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.     

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

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

A subpopulation of adherent accessory cells bearing both I-A and I-E or C subregion antigens is required for antigen-specific murine T lymphocyte proliferation.

A murine T lymphocyte proliferation assay that used antigen-primed lymph node T cells, was antigen specific, and required exogenous accessory cells was used to characterize the accessory cells that supported proliferation. These cells were Thy 1.2 negative, radioresistant, glass-adherent, and were functional only if alive. The accessory cell function of spleen adherent cells was much greater than that of peritoneal cells. Also, the accessory cell function of spleen adherent cells was proportional to the length of time such cells were incubated with antigen and very small numbers of such cells provided accessory cell function. Cytotoxic studies with subregion-restricted anti-Ia antibodies and complement indicated that accessory cell function resided in a subpopulation of spleen adherent cells that bore both I-A and I-E or C subregion antigens. The function of such cells was not related to a selective ability (vs other spleen adherent cells) to take up antigen. These data indicate that antigen-specific stimulation of T lymphocyte proliferation requires at least one specific subpopulation of spleen adherent cells that can be phenotypically identified by its expression of Ia antigens and are consistent with the possibility that Ia antigens may be Ir gene products.     

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

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

Role of accessory cells in B cell activation. IV. Ia+ accessory cells are required for the in vitro generation of thymic independent type 2 antibody responses to polysaccharide antigens

It has been previously shown that the in vitro antibody response to TNP-Ficoll requires the presence of adherent accessory cells. In order to determine if this characteristic was unique to TNP-Ficoll or a general feature of the TI-2 antibody responses, responses to the polysaccharide antigens TNP-Levan and TNP-Dextran were studied. Also, it was determined if the functionally relevant accessory cell expresses Ia determinants. Passage of spleen cells over Sephadex G-10 abrogated the response to TNP-Levan and TNP-Dextran as well as to TNP-Ficoll. Addition of adherent accessory cells to the G-10 passed spleen cells reconstituted the response to all 3 antigens. Pretreatment of the adherent accessory cells with a specific anti-Ia serum plus complement abrogated the ability of these cells to provide accessory cell function in the responses to all 3 antigens. Thus, an Ia-positive adherent accessory cell is required for the generation of TI-2 antibody responses to these polysaccharide antigens. This raises the possibility that genetic restrictions may exist between the Ia-positive accessory cell and the lymphocytes involved in the responses to TNP-Ficoll, TNP-Dextran, and TNP-Levan.     

The immunobiology of T cell responses to Mls-locus-disparate stimulator cells. II. Effects of Mls-locus-disparate stimulator cells on cloned, protein antigen-specific, Ia-restricted T cell lines

Cloned, protein antigen-specific, Ia-restricted T cell lines frequently (approximately 20%) also respond strongly to stimulator cells from strains expressing stimulatory alleles at the chromosome 1-encoded Mls-locus. Furthermore, such responses are blocked by monoclonal antibodies specific for Ia antigens expressed by the stimulator rather than the responder cells. However, such responses show no specificity for polymorphic determinants on Ia molecules, although in such responses, as in primary and secondary T cell responses to stimulating Mls-locus alleles, I-E molecules appear to play a central role. These results, combined with the unique immunobiology of the primary T cell proliferative response to Mls-locus-disparate stimulator cells, suggest to us that this response involves the interaction of the receptor on T cells for antigen:self Ia with a relatively nonpolymorphic region of Ia glycoproteins. This hypothesis is supported by the observation that a monoclonal antibody to the T cell receptor will inhibit both responses, although the response to Mls-locus-disparate stimulators appears to be more sensitive to these antibodies. We propose that the interaction of the T cell receptor with Ia is stabilized by a cell interaction molecule encoded or regulated by the Mls-locus gene product permitting the T cell receptor:Ia glycoprotein interaction to lead to T cell activation.     

Nature of the antigenic complex recognized by T lymphocytes. IV. Inhibition of antigen-specific T cell proliferation by antibodies to stimulator macrophage Ia antigens.

We have previously demonstrated that nonimmune guinea pig T lymphocytes could be specifically sensitized with TNP-modified allogeneic macrophages after eliminating the alloreactive T cells with bromodeoxyuridine (BUdR) and light treatment. This procedure allowed the unique opportunity to use anti-Ia sera directed against the Ia antigens of only the stimulator macrophages or responder T cells to determine against which cell type anti-Ia would block TNP-specific stimulation. It was found that the TNP-specific DNA synthetic response of BUdR and light-treated T cells stimulated with TNP-modified allogeneic macrophages was totally eliminated by anti-Ia sera directed solely against the allogeneic stimulator macrophage. In contrast, anti-Ia sera directed only against the responder T cells had no effect on their response to TNP-modified allogeneic macrophages. These findings indicate that macrophage Ia antigens are required for efficient T cell-macrophage interactions and raise the possibility that T cell Ia antigens may not be required for collaboration with macrophages. This latter possibility was substantiated by experiments in which we show that treating T cells with anti-Ia sera and complement to remove the Ia-positive cells either before or after priming, or both, had no effect on their ability to be primed and restimulated with TNP-modified macrophages.     

Primary type II alveolar epithelial cells present microbial antigens to antigen-specific CD4+ T cells

Type II alveolar epithelial cells (AEC) can produce various antimicrobial and proinflammatory effector molecules. This, together with their abundance and strategic location, suggests a role in host defense against pulmonary pathogens. We report that murine type II AEC, like their human counterparts, express class II major histocompatibility complex (MHC). Using a murine model of pulmonary tuberculosis, we find that type II AEC become activated and have increased cell surface expression of class II MHC, CD54, and CD95 following infection. Type II AEC use the class II MHC pathway to process and present mycobacterial antigens to immune CD4+ T cells isolated from mice infected with Mycobacterium tuberculosis. Therefore, not only can type II AEC contribute to the pulmonary immunity by secreting chemokines that recruit inflammatory cells to the lung, but they can also serve as antigen-presenting cells. Although type II AEC are unlikely to prime na?ve T cells, their ability to present antigens to T cells demonstrates that they can participate in the effector phase of the immune response. This represents a novel role for type II AEC in the immunological response to pulmonary pathogens.     

The expression of Ia antigens on immunocompetent cells in the quinea pig. I the differential expression of Ia antigens on T cell subpopulations.

We have examined the effect of negative selection with anti-Ia serum and C on a number of T cell functions and have clearly defined two subpopulations of guinea pig T lymphocytes. One subpopulation is susceptible to the lytic effects on anti-Ia serum and C and includes the majority of the primed T cells which proliferate and which produce migration inhibition factor in response to specific antigen stimulation in vitro. The lytic effects of anti-Ia serum were directed against the antigen-specific T cell and not an accessory cell such as a macrophage or nonantigen-specific T cell. No evidence for allelic exclusion of the Ia antigens of the antigen-responsive cell could be demonstrated. The susceptibility of the mitogen-responsive T cell to lysis by anti-Ia serum and C varied with the mitogen used, anatomic origin of the T cell, and the strain of animals studied. A second subpopulation of T cells is completely resistant to the lytic effects of anti-Ia serum and C and includes the primed T helper cell and the T cell that proliferates in response to alloantigenic stimulation in the MLR.     

T cell surface markers expression by immature human thymocytes in in vitro culture: role of Ia+ accessory cells

Previous studies have indicated that the human thymus is composed of several discrete compartments. Cortical thymocytes are reactive with the monoclonal antibody anti-T6, whereas most medullary cells, unreactive with anti-T6, stain brightly with anti-T3 antibody, which defines mature T cell populations. By using an indirect immune rosette method, we isolated the minor thymocyte population (1 to 2% of all thymocytes) lacking both T3 and T6 but expressing T11 antigens. These cells could be maintained in culture supplemented with recombinant IL 2 (Rec-IL 2) for several days. Under these conditions, T3-T6- cells were shown to undergo phenotypic changes. In the absence of thymic macrophage (Mo), T3+ and T8+ thymocytes appeared in culture, whereas the development of T4+ cells strictly required the presence of Mo. The expression of T4 antigen could be largely prevented by the addition of anti-HLA-DR antibody, further indicating that Ia+ accessory cells had the ability to promote in vitro development of T4+ thymocytes. In the presence of Mo, not only T4+ but also T8+ cells were obtained. Double fluorescence staining with anti-T8-FITC and anti-T4-biotin demonstrated that after 12 days of culture, T4 and T8 antigens were mutually exclusive. Furthermore, during the course of these studies, we observed that under the culture conditions utilized (e.g., presence or absence of Mo), T3-T6-thymocytes failed to express the T6 antigen. Thus, the in vitro development of T cells bearing a mature phenotype could be obtained in the absence of intermediate expression of cortical (T6+) thymocytes.     

Noncovalent association with stress protein facilitates cross-priming of CD8+ T cells to tumor cell antigens by dendritic cells.

A viral oncogene carrying well-defined K(b)/D(b)-restricted epitopes was expressed in a heat shock protein (hsp)-associated or nonassociated form in the murine tumor cells P815 and Meth-A. Wild-type SV40 large T-Ag (wtT-Ag) is expressed without stable hsp association; mutant (cytoplasmic cT-Ag) or chimeric (cT272-green fluorescent fusion protein) T-Ag is expressed in stable association with the constitutively expressed, cytosolic hsp73 (hsc70) protein. In vitro, remnants from apoptotic wtT-Ag- or cT-Ag-expressing tumor cells are taken up and processed by immature dendritic cells (DC), and the K(b)/D(b)-binding epitopes T1, T2/3, and T4 of the T-Ag are cross-presented to CTL in a TAP-independent way. DC pulsed with remnants of transfected, apoptotic tumor cells cross-presented the three T-Ag epitopes more efficiently when they processed ATP-sensitive hsp73/cT-Ag complexes than when they processed hsp-nonassociated (native) T-Ag. In vivo, more IFN-gamma-producing CD8+ T cells were elicited by a DNA vaccine that encoded hsp73-binding mutant T-Ag than by a DNA vaccine that encoded native, non-hsp-binding T-Ag. Three- to 5-fold higher numbers of T-Ag (T1-, T2/3-, or T4-) specific, D(b)/K(b)-restricted IFN-gamma-producing CD8+ T cells were primed during the growth of transfected H-2(d) Meth-A/cT tumors than during the growth of transfected Meth-A/T tumors in F(1)(b x d) hosts. Hence, the association of an oncogene with constitutively expressed, cytosolic hsp73 facilitates cross-priming in vitro and in vivo of CTL by DC that process material from apoptotic cells.     

Recognition of alloantigen by cytotoxic T cell precursors is independent of the function of Ia+ cells

The relationship between the requirement for Ia+ accessory cells (IaAC) and the differentiation of the precursor of CTL (PCTL) in the primary in vitro induction of allogeneic CTL was studied. The following observations were made PCTL can complete the antigen recognition step in the absence of IaAC. This process probably does not involve cell division. The sensitized PCTL require additional signals, which are provided through an IaAC-dependent process to differentiate into the effector CTL. IaAC from any of the responder, the stimulator, or even the third-party strains can fulfill the requirement. However, IaAC of the responder strain failed to generate the helper signal in the absence of allogeneic antigens. The cellular events taking place in the induction of CTL are discussed.     

Antigen presentation in the rat. II. An Ia+ radiosensitive T cell can present antigen to primed Ia- T cells

We demonstrated previously the presence of an Ia+ (OX-6+) antigen-presenting cell within the rat T cell fraction that is capable of presenting antigen to antigen-primed OX-6-T cells. This antigen-presenting cell (T-APC) reacted with the monoclonal antibodies W3/25 and W3/13, which is known to react mainly with rat T cells. Further characterization of the T-APC indicated that the cell also reacted with the monoclonal antibody OX-19, which is highly specific for rat T cells. Moreover, the antigen-presenting function of the T-APC was sensitive to treatment with mitomycin C or gamma-irradiation (2000 rad). Under similar conditions, antigen presentation by partially purified dendritic cells or macrophages was totally resistant to these treatments. The antigen-presenting activity of gamma-irradiated T-APC was not reconstituted by the addition of the lymphokines IL 1, IL 2, or Con A supernatants. Although unirradiated T-APC were able to stimulate an MLR response, this function was also sensitive to gamma-irradiation, whereas the MLR-stimulating ability of macrophages and dendritic cells was resistant to gamma-irradiation. These data indicate that Ia+ T cells from the rat are capable of presenting antigen to antigen-primed T lymphocytes and that, in contrast to antigen presentation by macrophages and dendritic cells, the function of T-APC is gamma-radiation sensitive.     

Nematode infection enhances survival of activated T cells by modulating accessory cell function.

The type of immune response generated following exposure to Ag depends on a variety of factors, including the nature of the Ag, the type of adjuvant used, the site of antigenic entry, and the immune status of the host. We have previously shown that infection of rodents with Nippostrongylus brasiliensis (Nb) shifts the development of type 1 allo-specific responses toward type 2 immunity, suggesting nematode modulation of T cell activation. In this report we explore the immunomodulatory effects of Nb on T cell activation. We found that spleen cells from Nb-infected mice exhibited dramatically increased proliferation in response to Con A and anti-CD3. This hyperproliferation could be transferred in vitro to naive splenocytes by coculture with mitomycin C-treated cells from Nb-infected animals. The transfer was mediated by non-T accessory cells and supernatants derived from Con A-activated non-T cells, suggesting the involvement of a soluble factor secreted by accessory cells. The accessory cells secreted high levels of IL-6, and anti-IL-6 treatment abrogated the supernatant-induced hyperproliferation, thus confirming that IL-6 was mediating the effect. Further, spleen cells from Nb-infected mice were more resistant to activation-induced cell death (AICD) following mitogenic stimulation. Reduced AICD was also transferable and IL-6 dependent. Thus, the hyperproliferation was in part due to enhanced activated T cell survival. These phenomena mediated by accessory cells may contribute to the powerful polyclonal activation of type 2 immunity caused by nematode infection.     

Ly-6A.2 expression regulates antigen-specific CD4+ T cell proliferation and cytokine production.

Ly-6 proteins appear to serve cell adhesion and cell signaling function, but the precise role of Ly-6A.2 in CD4+ T lymphocytes is still unclear. Overexpression of Ly-6A.2 in T lymphocytes has allowed us to analyze the influence of elevated Ly-6A.2 expression on T cell function. In this study we report reduced proliferation of CD4+ T cells overexpressing Ly-6A.2 in response to a peptide Ag. Moreover, the Ly-6A.2-overexpressing CD4+ cells generated elevated levels of IL-4, a key factor that propels the differentiation of naive CD4+ T cells into Th2 subset. The hyporesponsiveness of Ly-6A.2 transgenic CD4+ T cells is dependent on the interaction of Ly-6A.2 T cells with the APCs and can be reversed by blocking the interaction between Ly-6A.2 and a recently reported candidate ligand. Overexpression of Ly-6A.2 in CD4+ T cells reduced their Ca(2+) responses to TCR stimulation, therefore suggesting effects of Ly-6A.2 signaling on membrane proximal activation events. In contrast to the observed Ag-specific hyporesponsiveness, the Ly-6A.2 transgenic CD4+ T cells produced IL-4 independent of the interactions between Ly-6A.2 and the candidate Ly-6A.2 ligand. Our results suggest that 1) interaction of Ly-6A.2 with a candidate ligand regulates clonal expansion of CD4+ Th cells in response to an Ag (these results also provide further functional evidence for presence of Ly-6A.2 ligand on APC); and 2) Ly-6A.2 expression on CD4+ T cells promotes production of IL-4, a Th2 differentiation factor.     

Innate immune cells contribute to the IFN-gamma-dependent regulation of antigen-specific CD8+ T cell homeostasis

IFN-gamma has a dual function in the regulation of T cell homeostasis. It promotes the expansion of effector T cells and simultaneously programs their contraction. The cellular mechanisms leading to this functional dichotomy of IFN-gamma have not been identified to date. In this study we show: 1) that expansion of wild-type CD8+ T cells is defective in IFN-gamma-deficient mice but increased in IFN-gammaR-deficient mice; and 2) that contraction of the effector CD8+ T cell pool is impaired in both mouse strains. Furthermore, we show that CD11b+ cells responding to IFN-gamma are sufficient to limit CD8+ T cell expansion and promote contraction. The data presented here reveal that IFN-gamma directly promotes CD8+ T cell expansion and simultaneously induces suppressive functions in CD11b+ cells that counter-regulate CD8+ T cell expansion, promote contraction, and limit memory formation. Thus, innate immune cells contribute to the IFN-gamma-dependent regulation of Ag-specific CD8+ T cell homeostasis.     

Macrophage-T cell interactions. IV. A new receptor on T cells: Ia antigens as receptors for Ig-antigen complexes which appear early after immunization.

The Fc-receptor-positive T cells have been shown to bear a new receptor which is involved in the uptake of cytophilic complexes of Ig and antigen which are detected in the serum of mice within 6 hr following immunization. It was shown that the Fc receptor on T cells is not necessary for the uptake of these complexes. This new receptor is labile during culture of the T cells in vitro and disappears together with the Fc receptors. The new receptor is taken up by the T cells from a macrophage supernate. The evidence presented suggests that the receptor for these complexes bears determinants coded by the I region of the major histocompatibility complex but is distinct from conventional Fc receptor in that it does not bind to Fc fragments of Ig.     

Myelin antigen-specific CD8+ T cells are encephalitogenic and produce severe disease in C57BL/6 mice

Encephalitogenic T cells that mediate experimental autoimmune encephalomyelitis (EAE) are commonly assumed to be exclusively CD4+, but formal proof is still lacking. In this study, we report that synthetic peptides 35-55 from myelin oligodendrocyte glycoprotein (pMOG(35-55)) consistently activate a high proportion of CD8+ alphabetaTCR+ T cells that are encephalitogenic in C57BL/6 (B6) mice. The encephalitogenic potential of CD8+ MOG-specific T cells was established by adoptive transfer of CD8-enriched MOG-specific T cells. These cells induced a much more severe and permanent disease than disease actively induced by immunization with pMOG(35-55). CNS lesions in pMOG(35-55) CD8+ T cell-induced EAE were progressive and more destructive. The CD8+ T cells were strongly pathogenic in syngeneic B6 and RAG-1(-/-) mice, but not in isogeneic beta2-microglobulin-deficient mice. MOG-specific CD8+ T cells could be repeatedly reisolated for up to 287 days from recipient B6 or RAG-1(-/-) mice in which disease was induced adoptively with <1 x 10(6) T cells sensitized to pMOG(35-55). It is postulated that MOG induces a relapsing and/or progressive pattern of EAE by eliciting a T cell response dominated by CD8+ autoreactive T cells. Such cells appear to have an enhanced tissue-damaging effect and persist in the animal for long periods.     

Native HIV-1 Tat protein targets monocyte-derived dendritic cells and enhances their maturation, function, and antigen-specific T cell responses.

Vaccination of cynomolgus monkeys with the biologically active HIV-1 Tat protein induces specific Th1 responses, including CTLs. Similar responses are also induced by vaccination with tat DNA, but not by vaccination with inactivated Tat or Tat peptides. This suggested that the native Tat protein may act differently on APC as compared with inactivated Tat or peptide Ag. In this study, we show that biologically active Tat is very efficiently taken up by monocyte-derived dendritic cells (MDDC) in a time (within minutes)- and dose-dependent (starting from 0.1 ng/ml) fashion, whereas uptake is very poor or absent with other APC, including T cell blasts and B lymphoblastoid cell lines. Although maturation of MDDC reduces their pino/phagocytic activity, mature MDDC take up Tat much more efficiently than immature cells. In addition, Tat uptake is abolished or greatly hampered by oxidation/inactivation of the protein or by performing the experiments at 4 degrees C, suggesting that MDDC take up native Tat by a receptor-mediated endocytosis. After uptake, active Tat protein induces up-regulation of MHC and costimulatory molecules and production of IL-12, TNF-alpha, and beta chemokines, which drive Th1-type immune response. In contrast, these effects are lost by oxidation and inactivation of the protein. Finally, native Tat enhances Ag presentation by MDDC, increasing Ag-specific T cell responses. These data indicate that native Tat selectively targets MDDC, is taken up by these cells via specialized pathways, and promotes their maturation and Ag-presenting functions, driving Th1-type immune responses. Thus, Tat can act as both Ag and adjuvant, capable of driving T cell-mediated immune responses.