Adherent Ia+ murine cell lines with characteristics of dendritic cells. II. Characteristics of I region-restricted antigen presentation

The ability of an adherent Ia+, interleukin 1+ (IL-1) tumor cell line (P388AD) to present turkey gamma-globulin (TGG) to primed T lymphocytes was demonstrated and compared with normal antigen-presenting cells (APC) found in mouse spleen. P388AD tumor cells presented TGG to long-term cultures of TGG-reactive T cells (LTTC) and to lymph node-derived T cells which were enriched on nylon wool columns and subsequently depleted of endogenous antigen-presenting cells with anti-Ia antisera and complement. MHC-restricted antigen presentation by P388AD was observed when long-term cultures of TGG-reactive T cells were used as the responding T-cell population. Furthermore, antisera directed against I-region determinants expressed on the P388AD tumor cells inhibited TGG-specific T-cell proliferation in a dose-related fashion, suggesting a functional role for the tumor cell-associated Ia molecules. The kinetics of antigen presentation to LTTC by P388AD were similar to the kinetics observed for splenic APC, although the magnitude of the proliferative response to LTTC to TGG was generally lower when antigen (Ag) was presented by the tumor cells compared to splenic antigen-presenting cells (APC). However, the magnitude of T-cell proliferation of immune lymph node (LN) T cells was comparable when Ag was presented on tumor cells or splenic APC. Several experiments suggested that Ag uptake and/or processing may be less effective in P388AD tumor cells as compared to normal splenic APC. A nonadherent Ia+, IL-1- tumor cell line (P388NA), which was isolated from the same parental tumor as P388AD, was also tested for the ability to present Ag to primed T lymphocytes and Ag-reactive LTTC. In contrast, to P388AD, the nonadherent tumor cell failed to present TGG under identical culture conditions even though Ia molecules were expressed on the tumor cells and Ag uptake had occurred. However, the defect in Ag presentation by P388NA could be corrected if an exogenous source of purified interleukin 1 was supplied to the cultures. A unique opportunity thus exists with both the P388AD and P388NA tumor cell lines to decipher some of the molecular interactions leading to T-cell proliferation during antigen presentation.     

Role of self carriers in the immune response and tolerance. XII. Effect of epitope density and antigen-presenting cell phenotype on the presentation of hapten-modified self for the induction of immunity or tolerance in vitro

The data presented in the accompanying paper (J. P. Cogswell, R. P. Phipps, and D. W. Scott, Cell. Immunol. 114, 55-70, 1988) indicate that certain macrophage-like and lymphoid dendritic-like (P388AD.2) tumor lines which express major histocompatibility encoded class II (Ia) antigens and produce interleukin 1 (IL-1) are uniquely able to present hapten-modified self (HMS) in an immunogenic fashion in vivo. In the current study, the relationship between phenotype and function has been confirmed utilizing a completely in vitro system. This investigation revealed that B-cell priming required T cells restricted to P388AD.2's I-A antigens. In addition, exogenous IL-1 reconstituted the response of an IL-1-deficient tumor (P388AD.2-ILd), although it had no effect on the other nonimmunogenic Ia+ tumor lines. Unlike the in vivo system, effective B-cell tolerance was induced when P388AD.2 was modified with high concentrations (10 mM) of hapten or when highly haptenated tumor was added to 0.1 mM TNBS-modified P388AD.2. These results suggest that positive regulation of in vitro immune responses to HMS is dependent upon the phenotype of the accessory cell carrier (with lymphoid dendritic-like cells being unusually potent), while negative regulation is associated with high epitope density. This system now allows the dissection of the properties of different accessory cells and the signals required for B-cell priming or tolerance induction.     

Accessory cell function in a Con A response: role of Ia and interleukin 1

Accessory cell (A-cell) function in a Con A response was analyzed. Irradiated P388D1 cells efficiently induced a proliferative response to Con A of T cells purified from spleen cells, whereas paraformaldehyde-fixed P388D1 cells failed to serve as A cells. Although IL-1 containing culture supernatant (SN) of a macrophage hybridoma induced the Con A response of the T-cell preparations, the depletion of Ia+ cells by the treatment with anti-Ia antibody and complement abrogated the response in the presence of IL-1. Fixed P388D1 cells and the hybridoma SN synergized in the reconstitution of the response. A 15,000-Da fraction of the hybridoma SN or human recombinant IL-1 alpha was able to substitute the hybridoma SN for the response. The reconstitution of the response by IL-1 and fixed P388D1 cells was inhibited by the addition of monoclonal anti-Ia antibody. These results indicate that IL-1 or fixed P388D1 cell does not exert a sufficient signal by itself and both of them are required for the reconstitution of a Con A response of highly purified T cells, and that Ia on fixed P388D1 cells play an important role.     

Role of self carriers in the immune response and tolerance. XI. Correlation of Ia expression and interleukin-1 production with delivery of immunogenic signals in vivo by hapten-modified accessory cell-like tumor lines

It was recently demonstrated that a lymphoid dendritic-like tumor, P388AD.2, presented hapten-modified self (HMS) in an immunogenic fashion even after injection via the normally "tolerogenic" intravenous (iv) route. To determine whether this property was unique to the P388AD.2 line, other hapten-modified tumors were administered iv and the result of their presentation was measured by changes in the number of splenic plaque-forming cells (PFC) following in vitro challenge with thymic-independent antigens. Of the six tumors tested, two (P388 and J774.5R) primed for augmented PFC responses, while four others (P388NA.10, P388D1, WEHI-231, and 70Z/3) did not. When these tumors were compared for Ia expression and production of interleukin-1 (IL-1), it was discovered that (1) all of the immunogenic tumors were Ia+ and IL-1 producing (IL-1+), although not all Ia+,IL-1+ tumors could elicit augmented PFC responses; (2) none of the tumors that were deficient in either Ia expression or IL-1 production could prime B-cell responses in vivo; and (3) the ability to augment PFC responses was proportional to the density of Ia on the immunogenic tumors. These results demonstrated that P388AD.2 was not the only tumor line capable of presenting HMS iv as an immunogen, and that the accessory cell phenotype is critical for the induction of an immunogenic response in vivo.     

Accessory cell function in the Con A response: role of Ia-positive and Ia-negative accessory cells

We have examined the role of Ia-positive and Ia-negative accessory cells (AC) and soluble factors in Con A-stimulated murine T cell activation. Supernatant fluids containing interleukin 1 (IL 1) derived from the P388D1 macrophage cell line and from a lipopolysaccharide (LPS)-stimulated macrophage hybridoma provided only partial reconstitution of the response of purified T cells (18 to 27%). The complete reconstitution obtained with gamma-irradiated spleen cells or LPS-activated B cells was inhibited by approximately 60 to 77% when anti-Ia antibody was included in the culture. Despite this apparent involvement of Ia+ spleen AC, Ia-negative L cell AC could also reconstitute the response of both Class I-restricted Lyt-2+ T cells and Class II-restricted L3T4+ T cells. When the Ia-negative AC were employed, the L3T4 antigen on L3T4+ T cells played a critical role because addition of anti-L3T4 antibody to the culture inhibited the response by 85 to 90%. In contrast, anti-L3T4 did not inhibit the response in the presence of spleen AC. These results suggest that the molecules involved in T cell-AC interactions may vary depending on the AC source. Moreover, at least one of the putative target ligands for L3T4 presumably is not Ia, because anti-L3T4 inhibited T cell stimulation when Ia-negative AC were used.     

The murine IL 2 receptor. III. Cellular requirements for the induction of IL 2 receptor expression on T cell subpopulations

The accessory cell requirements for the induction of the IL 2 receptor by the lectin Con A on murine T cell subsets were directly assayed with anti-IL 2 receptor monoclonal antibodies. Substantial levels of IL 2 receptor expression were induced on T lymphocytes of the MHC class I-restricted, suppressor/cytotoxic phenotype (L3T4-, Ly-2+) in the presence and absence of accessory cells. In contrast, high levels of IL 2 receptor expression could only be induced on T cells of the MHC class II-restricted, helper/inducer phenotype (L3T4+, LY-2-) in the presence, but not in the absence, of accessory cells. Ia- cells such as the P388D1 macrophage line or cultured fibroblasts (DAP X 3) were as efficient as the Ia+ B cell hybridoma LB in providing accessory cell function for the L3T4+, Ly-2- subset. PMA, but not purified human IL 1, could substitute for accessory cells for both IL 2 receptor expression and IL 2 secretion by the L3T4+, Ly-2- subset. These data suggest that IL 2 receptor induction on the L3T4+, Ly-2- subset is complex, possibly requiring a T cell-accessory cell interaction, whereas the lectin may directly trigger IL 2 receptor expression on L3T4-, Ly-2+ T cells.     

Antigen presentation by interferon-gamma-treated endothelial cells and fibroblasts: differential ability to function as antigen-presenting cells despite comparable Ia expression

The effect of interferon-gamma (IFN-gamma) on endothelial cell (EC) and fibroblast (FB) class II major histocompatibility complex (MHC) gene product expression and antigen presenting ability was examined. Control FB did not express class II MHC gene products, whereas a small (less than 1%) population of passaged EC expressed class II gene products. IFN-gamma induced a comparable density of HLA-DR expression on nearly all EC and FB. IFN-gamma-treated EC and FB also expressed HLA-DP but at a lower density, whereas HLA-DQ expression was barely detectable on either cell type. Control FB were not able to stimulate allogeneic T4 cell DNA synthesis or function as antigen-presenting cells (APC). Control EC were also unable to stimulate allogeneic T4 cell DNA synthesis unless large numbers of stimulator cells were used. Small numbers of IFN-gamma-treated EC were able to stimulate allogeneic T4 cell DNA synthesis, whereas larger numbers were markedly more effective than control EC. In contrast, IFN-gamma-treated FB were ineffective stimulators of allogeneic T4 cell DNA synthesis. IFN-gamma-treated FB were able to present the exogenous antigen SKSD to autologous but not allogeneic T4 cells, but they were extremely inefficient APC. The inability of IFN-gamma-treated FB to function as APC could not be explained by FB-mediated immunosuppression, Ia density, or HLA-DQ expression. This limited capacity of IFN-gamma-treated FB to participate in Ia-restricted functional interactions with T4 cells correlated with a similar diminished capacity to support nonspecific mitogen-induced proliferation of T4 cells before IFN-gamma-induced Ia expression. This accessory cell function was not enhanced by IFN-gamma treatment. Monocytes syngeneic to the responding T4 cells but not interleukin 1 (IL 1) permitted IFN-gamma-treated FB but not control FB to stimulate allogeneic T4 cell DNA synthesis, but they remained markedly less effective stimulators than monocytes. Moreover, IFN-gamma-treated FB were effective stimulators of alloprimed T4 cells, in contrast to their inability to stimulate fresh T4 cells. Furthermore, monocytes and IFN-gamma-treated FB were comparably effective stimulators of alloreactive T cell lines. These data suggest that accessory cells perform functions unrelated to Ia and IL 1 that are necessary for mitogen-, alloantigen-, and antigen-induced proliferation of freshly isolated T cells. Monocytes and EC effectively perform this function, but FB do not. This accessory cell function does not seem to be as important for the activation of primed T cells.     

Evidence for effects of interleukin 4 (B cell stimulatory factor 1) on macrophages: enhancement of antigen presenting ability of bone marrow-derived macrophages

We have studied the effects of recombinant mouse interleukin 4 (IL 4) (previously known as B cell stimulatory factor 1) on the antigen-presenting ability of murine splenic B cells and bone marrow macrophages. Our assay is based on the induction of antigen-presenting ability in these cells after incubation with IL 4 for 24 hr. The presenting cells were then used to stimulate IL 2 production by antigen-specific, I-Ad-restricted T cell hybridomas, a response mainly dependent on the induction of Ia antigens. Consistent with our previously published data using partially purified natural IL 4, we show here that recombinant IL 4 (but not interferon-gamma (IFN-gamma) or IL 1) induces antigen-presenting ability in B cells. Recombinant IL 4 was also found to induce antigen-presenting ability in a cloned, bone marrow derived-macrophage cell line (14M1.4), and in normal bone marrow-derived macrophages. These macrophage populations also respond to IFN-gamma showing enhanced antigen-presenting ability (mediated by increased Ia antigen expression). A small but significant increase in Ia antigen expression was also detected in 14M1.4 macrophages induced with IL 4. However, additional analysis suggested that the effect of IL 4 on 14M1.4 is different from that of IFN-gamma, because IL 4 (but not IFN-gamma) is able to maintain the viability and increase the size of and metabolic activity of bone marrow macrophages. However, IL 4 may not affect all macrophages because the macrophage cell line P388D1, which responds to IFN-gamma, failed to show enhanced antigen-presenting function after stimulation with IL 4. These observations indicate that IL 4, a lymphokine previously considered to be B cell lineage specific, has effects on macrophages and may be involved in their activation.     

Conversion of a tolerogenic to an immunogenic signal by P388AD.2 cells, a lymphoid dendritic cell-like tumor line

The lymphoid dendritic cell-like tumor P388AD.2 is capable of converting a tolerogenic signal into an immunogenic one. In the present study, adherent P388AD.2 cells were pulsed with the tolerogen, fluoresceinated (FL) sheep gamma-globulin (SGG), washed, and incubated with normal spleen cells. After 24 hr, the spleen cells were harvested and challenged in secondary cultures with immunogenic doses of FL-thymic independent (TI) antigens. The plaque-forming cell response on day 3 of secondary culture was increased as much as 400% compared with control spleen cultures exposed to untreated P388AD.2 cells. The increased response was specific for the FL-hapten and occurred only when P388AD.2 cells were pulsed with FL-Ig or FL-F(ab')2 fragments, but not with FL-synthetic tolerogens or other FL-antigens. Furthermore, the augmentation required histocompatible T cells in the primary cultures. Additional experiments showed that if cultures were devoid of Lyt-1+ cells but not Lyt-2+ cells, no augmentation occurred. A variety of other macrophage-like tumor cells, some with known antigen presenting properties, were tested for the ability to present tolerogen in an immunogenic fashion. Only an I-A+ J774 clone was able to present tolerogen in an immunogenic fashion. However, we failed to find a correlation between the presence of surface Ia antigen and tolerogen-presenting ability. The results suggest that certain types of cells may play a role in immune regulation by abrogating the tolerogenicity of Ig tolerogens via their presentation in an immunogenic mode.     

Evaluation of Ia+ tumor cell lines and peritoneal exudate macrophages as accessory cells: differential requirements for the activation of certain T cell functions

Several Ia+ (BC3A, TA3, D1B) or Ia-inducible (WEHI-3, P388D1) tumor lines were tested for accessory cell function for the activation of antigen-specific T cell proliferation and for the induction of T helper cells that help B cells in antibody production. All lines were able to induce antigen-specific T cell proliferation in an MHC-restricted way, but none activated T helper cells to soluble antigens under all conditions tested. In comparison, starch-induced peritoneal exudate macrophages induced T cell proliferation as well as T cell help. Some of the lines tested induced nonspecific suppressor cells that were Ly-2-positive and partially or completely inhibited antibody responses. The induction of suppressor cells, however, is not the reason for the failure of the tumor lines to activate T helper cells. These data indicate that antigen-specific T cell proliferation and helper activity do not necessarily correlate.     

Differential requirement for accessory cells in polyclonal T-cell activation

Highly purified rat Ia-negative (OX-6-) and Ia-positive (OX-6+) T cells were employed to examine the requirement for accessory cells (AC) and/or soluble factors in the activation of resting T cells with Con A, PHA, sodium periodate, or antigen. A variety of cells were employed as AC, including Ia-positive and Ia-negative macrophages (M phi), gamma-irradiated (2000 rad) or non-irradiated OX-6+ T cells, and several Ia-negative adenovirus-transformed rat embryo fibroblast cell lines. Our results suggested that for the expression of IL-2 receptors (IL-2R) and proliferation of OX-6- T cells in response to Con A, PHA, or antigen, there was an obligatory requirement for the presence of AC which could not be overcome by the addition of IL-1 and/or IL-2. Activation of OX-6- T cells with antigen required the presence of Ia+ AC, while activation with mitogens could be initiated with Ia- AC. M phi were efficient in AC function in all responses tested, while the AC function of OX-6+ T cells (TAPC) proved discriminatory under different conditions. The optimal response to PHA required much higher concentrations of TAPC as AC than for the Con A response. TAPC failed to stimulate sodium periodate-treated T cells under any conditions tested. Furthermore, when TAPC were employed as AC, their antigen-presenting ability was radiosensitive, while their AC function for Con A and PHA was radioresistant. These results suggest that molecules involved in T cell-AC interactions may differ, depending on the source of AC and/or type of the proliferative stimulus provided to T cells. This data has been discussed in the context of T-cell activation.     

Role of self carriers in the immune response and tolerance. X. A lymphoid dendritic-like tumor, P388AD.2, acts as a novel immunogenic carrier for hapten

Hapten-modified spleen cells, peritoneal exudate cells, and certain lymphoid tumors preferentially induce specific tolerance after i.v. administration. In contrast to these tolerogenic carrier cells, we found that a haptenated lymphoid dendritic-like tumor, P388AD.2, acts as a potent immunogen after i.v. injection. The immunogenicity of P388AD.2 was analyzed by measuring the specific augmentation of plaque-forming cell (PFC) responses when spleen cells from mice previously injected with haptenated tumor cells were challenged in vitro with thymus-independent antigens. Optimal immunization was found to be dependent on cell dose and hapten concentrations. Further studies indicated that P388AD.2 elicited a response which was T cell-dependent and which involved both the so-called Lyb-3,5,7- and Lyb-3,5,7+ B cell populations. Injection of haptenated tumor into different mouse strains suggested that H-2 compatibility was required to prime B cells in vivo, although significant augmentation could also be achieved in allogeneic C57B1/6J mice. The enhanced PFC responses elicited in H-2b mice could not be explained by allo-recognition of class I or II MHC determinants. In toto, these results suggest that P388AD.2 acts as a unique accessory cell for the presentation of hapten-modified self.     

Qualitative and quantitative studies of antigen-presenting cell function by using I-A-expressing L cells

I-A-expressing transfected murine L cells were analyzed as model antigen-presenting cells. Four features of accessory cell function were explored: antigen processing, interaction with accessory molecules (LFA-1, L3T4), influence of Ia density, and ability to stimulate resting, unprimed T lymphocytes. I-A+ L cells could present complex protein antigens to a variety of T cell hybridomas and clones. Paraformaldehyde fixation before but not subsequent to antigen exposure rendered I-A+ L cells unable to present intact antigen. These results are consistent with earlier studies that made use of these methods to inhibit "processing" by conventional antigen-presenting cells. The ability of anti-L3T4 antibody to inhibit T cell activation was the same for either B lymphoma or L cell antigen-presenting cells. In striking contrast, anti-LFA-1 antibody, which totally blocked B lymphoma-induced responses, had no effect on L cell antigen presentation, measured as interleukin 2 (IL 2) release by T hybridomas, proliferation, IL 2 release, or IL 2 receptor upregulation by a T cell clone. I-A+ L cell transfectants were found to have a stable level of membrane I-A and I-A mRNA, even after exposure to interferon-gamma-containing T cell supernatants. In agreement with earlier reports, a proportional relationship between the (Ia) X (Ag) product and T cell response was found for medium or bright I-A+ cells. However, dull I-A+ cells had a disproportionately low stimulatory capacity, suggesting that there may be a threshold density of Ia per antigen-presenting cell necessary for effective T cell stimulation. Finally, I-A-bearing L cells were shown to trigger low, but reproducible primary allogeneic mixed lymphocyte responses with the use of purified responder T cells, indicating that they are capable of triggering even resting T cells. These studies confirm the importance of antigen processing and I-A density in antigen-presenting cell function, but raise questions about the postulated role of the LFA-1 accessory molecule in T cell-antigen-presenting cell interaction. They also illustrate the utility of the L cell transfection model for analysis and dissection of antigen-presenting cell function.     

Recombinant granulocyte-macrophage colony-stimulating factor restores deficient immune responses in mice with chronic Trypanosoma cruzi infections

Spleen cells from mice with chronic Trypanosoma cruzi infection generate a minimal plaque-forming response to SRBC in vitro. Addition of granulocyte-macrophage (GM)-CSF to cultures of spleen cells from chronically infected mice restored the plaque-forming cells (PFC) response to normal levels. Splenic adherent cells from chronically infected mice were deficient in their ability to reconstitute the PFC response of accessory cell-depleted normal spleen cells. Preincubation of splenic adherent cells from infected mice with GM-CSF restored their ability to reconstitute the PFC response of adherent cell depleted cultures. Ia Ag expression by splenic adherent cells from chronically infected mice was significantly lower compared to Ia Ag expression of cells from normal mice. Incubation of splenic adherent cells from chronically infected mice for 48 h with GM-CSF increased levels of Ia Ag expression to approximately those of uninfected mice. Peritoneal macrophages from infected mice produced IL-1 after incubation with GM-CSF at levels equivalent to those produced by similarly treated control macrophages. Spleen cells from chronically infected mice showed significant induction of IL-2 mRNA after GM-CSF treatment, and the addition of the anti-IL-2 mAb to GM-CSF supplemented cultures of spleen cells from infected mice blocked the restoration of the anti-SRBC PFC response. Thus, the ability of GM-CSF to restore the anti-PFC response to SRBC appears to involve the up-regulation of accessory cell function that includes increased Ia Ag expression and the induction of IL-1 production. These events also involve increased IL-2 production with resultant up-regulation of the response to SRBC by spleen cells from infected mice. Finally, it was shown that treatment of infected mice with rGM-CSF completely restored their depressed PFC production in vivo.     

Influence of accessory cell and T cell surface antigens on mitogen-induced IL 2 receptor expression

We have assessed the inhibitory effects of various monoclonal antibodies on the expression of the IL 2 receptor. Anti-LFA-1, but not anti-Ly-2, markedly inhibited the induction of the IL 2 receptor on the Ly-2+ subset. T-depleted spleen cells, L cells, and B lymphoma cells all functioned as potent accessory cells (AC) for the induction of the IL 2 receptor on L3T4+ T cells. Anti-LFA-1 inhibited the induction of the IL 2 receptor irrespective of the type of AC used. Anti-L3T4 only inhibited the induction of IL 2 receptor expression when L cells were the source of AC. The inhibitory capacity of anti-L3T4 was not related to the expression of Ia on the AC population, because the magnitude of inhibition was comparable in cultures containing either Ia+ or Ia- L cells, whereas no inhibition was seen with either Ia+ or Ia-B lymphoma cells. We conclude from these studies that LFA-1 plays a critical role in mitogen-induced activation of both T cell subsets by promoting both T-AC and T-T interactions. Although anti-L3T4 can inhibit T cell activation in the absence of the recognition of Ia, the mechanism of inhibition and the proposed target molecule for L3T4 on the AC or the T cell have not been determined in our studies. A number of different models for the function of this cell surface antigen are discussed.     

Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). V. T cell proliferative response and cellular interactions

We previously demonstrated that in vivo antibody production to HBsAg in the mouse is regulated by at least two immune response (Ir) genes mapping in the I-A (HBs-Ir-1) and I-C (HBs-Ir-2) subregions of the H-2 locus. To confirm that H-2-linked Ir genes regulate the immune response to HBsAg at the T cell level and to determine if the same Ir genes function in T cell activation as in B cell activation, the HBsAg-specific T cell responses of H-2 congenic and intra-H-2 recombinant strains were analyzed. HBsAg-specific T cell proliferation, IL 2 production, and the surface marker phenotype of the proliferating T cells were evaluated. Additionally, T cell-antigen-presenting cell (APC) interactions were examined with respect to genetic restriction and the role of Ia molecules in HBsAg presentation. The HBsAg-specific T cell proliferative responses of H-2 congenic and intra-H-2 recombinant strains generally paralleled in vivo anti-HBs production in terms of the Ir genes involved, the hierarchy of responses status among H-2 haplotypes, antigen specificity, and kinetics. However, the correlation was not absolute in that several strains capable of producing group-specific anti-HBs in vivo did not demonstrate a group-specific T cell proliferative response to HBsAg. The proliferative responses to subtype- and group-specific determinants of HBsAg were mediated by Thy-1+, Lyt-1+2- T cells, and a possible suppressive role for Lyt-1-2+ T cells was observed. In addition to T cell proliferation, HBsAg-specific T cell activation could be measured in terms of IL 2 production, because anti-HBs responder but not nonresponder HBs-Ag-primed T cells quantitatively produced Il 2 in vitro. Finally, the T cell proliferative response to HBsAg was APC dependent and genetically restricted in that responder but not nonresponder parental APC could reconstitute the T cell response of (responder X nonresponder)F1 mice, and Ia molecules encoded in both the I-A and I-E subregion are involved in HBsAg-presenting cell function.     

Activation requirements for normal T cells: accessory cell-dependent and -independent stimulation by anti-receptor antibodies

We have examined the requirements for the activation of normal T cells by two anti-T cell receptor antibody preparations, including a rabbit antiserum, R3497, which binds to all normal T cells, and a rat monoclonal antibody, KJ16-133, which binds to about 20% of T cells. The requirements for stimulation of T cells by both antibodies were similar. Soluble antibodies in the absence of accessory cells (AC) failed to induce either proliferation or the expression of IL 2 receptors, and the addition of either IL 2 or PMA failed to synergize with these soluble antibodies for an AC-independent proliferative response. Activation could only be achieved in the presence of Fc receptor-positive AC, although Fc receptor expression alone appeared not to be sufficient for AC activity because some Fc receptor-positive cells did not function in this capacity. Activation with anti-receptor antibody conjugated to Sepharose 4B beads could be demonstrated in the presence of some exogenous cofactors, such as IL 2 and PMA, but not in the presence of recombinant IL 1. When activation by soluble antibody plus AC was compared to activation by bead-conjugated antibody + recombinant IL 2, it was found that the former favored the stimulation of Lyt-2+ cells. The effects of the addition of anti-L3T4 monoclonal antibody was also examined in this system. Anti-L3T4 inhibited the response of L3T4+ cells when used in the presence of Ia+ as well as Ia- AC, and it also inhibited activation in a system in which KJ16-133 conjugated to Sepharose was used in the absence of AC. Because anti-L3T4 had an inhibitory effect in the presence of Ia- AC as well as in the absence of any AC, it is concluded that L3T4 does not necessarily function by interacting with Ia on the surface of AC, and may directly transmit down-regulatory signals when bound by anti-L3T4.     

Antigen presentation to human T lymphocytes. II. Requirements for Mac-120+ macrophages and responsiveness to interleukin 2

To investigate mechanisms by which antigen, macrophages, and interleukin 2 (IL2) participate in the induction of secondary T-cell proliferative responses, trinitrophenyl (TNP) was presented in three distinct modes: (i) TNP-modified peripheral blood mononuclear cells (TNP-PBMC), (ii) TNP-PBMC cell sonicates, and (iii) TNP-ovalbumin (TNP-OVA). Stimulators were depleted of Mac-120+ macrophages using Mac-120 monoclonal antibody plus complement. TNP-Mac-120 macrophages stimulated primed T cells nearly as well as TNP-unfractionated macrophages (which were about 40% Mac-120+). In contrast, although greater than 70% DR+, Mac-120- macrophages plus either TNP-OVA or TNP-PBMC sonicate elicited minimal responses compared to unfractionated macrophages plus antigen. After 21-28 days of in vitro priming, macrophage-depleted T cells were not stimulated to proliferate by either IL2 alone or sonicates alone. IL2 plus TNP-PBMC sonicates, however, stimulated significant proliferation. Furthermore, this response was considerably greater than that to IL2 plus either TNP-T cell sonicates or TNP-mouse spleen sonicates. Thus, the Mac-120+ macrophage population may have an important antigen-presenting and/or accessory function in the stimulation of primed T cells by soluble or particulate antigen, although it is unnecessary for responses to intact TNP-Ia+ PBMC. In addition, the data suggest that Ia+ sonicates alone may suffice for induction of IL2 responsiveness, but not for endogenous IL2 production and subsequent proliferation by primed T cells.     

The role of Ia molecules in the activation of T lymphocytes. IV. The basis of the thymocyte IL 1 response and its possible role in the generation of the T cell repertoire

The activation requirements for thymocyte proliferation were investigated. Thymocytes proliferate in the presence of exogenous interleukin 1, which has been used as the classic assay for this factor. This response, however, is greatly decreased in cultures of purified thymic T cells. Purified thymic T cells will proliferate in the presence of IL 1 if accessory cells are added to culture. The requisite accessory cell is a non-T, adherent, radioresistant cell found in macrophage/dendritic cell-enriched fractions of both thymus and spleen. This cell bears Ia molecules, which are critically involved in the activation of thymocytes. This thymocyte-accessory cell interaction is not dependent on exogenous nominal antigens. Therefore, it appears that IL 1 allows the expansion of thymocytes with specificity for self-class II MHC antigens. This response was found to be unique to this stage of T cell development and can be observed with both mature and immature thymic T cell subsets. The implications of these findings for the physiologic expansion of self-restricted T cells in the thymus are discussed.     

Cloned murine Ia+ BK-BI-2.6.C6 T cells function as accessory cells presenting protein antigens to long-term-cultured antigen-specific T cell lines

A variant clone, BK-BI-2.6.C6, was derived from the murine bovine insulin-reactive T cell line BK-BI-2.6 with helper/amplifier phenotype. Variant cells have lost reactivity to insulin, but have acquired constitutive IL 2 receptor expression, growing in IL 2-containing medium without feeder cells. In contrast to their ancestor line, variant cells synthesize and express I-A and I-E region-dependent class II molecules as indicated by metabolic radiolabeling, immunoprecipitation with subregion-specific monoclonal antibodies and two-dimensional (2D) gel electrophoresis (1D isoelectric focusing, 2D SDS-PAGE). BK-BI-2.6.C6 cells can act as accessory cells, presenting the protein antigens bovine insulin and ovalbumin to antigen-dependent long-term cultured T cell lines BK-BI-1.2 and BK-OVA-1 in the context of I-A restriction elements. Antigen recognition on presenting BK-BI-2.6.C6 accessory cells resulted in highly efficient IL 2 production. However, in contrast to splenic antigen-presenting cells, BK-BI-2.6.C6 cells did not initiate antigen-specific [3H]thymidine incorporation by the T cell lines tested. Further study of accessory function of Ia+ T cell clones might provide insight into processes regulating T cell responses to antigen.