Adherent cell function in murine T lymphocyte antigen recognition. II. Definition of genetically restricted and nonrestricted macrophage functions in T cell proliferation.

The mechanisms by which adherent cells, presumably of mononuclear phagocytic lineage, influence in vitro antigen-specific activation of murine T lymphocytes was examined. Two distinct functions for macrophages could be discerned. One macrophage function is dependent on a soluble factor produced by cultured adherent cells and is most easily studied with complex multideterminant antigens. This factor is neither antigen-specific nor MHC-restricted in its action in that PEC, regardless of haplotype, produce factor in the absence of antigen. A second function, antigen-specific T cell activation, is seen when antigens of more restricted heterogeneity are used, such as those under the control of Ir genes. This latter activity demands identity or partial identity between the antigen-presenting cell and the primed T cell, thus suggesting an additional specific, genetically restricted function for macrophages in in vitro antigen recognition. Whether these adherent cell functions are mediated by all or distinct subsets of cells was not established.     

The activation of guinea pig T lymphocytes by anti-beta 2-microglobulin serum.

In order to study further the role of beta 2-m in the regulation of the immune response, we have examined the effects of a goat anti-guinea pig beta 2-m serum on a number of T lymphocyte functions in vitro. Anti-beta 2-m serum produced a marked inhibition of the response of peritoneal exudate T cells to antigen and mitogen stimulation. Surprisingly, a marked activation of lymph node T lymphocyte proliferation was observed in the absence of antigen or mitogen stimulation. This stimulatory effect of anti-beta 2-m serum was shown to be specific for beta 2-m and required the presence of macrophages. The T cell proliferative response induced by anti-beta 2-m could not be blocked by antisera to the antigens of the guinea pig MHC. These studies suggest that beta2-m may play some critical role in the immune response at the level of T cell activation.     

Identification and characterization of gp TFA-1, a guinea pig T cell surface antigen associated with T cell function

Monoclonal antibodies (Ab) were produced that specifically recognized guinea pig T cells. FACS analysis revealed that Ab 188 bound to the majority of peripheral T lymphocytes of strain 2 and strain 13 guinea pigs and to a minor population of thymocytes. It failed to react with the Ia-bearing guinea pig B cell leukemia line EN-L2C, with macrophages, bone marrow cells, erythrocytes, or thrombocytes. Treatment of T cells with Ab 188 and complement prevented T cell activation. Culturing primed T cells with antigen- or mitogen-pulsed syngeneic or with allogeneic macrophages in the continuous presence of Ab 188 produced a marked, dose-dependent inhibition of T cell proliferation. The antigen defined by Ab 188 was therefore designated guinea pig T lymphocyte function-associated antigen-1, gp TFA-1. The magnitude of inhibition by Ab 188 varied between 65 and 85% whereas three other antibodies to guinea pig T cells had no inhibitory effect on T cell proliferation. Time course experiments revealed that gp TFA-1 is critically involved in an early phase of T cell activation. Maximal inhibition was achieved only if the antibody was present from the beginning of the cell culture; the addition of antibody after 24 hr of culture no longer had an inhibitory effect. Ab 188 did not induce T cell mitogenesis. Two-dimensional analysis (one-dimensional, IEF; two-dimensional, SDS-PAGE) of immunoprecipitates obtained from NP40 lysates of [35S]methionine-labeled T cell blasts indicated that a molecule was specifically precipitated that consisted of two noncovalently associated polypeptide chains with apparent m.w. of 43,000 and 38,000. Both subunits displayed extensive charge heterogeneity focusing at an average isoelectric point of 5.0 and 6.5, respectively. The gp TFA-1 molecule exhibits striking similarities in its functional and structural properties to recently described clonotypically expressed T cell glycoproteins, which were shown to be involved in antigen recognition by T cells in the murine and human systems.     

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.     

Phytohemagglutinin-induced proliferation of guinea pig thymus-derived lymphocytes. I. Accessory cell dependence.

The accessory cell requirement for mitogen-induced T lymphocyte proliferation has been investigated by using a population of guinea pig lymph node lymphocytes enriched in T cells and markedly depleted of macrophages and B lymphocytes. We have found that effective phytohemagglutinin-induced proliferation of T cells is dependent on the participation of accessory cells. Augmentation of PHA responsiveness was noted when cultural conditions were manipulated to increase cell density, suggesting that physical proximity between T cell and accessory cell is required for efficient triggering. Both syngeneic and allogeneic macrophages, as well as syngeneic fibroblasts, serve as accessory cells in this response whereas polymorphonuclear leukocytes or thymocytes do not. Thus, although PHA-induced T lymphocyte proliferation requires accessory cells, the specificity of these cells is strikingly less stringent than for antigen-mediated triggering of immune guinea pig T cells, a response which is dependent upon participation of syngeneic macrophages.     

Antigen handling by guinea pig macrophages: further evidence for the sequestration of antigen relevant for activation of primed T lymphocytes.

Guinea pig macrophages can take up sufficient 2,4 dinitrophenyl guinea pig albumin during a brief in vitro exposure at 37 degrees C to trigger proliferation and lymphokine production with primed T lymphocytes on subsequent co-culture. Treatment of such antigen-bearing macrophages with trypsin, a procedure which removes surface antigen, does not alter the ability of such macrophage to initiate the release of migration inhibition factor from sensitized T lymphocytes. In addition, formation of antigen-specific rosettes between primed T cells and antigen-bearing macrophages is not blocked by high concentrations of antibody directed against the antigen mediating this interaction. Similarly, primed T lymphocyte DNA synthesis induced by antigen-bearing macrophages is not inhibited by specific antibody to that antigen. These data support the conclusion that the fraction of macrophage-associated antigen which is relevant to T lymphocyte activation does not reside on the macrophage surface but rather remains in a restricted compartment from which it is accessible to the T cell but unavailable to either blockade by specific antibody or removal by proteolytic enzymes.     

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.     

Antigen recognition: the specificity of an isolated T lymphocyte population.

Guinea pig lymph node lymphocytes were separated into T and B cell fractions on immunoabsorbent columns. Separated cells were functionally distinct: T cells proliferated in response to ConA, PHA, soluble and alloantigen, whereas anti-Ig reagents only stimulated B cells. The in vitro proliferative response of guinea pig lymph node T lymphocytes was then shown to be highly discriminating when elicited by a series of structurally similar synthetic DNP-oligolysine antigens. Proliferation was always most extensive in response to the homologous, immunizing antigen, and less intense to cross-reacting DNP-oligolysines. Specificity of proliferation was maintained in the absence of both B lymphocytes and antibody secreting cells, suggesting that T cell recognition is not "acquired" from B cells or secreted antibody, but is a property inherent to the T cell.     

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.     

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.     

T cell induction of membrane IL 1 on macrophages

We have studied the role of T cells in the induction of a membrane-associated form of interleukin 1 (mIL 1) in murine macrophages. T helper cell clones and a T cell hybridoma induced macrophages to express mIL 1 after an antigen-specific, Ia-restricted interaction. Induction of mIL 1 was proportional to antigen concentration and was increased in the early course of the response in macrophages pretreated in culture with interferon-gamma. mIL 1 activity was detectable 4 hr after interaction with T cells. mIL 1 induction was inhibited by antibodies to either class II molecules or the T cell receptor. Two pathways of T cell-mediated mIL 1 induction could be defined. In the first, T cells, whose protein synthesizing capacity was completely eliminated by pretreatment with the irreversible protein synthesis inhibitor emetine, induced levels of mIL 1 expression indistinguishable from controls. In the second, T cells stimulated by paraformaldehyde-fixed macrophages in the presence of concanavalin A or antigen secreted a soluble factor that induced macrophage mIL 1 expression. Thus, it appears that T cells may induce macrophages to express mIL 1 both by direct cell-cell contact mediated through binding of T cell receptor to the Ia/antigen complex, and through the release of a lymphokine after activation. This lymphokine does not appear to be IL 2, IFN-gamma, BSF-1, or CSF-1.     

Antigen presentation by liver sinusoidal lining cells after antigen exposure in vivo

The ability of liver sinusoidal lining cells (LSLC), a mixture of Kupffer cells and endothelial cells, to function as antigen-presenting cells (APC) was examined. Guinea pig LSLC were found to present antigen in vitro, albeit somewhat less effectively than a reference population of peritoneal exudate macrophages. The difference in APC function could not be explained by a deficiency of interleukin 1 (IL 1), as LSLC secreted IL 1 and expressed membrane-bound thymocyte stimulatory activity. The ability of LSLC to take up antigen from the portal blood in vivo and present it to primed T lymphocytes in vitro was also investigated. Trinitrophenyl-ovalbumin was injected intraportally into either strain 13 or strain 2 guinea pigs. The LSLC were subsequently isolated by collagenase digestion and density separation and assessed for the ability to induce proliferation of antigen-primed accessory cell-depleted syngeneic peritoneal exudate T lymphocytes in vitro. The in vivo antigen-pulsed LSLC were found to present antigen in vitro to primed T cells in an antigen-specific and genetically restricted manner. T cell DNA synthesis induced by antigen-bearing LSLC could be augmented by coculture with additional accessory cells, but not IL 1-containing macrophage supernatants. Enhancement of responsiveness was not genetically restricted. The demonstration that LSLC can take up, process, and retain antigen in vivo and present it to primed T cells in vitro suggests that LSLC are capable of contributing to the immune response to antigens appearing in portal blood.     

Cell-cell interactions in the T cell proliferative response. I. Analysis of the cell types involved and evidence for nonspecific T cell recruitment

In the antigen-induced T cell proliferative response, it has been firmly established that antigen-specific T cell activation signals are provided by a specialized antigen-presenting cell (APC). However, the number of responding T cell populations involved has not been clearly delineated. This problem can be analyzed by plotting the logarithm of the number of cultured cells against the logarithm of the response. This yields a straight line, the slope of which indicates the minimum number of interacting cell populations that are required to give the response. In the antigen-induced T cell proliferative response, this number is 3. Based on their ability to shift the slopes of the log cell number-log response line, two of the populations were identified to be T cells. The third cell, which was present in irradiated spleens, possessed certain properties of the APC. Of the two T cells, one was antigen-specific and the other could come from normal unprimed animals. The frequency of antigen-specific proliferating T cells in primed animals was estimated to be only 1 in 1.3 x 10(3), and a large proportion of the proliferation was shown to be due to unprimed cells. Furthermore, without the need to use antigen-pulsed macrophages, this slope analysis demonstrated convincingly that the successful interaction between APCs and proliferating T lymphocytes is determined by products of the I-region immune response genes.     

Granulocyte-macrophage colony-stimulating factor augments the primary antibody response by enhancing the function of antigen-presenting cells

Purified, recombinant-derived murine granulocyte-monocyte colony-stimulating factor was found to enhance the primary in vitro immune response to SRBC by murine spleen cells. In determining the mechanism of this augmentation, it was found that only splenic adherent cells and neither resting nor activated T cells nor B cells expressed specific receptors for GM-CSF. When splenic adherent cells were pulsed briefly with GM-CSF before addition to macrophage-depleted cultures, they reconstituted the PFC response to a significantly greater degree than did control macrophages. Splenic adherent cells incubated overnight with SRBC plus GM-CSF were also more efficient antigen-presenting cells than splenic adherent cells incubated with antigen alone. The mechanism of this enhanced antigen presentation was found to be due to a GM-CSF-dependent increase in the level of IL 1 secretion and Ia antigen expression. Consistent with these data was the finding that GM-CSF augmented IL 2 production by splenic T cells in response to suboptimal concentrations of Con A. Finally, the day 5 in vivo antibody response (as measured by serum titers) of mice immunized with a low dose of SRBC was enhanced by two daily inoculations of GM-CSF. Thus, the role that GM-CSF plays in augmenting immune responses may not be solely accounted for by its ability to cause the proliferation or differentiation of macrophages, but more than likely includes its ability to enhance the function of antigen-presenting macrophages.     

Determinant specific suppression of antigen-induced T cell proliferation in the guinea pig. II. Determinant specific suppression of in vitro T cell responsiveness parallels a selective suppression of anti-hapten but not anti-carrier antibody responses

Using an antigen of defined physical structure with precisely mapped immunogenic sites, we asked whether those molecular sites previously shown to be critical for immune response gene-mediated initiation of T cell proliferation and T help correspond to the same molecular regions capable of inducing antigen-specific suppression of T cell proliferation and antibody production. Inbred strain 2, 13, and 2 x 13 F1 hybrid guinea pigs were immunized with various species variants or fragments of insulin adjuvant before subsequent immunization with antigen in complete Freund's adjuvant. Analysis of the patterns of depressed T cell responsiveness showed a striking correspondence to the Ir gene-dependent mechanism that controls the recognition of discrete regions within the insulin molecule observed in T cell help in antibody production. In addition, suppression of carrier-specific T cells parallels suppression of anti-hapten antibody responses when hapten is presented on the suppressed carrier without a concomitant suppression of the anti-carrier antibody response.     

Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity. IV. Role of macrophage and its soluble factor in antigen-induced MIF production of immune T lymphocytes.

Histocompatibility-linked restriction of macrophage-T lymphocyte interaction in antigen-induced MIF production by sensitized lymphocytes was examined, by using combinations of inbred strain 2, strain 13, and JY-1 guinea pigs. The effective interaction of the antigen-bearing macrophages with the immune T lymphocytes was observed when the donor of the antigen-bearing macrophages and that of the immune lymphocytes shared Ia antigens of the major histocompatibility complex. Identities of B antigens and S antigens were not important for this cooperation. It was further demonstrated that the previously reported soluble factor derived from LPS-stimulated peritoneal adherent cells (macrophages) could help antigenic activation of the immune lymphocytes across the strain barrier provided a small number of macrophages (0.01%) from syngeneic strain were present. These results show that the presence of macrophages is absolutely required to present antigen to immune T lymphocytes in a genetically restricted manner and the soluble factor from macrophages appears to give a nonspecific effect on the lymphocyte activation in addition to or in collaboration with antigenic stimulation.     

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.     

The murine Kupffer cell. I. Characterization of the cell serving accessory function in antigen-specific T cell proliferation.

Murine Kupffer cells, the tissue macrophages of the liver, were isolated by collagenase digestion, differential sedimentation over Metrizamide, and glass adherence. The resultant cell population was more than 86% phagocytic, and 95% of cells stained positively for alpha-naphthyl butyrate esterase activity. The cells also had cell surface receptors for complement (C) and the Fc portion of IgG. In addition, a large proportion of Kupffer cells was shown to bear Ia antigens: about half of the cells bore I-A subregion-encoded antigens and about half bore I-BJE or I-EC subregion-encoded antigens. Kupffer cell populations were capable of reconstituting antigen-stimulated proliferative responses of antigen-primed, macrophage-depleted, lymph node T cells. The ability to reconstitute proliferation was enriched in the adherent population and was resistant to radiation and treatment with an anti-Thy antiserum and C. We conclude that isolated murine Kupffer cells bear the Ia phenotype of accessory cells that function in antigen presentation and that Kupffer cells can participate in the induction of antigen-specific immune responses. These data suggest that Kupffer cells may play a role in modulating responses to enterically derived antigens.     

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.     

T lymphocyte-enriched murine peritoneal exudate cells. I. A reliable assay for antigen-induced T lymphocyte proliferation.

The in vitro activation of murine thymus-derived (T) lymphocytes by soluble protein and synthetic antigens has been difficult to assess because of the lack of a specific and reliable proliferation assay. The present report describes the development of an assay system which overcomes these problems by making use of a population of nylon wool column-purified T lymphocytes obtained from thioglycollate-induced peritoneal exudates of immunized mice. PETLES (peritoneal exudate, T lymphocyte-enriched cells) were composed mainly of T lymphocytes, eosinophils and small numbers of macrophages. Contamination with bone marrow-derived (B) lymphocytes averaged only 2%. When PETLES from immunized mice were stimulated in microtiter cultures with the immunizing antigen, large degrees of proliferation ensued as measured by incorporation of 3H-methyl-thymidine 5 days after initiation. As few as 1.25 x 10(4) cells and as little as 50 ng/ml of antigen gave significant stimulation. Maximum responses were obtained witn a series of 10 experiments under these optimal conditions, gave a mean incorporation of 70,900 cpm while the controls cultured without antigen showed only 3,600 cpm. PETLES from nonimmunized mice or from mice immunized to other antigens did not respond to DNP5OVA although they did respond to mitogens. The antigen-induced proliferation was shown to require the presence of immune T lymphocytes by two criteria: elimination of the response by treatment with anti-Thy 1.2 serum plus complement and failure to reconstitute the response when the few remaining immune B lymphocytes left after anti-Thy 1.2 treatment were added to nonimmune T lymphocytes. In addition, the system exhibited carrier specificity. Because of the paucity of B lymphocytes in the population, their contribution to the overall magnitude of the proliferative response was negligible as demonstrated by the small response to B cell mitogens. Thus, the assay appears to be a quantitative as well as a qualitative assay for one aspect of T lymphocyte function. This technique should prove useful for the study of murine T lymphocytes in vitro.