A comparison of the stimulatory activities of lymphoid dendritic cells and macrophages in T proliferative responses to various antigens

The identities of murine accessory cells and the mechanism by which they process antigen and stimulate T cell proliferation have been examined with cell separation techniques and specific agents to block antigen catabolism. Using preparations of splenic dendritic cells (DC) and macrophages (M phi) with minimal cross-contamination, we found that only DC could induce syngeneic mixed leukocyte reaction (MLR), whereas both DC and M phi could initiate allogeneic MLR. This observation may have significant implications for syngeneic MLR as a manifestation of self Ia recognition, and for the cell type that defines self Ia during ontogeny. DC and M phi could present soluble antigens such as purified protein derivative of tuberculin (PPD) and Salmonella flagellin about equally well to antigen-specific T cell lines. M phi, however, were much more effective than the non-phagocytic DC at inducing T cell proliferation to whole Corynebacterium parvum organisms. These differences could not be attributed to differences in antigen uptake. The results suggest that the bacteria must be ingested and processed by phagocytes before T cell activation. Using the lysosomotropic agent chloroquine to inhibit antigen catabolism in accessory cells, we found that the presentation of large antigens by M phi and DC was abolished by chloroquine treatment, whereas T cell activation by antigens (such as PPD or integral membrane Ia for MLR) that apparently required no processing was relatively insensitive to chloroquine. Thus, in addition to differences between cells, discrete functions within each cell type can also be distinguished.     

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.     

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)     

Enhancement of antigen-presenting function of dendritic cells with culture supernatants of mouse peritoneal macrophages stimulated with certain particulate substances

The production from murine resident peritoneal macrophages (M phi) of a soluble factor, which was capable of enhancing the antigen-presenting (AP) function of dendritic cells (DC), was examined. The supernatants of peritoneal M phi (M phi sup) were prepared by culturing peritoneal M phi with particles, i.e., zymosan A, latex, and sheep red blood cells (SRBC), or antigen-antibody (Ag-Ab) complexes such as keyhole limpet hemocyanin (KLH)-anti-KLH, ovalbumin (OVA)-anti-OVA, and SRBC-anti-SRBC complexes. When exposed to M phi sup during antigen pulsing DC induced a marked antigen-specific T cell proliferation, relative to DC treated with the supernatants from M phi cultured without stimuli (control sup). On the other hand, M phi sup-treated splenic M phi stimulated antigen-specific T cell activation to almost the same extent as did splenic M phi treated with control sup. These results indicated that peritoneal M phi elaborated a soluble factor which preferentially enhanced the AP capacity of DC when stimulated with particles or Ag-Ab complexes. Analytical gel filtration of M phi sup revealed that the factor had an apparent molecular weight of 27,000 daltons which was distinct from interleukin 1.     

Cellular requirements for induction of human primary proliferative responses to trinitrophenyl-modified cells

Cellular requirements for induction of primary proliferative responses by human T cells to trinitrophenylated autologous stimulators have been characterized. Substantial proliferative responses were observed with each of the Ia+ stimulator populations tested. Nevertheless, major differences in the hapten specificity of such responses were observed. Thus purified macrophages/monocytes (M phi) when TNP-modified induced responses that were relatively modest in absolute magnitude, but were highly hapten specific. This reflected the very limited capacity of purified M phi to induce proliferation when unmodified, i.e., an autologous mixed leukocyte response (AMLR). In contrast, unmodified M phi-depleted B plus null cells were potent stimulators of AMLR, but hapten modification did not significantly enhance the responses induced by these cells. Moreover, when M phi were added to B plus null cell stimulators AMLR responses were reduced and, with TNP-modified stimulators, hapten-specific responses were restored. The data thus suggest that M phi may have important roles in induction of primary T cell responses to conventional antigens but function largely as regulators rather than stimulators of AMLR. Finally, we have introduced a novel antigen-presenting cell population, the irradiated Ia+ TNP-specific cloned T cell. The possibility that such cells may utilize autostimulatory positive feedback circuits for activation of naive T cells and in interactions between subpopulations of hapten-reactive T cells is discussed.     

LPS and specific T cell responses: interleukin 1 (IL 1)-independent amplification of antigen-specific T helper (TH) cell proliferation

Lipopolysaccharide (LPS) fraction of endotoxin induces a significant potentiation of the antigen-specific proliferative response of T helper (TH) cell lines. This effect was obtained with LPS from different bacterial sources and reproduced with the lipid A moiety of endotoxin. Purified adherent spleen cells used as antigen-presenting cells (APC) support this LPS-enhanced TH cell proliferation. In addition, the effect of endotoxin on specific TH cell responses was found to be absolutely dependent on the interaction between TH lymphocytes and APC through antigen-specific recognition. Thus, it was not observed in the absence of specific antigen or when monoclonal antibodies against class II MHC products or against L3T4 antigens were used to inhibit the T cell-APC interaction. Similarly, it was found that APC from the B6.CH-2bm12 mutant do not support the LPS-mediated enhancing effect. Furthermore, interleukin 1 (IL 1) appears not to be involved in LPS-mediated enhancement, and this effect is not reproduced by muramyl dipeptide (MDP)-mediated activation of APC.     

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.     

A single monoclonal anti-Ia antibody inhibits antigen-specific T cell proliferation controlled by distinct Ir genes mapping in different H-2 I subregions

A xenogeneic rat anti-mouse Ia monoclonal antibody, M5/114 (gamma 2b, kappa), was studied for its effects in vitro on T cell proliferative responses. Strain distribution studies revealed that M5/114 could inhibit I-A subregion-restricted T cell responses of the H-2b,d,q,u but not the H-2f,k,s haplotypes, indicating that this xenoantibody recognizes a polymorphic determinant on mouse Ia molecules. This same monoclonal antibody was found to inhibit BALB/c (H-2d) T cell proliferation to both G60A30T10 and G58L38 phi 4. The Ir genes regulating responses to these antigens map to either the I-A subregion (GAT), or the I-A and I-E subregions (GL phi), raising the possibility that M5/114 recognizes both I-A and I-E subregion-encoded Ia glycoproteins. It could be shown, using appropriate F1 responding cells, that M5/114 does in fact affect GAT and GL phi responses by interaction with both the I-A and the I-E subregion products, and not by any nonspecific effect resulting from binding to the I-A subregion product alone. These results are consistent with genetic and biochemical studies directly demonstrating that M5/114 recognizes A alpha A beta and E alpha E beta molecular complexes. The existence of a shared epitope on I-A and I-E subregion products suggests the possibility that these molecules arose by gene duplication. Finally, the precise correlation between the Ia molecules recognized by M5/114 and the ability of this antibody to block T cell responses under Ir gene control strengthens the hypothesis that Ia antigens are Ir gene products.     

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

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

Antigen processing requirements for T cell activation: differential requirements for presentation of soluble conventional antigen vs cell surface MHC determinants

The present studies were undertaken to characterize the antigen-processing requirements involved in the responses to T cells to soluble antigen (antigen specific), to allogeneic cell surface MHC determinants (alloreactive), and to syngeneic MHC determinants (autoreactive). T cell clones were used that have dual cross-reactive specificities either 1) for self MHC plus soluble antigen and for allogeneic MHC products or 2) for syngeneic MHC and for allogeneic MHC, in order to permit comparison of the processing requirements for responses of the same T cell to distinct antigenic stimuli. The proliferative responses of antigen-specific, Ia-restricted T cell clones to soluble antigens were sensitive to treatment of antigen-presenting cells (APC) with 125 to 250 microM chloroquine, a lysosomotropic agent previously shown to inhibit the processing of soluble antigens. In contrast, the same T cell clones were only minimally affected in their ability to respond to similarly chloroquine-treated APC expressing allogeneic MHC products. The responses of autoreactive T cell clones to syngeneic stimulating cells and their cross-reactive responses to allogeneic cells were both resistant to chloroquine treatment of stimulating cells. The failure of chloroquine to inhibit antigen presentation to autoreactive T cell clones suggests that these clones are specific for self Ia not associated with in vitro processed foreign antigen. Thus, chloroquine sensitivity distinguishes the in vitro antigen-processing requirements for presentation of the soluble antigens tested from the requirements for presentation of syngeneic or allogeneic cell surface MHC determinants to the same T cells.     

Development and characterization of interleukin-2-independent antigen-specific human T cell clones that produce multiple lymphokines

The development of antigen-specific T lymphocyte lines and clones has greatly facilitated the investigation of T-cell recognition of and response to foreign antigens. In the present study, human antigen-specific helper T cell lines and clones which are completely independent of exogenous interleukin-2 (IL-2) have been developed by cyclic restimulation with the soluble antigen keyhole limpet hemocyanin (KLH) to which the T cell donor had previously been immunized. These T cells uniformly bear the OKT4 phenotype and were shown to require both histocompatible antigen-presenting cells (APC) and antigen for optimal proliferation. The T cell line was composed of a highly antigen-specific and clonable T cell population. Following four cycles of antigen stimulation, limiting dilution cloning analysis showed a Poisson distribution of clonable T cells with a precursor frequency of 0.62, and from 88 to 92% of viable clones were specific for the stimulating antigen. Individual clones were obtained which recognized KLH with either DR 1 (one parental Ia haplotype of the donor) or DR 2 (the other parental Ia haplotype) allogeneic APC, but not both. Following stimulation with KLH, the T cell clones produced IL-2. Peak amounts of IL-2 were assayable in the first 6 to 24 hr after stimulation. In contrast, virtually no IL-2 was detectable in supernatants at 72 to 96 hr, suggesting autoutilization by the proliferating T cells. In addition, some clones were also capable of producing both B cell growth factor and IL-2 following KLH stimulation. These IL-2-independent T cells appeared to be derived from a discrete Leu 8-negative subclass of T4⁺ cells and expressed the full complement of Ia antigen of the donor. Thus, soluble antigen-specific human helper T cell clones have been produced which can be maintained in the absence of exogenous IL-2, elaborate their own growth factors and other immunoregulatory lymphokines, and show fine DR-related restriction to either one or the other parental DR haplotypes in antigen-stimulated proliferative responses.     

In vivo treatment with anti-I-A antibodies: differential effects on Ia antigens and antigen-presenting cell function of spleen cells and epidermal Langerhans cells

The in vivo activation of T cells by a variety of antigens can be inhibited by the administration of anti-I-A antibodies (Ab) at the time of antigen priming. This inhibition can partially be explained by the temporary loss of Ia molecules from Ia-bearing antigen-presenting cells (APC) in the spleen. In this study, the effects of i.p. injected monoclonal Ab specific for I-A glycoproteins of different H-2 haplotypes on Ia antigen expression and APC function of spleen cells and epidermal Langerhans cells were compared. It was found that anti-I-A Ab quickly bound to both spleen cell and Langerhans cell Ia antigens. Although spleen cell Ia antigens were modulated and thus temporarily disappeared, Ia antigen expression by epidermal Langerhans cells was not modulated. In functional studies, the capacity of spleen cells and epidermal cells from anti-I-A Ab treated vs control animals to function as APC for antigen-specific, I-A- or I-E-restricted T cell clones was tested. A single injection of anti-I-A Ab completely abolished the APC function of spleen cells as shown in several inbred mouse strains, F1 animals, and with the use of several different Ab and T cell clones. In contrast, Langerhans cell-dependent APC function of epidermal cells remained completely unaltered. Even multiple injections of high doses of Ab never caused any inhibition of Langerhans cell function. Experiments with anti-I-Ak or anti-I-Ad Ab in an (H-2k X H-2d)F1 animal showed abrogation of APC function of spleen cells, but again not of Langerhans cells. Thus in vivo anti-I-A Ab administration appears to differentially affect Ia antigen expression and APC function from spleen and epidermis: Ia antigens are modulated from spleen cells but not from epidermis, and APC function disappears in the spleen but not in the epidermis. The abrogation of splenic but not of Langerhans cell APC function with anti-I-A Ab will facilitate the dissection of the relative contributions of Langerhans cells as compared with other APC in the generation of cutaneous immune responses.     

Functional defects of culture-grown bone marrow-derived macrophages from autoimmune MRL/MpJ-lpr/lpr mice

To investigate the primary defects and development of macrophages in MRL/MpJ-/pr/lpr (MRL/l) mice, we used a pure population of macrophages derived from bone marrow precursor cells cultured in the presence of L-cell conditioned medium (LCM) as a source of colony stimulating factor. Bone marrow-derived macrophages (BMM phi) from MRL/l mice had lower antigen presenting activity as detected by the induction of antigen-specific T cell proliferation, than age- and sex-matched control mice (CBA/J). Cell surface antigens (Ia and Mac-1) were determined quantitatively by a cell sorter as markers of macrophage differentiation. The BMM phi from MRL/l contained a much smaller number of Ia antigen-positive macrophages than those from normal mice. Treatment of BMM phi with an Ia-inducing of factor (IFN-gamma) markedly increased the expression of Ia antigens. This increase was significantly greater in BMM phi from MRL/l mice than in BMM phi from control mice. Expression of Mac-1 antigen was not different in BMM phi from the two strains. The Fc-mediated phagocytosis of IgG-coated sheep red blood cells was decreased in BMM phi from MRL/l mice compared with those from control mice. The function of nonspecific phagocytosis as measured by latex-bead incorporation was also impaired in MRL/l mice. The functional defects of MRL/l BMM phi found in these experiments are not secondary defects acquired under the influence of environmental signals during development, but are derived from the primary abnormalities which already exist in myeloid stem cells.     

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.     

Liver sinusoidal lining cells express class II major histocompatibility antigens but are poor stimulators of fresh allogeneic T lymphocytes

Guinea pig liver sinusoidal lining cells (LSLC), a mixture of Kupffer cells (KC) and sinusoidal endothelial cells (EC), were examined for their capacity to function as antigen-presenting cells (APC). LSLC were extremely poor stimulators of freshly isolated allogeneic T lymphocytes even though a large number of them expressed class II major histocompatibility complex (MHC) antigens (Ia). This deficiency could not be explained by a lack of soluble factor production by LSLC, because an interleukin 1-containing macrophage (M phi) supernatant could not restore the capacity of LSLC to stimulate allogeneic T cells. Moreover, LSLC were able to promote mitogen-induced proliferation of accessory cell-depleted T lymphocytes. No evidence of suppression was apparent in experiments in which LSLC were added to cultures of T cells stimulated by allogeneic peritoneal exudate M phi (PEM). The Ia expressed by LSLC was functional because they were able to stimulate an alloreactive T cell line. When LSLC were mixed and co-cultured with either PEM syngeneic to the responding lymphocytes or Ia-negative fibroblasts, the allostimulatory ability of LSLC was greatly augmented. In contrast, the addition of mitogen-activated T cell supernatants had only a minimal effect on the capacity of LSLC to stimulate allogeneic T cells. The data suggest that LSLC lack a biologic property that is necessary for recognition of class II MHC determinants by fresh but not primed allogeneic T cells and that is not required to support T cell activation induced by nonspecific mitogenic lectins. These findings may be important in understanding the reason that antigen introduced into the portal blood appears not to initiate an immune response.     

Dissection of the functions of antigen-presenting cells in the induction of T cell activation

The functions of antigen-presenting cells (APC) in the initiation of T cell activation was examined by culturing antigen-bearing guinea pig macrophages (M phi) with T cells obtained from antigen-primed animals. Although such antigen-bearing M phi stimulated primed syngeneic T cell DNA synthesis, as assessed by tritiated thymidine incorporation, paraformaldehyde fixation (0.15% for 1 min at 37 degrees C) abolished this capacity. Analysis with acridine orange staining indicated that fixed antigen-bearing M phi could not trigger primed syngeneic T cells to progress from the G0 to the G1 phase of the cell cycle. The addition of control non-antigen-bearing syngeneic or allogeneic M phi but not interleukin 1 or 2 to cultures of T cells and fixed APC permitted a proliferative response. Although the interaction between fixed antigen-bearing M phi and responding T cells was genetically restricted, there was no similar restriction for the supplemental control M phi. In fact, completely Ia-negative endothelial cells (EC) and fibroblasts (FB) could restore antigen responsiveness to cultures of fixed antigen-bearing M phi and syngeneic responding T cells, although they could not directly present antigen. Moreover, metabolically intact accessory cells, including Ia-negative EC and FB, could take up and process antigen to an immunogenic moiety, which fixed Ia-positive M phi could present to primed T cells. These data indicate that recognition of the antigen-Ia complex on an APC is necessary but not sufficient to trigger proliferation of freshly obtained primed T cells. The results additionally support the conclusion that APC carry out at least two separate functions necessary for the initiation of antigen-induced T cell activation. Not only must the APC display the antigen-Ia complex, but it must also convey another required effect. This influence, which apparently involved the establishment of cell to cell contact, was neither Ia nor antigen dependent and could only be provided by a metabolically intact cell. By contrast, genetically restricted antigen presentation could be accomplished by a fixed Ia-positive cell. Only when both the antigen-Ia complex and the influence of an intact accessory cell were provided by the same or different accessory cell were T cells triggered to enter the cell cycle.     

Ability of fixed B-lymphoma cells to present foreign protein antigen fragments and allogenic MHC molecules to a cloned helper-T-cell line

Cloned, L3T4+ T cells have been shown to respond to foreign protein antigens in the context of self-Ia glycoproteins and to non-self Ia glycoproteins. In the case of responses to foreign proteins, fixed antigen-presenting cells can present antigen fragments, but cannot present native proteins. Whether fixed allogenic cells can stimulate has been controversial. We have examined this question using a dual-reactive cloned helper-T-cell line. We find that conditions of fixation that block the presentation of native antigen to this cloned line, but which allow the presentation of antigen fragments, also allow presentation of allogeneic Ia molecules, leading to stimulation of the cloned line. This study also revealed an occult alloreactivity in the cloned T-cell line, which was expressed by fixed, but not by normal, antigen-presenting B lymphoma cells. All of these stimuli proceeded via the same clonotypic receptor, as determined by blocking with anti-T-cell receptor monoclonal antibody. These data suggest that responses to non-self Ia glycoproteins involve direct recognition of the allogeneic Ia molecules and do not require processing and presentation of these antigens by self Ia molecules.     

Astrocytes as antigen-presenting cells. I. Induction of Ia antigen expression on astrocytes by T cells via immune interferon and its effect on antigen presentation

Ia antigens seem to control immune responses on at least two levels. First, they influence the antigen recognition repertoire of the T cells. Second, their variable expression on certain antigen-presenting cells is a powerful regulatory mechanism for the local immune reaction. This is particularly important in the central nervous system (CNS) in which no Ia antigens are normally expressed. Recent experiments in this context have shown that astrocytes are able to express Ia antigens during interaction with T cells, and that they function as antigen-presenting cells. The Ia-inducing activity is produced by activated T cells, and can be replaced by immune interferon (IFN-gamma). In this study we report on the functional and kinetic relationship between Ia antigen expression on astrocytes and the immune-specific activation of T cells by astrocytes. Normal resting astrocytes were found to be negative for Ia antigens by immunofluorescence and by biochemical criteria. Moreover, they are only able to stimulate T cells after they have been induced to express Ia antigens by a signal from the T cells, which is probably mediated by IFN-gamma. In conclusion, the immune-specific interaction between astrocytes and T lymphocytes is a sensitively controlled system that might be pivotal to the development of immune responses in the brain. Malfunction of the system could be an important factor in the pathogenesis of aberrant immune reactions in the CNS, e.g., in multiple sclerosis.     

Functional subsets of human monocytes defined by monoclonal antibodies: a distinct subset of monocytes contains the cells capable of inducing the autologous mixed lymphocyte culture

The induction of most immune responses requires the close cooperation between T cells and antigen-presenting cells (APC), presumably of monocyte/macrophage (M phi) lineage. To characterize human APC further, we used two monoclonal antibodies, OKM1 and OKM5, to isolate and identify M phi subsets. OKM1 has been described and recognizes cell surface antigens on most M phi and granulocytes. OKM5 recognizes cell surface determinants present on the majority of human M phi but does not recognize other hematopoietic cell types. A small subset of peripheral blood M phi is OKM1-OKM5+. Human peripheral blood E- cells were separated into OKM1+ and OKM1- subsets by a rosetting technique utilizing anti-Ig-coated red cells. The capacity to present self antigens in the autologous mixed lymphocyte culture (AMLC) resided predominantly within the E-OKM1- subset, even if surface membrane Ig-positive cells were eliminated. Similar experiments showed that the ability to stimulate in AMLC was contained in the E-OKM5+ population and in fact resided primarily within the E-OKM1-OKM5+ subset. All of these subsets were able to trigger allogeneic T cells to proliferate. The capacity of these APC subsets to present soluble antigens (mumps, tetanus toxoid) was also examined. The data demonstrated that although the majority of these APC are E-OKM1+, E-OKM1-OKM5+ cells can also present foreign antigen. Taken together, these data suggest OKM1 and OKM5 can be used to isolate two functionally distinct human M phi subsets. One subset (E-OKM1+) is capable of presenting soluble antigens but shows minimal ability to trigger AMLC. The other subset (E-OKM1-OKM5+) can also present soluble antigens but is the predominant subset that can trigger AMLC.     

Interleukin 4 enhances the ability of antigen-specific B cells to form conjugates with T cells

T cell-B cell conjugates are formed when trinitrophenyl-specific B cells are exposed to trinitrophenyl-ovalbumin and ovalbumin-specific T hybridoma cells. The proportion of conjugates was increased two- to threefold when antigen-pulsed trinitrophenyl-specific B cells, but not T cells, were pre-exposed to interleukin 4. Antigen-specific B cells pretreated with antigen and interleukin 4 and cultured in the presence of specific T helper cells also produced a larger proportion of antibody-secreting cells as compared to cells pretreated with antigen alone. The interleukin 4-induced enhancement of T/B conjugate formation occurred over a wide range of antigen concentrations, was dependent on the concentration of interleukin 4, and was inhibited by the monoclonal anti-interleukin 4 antibody, 11B11. The importance of Ia antigens in the enhancement of conjugate formation and generation of antibody-secreting cells is suggested by a) the fact that the interleukin 4-mediated increase in the density of Ia antigens on the antigen-specific B cells correlated with their enhanced ability to form T/B conjugates, b) the kinetics of the interleukin 4-mediated increase in conjugate formation and surface Ia expression were similar, c) 10- to 20-fold higher concentrations of anti-I-A antibody were required to inhibit T/B conjugate formation by 50% with interleukin 4-treated antigen-specific B cells compared with untreated antigen-specific B cells, and d) interferon-gamma, which inhibits the interleukin 4-mediated increase in Ia antigens, inhibited the interleukin 4-induced enhancement of T/B conjugate formation. These results indicate that the interleukin 4-induced increase in the expression of Ia antigens on B cells plays an important role in the enhancement of T/B cell interactions and the subsequent differentiation of antigen-specific B cells into antibody-secreting cells.