Human macrophage-lymphocyte interaction in proliferation to soluble antigen: I. Specific deletion of lymphocyte proliferative activity on macrophage monolayers

The interaction between human T lymphocytes and autologous macrophages (Mφ) in the proliferative response to soluble antigen was investigated. The presence of Mφ was a requirement for maximal lymphocyte proliferation to tetanus toxoid antigen. Brief exposure of Mφ to TT resulted in “antigen-pulsed” Mφ which could stimulate proliferation by lymphocytes in the absence of free antigen. Taking advantage of the adherent nature of Mφ, monolayers of antigen-pulsed Mφ were used to specifically adsorb antigen-reactive lymphocytes. This immunoadsorption was manifested by a specific deletion of proliferative activity in the fraction of lymphocytes failing to bind to the monolayers.     

Specific binding of T lymphocytes to macrophages. I. Kinetics of binding.

Peritoneal exudate lymphocytes obtained from immune guinea pigs and cultured for 1 week on antigen-pulsed autologous macrophages were tested for their ability to bind to fresh antigen-pulsed autologous macrophages or to macrophages pulsed with an irrelevant antigen. Up to 30% of the lymphocytes bound to macrophages bearing the relevant antigen whereas only 2 to 5% remained nonspecifically bound to macrophages after vigorous washing. Specific binding was observed in cultures as early as 1 hr. Analysis of the kinetics of binding suggests that the observed nonspecific binding is not a step in specific binding. The possibility that weaker antigen-independent association between lymphocytes and macrophages precedes specific binding cannot be excluded. No evidence was obtained that serum antibody adsorbed to the macrophage or T cell plays a role in this cell interaction or that the T cell can bind antigen directly. We suggest that the observed specific binding represents the initial event in stimulation of T lymphocytes by antigen.     

Role of nominal antigen and Ia antigen in the binding of antigen-specific T lymphocytes to macrophages.

We have previously demonstrated that when primed T lymphocytes were repeatedly incubated on monolayers of antigen-pulsed macrophages (M phi), the cells that failed to adhere to the monolayer demonstrated a marked depletion of their proliferative response that was specific both for the antigen used for pulsing the M phi and for Ia determinants on the M phi. In order to further analyze the contribution of the nominal antigen and Ia antigens to the physical binding of T lymphocytes to M phi, we have attempted to block the absorption of T lymphocytes to M phi with a large excess of soluble antigen and with anti-Ia sera. Our results demonstrate that anti-Ia sera inhibit but that soluble antigen augments the binding of specific T lymphocytes to M phi. The implications of these findings for "dual recognition" and "linked recognition" models of T lymphocyte receptors are discussed.     

Specific absorption of T lymphocytes committed to soluble protein antigens by incubation on antigen-pulsed macrophage monolayers.

Monolayers of macrophages (Mphi) pulsed with antigen were used as immunosorbents for T lymphocytes from guinea pigs primed to soluble protein antigens. T lymphocytes were cultured on the Mphi monolayers for 4 hr, then aspirated and reincubated on a fresh monolayer pulsed with the same antigen for a second and a third step. T lymphocytes so treated were selectively deprived of cells responding in assay for antigen-dependent proliferation against the antigen used for pulsing the absorbing monolayer, but maintained their response to other antigens. The lymphocytes adhering to the Mphi of the absorbing monolayer were capable of giving a full response to the antigen used for pulsing the Mphi of the monolyers. The proliferative response of F1 T lymphocytes to antigen in association with Mphi of either parental strain could be absorbed leaving the response to antigen in association with Mphi of the other parental strain. The absorption of the proliferative response was not inhibited by addition of excess soluble antigen to the medium of the absorption culture. Our results indicate that specific guinea pig T lymphocytes responding by proliferation to soluble protein antigens recognize and bind specifically to a complex of Ia antigen and protein antigen at the surface of the Mphi.     

Binding of antigen-specific, H-2-restricted T cell hybridomas to antigen-pulsed adherent cell monolayers

Two methods were investigated for studying the binding of radiolabeled hybridoma T cells to antigen (Ag) and H-2 products for which they bore receptors. In both cases hybridoma T cells were labeled with tritiated thymidine. In one method labeled cells were added to adherent splenic cells prepulsed with antigen, and the mixture was incubated overnight at 37 degrees C before nonadherent cells were gently washed away. The percent of adherent hybridoma T cells was then estimated by harvesting the adherent monolayers and measuring tritium counts bound. In a second method radiolabeled hybridoma T cells were added to adherent antigen-pulsed B cell lymphomas or hybridomas for between 15 min and 1 hr at 37 degrees C before removal of nonadherent cells and harvesting of the adherent monolayers. In both cases binding was both antigen- and I-region specific. In the second case binding was also rapid; significant binding could be measured after 15 min incubation. These techniques were used to study subclones of one of our T cell hybridomas that were thought by a functional assay (interleukin 2 release) to have lost receptors for Ag/H-2. It was found that subclones of the hybridoma that no longer secreted interleukin 2 in response to Ag/H-2, even though they continued to secrete interleukin 2 in response to concanavalin A, also no longer bound specifically to Ag-pulsed monolayers of the appropriate H-2 type. This confirmed the idea that these subclones had lost the ability to synthesize receptors for Ag/H-2. It is hoped that assays of this type will be useful in the future for the study of Ag/H-2 receptors on T cells.     

Specific binding of T lymphocytes to macrophages. II. Role of macrophage-associated antigen.

Peritoneal exudate lymphocytes from immune guinea pigs that bind in vitro to autologous antigen-pulsed macrophages were allowed to proliferate for 1 week to give a population markedly enriched in antigen-specific T cells. This enriched population was then studied with regard to its binding to fresh autologous antigen-pulsed macrophages. Specific binding was not inhibited by a large excess of antigen in the media (5000-fold greater than the amount of antigen associated with the macrophages) either soluble or bound to Sepharose beads, or by coating the antigen-pulsed macrophags with antibody to the exogenous antigen, by reacting a second layer of antibody to the heterologous antibody, or by haptenating the antigen and treating the hapten-antigen macrophage complex with excess anti-hapten antibody. Results of treating antigen-pulsed macrophages with the proteolytic enzymes trypsin and pronase indicate that exogenous antigen is on the macrophage surface, but the experiments failed to prove that the removable antigen is essential for binding. The simplest interpretation of these results is that the T cell receptor is not specific for native exogenous antigen.     

In vitro selection and extended culture of antigen-specific T lymphocytes. II. Mechanisms of selection.

Functional selection of antigen-specific T lymphocytes can be achieved by culturing thymus-dependent (T) lymphocytes from immunized guinea pigs on "monolayers" of antigen-pulsed adherent peritoneal exudate cells (PEC) from nonimmune syngeneic donors. Several aspects of the in vitro selection of T lymphocyte-rich peritoneal exudate lymphocytes (PEL) were studied. It was shown that irradiated adherent PEC were equivalent to nonirradiated adherent PEC in supporting selection cultures, indicating that the lymphocytes harvested at the end of the selection culture derive from the immune donors of the PEL and not from the nonimmune donor of the adherent PEC. The relative importance of specific adherence and specific proliferation for achieving selection was determined by comparing the degree of selection obtained when nonadherent cells were discarded at 24 hr with that noted when the discard step was omitted. It was found that omitting the discard step markedly diminished the degree of selection. On the other hand, blocking proliferation with specific alloantisera after the discard step did not diminish the degree of selection, although it did diminish the cell yield. Thus, specific adherence to antigen-pulsed PEC appeared to be critical in the selection culture procedure. An estimate of the degree of enrichment obtained by the selection culture procedure was obtained by culturing selected cells in an excess of nonprimed PEL, so that auxiliary cells would not be limiting. Under these conditions, it appeared that selected cells were enrichied from 4- to 10-fold in antigen-responsive cells with respect to the initial cell population.     

Specific binding of T lymphocytes to macrophages. III. Spontaneous dissociation of T cells from antigen-pulsed macrophages.

Peritoneal exudate lymphocytes (PEL) from immune guinea pigs that adhere to antigen-pulsed macrophages (MO) were cultured for 1 week to yield a population enriched in antigen-specific (selected) T cells. These cells bind specifically within hours to fresh autologous antigen-pulsed MO. Thd dissociation of these selected PEL from antigen-pulsed MO was studied. No evidence was obtained that factors in the culture medium play a role in dissociation. Lymphocytes that have dissociated from antigen-pulsed MO are usually fully capable of rebinding to MO freshly pulsed with antigen, suggesting that there is no deficiency in the lymphocytes ability to bind. In contrast, readding antigen to cultures during incubation prevents the predicted dissociation. Moreover, repulsing MO cultured without selected PEL restores their capacity to bind fresh selected PEL. These findings indicate that decay of antigen associated with with MO is the major mechanism underlying the observed dissociation.     

Alveolar macrophages in pulmonary immune responses. I. Role in the initiation of primary immune responses and in the selective recruitment of T lymphocytes to the lung

Antigen inoculated intratracheally (IT) into animals can induce primary immune responses and selectively recruit specific T cells to the lung. In the current study, the role of alveolar macrophages (AM) in these two responses was investigated. Antigen-pulsed bronchoalveolar cells (BAC) inoculated IT into guinea pigs generated a population of immune T cells that proliferated in vitro on reexposure to antigen-pulsed macrophages (M?). The possibility that antigen-pulsed donor BAC shed antigen that was subsequently processed and presented by host M? was ruled out by genetic experiments. Thus, peritoneal exudate lymphocytes (PEL) from (2 X 13)F1 guinea pigs primed with antigen-pulsed BAC from strain 2 animals responded preferentially to antigen-pulsed strain 2 M? rather than to antigen-pulsed strain 13 M?. In a second set of studies, antigen-pulsed BAC inoculated IT into guinea pigs selectively recruited antigen-specific T cells to the lung. Genetic experiments verified that inoculated BAC were the source of the antigen-presenting cells responsible for selective recruitment. Thus, antigen-pulsed strain 2 BAC inoculated IT recruited a greater proportion of (2 X 13)F1 T cells that recognized antigen in the context of strain 2 M? than F1 T cells that recognized antigen on strain 13 M?. Taken together, these studies suggest that AM contribute to the regulation of pulmonary immunity by both inducing T lymphocyte immunity and selectively recruiting specific T cells to the lung.     

Modulation of the helper activity of educated T cells to the synthetic polypeptide poly(Tyr,Glu)-poly(dlAla)-poly(Lys) by adherent cell-bound antigen

The effects of culturing in vivo- or in vitro-activated helper cells on antigen-pulsed splenic adherent cells were studied. In vivo T cells educated to the synthetic polypeptide (T,G)-A-L were obtained by the transfer of thymocytes into lethally irradiated mice. When the activation process was suboptimal, resulting in a low helper function of the cell preparation, incubation of the educated cells on (T,G)-A-L-pulsed splenic adherent cells for 24 hr potentiated their activity, and efficient helper cells were obtained. This process was found to be antigen specific, it did not involve de novo education of naive cells or selection of specific T lymphocytes, but rather completion of the education procedure, which had already started in vivo. It seems that a physical contact between the educated T cells and the antigen-presenting cells is essential for inducing the enhanced helper effect. It is also apparent that during this 24-hr culture on antigen-pulsed macrophages T cells did not proliferate, but rather differentiated into immunocompetent helper cells. On the contrary, when the initial education step was efficient the subsequent culture of the activated T cells on antigen-pulsed splenic adherent cells resulted in a marked decay in the helper function of the cells, while control monolayers were inert. Thus, macrophage-bound antigen differentially modulates the helper function of educated T cells, a procedure which is probably dependent on the degree of maturation or differentiation of the T lymphocyte.     

Specific binding of T lymphocytes to macrophages. V. The role of Ia antigens on Mphi in the binding.

The effect of anti-Ia alloantiserum on the capacity of selected peritoneal exudate lymphocytes (selected PEL) to bind to antigen-pulsed F1 (responder x nonresponder) macrophages was investigated. With the use of selected PEL for antigens under Ir gene control, it was shown that anti-Ia serum to the responder haplotype blocked adherence of selected PEL to antigen-pulsed macrophages whereas anti-Ia serum to the nonresponder haplotype did not. The target cell of the anti-Ia alloantiserum appeared to be the macrophage because anti-13 Ia in contrast to anti-2 Ia did not inhibit binding of F1 (2 x 13) DNP-GL selected PEL to DNP-GL pulsed strain-2 Mphi (responder strain). Taken together with previous experiments that indicate that an antibody to the native protein antigen employed is unable to block specific binding, the present results suggest that T cells may recognize fragments of exogenous antigen in association with Ia molecules.     

Role of macrophages as modulators but not as autonomous accessory cells in the proliferative response of immune T cells to soluble antigen

The role of murine macrophages (M phi) and that of splenic dendritic cells (DC) were investigated in the antigen-specific proliferative response of memory T cells of mice primed with key-hole limpet hemocyanin (KLH) 6 weeks or more before. Peritoneal M phi, whether expressing Ia antigens or not, did not function as autonomous accessory cells (A cells). A-cell activity of the spleen adherent cell population, which comprised M phi in the majority and DC in the minority, was abolished by eliminating DC with a DC-specific monoclonal antibody and complement, and regained by the addition of a small number of DC. Though M phi did not function as autonomous A cells, they augmented the proliferative response in the presence of a small number of DC. This occurred not only in the presence of free antigen, but also when DC and/or M phi were pulsed with antigen. A culture supernatant of M phi having interleukin-1 activity was effective in enhancing the proliferation of T cells which responded to antigen-pulsed DC. On the other hand, interleukin-2 did not replace DC even in the presence of antigen-pulsed Ia+ M phi. We also investigated recently primed T cells, but no evidence was obtained in favor of the competence of M phi as autonomous A cells.     

Antigen-specific T cell cluster formation on antigen-pulsed macrophage monolayers in mice

We describe the quantitative measurement of antigen-specific clusters formed by antigen-pulsed macrophages and immunized T cells in mice. We have found the peripheral blood T cells show very little non-specific adhesion to macrophages in mice. By using this population of lymphocytes in the peripheral blood as the source of immunized T cells, we could quantitate antigen-specific cluster formation. On OVA-pulsed monolayers of peritoneal exudate macrophages from normal BALB/c mice, syngeneic peripheral blood T cells from donors immunized with the same antigen develop 20-40 clusters per 1,000 macrophages, whereas the same T cells on non-pulsed monolayers develop only 0-5 cluster-like accumulations of cells. On antigen-pulsed monolayers of macrophages from allogeneic (C57BL/6 or A/J) mice, clusters are developed only in the negative range (0-5/1,000 macrophages). Considering the observation by Braendstrup et al, these data seem to suggest that histocompatibility between macrophages and T cells is required to develop antigen-specific T cell clusters on antigen-pulsed macrophage monolayers, and that the genetic restriction of immune responsiveness may be directly expressed in this initial form of cellular interaction between antigen-bearing macrophages and specific T cells.     

Stimulation of T-cell activation by UV-treated, antigen-pulsed macrophages: evidence for a requirement for antigen processing and interleukin 1 secretion

The nature of the defect(s) in the ability of UV-treated guinea pig macrophages to stimulate the proliferative response of guinea pig T cells to soluble protein antigens was investigated. T cells proliferated vigorously when cultured with peritoneal exudate cells (PEC) which had been pulsed with soluble protein antigens, but failed to proliferate when cultured with soluble antigen or with antigen-pulsed, UV-treated PEC. UV-treated macrophages were unable to secrete interleukin 1 (IL-1). Addition of IL-1 partially restored the T-cell proliferative response stimulated by antigen-pulsed, UV-treated PEC. However, IL-1 was able to restore such a response only when the PEC were pulsed with antigen before being exposed to UV. Similar results were obtained when antigen-pulsed PEC were used to stimulate T cells to secrete interleukin 2 (IL-2). These results demonstrate that UV-treated macrophages are defective both in their ability to properly process and present antigen for T-cell recognition and in their ability to secrete IL-1.     

Specific partial depletion of graft-vs-host activity by incubation and centrifugation of mouse spleen cells on allogeneic spleen cell monolayers.

Spleen cells (from BALB/c mice immunized with the C57BL/6 lymphoma EL4, or from non-immune BALB/c) were incubated on monolayers of [C57BL/6 times BALB/cF1 (B6CF1) spleen cells on polylysine-coated polystyrene Petri plate, for 1/2 hr or for 1 hr at 37 degrees C followed by centrifugation of the monolayers for 5 min at 70 times G to 110 times G at 34 to 37 degrees C. Control monolayers were BALB/c spleen cells. As measured by the Simonsen spleen weight assay in neonatal mice, graft-vs-host (GVH) activity was partially depleted in cell populations nonadherent to B6CF1 monolayers. Residual GVH activity of these nonadherent cells was about half that of cells incubated on the control syngeneic monolayers (the mean of eight experiments was 49% +/- 11% S.D.). Two or three consecutive cycles of incubation and centrifugation did not significantly diminish the residual GVH activity, suggesting that spleen cells with GVH activity are heterogeneous with respect to binding to allogeneic target cells under the above conditions. Cell populations nonadherent to third-part [A times AL]F1 monolayers retained full activity, and cell populations partially depleted of GVH activity in B6CF1 neonates had full activity in third-party [BALB/c times AL]F1 neonates.     

Human lymphocyte dependent antibody mediated cytotoxicity: Adherence of lymphocytes to antibody treated target cells

An in vitro human antibody dependent cell mediated cytotoxicity system was used to study the adherence of nonsensitized attacking lymphocytes from peripheral blood to antibody coated melanoma target cells. Specific adherence of attacking cells was documented by labeling the lymphocytes with ⁵¹Cr. The degree of specific adherence was proportional to antibody concentration and incubation time and could be detected before the lysis of target cells. Adsorption of attacking lymphocytes on immune serum treated target cells depleted B cells, enriched T cells, and removed most cytotoxic activity of nonadherent lymphocytes in this system. These results were not found when attacking lymphocytes were adsorbed on normal serum treated target cells.     

Macrophage-T cell interaction mediated by immunogenic and non-immunogenic forms of a monofunctional antigen

Summary As an approach to the elucidation of the essential steps in the immune pathway, the uptake and retention of immunogenic and non-immunogenic analogs of a monofunctional antigen by guinea pig macrophages and the efficiency of macrophages pulsed with the compounds to present antigen to sensitized T lymphocytes were compared. L-Tyrosine-azobenzene-p-arsonate (RAT) and its non-immunogenic analog, 4-hydroxyphenyl-n-propane-3-azobenzene-p-arsonate (RAN), react similarly with antiarsonate antibody, but RAN, unlike RAT, is unable to induce cellular immunity in guinea pigs. The uptake and retention patterns of the two compounds by macrophages differed in that, at a given time, more RAN than RAT was retained and detectable on cell surfaces by anti-arsonate antibody. Equivalent numbers of T lymphocytes from guinea pigs sensitized to RAT formed antigen-dependent clusters with macrophages pulsed with either RAT or RAN after 24 hr in culture, but not with macrophages pulsed with an azobenzenoid compound of unrelated specificity. On the other hand, T lymphocytes from guinea pigs immunized with RAN showed no significant capacity to bind to macrophages which had been pulsed with any of the compounds. The number of lymphocytes from RAT-sensitized animals which bound to RAT-pulsed macrophages remained relatively stable over a 48 hr period, whereas clusters of the same lymphocytes with RAN-pulsed macrophages dissocitated to background levels within that time. Early cluster formation mediated by RAN, as well as its ability to induce transient specific T cell unresponsiveness to RAT in vivo, indicate that T cells are capable of recognizing (binding) the non-immunogen. However, such early, and perhaps weak, interaction with RAN-pulsed macrophages did not induce DNA synthesis by T cells. Anti-Ia serum completely blocked cluster formation mediated by either RAT or RAN. Thus, the only significant distinction disclosed by these studies between the immunogenic and non-immunogenic compounds was the stability of macrophage-T cell interaction as determined by the persistence of antigen mediated cell clusters in culture, suggesting that this may be a factor in immunogenic discrimination.Abbreviations ABA azobenzenearsonate - BSA bovine serum albumin - CFA complete Freund's adjuvant - IFA incomplete Freund's adjuvant - KLH keyhole limpet hemocyanin - LNC Lymph node cells - MHC major histocompatibility complex - PEC peritoneal exudate cells - PEL peritoneal exudate lymphocytes - RAN 4-hydroxyphenyl-n-propane-3-azobenzene-p-arsonate - RAT L-tyrosine-azobenzene-p-arsonate - TAT L-tyrosine-azobenzene-p-trimethylammonium chloride Aided by USPHS Grant AI 05664.     

Inhibition of mitogen-stimulated T lymphocyte proliferation by calcitonin gene-related peptide

The effect of calcitonin gene-related peptide (CGRP) on mouse lymphocyte proliferation stimulated by mitogens was studied. CGRP (10(-10)-10(-7) M) dose-dependently inhibited the proliferative response of mouse lymph node cells and spleen cells stimulated by T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA), whereas a B cell mitogen lipopolysaccharide (LPS) did not inhibit this response. The maximal inhibition by this peptide was 50% to 80% at 10(-8) and 10(-7) M. The addition of 10(-8) and 10(-7) M CGRP to lymph node cell cultures 24 hr after stimulation with Con A or PHA also had a significant inhibitory effect on the proliferative response. Furthermore, in the same concentration range (10(-10)-10(-7) M) CGRP increased intracellular cyclic AMP concentration in nylon wool nonadherent cells, but not in nylon wool adherent cells. CGRP had no significant effect on intracellular cyclic GMP concentration. In addition, specific binding of CGRP was observed in mouse spleen cells. Our present study suggests that CGRP inhibits the proliferative response of T lymphocytes to the mitogens by interacting with cell receptors coupled with adenylate cyclase. CGRP may be implicated in the regulation of T cell function.     

Immunologic function of endothelial cells: guinea pig aortic endothelial cells support mitogen-induced T lymphocyte activation, but do not function as antigen-presenting cells

The possibility that vascular endothelial cells (EC), like macrophages (M phi), can function as accessory cells necessary for mitogen- and antigen-induced T cell activation was examined. EC were enzymatically detached from the luminal surfaces of guinea pig aortas and then propagated in culture. Lymph node T lymphocytes were rigorously depleted of adherent cells, such that they completely lost the capacity to respond to mitogenic stimulation with phytohemagglutinin or concanavalin A. In this system, EC restored mitogen-induced T cell DNA synthesis as effectively as did M phi. This effect could not be explained by a facilitation of residual accessory cell activity within the responding T cell population, because EC restored mitogen responsiveness to T cells that had been treated with anti-Ia antibody and complement. Support of mitogen responsiveness could not be accounted for by secreted products of M phi or EC in the absence of intact accessory cells. In addition to the capacity to serve as fully sufficient accessory cells for the induction of mitogen-stimulated T cell proliferation, EC exerted a number of modulatory influences on T lymphocyte responses in cultures supported by M phi. When such cultures were supplemented with small numbers of EC, responses were dramatically augmented; larger numbers of EC resulted in marked suppression. At least part of these immunomodulatory effects could be accounted for by the activity of secreted products of EC. EC did not express detectable Ia antigens assayed either by indirect immunofluorescence, with the use of the fluorescence-activated cell sorter, or by complement-mediated cytotoxicity. Moreover, treating the EC population with anti-Ia antibody and complement had no effect on its capacity to support mitogen-induced T cell DNA synthesis. As would be expected from the lack of Ia antigen expression, EC were incapable of presenting antigen to primed T cells. They did, however, carry enough antigen into the cultures such that effective antigen presentation could occur when the cultures were supplemented with M phi that were syngeneic but not allogeneic to the responding T cells. Moreover, EC were capable of dramatically augmenting antigen-specific responses stimulated by antigen-pulsed M phi. There was no genetic restriction for this EC-mediated augmentation of antigen responsiveness. These results indicate that EC are capable of functioning as completely sufficient accessory cells for mitogen-induced T cell DNA synthesis and, in addition, are able to modulate ongoing M phi-supported T lymphocyte responses in both a positive and negative manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro and in vivo analyses of a genetically—restricted antigen specific factor from mixed cell cultures of macrophage,T and B lymphocytes

An immunostimulatory factor was identified to be secreted by antigen-pulsed macrophages.This factor was able to induce the generation of antigen specific T helper lymphocytes in vitro as well as in vivo.Further in vitro experiments testing for the genetic restriction of this factor indicated that it is a geneticallyrestricted antigen specific factor (ASF).The Cunningham plaque assay was used to quantify the generation of T helper lymphocytes by measuring the number of plaque forming cells after sequential incubations of antigen-qulsed macrophages with T lymphocytes,and then spleen cells,and finally the TNP-coated sheep red blood cells.