specific tolerance after OVA feeding.