Nature of T lymphocyte recognition of macrophage-associated antigens. IV. Inhibition of peptide antigen presentation by brief treatment of macrophages with anti-Ia serum

To examine the role of macrophage la antigens in T-lymphocyte stimulation, guinea pig macrophages were briefly treated with anti-Ia serum before or after antigen pulsing with the peptide antigen human fibrinopeptide B (hFPB). To assess their antigen-specific stimulatory capacity, the variously treated macrophages were added to culture with hFPB-immune guinea pig T cells and stimulation was determined by the incorporation of [³H]thymidine. Macrophages treated with anti-Ia serum before antigen pulsing stimulated T-cell responses equivalent to those observed with antigen-pulsed macrophages treated with normal serum. By contrast, brief anti-Ia treatment of macrophages immediately following a 2-hr antigen pulse reduced subsequent T-cell responses by 45 to 70%. Similar treatment of macrophages pulsed with antigen for only 1 hr produced only modest inhibition of T-cell responses. However, if macrophages pulsed for 1 hr with antigen were incubated several hours before brief anti-Ia serum treatment, the subsequent T-cell responses were reduced by 40 to 60%. This inhibition was specific for antiserum directed against Ia antigens of the guinea pig MHC, since brief macrophage treatment with antiserum directed against B.1 antigens, the guinea pig equivalent of murine H-2K and H-2D antigens, produced no inhibition of their T-cell stimulatory capacity. These results are discussed with respect to the formation of the immunogen presented by macrophages for T-cell recognition.     

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.     

Signal requirements for lymphocyte activation: Role of a T-cell growth factor produced by guinea pig peritoneal exudate lymphocytes

Peritoneal exudate lymphocytes (PEL) from immunized guinea pigs, when pulsed with antigen, rapidly release a T-cell stimulatory factor (TSF). TSF nonspecifically enhances the proliferation of purified guinea pig T cells in the presence of another signal such as PMA, PHA, or Con A. On a per cell basis, antigen-pulsed PEL produce about 14 times more activity than similarly stimulated lymph node lymphocytes.Several lines of evidence support the view that TSF is the guinea pig equivalent of TCGF (IL-2). TSF containing supernatants have IL-2 activity when assayed on the IL-2-dependent CT6 cell line. When TSF containing supernatants were absorbed with CT6 cells, there was a significant decrease of both TSF activity as assessed on guinea pig T cells as well as IL-2 activity as assessed by the CT6 assay. Additionally, partially purified human and mouse IL-2 have TSF activity, while the macrophage product, IL-1, has no TSF activity. After chromatography on a S-200 column, TSF activity and IL-2 activity coelute at an apparent molecular weight of 19,000.     

Hapten-specific T lymphocyte activation by glutaraldehyde-treated macrophages: an argument against antigen processing by macrophages.

In this communication the effects of glutaraldehyde treatment of trinitrophenyl-(TNP) modified macrophages on their ability to stimulate TNP-specific guinea pig T lymphocyte proliferation were studied. TNP-modified macrophages briefly treated with glutaraldehyde retained much of their ability to stimulate TNP-primed T cells. In contrast, similar treatment of allogeneic macrophages or soluble protein antigen-pulsed syngeneic macrophages completely eliminated their ability to stimulate a mixed leukocyte reaction or protein antigen-specific proliferation, respectively. TNP-modification did not appear to interfere with glutaraldehyde reactivity since macrophages treated with glutaraldehyde before or after TNP-modification stimulated equivalent T cell responses. However, glutaraldehyde treatment of TNP-modified macrophages that had been cultured overnight dramatically reduced their ability to stimulate TNP-specific T cells. Glutaraldehyde-treated TNP-modified macrophages also expressed the same genetic restrictions of T cell activation as untreated stimulators. Thus, T cells primed with syngeneic TNP-modified macrophages were restimulated only by glutaraldehyde-treated TNP-modified syngeneic, but not by allogeneic, macrophages. These results are discussed with respect to the nature of the TNP-specific immunogen recognized by T cells.     

Nature of the antigenic complex recognized by T lymphocytes. VI. The effect of anti-TNP antibody on T cell responses to TNP-conjugated macrophages.

In this paper we examined the effect of anti-TNP antibody on guinea pig T cell proliferation in response to TNP-modified macrophages in vitro. The addition of anti-TNP to TNP-modified macrophages immediately after conjugation inhibited their ability to stimulate TNP-specific T cell proliferation. This inhibition appeared to be specific for the TNP response since anti-TNP had no effect on the ability of TNP-modified macrophages pulsed with either PPD or TNP-Ova to stimulate efficient PPD or Ova T cell responses. On the other hand, anti-TNP had no effect on the TNP-specific response to TNP-modified macrophages that had been cultured overnight before addition to primed T cells or to macrophages which had been pulsed with TNP-Ova. We also demonstrated that the same TNP-specific T cell subpopulation responds to both freshly TNP-modified macrophages and overnight cultured TNP-modified macrophages. These results suggest that the relevant TNP-determinants recognized by T cells are not exposed on the macrophage surface and raise the possibility that macrophages must process membrane-conjugated TNP to create the immunogen recognized by T cells.     

IL-2 secretion by soluble antigen-reactive human T-cell clones

Human tetanus toxoid specific T-cell lines and clones capable of producing IL-2 were established. IL-2 production occurred only when the antigen-specific T cells were cultured with both tetanus toxoid antigen and an autologous, irradiated adherent cell population. The T-cell lines and clones remained strictly dependent on exogenous IL-2 for proliferation at all other times. Phenotypic characterization with monoclonal antibodies recognizing T-cell subsets revealed that the antigen-specific lines and clones bore predominantly OKT3 and OKT4 markers with essentially no OKT8 positive cells present. T-cell clones which were demonstrated to secrete IL-2 activity could also partially deplete media of IL-2 if cultured in the absence of soluble antigen and irradiated adherent cells.     

Adherent cell function in murine T lymphocyte antigen recognition. I. A. macrophage-dependent T cell proliferation assay in the mouse.

The data in this report describe a T cell proliferation assay with nylon wool column-purified murine lymph node lymphocyte from animals immunized by footpad injection of antigen in CFA. It was found that the in vitro immune response of sensitized T cells to soluble protein antigens was functionally dependent on the presence of adherent cells, more specifically macrophages, at all concentrations of in vitro antigen challenge. The response was due to T cells in that cytotoxic treatment of the immune lymphocyte cells with anti-Thy 1.2 serum and complement effectively eliminated the antigen-specific DNA synthetic responses. The antigen-specific proliferation of murine lymphocytes depleted of adhereent cells could not be reconstituted with either guinea pig macrophages nor murine fibroblasts, indicating the existence of species and cell type specificity. In contrast to previous observations in the guinea pig, soluble products of cultured adherent cells could at least partially replace the function of intact macrophages in the response to antigen.     

Antigen-specific murine T-cell proliferation: role of macrophage surface Ia and factors.

The proliferation of Mycobacterium-primed murine lymph node T cells to purified protein derivative of tuberculin (PPD), as measured by the uptake of tritiated thymidine, requires the obligatory participiation of macrophages which stimulate the T cells either directly with antigen in association with cell surface Ia (I region-defined antigens), or indirectly by means of soluble factors. We have examined the possibility that this functional dichotomy is due to heterogeneity within the macrophage population. Since the maturation of macrophages from the precursor monocytes is associated with cell enlargement, macrophage subpopulations differing in developmental stage are obtained by cell fractionation according to size by velocity sedimentation. Nylon-wool-purified T cells which have been depleted of macrophages and B cells are stimulated with PPD either in a free form or bound to macrophages which have been incubated for a short time (i.e., pulsed) with PPD. We found that for PPD-pulsed macrophages, only the smallest (and probably the most immature) are capable of inducing T-cell proliferation. This antigen presentation function is mediated by cell surface Ia since it is abolished by pretreatment of the macrophages with anti-Ia serum and complement. On the other hand, all macrophages, irrespective of sensitivity to anti-Ia serum, secrete factors which will stimulate T-cell proliferation in the presence of free PPD. Thus the maturation of macrophages is accompanied by a shift from Ia-dependent to Ia-independent mechanisms of immunostimulation.     

Genetically restricted immune responses in guinea pigs primed in vivo with antigen-bearing macrophages.

Guinea pigs injected intradermally with antigen pulsed macrophages generate a population of immune T cells that proliferate in vitro on second exposure to antigen. T cells from F1 (2 X 13) guinea pigs immunized with DNP-OVA on one parental macrophage respond in vitro only to DNP-OVA on macrophages identical to those used for immunization and not to DNP-OVA associated with the other parental macrophages. These results demonstrate that the immunogenicity of antigen is dependent upon the macrophages used for priming in that, with this approach, strain 2 or 13 guinea pigs immunized with allogeneic macrophages pulsed with antigen do not respond to either allogeneic or syngeneic antigen-bearing macrophages. However, lysates of antigen-pulsed macrophages can still immunize either allogeneic or syngeneic recipient via their own macrophages. F1 (2 X 13) guinea pigs are immunized by insulin B chain pulsed strain 13 macrophages (responder) but not by strain 2 macrophages (nonresponder) suggesting that whether a F1 (nonresponder X responder) guinea pig recognizes antigen bound to a parental macrophage is genetically restricted before immunization to the same extent as the donor parental macrophages used for immunization.     

Alloreactive T cells from individual soft agar colonies specific for guinea pig Ia antigens. II. Cellular and molecular requirements for optimal proliferative responses

We have investigated the cellular and molecular requirement for optimal proliferative responses of several alloreactive T cell lines that were derived from individual soft agar colonies and were specific for guinea pig Ia antigens. Optimal proliferation of several colonies was observed in cultures containing purified allogeneic macrophages and growth factor(s) present in supernatant fluids of Con A-activated T cells (Con A-S). Significant proliferative responses of these alloreactive T cell colonies were also routinely detected in cultures only supplemented with unfractionated irradiated allogeneic peritoneal exudate cell (PEC). The T cell component of the stimulator cell population was crucial for these responses by producing necessary growth factor(s) endogenously in the culture. Thus, 2 signals, allogeneic Ia antigens and growth factor(s), were required for optimal proliferative responses of these alloreactive T cell colonies. Furthermore, macrophage-associated Ia antigen was more efficient than B cell-associated Ia for these responses. The requirement for allogeneic Ia antigen was not absolute, since the colonies could easily be expanded when the cultures were supplemented with irradiated syngeneic PEC and the T cell mitogens, Con A or PHA. The effect of the mitogen was mediated via the T cells in the irradiated PEC, since removal of the T cells from these PEC markedly reduced the responses. Thus, it is likely that a nonspecific signal(s) presumably from T cells can promote proliferation of alloreactive T cell colonies in the absence of allogeneic Ia antigen. These results suggest 2 mechanisms of activation of these alloreactive T cells.     

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.     

The expression of Ia antigens on immunocompetent cells in the guinea pig. III. Functional selection of Ia-negative T cells by in vitro culture.

In the present study we examined the expression of I-region-associated (Ia) antigens by guinea pig T lymphocytes stimulated in vitro with antigen-pulsed macrophages. Treatment of lymph node (LNL) or peritoneal exudate (PEL) T cells taken directly from immune animals with anti-Ia serum and complement (C) dramatically reduced their proliferative response to antigen-pulsed macrophages when determined on the 4th day of culture. In contrast, the response of immune T cells that had been selected by culture for a week with antigen-pulsed macrophages and restimulated in a second culture was not affected by anti-Ia and C treatment. This same result occurred with selected LNL or PEL that were initially treated before the selection culture with either normal serum or anti-Ia serum and C. LNL became resistant to anti-Ia serum and C treatment by 3 days of culture whereas antigen-specific PEL were still sensitive at that time. These results indicate that in an immune animal two antigen-specific T cell subpopulations are generated based on their sensitivity to anti-Ia serum and C treatment, but that only the resistant population is selected by in vitro culture. In addition, we demonstrated that the Ig-negative T cell population can only be activated by histocompatible antigen-pulsed macrophages.     

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.     

Activated human T cells can present alloantigens but cannot present soluble antigens

Human T cells, when activated by antigen or mitogen, express Ia antigens. We have examined the capacity of activated T cells to stimulate autologous and allogeneic T cells and their ability to present soluble antigen. Interleukin 2-dependent T-cell lines (TCL), free of accessory cells, were used for antigen-presenting cells. These activated T cells were potent stimulators in an autologous mixed lymphocyte reaction (AMLR), more so than autologous irradiated non-T mononuclear cells. Activated T cells were also able to stimulate proliferation of allogeneic T cells in the absence of any other accessory cells, and this stimulation was blocked by anti-Ia antibodies. Resting unstimulated T cells were unable to stimulate autologous or allogeneic responses. Thus, activated T cells were able to present self antigens and alloantigens. However, activated T cells could not present soluble antigens to autologous T cells or to antigen-specific TCL even if exogenous interleukin 1 was added to cultures. The ability of activated T cells to stimulate an AMLR in vitro may reflect an important immunologic amplification mechanism in vivo. The ability of activated T cells to present alloantigens but not soluble antigens suggests an inability to process antigen, and this may provide further insights into the complexities of antigen presentation.     

Interleukin 2-driven T lymphocyte proliferation is dependent upon a surface antigen distinct from the interleukin 2 receptor: requirements for inhibition of T-cell proliferation by monoclonal antibody 5C3

A monoclonal antibody (5C3) to an antigen expressed on activated guinea pig T lymphocytes that did not react with the interleukin 2 (IL-2) receptor, but inhibited IL-2-driven proliferative responses has been previously characterized. The present study provides further analysis of the inhibitory capacity of 5C3 for T-cell proliferation and of the relationship between the expression of the antigen defined by 5C3 and the capacity of cells to respond to IL-2. 5C3 inhibited proliferation of T-cell blasts to IL-2-containing fluids when added as late as 8 hr prior to termination of a 26-hr culture. 5C3 pretreatment of the IL-2-responsive blast cells was also sufficient to detect significant inhibition of proliferation. FACS analysis of these blasts indicated that maximal 5C3 binding was required for pretreatment to result in inhibition of IL-2-driven proliferation. Delayed addition of 5C3 to culture or pretreatment with 5C3 of responding cells also resulted in inhibition of proliferation of immune T lymphocytes to antigen-pulsed-presenting cells. Lastly, although modulated 5C3- blasts failed to proliferate to IL-2, induction of the 5C3-bearing molecule on these 5C3- blasts correlated with restoration of the ability of these cells to proliferate to IL-2. Collectively, these results further support the hypothesis that monoclonal antibody 5C3 interferes with a critical signal in the IL-2 growth pathway.     

Functional interactions of human and murine lymphoid cells. I. Analysis of primary and secondary responses

In this study human T-cell responses against murine alloantigens were analyzed. The results show that optimal primary responses are obtained from peripheral blood mononuclear cells only when murine splenic adherent cells (SAC) were used as antigen. Further analysis revealed that human T cells were able to respond directly to murine cells without the need for antigen reprocessing; however, human interleukin 1 (IL-1) was required for optimal stimulation. In contrast, secondary proliferative responses to murine cellular antigens could be induced from primed T cells even in the absence of SAC and/or IL-1. These proliferative responses, and in addition, cytotoxic T-cell responses, were specific for the priming antigen. Long-term human T-cell lines specific for murine alloantigens were found to replace the need for murine T cells in antigen-specific murine B-cell responses to sheep red blood cells. The mechanism of help delivered by the human T cells appeared to be by the release of nonspecific helper-T-cell factors. The evidence presented for this is the inability of these cells to stimulate cells from mice that express the X chromosome B-cell defect xid.     

Interleukin 1 secretion is not required for human macrophage support of T-cell proliferation

Interleukin 1 (IL-1) is a soluble factor secreted by stimulated monocytes (Mo) and animal macrophages (Mx). We have previously demonstrated that human Mo cultured in vitro for 1-6 days transform to Mx, and retain their ability to support concanavalin A (Con A)-driven T-cell proliferation. We have also shown that, paradoxically, these Mx do not secrete IL-1, when stimulated by endotoxin (LPS). In this study we examined two alternative hypotheses: T cells plus mitogen induce Mx IL-1 production, and human Mx deliver a second signal to T cells via a non-IL-1 mechanism. IL-1 was assayed in a mouse CD-1 thymocyte system without concanavalin A. Mo/Mx were cultured with T cells at low (2 X 10(4)/200 microliters) or high (1 X 10(5)/200 microliters) concentrations for 2 or 4 days, in the presence of Con A. Six hours prior to quantitation of proliferation, 50 microliters of supernatant was removed and assayed for IL-1. As expected both Mo and Mx enhanced T-cell proliferation eight- to tenfold. Mo secreted large amounts of IL-1; there was no demonstrable IL-1 activity present in supernatants from cultures containing either T cells and Mx, or Mx alone. Similar results were obtained by preincubating the cells (Mo, Mx, and T cells) with Con A for 12 hr and removing Con A prior to a 36-hr coculture. We examined the possibility that a small amount of IL-1 may be able to support Con A-stimulated T-cell proliferation and yet may not induce thymocyte proliferation. The highest dilutions of Mo supernatant (1:125) which supported T-cell proliferation also caused a fivefold increase in thymocyte proliferation. Supernatants from Mx failed to stimulate thymocyte proliferation or support Con A-driven T-cell proliferation. However, Mo and Mx lysates contain Il-1 activity. We conclude that human Mx support Con A-induced T-cell proliferation in the absence of IL-1 secretion. Mx may support T-cell proliferation by cell-bound IL-1 or by a non-IL-1 mechanism.     

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.     

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.     

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.