Studies on cellular immunity toSalmonella typhi in vitro

Salmonella typhi (strain Ty2—4446) cultivatedin vitro within the macrophage of mice immunised twice specifically with a high dose of killed vaccine, multiplied less intensively than in the cells of non-immunised control animals during 24 hours cultivation. In addition, a certain suppression of growth ofSalmonella enteritidis andSalmonella suis var. Kunzendorf was observed in the macrophages of mice immunised withSalmonella typhi vaccine. Double immunisation of mice with high doses of killed vaccine fromSalmonella enteritidis andSalmonella suis led to the mice macrophages being able to suppress multiplication of both homologous microbes andSalmonella typhi. The formation of such cross resistance in mice immunised with salmonella vaccines excludes the possible participation of O, H, and Vi antibodies in this phenomenon.     

TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: role of IL-17-producing T cells

Whole cell pertussis vaccines (Pw) induce Th1 responses and protect against Bordetella pertussis infection, whereas pertussis acellular vaccines (Pa) induce Ab and Th2-biased responses and also protect against severe disease. In this study, we show that Pw failed to generate protective immunity in TLR4-defective C3H/HeJ mice. In contrast, protection induced with Pa was compromised, but not completely abrogated, in C3H/HeJ mice. Immunization with Pw, but not Pa, induced a population of IL-17-producing T cells (Th-17), as well as Th1 cells. Ag-specific IL-17 and IFN-gamma production was significantly lower in Pw-immunized TLR4-defective mice. Furthermore, treatment with neutralizing anti-IL-17 Ab immediately before and after B. pertussis challenge significantly reduced the protective efficacy of Pw. Stimulation of dendritic cells (DC) with Pw promoted IL-23, IL-12, IL-1beta, and TNF-alpha production, which was impaired in DC from TLR4-defective mice. B. pertussis LPS, which is present in high concentrations in Pw, induced IL-23 production by DC, which enhanced IL-17 secretion by T cells, but the induction of Th-17 cells was also dependent on IL-1. In addition, we identified a new effector function for IL-17, activating macrophage killing of B. pertussis, and this bactericidal activity was less efficient in macrophages from TLR4-defective mice. These data provide the first definitive evidence of a role for TLRs in protective immunity induced by a human vaccine. Our findings also demonstrate that activation of innate immune cells through TLR4 helps to direct the induction of Th1 and Th-17 cells, which mediate protective cellular immunity to B. pertussis.     

T lymphocyte immunity to reovirus: cellular requirements for generation and role in clearance of primary infections

The cellular requirements for the development of an immune response to reovirus type 1 and the role of such a response in the clearance of a primary infection with that virus were explored. An Ia-bearing antigen-presenting cell requirement is demonstrated for the in vitro generation of secondary anti-reovirus cytolytic T lymphocytes (CTL). It is then shown that mice whose spleens are depleted of Ia-bearing adherent cells by exposure in vivo to ultraviolet (UV) radiation exhibit depressed priming for reovirus-specific T lymphocyte function-CTL generation, delayed-type hypersensitivity reactivity, and T cell proliferative responsiveness. These UV-irradiated mice clear primary systemic reovirus infections as readily as normal mice. Further, athymic 'nude' mice show no defect in their ability to clear a reovirus infection. The implications of these findings for our understanding of the role of virus-specific T cell function in the clearance of systemic viral infections are discussed.     

The role of antigen-presenting B cells in T cell priming in vivo. Studies of B cell-deficient mice

Mice rendered B cell deficient by treatment with rabbit anti-mouse IgM (anti-mu) antibodies from birth fail to respond when primed with soluble protein antigens in CFA, as measured by T cell proliferation when challenged with antigen in vitro. The role of B cells in T cell priming in vivo was examined by adoptively transferring hapten-specific B cells into anti-mu mice, followed by immunization with haptenated Ag in CFA. The T cell proliferative response to OVA of anti-mu BALB/c mice was partially restored by the administration of TNP or FITC-specific B cells and immunization with TNP-OVA or FITC-OVA, respectively. This reconstitution was Ag-specific, inasmuch as hapten-binding B cells restored the T cell responses to OVA in mice immunized with the same hapten coupled to OVA. The mechanism of B cell reconstitution of T cell priming in anti-mu mice was addressed using parental to F1 B cell transfers. The Ia restriction pattern of the activated T cells from these mice indicated that both direct presentation of Ag by transferred B cells and antibody-mediated enhancement of Ag presentation by non-B, host Ag-presenting cells occurred. Thus, Ag-specific B lymphocytes play a critical role in priming of T cells in vivo.     

A pivotal role of vitamin B9 in the maintenance of regulatory T cells in vitro and in vivo

Dietary factors regulate immunological function, but the underlying mechanisms remain elusive. Here we show that vitamin B9 is a survival factor for regulatory T (Treg) cells expressing high levels of vitamin B9 receptor (folate receptor 4). In vitamin B9-reduced condition in vitro, Treg cells could be differentiated from naïve T cells but failed to survive. The impaired survival of Treg cells was associated with decreased expression of anti-apoptotic Bcl2 and independent of IL-2. In vivo depletion of dietary vitamin B9 resulted in the reduction of Treg cells in the small intestine, a site for the absorption of dietary vitamin B9. These findings provide a new link between diet and the immune system, which could maintain the immunological homeostasis in the intestine.     

Cytotoxic cells suppress in vitro generation of cellular immunity by stimulator cell lysis

Irradiated BALB/c spleen-cell populations actively cytotoxic to BL/6 alloantigens (modulator cells) were capable of suppression of the in vitro generation of BALB/c anti-BL/6 cellular cytotoxicity. This suppression was abrogated by anti-θ serum plus complement. The suppression was dose dependent on the number of modulator cells and correlated directly with the magnitude of their cytotoxicity. By varying the number of stimulator cells, specific suppression for a relevant stimulator cell and nonspecific suppression for an irrelevant stimulator cell were demonstrated in the same cultures. These data suggest that cytotoxic cells caused specific suppression in mixed lymphocyte culture by lysing stimulator cells although evidence for other nonspecific suppressor factors was seen. A model was proposed suggesting that cell populations possessing high levels of cytotoxicity may feed back negatively on an ongoing immune response by competing with proliferating T cells for cellular antigen.     

Expression of surface lymphotoxin and tumor necrosis factor on activated T, B, and natural killer cells.

The expression of membrane-associated forms of lymphotoxin (LT) and TNF were examined on cell lines of T, B, and myeloid origin, IL-2 dependent T cell clones, and peripheral blood lymphocytes. Inducible and constitutive patterns of surface LT expression were found on T cells as exemplified by the II-23.D7, a CD4+T cell hybridoma, and HUT-78, a T cell lymphoma. Phorbol ester induced surface LT expression on Ramos, an EBV transformed B cell line, but at a slower rate of appearance when compared to the II-23.D7. Secretion of LT was rapidly inducible by phorbol ester in II-23.D7 and also in HUT-78 but with slower kinetics; surface LT expression continued in both lines after secretion had ceased. Low levels of membrane TNF were transiently induced on II-23.D7 and HUT-78, but none was observed on Ramos. Peripheral blood monocytes and some myeloid tumor lines did not express surface LT. Several T cell clones expressed surface LT after Ag-specific stimulation, and expression persisted several days. Stimulation through the TCR or by IL-2 rapidly induced surface LT on resting peripheral T cells and CD56+ NK cells; pokeweed mitogen activation induced expression on CD20+ B cells. Consistent with previous results, immunoprecipitation with anti-LT mAb showed that LT was complexed with a distinct 33 kDa glycoprotein (p33) on cells that expressed surface LT, whereas secreted LT was not associated with p33. Surface and secreted modes of LT expression by activated T, B, and NK cells suggests that LT can be utilized as either a localized or diffusible mediator in immune responses.     

Relationship of lymphotoxin secretion and DNA synthesis in the human mixed lymphocytes reaction in vitro.

Influenza virus particles, inactivated with formalin, have been covalently bound to cyanogen bromide-activated Sepharose beads (Se-vi beads). Preservation of the hemagglutination properties of the viral particles enabled a strong binding of pigeon or human group O erythrocytes (PRBC or HoRBC) to these Se-vi beads. The conditions for preparation of PRBC- or HoRBC-Se-vi columns are described.Spleen cell suspensions from mice immunized with the above erythrocytes were considerably depleted of cells forming hemolytic plaques (PFC) against the corresponding erythrocytes after passage through these columns. In the case of cells from nonimmunized mice, the depletion is still greater and reaches up to 95–100%. However, the number of PFC reactive to unrelated erythrocytes is not affected in the filtered population. Specifically attached cells recovered from the Se-vi-RBC columns passed with normal spleen cells are considerably enriched in the number of PFC against homologous erythrocytes. Syngeneic irradiated hosts transferred with filtered cells are able to give a normal primary PFC response against heterologous, but not against homologous RBC up to the 12th day after immunization. These results are discussed in relation to the problem of precommitment of specific PFC precursor cells.     

T cells recognizing polysaccharide-specific B cells function as contrasuppressor cells in the generation of T cell immunity to Pseudomonas aeruginosa

The cellular interactions involved in the development of T cell-mediated immunity to Pseudomonas aeruginosa have been examined. T cell immunity can be generated by immunizing mice with 10 micrograms of P. aeruginosa polysaccharide (PS) plus the antimitotic agent vinblastine sulfate. Vinblastine is required to inactivate a population of Ts cells generated by immunization with 10 micrograms of PS alone. Immunization with either live bacteria or with higher dose (50 micrograms) of PS without vinblastine also generates T cell immunity; these protocols activate a population of Lyt-1+, 2-, I-J+ T cells which, like vinblastine, counteract the effect of Ts cells. Immunization with 10 micrograms PS alone fails to activate this T cell subpopulation. When administered at the time of immunization, this subpopulation can render the tolerogenic 10-micrograms immunization protocols immunogenic. Like previously described contrasuppressor T cells, this T cell subpopulation exhibits an affinity for the lectin Vicia villosa. We have determined, however, that the T cells that act as contrasuppressor cells in this system are directly activated by PS-immune B cells and not by PS Ag. Furthermore, their activity can be removed by adsorption to PS-specific B cell hybridomas. Our studies indicate an important role for B cells in the development of T cell immunity to P. aeruginosa and suggest that a complex idiotype network controls the development of this response.     

In vitro expression of secondary antitumor immunity by in vivo tumor-sensitized T cells: Inhibition by tumor-induced suppressor T cells

Summary In vitro cultivation of memory immune cells from P815- or P388-immune mice with corresponding irradiated tumor cells induced generation of cytolytic T cells (CTL). The induction of CTL generation, as well as the cytolytic activity itself, was tumor-specific. The in vitro generation of CTL from P815- or P388-immune cells was suppressed by spleen cells from mice bearing corresponding progressive tumors (tumor size 15 mm). The tumor-induced suppressor cells suppressed the in vitro generation of CTL, but did not affect their cytolytic function. The suppression was tumor-specific and was mediated by Ly1⁺2^–L3T4⁺ T cells. Treatment of suppressor cell donors with cyclophosphamide or sublethal -radiation completely abolished the ability of their spleen cells to inhibit the in vitro CTL generation.     

NKT细胞在抗肿瘤免疫中的作用及其机制

NKT细胞是免疫细胞中一类具有NK细胞特定标志的T细胞亚群,经活化,既可直接作为抗肿瘤效应细胞发挥杀伤作用,又能通过激活其他免疫效应细胞,如NK细胞,间接实现抗肿瘤作用。NKT细胞在抗肿瘤免疫、获得性免疫应答及免疫调节中起着重要作用。     

In vitro depression of cellular immunity by Friend virus leukemic spleen cells.

Four distinct sublines of mouse L 929 cells (termed alpha, beta, gamma, and delta) were derived and shown to differ markedly in their in vitro sensitivity to human lymphotoxin (LT). The alpha L cell is most sensitive and is rapidly destroyed by very low dilutions of LT. This cell is 100 times more sensitive to LT than the most resistant (delta) L cell. The highly lymphotoxin-sensitive alpha cell makes it possible to reproducibly detect LT activity in as little as 0.0005 ml of supernatant medium. Additional studies revealed a direct correlation between the sensitivities of the four L cell sublines to LT and to direct cytolysis mediated by mitogen-stimulated human lymphocytes. The alpha, beta, gamma, and delta L cells were shown to be equally sensitive to antibody-mediated complement-dependent lysis, indicating that the sequence of sensitivities of these L cell sublines to the direct lymphocyte and to LT does not merely reflect a general susceptibility to cell destruction. These results lend further support to the view that lymphotoxin is an important mediator of in vitro target cell destruction by human effector lymphocytes.     

Immunomodulatory role of IL-4 on the secretion of Ig by human B cells

The effect of IL-4 on the production of Ig by human B cells was examined. Highly purified B cells were stimulated with Staphylococcus aureus (SA) and IL-4 alone or in combination with various other cytokines and the supernatants assayed for Ig by isotype-specific ELISA. IL-4 (10 to 100 U/ml) did not support Ig secretion by SA-stimulated blood, spleen, or lymph node B cells, whereas IL-2 supported the production of all isotypes including IgE. Moreover, IL-4 suppressed the production of all isotypes of Ig by B cells stimulated with SA and IL-2 including IgG1, IgG2, and IgE. IL-4-mediated suppression was partially reversed by IFN-gamma or -alpha and low m.w. B cell growth factor. TNF-alpha and IL-6 did not reverse the IL-4-induced suppression of Ig production. The inhibitory action of IL-4 on Ig production appeared to depend on the polyclonal activator used to stimulate the B cells. Thus, Ig secretion by B cells activated by LPS and supported by IL-2 was not inhibited by IL-4. Whereas IL-4 alone supported minimal Ig production by LPS-activated B cells, it augmented production of all Ig isotypes in cultures stimulated with LPS and supported by IL-2. IFN-gamma further enhanced production of Ig in these cultures. When the effect of IL-4 on the responsiveness of B cells preactivated with SA and IL-2 was examined, it was found not to inhibit but rather to promote Ig production modestly. A direct effect of IL-4 on the terminal differentiation of B cells was demonstrated using B lymphoblastoid cell lines. IL-4 was able to enhance the Ig secreted by an IgA-secreting hybridoma, 219 and by SKW6-CL-4, an IL-6-responsive IgM-secreting EBV transformed B cell line. These results indicate that IL-4 exerts a number of immunoregulatory actions on human B cell differentiation. It interferes with the activation of B cells by SA and IL-2, but promotes the differentiation of preactivated B cells, B cell lines, and B cells activated by LPS without apparent isotype specificity.     

Studies on in vitro effect of serotonin on calcitonin secretion by rat thyroid C cells

Summary Thyroid glands of young rats were incubated for 3 h in Eagle's solution supplemented with 5-hydroxy-l-tryptophan (5-HTP) or with serotonin. Following control incubations or incubations with serotonin, no serotonin could be demonstrated in C cells using immunocytochemical techniques. However, serotonin was demonstrated in the secretory granules of all C cells following incubation with 5-HTP. The secretory function of C cells was evaluated by ultrastructural and immunocytochemical studies, and by calcitonin radioimmunoassays of the incubation medium. Following incubation with 5-HTP, the secretory function of the majority of C cells was inhibited, and calcitonin levels in the media were decreased. Incubation with serotonin produced an increased secretory function of C cells and higher calcitonin levels in the media. The results indicate that serotonin and its direct precursor, 5-HTP, affect calcitonin secretion by rat thyroid C cells by distinct mechanisms.