Feedback suppression of the immune response in vivo. I. Immune B cells induce antigen-specific suppressor T cells

Stimulated lymphocytes are capable of synthesizing and secreting a variety of lymphokines which can affect the functions of several types of target cells. We report here the existence of a soluble factor released by activated human mononuclear leukocytes which produces a selective inhibition of human pulmonary fibroblast migration. This fibroblast migration inhibitory factor (FIF) was produced by antigen- or mitogen-stimulated human peripheral blood mononuclear leukocytes (PBML) and purified T cells. It inhibited the migration of ⁵¹Cr-labeled fibroblasts in a dose-dependent fashion with optimal effect (65–70% inhibition) obtained at 1:10 dilution and 8–20 hr of incubation. Sephadex G-100 fractionation revealed most activity to be found between 28,000 and 34,000 daltons. FIF was stable at 56 °C for 15 min, but destroyed at 80 °C or at low pH. This factor may play an important role in the modulation of fibrogenesis and healing processes by the immune system.     

Immunologic suppression after oral administration of antigen. III. Activation of suppressor-inducer cells in the Peyer's patches

Mice were orally administered sheep erythrocytes (SRBC) in a regimen previously known to produce systemic tolerance to SRBC. Cellular interactions and movement from the gut-associated lymphoid tissue (GALT) to the spleen were found to occur using both in vivo and in vitro transfer systems. The cell in the GALT which initiates the suppression circuit migrates from the GALT to the spleen shortly after contacting antigen. This cell is a T suppressor-inducer (Tsi) cell which interacts with splenic lymphocytes to induce the formation of an effector T suppressor cell (Ts). The Tsi and Ts can be separated from each other by their differential sensitivities to cyclophosphamide. In addition, the Tsi can be separated from other GALT T cells by its inability to bind the lectin, peanut agglutinin. Thus, cell migration and cellular interaction among T cells must occur to result in orally induced tolerance.     

In vivo immune response suppression by the supernatant from concanavalin A-activated spleen cells.

Supernatants from concanavalin A- (Con A) activated murine spleen cells have been shown to suppress the in vitro plaque-forming cell (PFC) response to sheep red blood cells (SRBC). The present study examined the effect of such Con A-activated spleen cell supernatants (herein termed CONS) on the in vivo immune response to SRBC in C57BL/6, BALB/c and CDF1 mice. CONS derived from BALB/c spleen cells suppressed direct PFC 4 and 8 days after immunization with 2 X 10(8) SRBC. CONS also suppressed indirect PFC 8 days after immunization, as well as serum hemagglutinins to SRBC. The PFC response of C57BL/6 (H-2b) mice was suppressed as much as that of BALB/c (H-2d) by CONS derived from BALB/c mice, indicating a lack of H-2 specificity of the CONS. In addition to suppression of the antibody response to SRBC, in vivo CONS administration resulted in reduction in spleen cell number. This reduction was not sufficient to explain the decreased PFC response. When the CONS was separated into less than 10,000 m.w. and greater than or equal to 10,000 m.w. fractions, the immunosuppressive activity was found in the less than 10,000 m.w. fraction. This observation suggests that intact interferon, SIRS, and MIF were not responsible for the results obtained.     

The role of tumor-specific Lyt-1+2- T cells in eradicating tumor cells in vivo. I. Lyt-1+2- T cells do not necessarily require recruitment of host's cytotoxic T cell precursors for implementation of in vivo immunity

The present study determines the Ly phenotype of T cells mediating tumor cell rejection in vivo and investigates some of cellular mechanisms involved in the in vivo protective immunity. C3H/HeN mice were immunized to syngeneic X5563 plasmacytoma by intradermal (i.d.) inoculation of viable X5563 tumor cells, followed by the surgical resection of the tumor. Spleen cells from these immune mice were fractionated by treatment with anti-Lyt antibodies plus complement, and each Lyt subpopulation was tested for the reconstituting potential of in vivo protective immunity in syngeneic T cell-depleted mice (B cell mice). When C3H/HeN B cell mice were adoptively transferred with Lyt-1-2+ T cells from the above tumor-immunized mice, these B cell mice exhibited an appreciable cytotoxic T lymphocyte (CTL) response to the X5563 tumor, whereas they failed to resist the i.d. challenge of X5563 tumor cells. In contrast, the adoptive transfer of Lyt-1+2- anti-X5563 immune T cells into B cell mice produced complete protection against the subsequent tumor cell challenge. Although no CTL or antibody response against X5563 tumors was detected in the above tumor-resistant B cell mice, these mice were able to retain Lyt-1+2- T cell-mediated delayed-type hypersensitivity (DTH) responses to the X5563 tumor. These results indicate that Lyt-1+2- T cells depleted of the Lyt-2+ T cell subpopulation containing CTL or CTL precursors are effective in in vivo protective immunity, and that these Lyt-1+2- T cells implement their in vivo anti-tumor activity without inducing CTL or antibody responses. The mechanism(s) by which Lyt-1+2- T cells function in vivo for the implementation of tumor-specific immunity is discussed in the context of DTH responses to the tumor-associated antigens and its related Lyt-1+2- T cell-mediated lymphokine production.     

The mechanism of tumor growth inhibition by tumor-specific Lyt-1+2-T cells. I. Antitumor effect of Lyt-1+2-T cells depends on the existence of adherent cells

In the present study we establish an assay system of tumor growth inhibition with the use of a diffusion chamber and investigate the mechanism by which tumor-specific Lyt-1+2-T cells exhibit their inhibiting effect on tumor cell growth. When a diffusion chamber containing X5563 plasmacytoma cells together with normal syngeneic C3H/HeN spleen cells was implanted in the peritoneal cavity of C3H/HeN mice, these tumor cells continued to proliferate at least 7 to 9 days. In contrast, spleen cells from C3H/HeN mice that had acquired X5563-specific immunity by intradermal (i.d.) inoculation of viable tumor cells, followed by surgical resection of the tumor, exhibited an appreciable inhibitory effect on the growth of X5563 tumor cells admixed in the chamber. This antitumor effect was mediated by Lyt-1+2-T cells and was tumor-specific, because the growth of X5563 or another syngeneic MH134 hepatoma cells was inhibited by spleen cells from C3H/HeN mice immunized to the respective tumor cell types. Most important, these tumor-specific Lyt-1+2-T cells lost their antitumor activity by depleting an adherent cell population contained in spleen cells, indicating that adherent cells are required for the Lyt-1+2-T cell-mediated antitumor effect. This was substantiated by the fact that immune spleen cells depleted of adherent cells could regain their tumor-inhibiting effect when normal spleen cells were added back as an adherent cell source, or more directly by adding back a splenic or peritoneal resident adherent cell population. These results indicate that tumor-specific Lyt-1+2-T cells mediate the tumor growth inhibition and that their antitumor effect depends on the coexistence of an adherent cell population.     

Lyt phenotype of cytotoxic T cells: shift from Lyt-1+2+3+ to a mixed population of Lyt-1+2+3+ and Lyt-1-2+3+ during in vitro culture

The Lyt phenotype of cytotoxic T cells generated in the primary H-2 response was investigated kinetically. The cytotoxicity generated in the early stage of culture was abolished by treatment with alpha Lyt-1,2,3, and complement (C), whereas that generated in the late stage was only partially eliminated by alpha Lyt-1, but was abolished by alpha Lyt-2, 3, and C. This suggested late expansion of the Lyt-1-2+3+ population. Lack of Lyt-1 antigen was confirmed with cells that were depleted of Lyt-1+ from primary culture and then stimulated in the secondary response by elimination of cytotoxicity and by direct Lyt typing. Results indicated that the response of proliferative and cytotoxic T cells of the Lyt-1+2+3+ phenotype in the early stage of culture was followed by activation of Lyt-1-2+3+ T cells. Cytotoxic T cells in the late stage were shown to be a mixture of Lyt-1+2+3+ and Lyt-1-2+3+ cells. This was confirmed with cytotoxic T cells from secondary culture and uncloned long-term T cell lines.     

Corticosteroid-induced suppression of murine B cell immune response antigens

The quantitative variation in expression of B cell surface immune response-associated antigens (sIa) that is induced by in vivo i.v. administration of dexamethasone was studied by flow microfluorometry. Injection of 40 micrograms of dexamethasone resulted in a 35 to 40% reduction in the expression of sIa within 3 hr, reached its maximum effect within 6 hr, which on average resulted in 75% suppression of control values of sIa, and by 12 hr after injection began returning towards baseline levels. The suppressive effect of dexamethasone on B cell sIa was dose dependent with respect to the length of time required to reach maximal suppression, as well as with respect to the duration of suppression that was attained. When injections of dexamethasone were repeated on consecutive days, no additional increase in the level of sIa suppression achieved was observed. B cell sIa was also diminished after injection of dexamethasone into athymic nude mice, which suggests that the suppressive effect of dexamethasone on B cell expression of sIa is not a T cell-dependent phenomenon. Taken together, these data suggest that the suppression of B cell sIa by corticosteroids may be a means whereby endogenous or exogenous corticosteroids are able to influence the normal as well as abnormal immunologic state.     

Human C3a-mediated suppression of the immune response. I. Suppression of murine in vitro antibody responses occurs through the generation of nonspecific Lyt-2+ suppressor T cell

C3a derived from the third component of human complement was found to suppress in vitro murine anti-SRBC responses. C3a-mediated suppression occurs through the generation of nonspecific Lyt-2+ suppressor T cells. The generation of suppressor cells occurs at an early phase in the response because incubation of naive T cells with C3a for as little as 30 min results in suppression of the anti-SRBC response. The generation of suppressor T cells requires the interaction of T cells, C3a, and a Sephadex G-10-adherent cell, presumably a macrophage. Although the mechanism of action of these suppressor cells has not been elucidated, several possibilities have been eliminated. C3a-suppressor T cells do not apparently release inhibitory lymphokines, nor is helper cell activity inhibited by a 2-day co-culture with these suppressor cells. The observation that interleukin 2 (IL 2)-containing lymphokine preparations could overcome C3a-induced suppression led us to investigate the interaction of the suppressors with IL 2 producer cells. However, neither C3a nor C3a-generated suppressor T cells can block the synthesis of IL 2.     

Enhancement and suppression of immunoglobulin G-producing B cells in the presence of immune T cells.

Mice were immunized one to three times with sheep red blood cells. Four to seven days after the last immunization, the spleens were removed and the cells were cultured in vitro in the absence of antigen. Removal of most T cells by anti-θ serum treatment prior to culture could increase the number of IgG-producing B cells without affecting the number of specific or nonspecific IgM-producing B cells detected after 2 days of culture. Addition of graded numbers of immune cells to pure immune B cells enhanced the number of IgG-producing B cells, whereas addition or higher number of immune cells caused suppression. Since removal of T cells could also enhance the proliferation of IgG-producing B cells induced by lipopolysaccharide (LPS), a polyclonal B-cell activator, it is suggested that the suppressive effects of high numbers of immune T cells are exerted directly on the B cells.     

Antigen-presenting cells for Lyt-2+ cells. I. Stimulation of unprimed Lyt-2+ cells by H-2 different Thy-1-Ia- cells prepared from spleen and bone marrow

In agreement with previous studies on Ia- tumor cells, evidence is presented that primary MLR of purified Lyt-2+ T cells to class I alloantigens can be elicited by a minor population of Thy 1- Ia- cells present in normal spleen, bone marrow, and day-13 fetal liver; these cells are non-stimulatory for L3T4+ T cells. The data strengthen the view that primary responses of Lyt-2+ cells do not require the presence of Ia+ cells.     

Accessory cells in immune suppression. III. Evidence for two distinct accessory cell-dependent mechanisms of T lymphocyte-mediated suppression

Suppression of antibody secretion by the 2,4,6-trinitrophenol (TNP)-binding BALB/c myeloma, MOPC 315, by idiotype- and hapten-reactive suppressor T cells is mediated by secreted factors (TsF) and requires the presence of accessory cells (AC). Idiotype-specific TsF functions only in the presence of Ia+ AC and is completely idiotype specific. Moreover, no suppression is observed when myeloma targets and AC are separated by cell-impermeable membranes, indicating that the role of AC may be to bind, focus, and/or present TsF to the myeloma cells. In contrast, TNP-specific TsF inhibits myeloma function in the presence of TNP-protein and activated macrophages that are not Ia+. This form of suppression is nonspecific at the effector stage; i.e., anti-TNP TsF inhibits a non-TNP binding cell line, TEPC 15, as long as TNP-protein and activated macrophages are present. Moreover, suppression occurs even when myeloma targets and AC are separated by cell-impermeable membranes. These results are consistent with the view that hapten-reactive TsF binds to antigen on the surface of macrophages and induces these cells to secrete nonspecific immunosuppressive molecules. Thus, different types of AC may play fundamentally different roles in TsF-mediated suppression; they may either bind and present TsF to targets (as in the case of idiotype-specific TsF) or secrete nonspecific immunosuppressants as a consequence of a TsF-antigen interaction (hapten-specific TsF). Autonomous, suppressible targets provide valuable experimental systems for analyzing the cellular interactions in T cell-mediated suppression.     

Accessory cells in immune suppression. I. Role of I-A+ accessory cells in effector phase idiotype-specific suppression of myeloma function

The suppression of MOPC 315 myeloma cells by idiotype-specific effector Ts requires the presence of non-immune AC. This requirement was demonstrated in cultures where myeloma targets and Ts were separated by cell-impermeable membranes or were in direct contact. The AC were adherent, radioresistant, and present in peritoneal exudates and in FcR+ as well as FcR- fractions of low density splenocytes; they bore cell surface I-A determinants and did not have to be H-2 compatible with myeloma cells and Ts. These studies demonstrate a novel role for Ia+ AC in immune regulation, and suggest that their accessory function may involve processing of T lymphocyte-derived suppressor factors or presentation of such factors to target cells.     

Tumor-infiltrating programmed death receptor-1+ dendritic cells mediate immune suppression in ovarian cancer

Within the ovarian cancer microenvironment, there are several mechanisms that suppress the actions of antitumor immune effectors. Delineating the complex immune microenvironment is an important goal toward developing effective immune-based therapies. A dominant pathway of immune suppression in ovarian cancer involves tumor-associated and dendritic cell (DC)-associated B7-H1. The interaction of B7-H1 with PD-1 on tumor-infiltrating T cells is a widely cited theory of immune suppression involving B7-H1 in ovarian cancer. Recent studies suggest that the B7-H1 ligand, programmed death receptor-1 (PD-1), is also expressed on myeloid cells, complicating interpretations of how B7-H1 regulates DC function in the tumor. In this study, we found that ovarian cancer-infiltrating DCs progressively expressed increased levels of PD-1 over time in addition to B7-H1. These dual-positive PD-1(+) B7-H1(+) DCs have a classical DC phenotype (i.e., CD11c(+)CD11b(+)CD8(-)), but are immature, suppressive, and respond poorly to danger signals. Accumulation of PD-1(+)B7-H1(+) DCs in the tumor was associated with suppression of T cell activity and decreased infiltrating T cells in advancing tumors. T cell suppressor function of these DCs appeared to be mediated by T cell-associated PD-1. In contrast, ligation of PD-1 expressed on the tumor-associated DCs suppressed NF-κB activation, release of immune regulatory cytokines, and upregulation of costimulatory molecules. PD-1 blockade in mice bearing ovarian cancer substantially reduced tumor burden and increased effector Ag-specific T cell responses. Our results reveal a novel role of tumor infiltrating PD-1(+)B7-H1(+) DCs in mediating immune suppression in ovarian cancer.     

Specific suppression of the immune response by HGG tolerant spleen cells. I. Parameters affecting the level of suppression.

After adoptive transfer, the spleen cells from mice made tolerant to human gamma-globulin (HGG) specifically suppress the immune response of normal spleen cells. However, this suppressive activity in the spleen cells of tolerant mice is only present for a brief period after treatment with tolerogen. Spleen cells from animals injected 10 days earlier with tolerogen reduce the immune response of an equal number of normal spleen cells by 75%. Spleen cells from mice made tolerant 40 days previously are, however, no longer suppressive, even though they remain completely unresponsive. These data suggest that active suppression of antigen-reactive cells is not the mechanism responsible for maintaining tolerance to HGG, but rather is only transiently associated with the tolerant state. Further evidence in favor of the separation of the tolerant state from suppressive activity is that complete suppression of the normal spleen cell response requires either a high ratio of tolerant to immune competent cells or a delay in the antigenic challenge of the reconstituted recipients. By contrast, such manipulations are not required to demonstrate the complete unresponsiveness of the tolerant cells after adoptive transfer.     

Characterization of responding cells in oxidative mitogen stimulation. I. Ia+ cells and Ly-1+2+ cells are required for the proliferative response

Addition of anti-Ia sera to cultures of mouse spleen cells stimulated with oxidative mitogens, neuraminidase/galactose oxidase (NaGO) or sodium periodate (NaIO4), inhibits the subsequent proliferative response 30 to 70%. Anti-H-2K or D sera were not specifically inhibitory. Similar inhibition was seen when cells were pretreated with anti-Ia sera and washed before exposure to the mitogenic enzymes. Treatment with anti-Ia serum and complement depletes greater than 89% of the NaGO and the NaIO4 responses but 2% or less of the phytohemagglutinin (PHA) response. The response to NaGO was sensitive to depletion with anti-Thy-1 serum, rabbit anti-mouse brain serum, anti-Ly-1, or anti-Ly-2 serum. Mixtures of Ly-1 and Ly-2-depleted populations did not restore responsiveness. Thus both an Ia+ cell and an Ly-1+2+ T cell are required for [3H]TdR incorporation in response to NaGO treatment.     

Regulation of the immune response to tumor antigens. IX. In vitro Lyt-1+2- cell proliferative responses to cellbound or subcellular tumor antigen

We investigated the cellular basis of immune reactivity to the S1509a fibrosarcoma in tumor-immune A/J mice. In a Winn assay, immune Lyt-1+2- T cells are capable of retarding S1509a tumor growth in naive A/J mice. In vitro proliferation to S1509a is also mediated by tumor-immune Lyt-1+2- T cells. This response is specific to the immunizing tumor and appears 5 to 7 days after reexposure to the tumor in vivo. Proliferation also requires the presence of a population of adherent cells. In fact, adherent peritoneal exudate cells pulsed with tumor membrane fragments derived from S1509a cells can stimulate proliferation. Proliferation is blocked by the addition of anti-I-Ak monoclonal antibody to the culture medium without complement or by treatment of the responder population with anti-I-Ak and complement. In vitro responsiveness is also inhibited by the presence of tumor-specific suppressor T cells in vivo. These observations suggest in vitro proliferation may provide a potential means of defining tumor antigens and cell-surface structures involved in tumor immunity.     

Neonatal splenic suppressor cells in the chicken. I. In vivo suppression of the immune response to bovine serum albumin by normal and tolerized neonatal spleen cells

The presence of active splenic suppressor cells in neonatal chickens, either normal or tolerant to bovine serum albumin (BSA), was examined by assessment of their effect on both primary and adoptively transferred secondary responses to BSA or sheep red blood cells (SRC). Both normal and BSA tolerized spleen cells were shown to be highly suppressive of secondary anti-BSA responses generated by specifically primed adult spleen cells in inert recipients. Suppression of the secondary anti-BSA response by normal spleen cells was slightly less effective than that seen with BSA tolerant spleen cells. Transfer of BSA tolerant spleen cells into normal recipients, followed by BSA challenge, prevented any significant primary anti-BSA response. In contrast, transfer of normal spleen cells into normal recipients, followed by BSA challenge, failed to show any suppression of the resulting primary response. Neither normal nor BSA tolerant neonatal spleen cells were capable of suppressing either primary or secondary responses to SRC. Thus, chickens tolerized to BSA have suppressor cells specific for the tolerizing antigen. We present evidence that both the tolerance associated suppressors and the suppressors detected in normal neonatal chickens are T cells.     

In vivo suppression of the primary immune response by a species of low density serum lipoprotein.

An apparent subspecies of normal human serum low density lipoprotein (LDL-In) has been identified with suppressive activity for early or facilitating events of human lymphocyte mitogen and allogenic cells stimulation in vitro. This report describes the effects of in vivo administration of LDL-In on the mouse anti-SRBC immune response. Human LDL-In is not species specific and was capable of suppressing the in vivo mouse anti-sheep erythrocyte (SRBC) hemagglutination response by 88% after the administration of 500 to 600 mug LDL-In IV, whereas human serum high density lipoproteins and fibrinogen had no effect. Maximal suppression occurred only when LDL-In was injected 24 to 48 hr before antigen administration. Simultaneous or subsequent injection of LDL-In had no effect. The activity of LDL-In was influenced by antigen dose and maximal at low antigen doses. The number of splenic plaque-forming cells was also reduced indicating a suppression of the clonal expansion of primary B cells to antibody-secreting cells rather than only suppression of antibody synthesis by differentiated B cells and their progeny. These observations suggest the hypothesis that endogenous LDL-In could play an important immunoregulatory role in the maintenance of immune homeostasis and the "natural" suppression of non-productive lymphocyte proliferation.     

T lymphocyte heterogeneity in the rat. III. Autoreactive T cells are activated by B cells

Evidence has been presented to show that CD4+ autoreactive T cell lines (ATs)2 in the rat require periodic stimulation with syngeneic spleen cells for in vitro proliferation. This proliferation can be blocked by treatment of the stimulator (spleen) cells with mAb to Ia antigens. Although ATs are Ia+ and can activate the allogeneic MLR, they fail to be autostimulatory. Fractionation of the spleen cells revealed that ATs can be stimulated with B cells and not by macrophages, although the latter were efficient in several accessory cell functions, including antigen presentation, lectin-dependent T cell activation and allogenic MLR response. Moreover, B cells proliferated and differentiated in response to AT cells. These data are compatible with a model in which ATs respond to hitherto undetermined B cell membrane antigen(s) in association with MHC class II antigens. These results may have important implications in understanding autoimmune responses.