Defective induction of antigen-reactive proliferating T cells in B cell-deprived mice. II. Anti-mu treatment affects the initiation and recruitment of T cells

Mice injected from day of birth onwards with rabbit anti-mouse IgM (antim-mu) antibodies were found to be B cell-deficient and defective for the induction of antigen-reactive proliferating T cells (TPRLF). This defective induction was not due to the absence of circulating antigen-specific antibodies since the daily injections of such antibodies during exposure to antigen did not restore the ability of anti-IgM treated animals to generate TPRLF. Analyzing the cellular events implicated in the induction of virgin antigen-reactive T cells, anti-mu-treated mice manifested impairment of the three interacting cell types involved in the induction of TPRLF. Thus, peritoneal and splenic antigen-presenting cells from such animals were impaired in their capacity to signal a primary antigen-specific T cell reaction. Their splenic lymphocytes could not function as initiator cells in transferring immunogenic signals to recruit TPRLF in normal recipients. Potent antigen-specific splenic initiator cells failed to induce the recruitment of specific TPRLF in anti-mu-treated mice. The defective induction of TPRLF in anti-mu-treated mice may be due to a functional impairment of cells expressing membrane-bound IgM molecules which seemingly play a central role in the transfer of immunogenic signals for the recruitment of antigen-specific circulating T cells. We suggest that splenic B cells function as initiators in the transfer of antigen-induced signals from peritoneal antigen-presenting cells to T cells. These seems to be the primary targets of anti-mu treatment.     

Antigen-induced feedback suppression: antigen-specific initiator T cells fail to recruit new antigen-reactive cells in immunized mice

Experiments were carried out to investigate the control exerted by memory cells on the activation of virgin antigen-reactive T cells. Spleen cells from normal mice, educated on human gamma-globulin (HGG)-fed macrophages, then injected into the footpads of syngeneic recipients, recruited antigen-specific reactive lymphocytes in normal mice, but failed to do so in HGG-tolerant or HGG-immunized recipients. This inhibition of recruitment was antigen-specific, since keyhole limpet hemocyanin (KLH) initiator lymphocytes did recruit KLH-reactive cells in HGG-immunized recipients. Spleen cells from immunized animals, unlike those from tolerant ones, were capable of supporting the generation in culture of HGG-initiator lymphocytes. Hence, the suppression we observed in immunized mice cannot be simply attributed to conventional Ly 23 suppressor T cells. To analyze the mechanism of the observed suppression, we tested the capacity of initiator cells educated on macrophages fed simultaneously with KLH and HGG to recruit antigen-reactive lymphocytes in normal as compared to immunized recipients. Such initiator lymphocytes recruited, in normal animals, both and HGG- and anti KLH-reactive cells. Yet, in HGG-immunized mice, initiator cells educated under similar conditions failed to recruit not only anti HGG-reactive cells but also anti KLH-reactive lymphocytes. This suggested that in HGG-immunized recipients, stimulated by HGG initiators (or by HGG antigen), HGG memory cells may lead to the generation of a factor (or factors) which may then nonspecifically suppress the recruitment and maturation of virgin antigen-reactive cells to unrelated antigens. This assumption is further strengthened by our findings which revealed that in the presence of HGG, spleen cells originating in HGG-immunized mice exert a weaker mixed lymphocyte culture reaction to alloantigens. Thus, it appears that an antigen may induce, in the presence of specific activated memory cells, the generation of factors which may suppress antigen-induced proliferation of certain T cell subsets. These results may contribute to our understanding of the possible homeostatic role played by memory T cells in feedback regulatory circuits existing among T cell subsets during immune processes. The possible role of such mechanisms in high- and low-zone tolerance is discussed.     

Evaluation of accessory cell heterogeneity. II. Failure of dendritic cells to activate antigen-specific T helper cells to soluble antigens

The activation of antigen-specific T cells requires Ia⁺, antigen-presenting accessory cells (AC). Dendritic cells (DC) and macrophages (M?) isolated from spleen and peritoneal exudate were tested as AC for the activation of T helper cells and the induction of T cell proliferation. The cell separations to obtain DC and splenic M? were performed by discontinuous bovine serum albumin gradients, adherence on petri dishes and rosetting with opsonized sheep erythrocytes. DC as well as the M? were able to induce antigen-specific T cell proliferation, but only the M? and not the DC activated antigen-specific T helper cells which help B cells for antibody production to soluble antigens. Keyhole limpet hemocyanin-specific T cells repeatedly stimulated with DC and antigen also did not express helper activity. The failure of DC to induce T helper cells was not due to the activation of a suppressor pathway. Thus, dendritic cells, although very efficient as AC in the induction of various T cell functions, are not able to activate T helper cells required for carrier-specific T-B cooperation and therefore cannot be the sole accessory cells. Based on these results and on previous data using Ia⁺ tumor cell line as AC, we confirm the existence of functional AC heterogeneity.     

Antigen presentation to T cell lines and clones by peritoneal macrophages, peritoneal B cells and antigen-specific B cell hybridomas

The antigen presenting cell (APC) activity of uninduced, resident peritoneal macrophages and B cells was compared to that of antigen-specific B cell hybridomas by measuring proliferative responses of antigen-specific, MHC-restricted T cell clones. The results demonstrate that peritoneal macrophages and B cells are much more efficient APC than irradiated splenic filler cells, and that unirradiated B cells were as good as, if not better than, macrophages. Both B cells and macrophages can be pulsed with antigen, although pulsed B cells were always found to be more efficient than pulsed macrophages. However, the APC activity of B cells was exquisitely sensitive to irradiation. The relative contribution of macrophages and B cells to the APC activity of mixed populations was easily distinguished by complement dependent lysis with monoclonal antibodies specific for unique differentiation antigens expressed by these cells. Normal peritoneal macrophages and B cells present the synthetic terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) to GAT-specific T cells clones and beef insulin to insulin-specific T cell lines nonspecifically. The APC activity of antigen-specific B cells was also examined by using novel GAT-specific, nonsecretor B cell hybridomas produced by fusing GAT-primed spleen cells to the HAT sensitive Balb/c lymphoma, M12.4.5. The hybridomas selected for these studies were GAT-specific, sIg+, Ia+ cells. These hybridomas presented GAT to GAT-specific T cells more efficiently than heterogeneous B cells suggesting that interaction with surface Ig receptors facilitated the uptake and/or processing of antigen. GAT-specific B cell hybridomas, like normal B cells, presented soluble beef insulin to an insulin-specific T cell clone nonspecifically. However, after pulsing with antigen overnight, the GAT-specific B cell hybridoma could activate only GAT-specific T cells.     

Cytophilic properties of surface immunoglobulin of thymus-derived lymphocytes

The cytophilic properties of released surface immunoglobulins of normal thymus lymphocytes and of activated thymus-derived lymphocytes (ATC) were analysed by lactoperoxidase-catalysed radioiodination in conjunction with immunological and autoradiographic techniques. Immunoglobulin from both normal T cells and ATC was cytophilic for macrophages (peritoneal exudate cells), but showed no detectable capacity to bind to either T lymphocytes or to bone-marrow-derived lymphocytes (B cells). Under the operative experimental conditions surface immunoglobulin of B cells did not show appreciable binding to macrophages. These results support the feasibility of models of collaboration between T cells and B cells which involve a soluble antigen-specific collaborative factor (T-cell Ig complexed with antigen) and show an obligatory requirement for macrophages.     

Effects of bilirubin on murine peritoneal and spleen cells

The effect of bilirubin on murine peritoneal and spleen cells was investigated. Bilirubin was found to have a strong and rapid effect on the expression of various kinds of Fc receptors on peritoneal macrophages. Significant changes were observed 30 min after the infection of bilirubin. The return to normal values was not observed earlier than after 24 h. The effect of bilirubin on Fc receptor expression of splenic macrophages was less pronounced. Expression of Ia antigen on macrophages was not influenced by bilirubin. The changes in percentage of sIg+ and Thy 1.2+ lymphocytes reflect a change in the ratio of T to B cells in the peritoneal cavity, as bilirubin caused 40% increase in numbers of B cells and a similar decrease in numbers of T cells. The percentage of splenic B lymphocytes was not influenced by bilirubin injection; but the ratio of T helper to suppressor cells was altered.     

Heavy-metal mitogenesis: Zn++ and Hg++ induce cellular cytotoxicity and interferon production in murine T lymphocytes

Splenic and lymph node lymphocytes from Balb/c mice were activated in vitro by the heavy-metal cations, Zn++ and Hg++, as noted by the several-fold increases in 3H-thymidine incorporation at 144 h of culture. Optimal mitogenic concentrations of Zn++ and Hg++ were 200 microM and 10 microM, respectively. Data from experiments using T or B splenic lymphocytes enriched by cell passage over nylon wool columns, through use of athymic Nu/Nu mouse spleen cells, or by cell lysis with monoclonal anti-Thy-1 and antibody plus complement, indicated than Zn++ and Hg++ are mitogens for T cells. Removal of macrophages from spleen cells by treatment with carbonyl iron, followed by cell passage through nylon wool, eliminated the lymphocyte responses to Zn++ and to Hg++. Moreover, addition of macrophage-depleted lymphocytes to monolayers of resident peritoneal macrophages restored the lymphocyte responses to these mitogens. Both Zn++ and Hg++ activated splenic lymphocytes to display lectin-dependent cytotoxicity and to produce acid-labile interferon.     

Evaluation of accessory cell heterogeneity. III. Role of dendritic cells in the in vitro activation of the antibody response to soluble antigens

Dendritic cells and macrophages obtained from spleen and peritoneal exudate were tested as accessory cells for the activation of lymphokine production by T cells, for supporting T-B cooperation and for the induction of antigen-specific T helper cells. Dendritic cells as well as macrophages were able to activate T cells for interleukin-2 secretion and functioned as accessory cells in T-B cooperation, but only macrophages induced T helper cells, which cooperate with B cells by a linked recognition interaction, to soluble antigens. Dendritic cell- and antigen-activated T cells also did not help B cells in the presence of Con A supernatants which contained various T cell- and B cell-stimulatory factors. The failure of dendritic cells to differentiate memory into functional T helper cells, but their efficient accessory cell function in T-B cooperation, where functional T helper cells are already present, can be best explained by a differential accessory cell requirement for T helper cell activation dependent on the differentiation stage of the T helper cell.     

Properties of antisera against lymphocytes of nude mice.

The activity of rabbit antisera against nu/nu BALB/c lymphocytes was estimated in vivo and in vitro. It was found that anti-lymphocyte serum (ALS) against nu/nu lymph node cells suppressed the alloantigen reaction and the spontaneous rosette-forming cell (sRFC) or plaque-forming cell (PFC) formation for T-dependent (sheep red blood cells) and T-independent (lipopolysaccharide) antigens. ALS against nu/nu spleen cells affected only the sRFC and PFC for T-independent antigen. The former serum exhibited a high cytotoxicity for the suspensions enriched or depleted in B cells, while the latter was more cytotoxic for the suspension enriched in B cells. This may indicate that ALS anti-nu/nu spleen cells is specific for B lymphocytes, and ALS anti nu/nu lymph node cells is directed not only to B cells but also to a subpopulation of T lymphocytes. It may suggest the existence of a subpopulation of T lymphocytes in nu/nu lymph node cells.     

Dengue virus-induced thymus-derived suppressor cells in the spleen of mice.

Adoptive transfer of spleen cells obtained from mice given three weekly i.p. doses of dengue type 2 virus (DV) suppressed DV antigen-specific antibody secretion as detected by the Jerne plaque technique. This suppression was produced by non-glass-adherent cells but not by glass-adherent cells. Immune spleen cells depleted of macrophages by carbonyl iron treatment had higher suppressor activity. Immune spleen cell homogenate could transfer the activity equally well. The immune spleen cells were separated into T and B lymphocytes by a nylon wool column. B lymphocytes had no suppressor activity; almost all the suppressor activity was present in T lymphocytes. Thus, macrophages and B lymphocytes had no suppressor activity; it was mediated by T lymphocytes through soluble factors.     

Involvement of the spleen in the control of the immunogenic and phagocytic function of thioglycollate-induced macrophages

The immunogenic capacity of thioglycollate-induced peritoneal macrophages of adult splenectomized animals was compared to that of macrophages of sham-operated controls. Macrophages from splenectomized animals were found to be impaired in their function as antigen-presenting cells, both in the education of virgin initiator T lymphocytes and in the stimulation of antigen-specific T memory cells. Macrophages from splenectomized animals were also severely impaired in their phagocytic capacity, as assessed in an opsonin-dependent bacterial phagocytosis assay. However, they were not impaired in their ability to pinocytose soluble keyhole limpet hemocyanin. These results indicate that the spleen may play a decisive role in controlling the differentiation of peritoneal macrophages.     

Stability of specific immunoglobulin secretion by EBV-transformed lymphoblastoid cells and human-murine heterohybridomas

We have examined variables leading to the generation of stable, antigen-specific, human immunoglobulin-secreting cell lines. Peripheral blood B lymphocytes enriched for Thomsen-Friedenreich antigen (T antigen)-specific cells were transformed with Epstein-Barr virus. Lymphoblastoid cells (LC) reactive with T antigen were either expanded without cloning or cloned at limiting dilution and then fused with murine 653 cells. Uncloned LCs from three transformations secreting polyclonal anti-T antibody (7-18 micrograms/ml/10(6) cells/24 hr total immunoglobulin) were subcultured at 100 cells/well, and T antigen-reactive cultures pooled. These cultures quickly lost specific antibody secretion, presumably due to overgrowth by clones of unknown specificity. T antigen-reactive LCs that were cloned three times at limiting dilution secreted 0.2 - 6.1 micrograms/ml/10(6) cells/24 hr but died or stopped secreting specific immunoglobulin after 77 to 155 days in culture. Pooling T antigen-reactive clones after each cloning step did not increase the long term stability compared to unpooled clones (p = 0.2). Fusions between cloned LCs and 653 cells failed to yield viable hybrids in nine of ten attempts with seven different LC lines. In contrast, fusion of uncloned LCs and 653 cells resulted in the generation of viable immunoglobulin-secreting heterohybrids in 22 of 24 fusions. The heterohybridomas produced from fusion of uncloned T antigen-reactive cultures with 653 cells secreted significantly more antibody (frequency of cell lines secreting greater than 2 micrograms/ml/10(6) cells/24 hr, p less than 0.01) and higher titers of antibody (frequency of cell lines secreting greater than four hemagglutination units of T antigen-specific antibody, p less than 0.03) than cloned lymphoblastoid cells. The hybrids maintained specific immunoglobulin secretion for longer in culture than either cloned or uncloned lymphoblastoid cell lines (p less than 0.001).     

Immunological senescence. I. The role of suppressor cells.

The in vitro anti-SRBC response of several murine strains declined markedly with age in parallel with an increase in the activity of suppressor cells in the spleen and bone marrow which prevented early events during the induction of the immune response. These suppressor cells released soluble mediators and lacked the characteristics of mature T cells or macrophages. In addition the suppressor cell in the bone marrow could be removed on anti-Ig columns and fractions of old splenic suppressor cells sedimenting at 0.32 cm/h were greatly enriched in surface Ig bearing cells. Old immunodepressed mice did not lack potentially immunocompetent cells since the antibody response of old spleen cells could be restored by specifically activated T cells or lipopolysaccharide which act on B cells. These results suggest that a rise in the activity of non-T suppressor cells in the spleen and bone marrow may account, in part, for the depression in humoral immunity observed in aging mice.     

Macrophages,but not dendritic cells,present collagen to T cells

Dendritic cells, such as epidermal Langerhans cells, play a crucial role for the antigen-specific priming of T cells. We have addressed the question whether dendritic cells present collagen, a major protein component in tissues through which dendritic cells migrate, i.e. the basement membrane, dermis, and synovial tissue. Langerhans cells, spleen cells and peritoneal macrophages were compared for antigen-presenting capacity using a panel of mouse T cell hybridomas reactive with different determinants on type II collagen, myelin basic protein, ovalbumin and pepsin. Langerhans cells did not present any of the type II collagen determinants, unless the antigen was administered as a 15-mer peptide, but did present myelin basic protein, ovalbumin and pepsin. Spleen cells and peritoneal macrophages, in contrast, presented all type II collagen determinants. This biased antigen presentation was also observed when Langerhans cells were pulsed with antigen in vivo. The inability to present type II collagen is related to the collagen sequence as such, since both native type II collagen, type II collagen α chains, as well as a type II collagen determinant incorporated in type I collagen, were not presented by Langerhans cells. In addition, granulocyte/macrophage colony-stimulating factor-expanded blood dendritic cells displayed the same biased antigen presentation, suggesting that the inability to present collagen is not restricted to dendritic cells localized in epidermis. B cell-deficient mice could prime a type II collagen-reactive T cell response, thus excluding B cells as obligatory antigen-presenting cells for the priming of collagen-reactive T cells. We suggest that neither Langerhans cells nor B cells, but macrophages are the primary antigen-presenting cells in the immune response towards type II collagen.     

Etiology of the liver granulomatous response in Schistosoma mansoni-infected athymic nude mice.

The effect of schistosome infection on the presence and maturation of splenic T lymphocytes in C3H/HeN nu/nu and nu/+ mice was examined. Spleens of uninfected nu/nu mice contained very low numbers (u to 2%) of T lymphocytes. This percentage did not increase throughout the 10 weeks of the infection. Spleens of uninfected nu/+ littermates contained 28.8% T cells, which decreased to 15.0% by week 10 of the infection. Similarly, whereas spleen cells of normal or infected nu/nu mice were nonresponsive to concanavalin A, the initial high response of nu/+ mice gradually diminished. Both nu/nu and nu/+ spleen cells responded well to lipopolysaccharide initially, but by 10 weeks their responsiveness declined. Sera of five infected nu/nu mice contained no antibodies to egg antigens, and one had a low titer (log2 5.0). In contrast, the mean titer of sera from six nu/+ mice was log2 10.7 Nu/+ mice had typical florid lesions, but nu/nu mice mounted sparse granulomatous reactions around eggs in the liver without evidence for hepatocellular damage. Dispersed liver granulomas of nu/nu mice contained 1.2% T and 20.3% B lymphocytes. Lesions of nu/+ mice contained 12.9% T and 18.4% B cells. Eighty percent of the macrophages from nu/nu and nu/+ granulomas displayed high density/avidity Fc receptors. Production of migration inhibition factor-active lymphokine by liver granulomas and spleens of schistosome-infected nu/nu mice is suggestive of the immune role of B cells in the granulomatous inflammation.     

T-dependent B-cell activation is signalled by an early increase in potassium influx

When splenic lymphocytes from RNC nu/ + mice were enriched for T cells by lectin purification, mixed to constant cell density with splenocytes from syngeneic nude mice, and cultured with Con A, a proliferative response ensued which was greater than that expected from the T cells alone. This was shown both by incorporation of ³H-thymidine after 48 h of culture and by uptake of potassium (measured as ⁸⁶Rb) after 15 h. Analysis of metaphase chromosomes stained with Hoechst dye 33258 from co-cultured T-enriched and nude lymphocytes (from female and male donors, respectively) mixed in the proportion 1:4 showed that 40 % of the mitotic cells came from nude spleen. About half of the blast cells in such mixtures stained strongly with fluorescein-coupled goat anti-mouse immunoglobulin; T-cell blasts did not stain under these conditions. Treatment of the nu/nu cells with anti-Thy 1.2 and complement had no effect on their subsequent proliferation in coculture.B lymphocytes from nude mouse spleen were therefore activated to proliferate in this system. This B-cell activation can be detected by increased potassium uptake 15 h after the initiation of co-culture. Thus the increased monovalent cation flux (previously demonstrated when B and T lymphocytes were separately stimulated) also occurs when B cells are stimulated through cooperation with mitogen-activated T cells, and is also detectable early in culture. Tdependent activation of B cells is therefore detectable considerably earlier than by conventional assays (such as plaque formation).     

Properties of antisera against lymphocytes of nude mice.

This paper reports results of a study on the activity of rabbit antisera against nu/nu Balb/c lymphocytes in vivo and in vitro. It was found that ALS against nu/nu lymph node cells suppressed the alloantigen reaction and the sRFC or PFC formation for T-dependent (SRBC) and T-independent (LPS) antigens. ALS against nu/nu spleen cells affected only the sRFC and PFC for T-independent antigen. The former serum exhibited a high cytotoxicity for the suspensions enriched or depleted in B cells while the latter one was more cytotoxic for the suspension enriched in B cells. It indicates that ALS anti nu/nu spleen cells is specific for B lymphocytes and ALS anti nu/nu lymph node cells is directed not only to B cells but also to a subpopulation of T lymphocytes. It suggests the existence of a subpopulation of T lymphocytes in nu/nu lymph node cells.     

Splenic macrophages and B cells mediate immunosuppression following abrupt withdrawal from morphine

We have previously shown that abrupt withdrawal (AW) from morphine induces greater than 80% immunosuppression in murine spleen cells, as assessed by the capacity to mount an in vitro plaque-forming cell response to sheep red blood cells. Present studies about the mechanisms of immunosuppression following AW showed that addition of highly enriched (CD11b+) splenic macrophages (obtained by cell sorting or magnetic separation) from AW mice to cultures of normal, unfractionated spleen cells suppressed immune responses. Further, addition of highly enriched (CD19+) B cells (but not T cells) from AW mice to normal cells was also immunosuppressive. B cells from AW mice were also able to inhibit the proliferative response of normal spleen cells to concanavalin A but not to lipopolysaccharide. Overall, the data suggest that immunosuppression by AW spleen cells is a result of active suppression by macrophages and B cells.     

Experimental autoimmune oophoritis. II. Both lymphoid cells and antibodies are successful in adoptive transfer.

Experimental autoimmune oophoritis can be readily induced by passive transfer of peripheral blood lymphocytes, lymph node cells, spleen cells, T- and B-enriched cell suspensions, immune serum and gamma globulins, from ovary antigen immunized rats to naive recipients. Adoptive transfer was markedly enhanced when recipient rats were injected simultaneously with sensitized lymphoid cells and anti-ovary antibodies. Histologically, this passively induced disease was much the same as the actively induced disease. By syngeneic lymph node assay it was shown that regional lymph nodes of neonatally thymectomized rats did not enlarge upon injection of EAOO lymphocytes which otherwise produced a marked effect in lymph nodes of normal recipient rats. Therefore, it appears that enlargement of the draining lymph node was dependent on the participation of host T cells. The possibility that development of EAOO may involve cooperation between antigen-reactive and effector classes of lymphocytes was discussed.     

Evaluation of accessory cell heterogeneity. I. Differential accessory cell requirement for T helper cell activation and for T-B cooperation

Several Ia+ tumor cell lines and peritoneal exudate macrophages were tested as accessory cells (AC) for the activation of antigen-specific T cells and for T-B cooperation. The macrophages and all the Ia+ tumor lines tested induced the release of lymphokines from T cells in a major histocompatibility complex (MHC)-restricted fashion and reconstituted the antibody responses of AC-depleted spleen cells or of purified T and B cells. However, only the normal macrophages but none of the tumor lines induced carrier-specific T helper (Th) cells which help B cells for specific antihapten antibody responses by linked recognition. For T-B cooperation accessory cells were also required, but in contrast to Th cell activation any type of Ia+ AC (e.g. macrophage or tumor line) was effective. Strong MHC-restriction between the lymphocytes and the AC was seen if antigen-pulsed AC were added into the AC-depleted T-B cooperation cultures. If the AC and antigen were concomitantly added to the AC-depleted T-B cultures, MHC-restriction was less obvious. Concanavalin A supernatant reconstituted the response of AC-depleted T-B cultures provided antigen-specific Th cells and the hapten-carrier conjugate were present. If, however, tumor line-activated T cells were added instead of macrophage-induced Th cells, no cooperation with B cells took place even in the presence of Con A supernatant. The results obtained demonstrate a differential AC requirement for the induction of Th cells depending on the differentiation stage of the Th cells.