Opposite modulation of lymph node cell and spleen cell responses to mitogens by muramyl dipeptide and its desmethyl analog

N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), the minimal structure necessary for adjuvant activity of mycobacterial cell wall preparations, was evaluated as an immunostimulant in mice. MDP treatment, which increased carbon clearance and nonspecific resistance to lethal Klebsiella challenge, induced lymph node cellular hyperplasia (4-fold). In contrast, spleen and resident peritoneal cell recovery was comparable to controls. Lymph node cells (LNC) from MDP-treated mice had enhanced [³H]thymidine uptake in unstimulated (4-fold) and lipopolysaccharide (LPS) (5-fold)-, concanavalin A (Con A) (2-fold)-, and phytohemagglutinin (PHA) (1.5-fold)-stimulated cultures. In contrast, spleen cells exhibited depressed responses when stimulated with LPS (2-fold), Con A (2- to 5-fold), and PHA (3-fold). Depressed responses of spleen cells to mitogens were demonstrated over a range of mitogen concentrations. The desmethyl analog produced similar effects, although spleen cells were not as hyporeactive. Opposing modulations of the immune system by MDP resembles that reported after BCG infection, and correlates with increased nonspecific host resistance to microbial challenge.     

In vitro immune blastogenesis during contact sensitivity in tumor-bearing mice: I. Description of progressive impairment and demonstration of splenic suppressor cells

T-cell responsiveness was measured by the DNA response of disassociated spleen and lymph node cells when exposed to antigen in vitro. Sensitized splenic lymphocytes from fibrosarcoma-bearing mice immunized with 2,4-dinitro-1,5-difluorobenzene (DN2FB) demonstrated a progressive decrease in T-cell responsiveness to the haptenprotein conjugate DNP-BSA. Hyporesponsiveness to the dinitrophenylated-protein conjugate appeared in the spleens but not lymph nodes of tumorous animals. Normal host lymph node cells (LNC) responded strongly 24 to 48 h after sensitization and subsequently declined with a corresponding increase in responsiveness in the spleen. Tumor-bearing hosts (TBH) had similar LNC kinetics during immunization, however, spleen cells were significantly suppressed when compared to normal BALB/c mice sensitization kinetics. Spleen cells from TBH were also capable of suppressing the in vitro response of normal primed lymphocytes to DNP-BSA when admixed. Results from these experiments suggest that in vitro measurement of contact sensitivity was affected by suppressor cells/products existing in the spleens but not lymph nodes of fibrosarcoma-bearing mice.     

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

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

20alpha-hydroxysteroid dehydrogenase: a T lymphocyte-associated enzyme.

20alpha-Hydroxysteroid dehydrogenase (20alpha-SDH), an enzyme which reduces progesterone to 20alpha-dihydroprogesterone, was found to be associated with T lymphocytes. 20alphaSDH activity was present in spleen cells bearing theta antigen, spleen cells nonadherent to nylon wool (T lymphocyte-enriched population), and in thymocytes. Almost no enzymatic activity was found in bone marrow cells from normal mice and in spleen cells from neonatally thymectomized or athymic nude mice. T cell mitogens (PHA and Con A), but not the B cell mitogen LPS, induced high levels of enzymatic activity 48 hr after addition to spleen cell cultures. The level of 20alphaSDH activity in lymphocytes was age dependent. At the age of 4 weeks 20alphaSDH activity in thymocytes, spleen cells, and lymph node lymphocytes was 3 to 5 times higher than at 8 and 16 weeks. Progesterone (5.0 X 10(-7) M) was found to inhibit thymocyte proliferation after exposure to mitogens, but not 20alpha-dihydroprogesterone (10(-6) M). 20alpha SDH may protect the embryonic thymocytes against high concentrations of progesterone.     

Neonatal infection with mouse thymic virus. Differential effects on T cells mediating the graft-versus-host reaction.

Neonatal infection with mouse thymic virus (TA), a murine herpes virus, produced extensive but temporary necrosis of the thymus which was maximal at 10 to 14 days of age. Studies of precursor and amplifier cells mediating graft-vs-host (GVH) reactivity of thymocytes, spleen cells (SC), and lymph node cells (LNC) of normal and TA-infected mice were made at 4 and 8 weeks of age. Infection with TA resulting in a profound reduction (70 to 80%) in the direct GVH reactivity of thymocytes at both ages; by comparison, the capacity of thymocytes to produce synergy when combined with normal LNC was normal at 8 weeks. Direct GVH reactivity of SC was depressed 90% 4 weeks after infection with TA but returned to near normal at 8 weeks. Direct GVH reactivity of LNC from TA-infected mice was normal at 4 and 8 weeks of age, but amplifier T cell activity in LNC was markedly depressed at 8 wekks. These results demonstrate that TA has highly selective effects upon subpopulations of T cells in thymus and lymph node.     

Activation of nonspecific killer cells by interleukin 2-containing supernatants

In the absence of specific antigen stimulation, nonspecific killer cells were induced by culturing C57BL/6 lymph node or spleen cells with interleukin 2-containing supernatants. These supernatants were obtained from stimulation of either rat spleen cells with concanavalin A or a variant of the T cell lymphoma, EL4 (H-2b) with phorbol myristic acetate. The ability of the EL4 supernatant to induce nonspecific killer cells was abrogated by absorption with an interleukin 2-dependent T cell line or by concanavalin A-stimulated spleen cell blasts, but not by lipopolysaccharide-stimulated spleen cell blasts or by a non-interleukin 2-producing EL4 line. Partially purified interleukin 2 from EL4 supernatants could also support nonspecific killer cell induction. The induction of cytolytic cells by interleukin 2 is sensitive to gamma-irradiation and has a D omicron of 120 rad. The nonspecific killer cells induced are likely cytotoxic T lymphocytes; the majority of the precursor and effector cells bear the Thy-1 alloantigen marker. These nonspecific killer cells killed a broad spectrum of target cells, including concanavalin A- and lipopolysaccharide-induced splenic blasts of syngeneic or allogeneic mice, a syngeneic tumor, and a cloned allogeneic cytotoxic T cell line. The frequency of precursors for nonspecific killer cells in C57BL/6 lymph node and spleen cells are 1/7000 and 1/12,000, respectively. Clonal analyses revealed that these nonspecific killers exhibit heterogeneity with respect to their target cell specificities. The induction of nonspecific killers by interleukin 2-containing supernatants is partially dependent on nylon wool-adherent cells; in antigen-stimulated cultures the most specific killer cells were obtained from cultures in which nylon wool-nonadherent lymph node responder cells were stimulated with nylon wool-nonadherent allogeneic splenic stimulator cells that were treated with anti-Thy-1 antibody and complement. The relevance of these findings with respect to the frequencies and fine specificities of cytotoxic T lymphocytes generated in interleukin 2-supplemented cultures is discussed.     

Absence of CD43 fails to alter T cell development and responsiveness

Genetic elimination of CD43 has been associated with increased T cell adhesiveness and T cell hyperresponsiveness to mitogens and alloantigens. Therefore, we investigated whether T cell development was perturbed in CD43-deficient mice by breeding CD43(null) mice with male Ag (Hy)-specific TCR-transgenic mice. Neither positive nor negative thymic selection of male Ag-specific T cells were affected by CD43 status. Furthermore, we did not observe a substantial or consistent hyperresponsive pattern in HY-CD43(null) lymph node cells compared with littermate HY-CD43(+/-) lymph node cells upon analysis of in vitro T cell stimulation with male Ag or mitogen. These observations challenged original conclusions associating absence of CD43 with T cell hyperresponsiveness and led us to re-examine this association. Reported phenotypes of CD43(null) mice have been based on mice with a mixed 129xC57BL/6 genetic background. To exclude a possible influence of genetic background differences among individual mice we analyzed CD43(null) littermates that had been back-bred onto the C57BL/6 background for seven to eight generations. We found that CD43(+) and CD43(null) littermates with the C57BL/6 background exhibited no differences in response to mitogen or alloantigen, thereby establishing that T cell hyperresponsiveness is not a general correlate of CD43 absence.     

Exogenous protease promotes specific antibody forming cell response in vitro

This report presents results from experiments which evaluated the effect of exogenous protease on the in vitro antibody-forming cell (AFC) response of hamster lymphocytes to sheep erythrocytes (SRBC). In the presence of fetal calf serum, trypsin and papain, but not thermolysin, α-chymotrypsin, thrombin, and submaxillary protease, were able to enhance the quantity of AFC which developed. Prior incubation of antigen with proteases had no effect on subsequent antigenicity. The following observations were made: (1) Addition of protease to the culture system enhanced the AFC response only if added in the first 48 hr of the assay. (2) Proteases were able to enhance the development of AFC in lymph node and spleen cell cultures lacking fetal calf serum for 24 hr. (3) When papain was added to spleen cell cultures which normally produce fewer AFC than lymph node cells (LNC) it promoted the development of a 6- to 10-fold increase in AFC causing the magnitude of the response to match the AFC response expected in LNC cultures. These data support a role for a proteolytic event in lymphocyte activation by specific antigens.     

Intratracheal priming with ovalbumin- and ovalbumin 323-339 peptide-pulsed dendritic cells induces airway hyperresponsiveness, lung eosinophilia, goblet cell hyperplasia, and inflammation

Dendritic cells (DC) are the primary APC responsible for the capture of allergens in the airways and the shuttling of processed allergens to the draining lymph nodes where Ag presentation and T cell activation take place. The mechanism of this Ag handling and presentation in asthma is poorly understood. In addition, the feasibility of asthma induction by DC priming has not been directly tested. In this report an asthma model using intratracheally (i.t.) injected splenic DC was used to address these issues. DC pulsed with a model Ag OVA or the major MHC class II-restricted OVA T epitope peptide OVA(323-339) and instilled i.t. primed mice to exhibit asthma-like diseases. With OVA as the Ag, mice exhibit airway hyperresponsiveness (AHR), lung eosinophilia and inflammation, and pulmonary goblet cell hyperplasia. In OVA(323-339)-immunized mice, AHR and goblet cell hyperplasia were noted, with little eosinophilia and parenchymal inflammation. The latter finding provides evidence for dissociating AHR from eosinophilia. In both cases mediastinal node hypertrophy occurred, and high levels of Th2 cytokines were produced by the lung and mediastinal lymph node cells (LNC). Interestingly, mediastinal LNC also produced high levels of Th1 cytokines. Lung cells produced much less Th1 cytokines than these LNC. These results demonstrate that DC when introduced i.t. are potent in inducing asthma-like diseases by recruiting lymphocytes to the lung-draining lymph nodes and by stimulating Th2 responses and also suggest that the lung environment strongly biases T cell responses to Th2.     

Mitogen responsiveness and inhibitory activity of mesenteric lymph node cells

Summary Blood-, lymph node-, and tumour-infiltrating lymphocytes (PBL, LNC, and TIL, respectively) from patients with colonic neoplasms were tested for responsiveness to phytohaemagglutinin (PHA). All populations responded, with LNC and PBL showing comparable reactivities while TIL were less reactive as assessed by incorporation of ³H-thymidine. Increased mitogen responsiveness was observed for T cells enriched by SRBC rosette formation or passage through nylon columns. Mitomycin C-treated LNC and TIL inhibited PHA induced ³H-thymidine incorporation of admixed autologous PBL, suggesting the presence of suppressor cells. Suppressor activity resided primarily in the SRBC rosetting population and was dose-dependent, with increasing numbers of LNC giving greater diminution of PHA response. Suppression by LNC was apparent only when they were added to PBL responders within 6 h of the initiation of stimulation assays, in common with the effects of Concanavalin A (Con A)-induced suppressors on PBL phytomitogen responsiveness. Con A-induced and LNC-suppressor activity could be reversed by addition of lymphocyte-conditioned medium (CM) containing T cell growth factor (TCGF; interleukin IL-2). These data provide further evidence that the suppressor phenomena observed in this system are a function of activated T cells present both in drainage lymph nodes and at the tumour site.     

Immunosuppression of experimental allergic encephalomyelitis. III. In vitro evidence for induction of suppressor T lymphocytes in draining lymph node cells of animals immunized with myelin basic protein complexed to lipopolysaccharides

We recently demonstrated that Lewis rats immunized with bacterial lipopolysaccharides (LPS) precomplexed to guinea pig myelin basic protein (BP) in complete Freund's adjuvant were less effective in inducing experimental allergic encephalomyelitis (EAE) than BP-immunized controls. When tested in vitro both lymph node cells (LNC) and spleen cells (SpC) of animals immunized with BP-LPS were less effective in proliferative responses to various mitogens, which included phytohemagglutinin, concanavalin A, purified protein derivative of tuberculin, LPS, and BP. Of importance immunization of rats with BP complexed to LPS results in the generation of cells in lymph nodes of these animals that suppress the mitogenic response of BP-immunized LNC and also SpC in mixed lymphocyte cultures. The suppressive effect of these cells in mixed lymphocyte culture reaction was found specifically in response to BP and to a lesser extent to LPS in LNC. SpC of BP-LPS immunized animals did not suppress the proliferative response to SpC of BP-immunized animals. Treatment of these LNC with antithymocyte serum and complement abolished this suppressive effect of LNC, suggesting that the immunoregulatory cells in LNC of BP-LPS immunized animals are suppressor T lymphocytes. The parallel between the in vitro induction of suppressor T lymphocytes in the draining LNC and the function of LPS in the development of EAE in Lewis rats suggests a possible immunologic significance of the effect.     

Novel virulence properties of the Salmonella typhimurium virulence-associated plasmid: immune suppression and stimulation of splenomegaly

Mice infected subcutaneously with wild-type Salmonella typhimurium, SR11, developed a significant splenomegaly when compared with mice infected with an equal number of a plasmid-cured strain. Further, the bacterial load in the spleen at 14 days after infection, measured as colony-forming units per gram tissue, was significantly higher in mice infected with the parent strain than in mice infected with the plasmid-cured strain. These data confirm the previously reported plasmid-associated ability of Salmonella to multiply within the spleen. In addition, lymph node cells (LNC) from mice infected with the parent strain had a significantly reduced ability to proliferate in response to concanavalin A, a T-cell mitogen, and to heat-killed S. typhimurium cells when compared with LNC isolated from mice infected with the plasmid-cured strain. Finally, reintroduction of a functional Tn5-tagged 90-kb plasmid into a plasmid-free strain restored its capacity to cause a marked splenomegaly and to suppress lymph node cell proliferation in BALB/c mice. These data demonstrate that the 90-kb plasmid of highly virulent S. typhimurium strains mediates several novel pathogenic properties in infected mice: (1) enhancement of the ability of Salmonella to multiply within the spleen; (2) stimulation of a splenic inflammatory response as displayed by marked splenomegaly; and (3) a general suppression of lymphocyte responsiveness to both T-cell mitogens and specific Salmonella antigens.     

Fractionation of lymphocyte subpopulations which regulate mixed lymphocyte reactions.

Four days after injection of allogeneic lymphocytes BALB/c splenic T cells suppress proliferation of syngeneic cells in mixed lymphocyte reactions (MLR). Conversely, lymph node cells from the same mice amplify MLR responses. To further characterize these functional subpopulations, alloantigen-primed lymphocyte suspensions from both organs were fractionated by velocity sedimentation at unit-gravity. After fractionation MLR suppressor cells from spleens localized exclusively in rapidlly sedimenting fractions of large cells. MLR suppressor activity of cells from these fractions, as well as that of unfractionated spleen cell suspensions, was abolished by treatment with anti-Thy-1.2 serum and complement. Spleen cell fractions of similar sedimentation velocity also secreted a soluble MLR suppressor into culture supernatants. Although inhibitory of MLR, spleen cells of rapid sedimentation velocity did not suppress responses to T cell mitogens. In marked contrast with the effects of spleen cells, large 4-day-alloantigen-primed lymph node cells had no suppressive activity in MLR. MLR amplifier cells of uncertain derivation were found in fractions of medium sedimentation velocity from both spleens and lymph nodes. Fractionation of alloantigen-primed lymph node cell suspensions did reveal, however, a subpopulation of small cells with MLR suppressor acitivty which was unaffected by treatment with anti-Thy-1 serum and complement. The data thus indicate that large alloantigen-activated lymphocytes are not intrinsically suppressive nor are cells which suppress MLR necessarily large. We consequently conclude that regulation of MLR responses by alloantigen-primed lymphocytes involves a complex interaction between distinct functional subpopulations of cells which are separable both by physical and biologic properties.     

Primary in vitro antibody response of rabbit lymphoid cells and T-B cell collaboration in the absence of detectable mitogens

To investigate whether the antibody response and T-B-cell collaboration in vitro can be obtained in the absence of mitogens, a method of obtaining an in vitro primary anti-sheep red blood cell antibody response by rabbit spleen and lymph node cells was developed. We used Marbrook culture vessels and a specially prepared medium containing 10% autologous serum and maintained at pH 7.4–7.6. The system was shown to be devoid of any polyclonal mitogens as assessed by [³H]thymidine incorporation and by direct examination for blast cells in stained smears. The primary response increased continuously over the 5-day cultivation period and only IgM but not IgG plaque-forming cells (PFC) were detected. In over 20 experiments, the response ranged from 357 ± 17 to 4425 ± 110 PFC/10⁷ cultured cells with a median stimulation index of 52. The spleen cells required less antigen than the lymph node cells and 2-mercaptoethanol inhibited the response of the spleen cells but not that of the lymph node cells. Lymphocytes were separated into highly pure T- and B-cell populations by negative selection using antibody-coated human erythrocytes to rosette either T or B cells and Ficoll-Hypaque centrifugation to remove rosetted cells. Upon cultivation, B cells alone gave a low IgM response, whereas B cells reconstituted with T cells gave a response similar to that obtained with unseparated lymphoid cells. We concluded that: (a) optimal conditions for obtaining primary in vitro antibody responses using rabbit spleen and lymph node cells were established, (b) T-B-cell collaboration was demonstrated in the rabbit primary antibody response to sheep erythrocytes, and (c) the primary antibody response in vitro and T-B-cell collaboration may occur in the absence of detectable polyclonal mitogens.     

Lymphoid cell subpopulations. I. Synergy between lymph node cells and thymocytes in response to alloantigens and mitogens.

Mixtures of isogenic thymocytes (TC) and lymph node cells (LNC) were shown to exhibit synergistic responsiveness to M and H-2 alloantigens in the mixed lymphocyte interaction (MLI). With respect to the kinetics and magnitude of proliferation and effector cell generation, the response occurring in synergizing cultures closely resembled that of optimal numbers of LNC or spleen cells (SC). In addition, the antigen specificity of effector cells generated by synergizing cultures was similar to that of effectors derived from cultures containing optimal numbers of responding SC. LNC-TC mixtures also exhibited synergy in response to the phytomitogens concanavalin A and pokeweed mitogen but not to phytohemagglutinin. Weakly positive synergy was observed in response to bacterial lipopolysaccharide. It is proposed that the phenomenon of synergy is not restricted to cultures containing mixtures of LNC and TC but also occurs in cultures containing optimal numbers of LNC or SC as a result of interactions between subpopulations of lymphocytes contained within these tissues.     

Immunosuppression in a murine B cell leukemia (BCL1): role of an adherent cell in the suppression of primary in vitro antibody responses

The ability of lymph node cells from mice bearing the BCL1 tumor to respond in vitro to mitogens, allogeneic cells, and both TI and TD antigens was investigated. The lymph nodes of such mice are not invaded with tumor cells and contain normal numbers of T and B cells. Nevertheless, at the peak of tumor burden in the spleen and blood (approximately 8 to 12 wk after injection with tumor cells), the lymph node cells from the tumor-bearing mice display markedly decreased responsiveness both to allogeneic cells and to antigens. In addition, small numbers of lymph node cells from the tumor-bearing mice suppress primary antibody responses of normal lymph node cells. This nonspecific suppression of antibody responses is mediated by a G-10 Sephadex adherent, non-T, non-B cell present in the nodes of the tumor-bearing mice. Since the BCL1 tumor model is in many respects similar to the prolymphocytic type of human chronic lymphocytic leukemia, the present results may be helpful in elucidating the mechanisms underlying the in vivo immunosuppression associated with lymphocytic neoplasms in humans.     

Immunogenetic differences between lymph node cell and bone marrow cell grafts in irradiated mice

C3H lymph node cell (LNC) grafts, but not bone marrow cell (BMC) grafts, were resisted by lethally irradiated NZB, (C57BL × NZB)F1, and (C57BL/6 × DBA/2)F1 mice. BALB/ c hosts did not resist C3H LNC, suggesting that Ir-like genes regulate resistance to such grafts. Cyclophosphamide, silica particles, and ⁸⁹Sr pretreatments of prospective host mice resulted in successful proliferation of C3H LNC in most instances. These agents were known to abrogate resistance to incompatible BMC grafts. The determinants for antigens recognized on LNC appear to map in or near the D region of H-2. LNC grafts of all H-2^k strains tested (C3H, CBA, C58, C57BR) were strongly resisted while A, C3H.A, B10.A(5R), A.TL, and A.Tla^b LNC grafts were not strongly resisted by NZB hosts. Grafts of H-2^b (C57BL/6, C57BL/10, 129) LNC, or BMC are resisted by NZB or (C57BL/6 × DBA/2)F1 hosts. (C3H × C57BL)F1 LNC but not BMC were resisted by similar hosts. (C57BL/6 × DBA/2)F1 mice were injected with C57BL/6 spleen cells four times to induce specific “unresponsiveness” to parental-strain Hemopoietic histocompatibility (Hh) antigens. Unresponsiveness was induced to C57BL/6 BMC, as expected, but C57BL/6 and C3H LNC grafts were resisted despite the spleen cell injections. The data suggest that the antigens recognized during rejection of C3H LNC are not expressed on C3H BMC. It is even conceivable that Hh antigens on C57BL/6 BMC and LNC have separate determinants. Alternatively, the injections of C57BL/6 spleen cells may have induced an anti-idiotypic response that was capable of eliminating C57BL/6 LNC by a different effector mechanism.     

RNA in the periphery of rapidly proliferating mouse lymphoid cells

RNA in the peripheries of various populations of lymph node cells (LNC) has been evaluated by measuring the electrophoretic mobilities of cells, before and after treatment with active or inactivated ribonucleases. Three different populations of LNC were studied: (1) “resting” normal age control LNC; (2) “syngeneic” LNC from irradiated (C3H × C57BL)F₁ or C3H mice four to six days following transplantation of syngeneic spleen cells; such cells were progeny of lymphopoietic progenitor cells of the spleen; and (3) “allogeneic” LNC from irradiated (C3H × C57BL)F₁ mice four to six days after grafting C3H (parental) spleen cells; such cells were progeny of lymphopoietic progenitor cells, but also alloantigen-sensitive cells of the spleen which proliferate in response to the host's alloantigens (a “graft-versus-host” immunological reaction). Whereas the normal LNC had no detectable peripheral RNA, the allogeneic and syngeneic LNC did, i.e., ribonuclease reduced their mean electrophoretic mobilities by 13.6 and 9.2 per cent, respectively. Since both allogeneic and syngeneic LNC had peripheral RNA, no specific correlation could be made with immunological activity. ³H-uridine and ¹⁴C-thymidine incorporation into lymph nodes was greatest in allogeneic, intermediate in syngeneic and least in age control lymph nodes, indicating a “population shift” in the spleen cell chimeras toward relatively immature, rapidly proliferating cells, which had a relatively high rate of RNA synthesis. Thus, rapidly proliferating lymphoid cells do have RNA in their peripheries, but its relation to specific immunological function has yet to be ascertained.     

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.     

Effects of antithymocyte serum on lymph node cells participating in the graft-vs-host reaction.

BALB/c mice were injected with 0.1 ml of antithymocyte serum (ATS) and tested at various times thereafter for graft-versus-host (GVH) reactivity of lymph node cells (LNC) and spleen cells (SC). Splenomegaly produced by LNC or SC injected alone was used as a measure of precursor T cell function and the capacity of such cells to produce synergy when combined with normal thymocytes was used to evaluate amplifier T cell activity. In lymph node, both precursor and amplifier function were strikingly depressed 2 days after ATS administration; by day 11, precursor function showed slight recovery while amplifier activity had recovered to supranormal levels. In spleen, precursor activity recovered two fold between 2 and 11 days after ATS inoculation but no amplifier activity could be detected at day 11. Since donors had not been deliberately stimulated with alloantigens prior to testing of LN and SC GVH acivity, these studies demonstrate (a) that precursors and amplifiers are disinct T cell populations that are committed to express unique functional activities before antigen exposure and (b) that, following depletion with ATS, these two populations recover independently at different rates in separate lymphoid compartments.