In vitro generation of suppressor T cells. Induction of CD3+, IgH-restricted suppressor cells

Previous studies of the immune response of C57BL/6 mice to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten determined that challenge with antigenic forms of hapten induces both immunity and suppression. The anti-NP plaque-forming cell response can be down regulated by an Ag-induced cascade consisting of three suppressor T cell subsets. These three populations, termed Ts1, Ts2, and Ts3 have been characterized to have inducer, transducer and effector functions, respectively. Although the functions of each of these subsets have been examined in vivo, the cellular requirements for in vitro Ts induction have only been investigated for the Ts3 population. The present study characterizes the cellular events that lead to the induction of the Ts2, suppressor transducer population. Culture of naive C57BL/6 spleen cells with Ts1-derived suppressor factor in the absence of exogenous Ag leads to the generation of Ts2 cells that mediate Ag-specific suppression of NP plaque-forming cell responses. Phenotypic analyses demonstrate that a CD3+, CD4-, CD5+, CD8+, and I-J+ precursor population is stimulated by TsF1 to become mature Ts2 cells that express CD3, CD8, and I-J but not CD5. Although previous studies have reported an essential role for B cells in the induction of other Ts populations, depletion of B cells from Ts2 induction cultures had no effect on Ts2 generation. Despite the absence of B cells in these cultures, the mature Ts2 cells were functionally IgH restricted. Studies with IgH congenic B.C-8 mice suggest that this restriction specificity was imposed by the idiotype-related determinants expressed on the TsF1, not the T cell genotype.     

In vitro T cell-mediated killing of Pseudomonas aeruginosa. IV. Nonresponsiveness in polysaccharide-immunized BALB/c mice is attributable to vinblastine-sensitive suppressor T cells

We reported previously that BALB/c mice immunized with a polysaccharide (PS) antigen isolated from immunotype 1 Pseudomonas aeruginosa and vinblastine sulfate develop T cell-mediated protective immunity, despite their failure to produce specific antibody. In vitro, Lyt-1-,2+, I-J+ T cells from vinblastine- and PS-immunized mice kill P. aeruginosa by secretion of a bactericidal lymphokine. BALB/c mice immunized with PS alone generate neither protective antibodies nor a protective T cell response. The current studies indicate that T cells from mice immunized with PS alone significantly suppress the bactericidal activity of T cells from mice immunized with vinblastine and PS. The suppressor T cells are of the same Lyt-1-,2+, I-J+ phenotype as the bactericidal T cells. Suppression is mediated by a soluble product of these suppressor T cells which both inhibits T cell proliferation and interferes with the production or release of the bactericidal lymphokine. Cyclophosphamide, used in other systems to remove suppressor T cells, fails to enhance bacterial killing and does not inhibit suppressor cell activity. These studies indicate that immunization with PS elicits responses in two functionally distinct subgroups of Lyt-1-,2+, I-J+ T cells, and that these cells are distinguishable by their sensitivity to vinblastine sulfate.     

In vitro induction of tumor-specific immunity. IV. Specific adoptive immunotherapy with cytotoxic T cells induced in vitro to plasmacytoma antigens

Summary Specifically activated cytotoxic T cells (CL's) were raised by cocultivation in vitro of normal spleen cells with syngeneic plasmacytoma cells. These CL's were fully active both in vitro in lysing ⁵¹Cr-labelled tumor cells and in in vivo Winn assays, in inhibiting the growth of tumor cells in syngeneic mice when inoculated admixed with the tumor cells, even at ratios of 2 CL's to 1 tumor cell. However the same CL populations were only marginally effective in an immunotherapy model in which CL's were injected intravenously and tumor cells subcutaneously or intramuscularly (at a ratio of up to 100 CL/1 tumor cell). It is suggested that the in vitro conditions alter cell surface properties, thereby interfering with normal cell traffic, and that if this problem can be overcome, in vitro educated CL's may constitute a potential source of activated cells for human tumor immunotherapy.National Health and Medical Research Council postgraduate research scholar     

Human suppressor T cells induced by concanavalin A: suppressor T cells belong to distinctive T cell subclasses.

Human peripheral blood lymphocytes were stimulated by concanavalin A (Con A) and then evaluated by their suppressive activity for thymus-derived (T) cell- and bone marrow-derived (B) cell-proliferative responses to mitogen and allogeneic cells. Con A-activated T cells markedly suppressed these responses, but Con A-activated B cells failed to demonstrate suppressor activity. Discontinuous bovine serum albumin (BSA) density gradient separation of T cells which had been activated by Con A demonstrated that a fraction containing blast cells as well as fractions containing unproliferated cells manifest the same degree of suppressor capabilities. However, when density gradient separation of T cells followed by subsequent incubation with Con A was performed, fractions of proliferating cells of low density exhibited no suppression; a fraction containing high density T cells produced marked suppression, but this fraction incorporated only little thymidine in response to Con A. Thus, these studies indicate that Con A-induced suppressor T cells belong to a distinctive subpopulation which has already been programmed to express this function before exposure to Con A and that cell proliferation may not be a prerequisite for the development of such suppressor T cells.     

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.     

Generation of suppressor T cells after local immunization with histocompatibility antigens

Subcutaneous (sc) hind-foot immunization (HFI) of mice with allogeneic spleen cells can induce a state of delayed-type hypersensitivity (DTH) as well as a state of suppression of DTH. This paper deals with the suppression induced by HFI. The state of suppression could be adoptively transferred by spleen cells and lymph node cells between Days 3 and 7 after HFI only. However, in the hind-foot-immunized mice the state of suppression lasted at least 25 days. The suppressor cells expressed the Thy-1+, Lyt-1-2+ phenotype and suppressed DTH antigen-specifically. The suppressor cells, however, also suppressed DTH responses to unrelated third-party alloantigens, provided the latter were administered during the induction of DTH together with the same alloantigens that were used for HFI. The HFI-induced T-suppressor cells suppressed the induction phase of DTH (i.e., the proliferative activity of the draining lymph node cells after secondary sc immunization), but not the expression phase of DTH (i.e., the activity of previously activated DTH effector T cells). H-2D compatibility between the donors of the HFI-induced T-suppressor cells and the recipients was required for the adoptive transfer of suppression. The differences in effect of local immunization versus systemic immunization on the induction and functional activity of T-suppressor cells are discussed.     

Hapten-specific responses to the phenyltrimethylamino hapten. IV. Occurrence of mechanistically distinct idiotypic suppressor T cells before the appearance of anti-idiotypic suppressor T cells induced by the monovalent antigen L-tyrosine-p-azophenyltrimethylammonium

We have previously shown that a single i.p. injection of the monovalent synthetic antigen, L-tyrosine-p-azophenyltrimethylammonium [tyr(TMA)] in complete Freund's adjuvant induces an anti-idiotypic T suppressor cell (Ts2) population that can be detected 6 wk later by its ability to shut down delayed-type hypersensitivity (DTH) specific for the TMA hapten. In this paper we present evidence that 2 wk after tyr(TMA) administration, a subset of Ts, termed Ts1, appears that is both functionally and phenotypically distinct from the late appearing Ts2 population. The early occurring Ts1 act only at the induction phase of the DTH response and can also suppress this response intrinsically. This latter point is in marked contrast to our previous observation that the tyr(TMA)-induced anti-idiotypic Ts2 fail to function intrinsically and can only be detected upon adoptive transfer into naive mice. Ts1 bear idiotypic receptors and are Ly-1+,2- in contrast to the anti-idiotypic Ly-1-,2+ Ts2 population. In addition, unlike the Ts2 population, Ts1 are comparatively nylon wool-adherent. Adsorption of Ts1 on either antigen- or idiotype-coated petri dishes indicate that the suppressor activity can be transferred only by antigen-binding cells. Cellfree factors prepared from spleens containing the Ts1 population can suppress DTH only if administered at the induction phase of the response, in contrast to the factors derived from the Ts2 population that act both at induction as well as effector phases, suggesting that Ts1 and Ts2 can function via soluble mediators. Finally, we show that when Ts1-bearing mice are primed and boosted for anti-TMA antibody formation, the resulting response was overall reduced with respect to the idiotype-positive and negative plaque-forming cells that differs from the Ts2-bearing hosts wherein the idiotypic component is preferentially suppressed. The appearance of Ts1 before the detection of Ts2 in the same experimental animals is discussed with reference to a normal physiologic sequence of events involved in suppressor pathways.     

Induction of specific suppressor T cells in vitro.

We describe conditions for generating sheep red blood cell-specific suppressor T cells in Mishell-Dutton cultures. The production of specific suppressor cells is favored by increasing antigen dose in the initial culture but can be produced by transferring more cells when lower doses of antigen are used. Transfer of small numbers of cells cultured with low doses of antigen leads to a specific helper effect. Transfer of large numbers of educated cells leads to nonspecific suppression. Suppression can be effected by the effluent cells from nylon wool columns which do not make detectable PFC. A fraction of these cells become resistant to treatment with anti-T cell sera and complement after culture. The suppressor cells are radiation sensitive and must be able to synthesize protein to suppress. They take 2 to 3 days of education to reach maximum suppressive efficiency and will not suppress cultures if added 2 to 3 days after culture initiation. Their production is favored by the absence of mercaptoethanol, suggesting that the observed suppression is not "too much help". The ability to generate specific suppressor cells in vitro should be of great benefit in determining the factors that regulate their appearance in vivo.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

Further studies on the tumor-specific suppressor cells induced by ultraviolet radiation.

Recent studies have suggested that the immunologic unresponsiveness of UV-irradiated mice against UV-induced fibrosarcomas might be due to the presence of suppressor lymphoid cells. In these experiments, we present additional evidence that suppressor lymphoid cells are present in lymph nodes and spleens of UV-irradiated mice and demonstrate that these cells are enriched after incubation on nylon wool columns, that they are inactive after treatment with anti-theta serum and complement, and that they are effective for at least 7 weeks after transfer to lethally x-irradiated mice. Splenectomy of UV-treated mice before tumor challenge did not restore their anti-tumor reactivity. The UV-induced suppressor cells appear to be specific for syngeneic UV-induced tumors, since they did not suppress the rejection of an allogeneic UV-induced tumor or two chemically induced syngeneic tumors.     

Mechanisms of contact photosensitivity in mice. IV. Antigen-specific suppressor T cells induced by preirradiation of photosensitizing site to UVB

Mice were successfully contact photosensitized with 3,3',4',5-tetrachlorosalicylanilide (TCSA) plus black light irradiation. Pre-exposure of the photosensitizing site (ca 5 cm2) to UVB (280 to 320 nm; 400 mJ/cm2) rendered mice unresponsive to a challenge reaction. Cell transfer experiments demonstrated that the spleens from the nonreactive mice contained suppressor T cells (Ts) that were antigen-specific and that blocked the afferent limb of contact photosensitivity to TCSA. To exert suppressive functions, Ts required another population of cyclophosphamide-sensitive T cells that resided in the spleens of nonsensitized mice. The results provide evidence that UVB-induced aberrant homeostasis of the skin caused a marked suppression of immune system that is associated with the generation of Ts.     

Induction of allospecific suppressor T cells in vitro.

Incubation of mouse thymic lymphocytes with irradiated allogeneic spleen cells gave rise to suppressor cells. The suppressor activity was assayed by adding the incubated cell mixture to a mixed lymphocyte culture (MLC) in which the responder cells were syngeneic with the sensitized thymocytes and the stimulator cells were syngeneic with the sensitizing spleen cells. Such addition suppressed significantly thymidine incorporation in the mixed lymphocyte reaction (MLR). The suppressor cells were found to carry the θ antigen and to function allospecifically, as shown by cross-testing in three allogeneic combinations. Our data suggest that these cells may originate from immature cortisone-sensitive thymic lymphocytes and also provide some preliminary information concerning their mode of action.     

Suppressor T cells and suppressor macrophages induced by Corynebacterium parvum

Corynebacterium parvum, injected intravenously into C57B1/6 mice (H-2^b) previously alloimmunized with P815 (H-2^d) mastocytoma cells, generated splenic suppressor cells that inhibited the development of primary cytotoxic lymphocytes in vitro. These suppressor cells differed from those generated by intravenous C. parvum injection of naive C57B1/6 mice. The former suppressor cells were effectively induced by administration of 700 μg of C. parvum whereas the latter suppressor cells were dependent upon higher doses (1400 μg) of adjuvant for their activation. Furthermore, suppressor cells generated in alloimmunized mice could only suppress C57B1/6 anti-P815 in vitro cytotoxic responses whereas suppressor cells generated in naive mice could suppress C57B1/6 anti-CBA (H-2^k) responses as well. Suppressor cells were not H-2 restricted in their action. Fractionation of spleens from alloimmunized, C. parvum-treated mice revealed the presence of suppressor T cells and suppressor macrophages. We were unable, however, to determine which cell was responsible for “antigen specificity” of suppression since the fractionation procedures seemed to trigger both suppressor cell types prior to adding them to the primary culture.     

Major histocompatibility complex-restricted augmenting T cells induced by in vitro cultivation of antigen-primed T cells. A new parallelism between suppressor and augmenting circuits

In vitro cultivation of primed T cells with antigen resulted in the induction of a regulatory T cell that nonspecifically augmented the in vitro antibody responses of H-2-compatible T and B cells. This T cell, designated as the augmenting T cell (Ta), was unable to help B cells by itself but enhanced the antibody response of B cells to several multitudes only when conventional helper T (Th) cells or cloned Th cells from the same H-2 haplotype coexisted. Ta was radioresistant and belonged to Lyt-1+, 2-, L3T4+, I-J- T cell lineage. Ta exhibited interesting H-2-restricted activities: when primed T cells from (A X B) F1 were cultured with the antigen in the presence of parent A type antigen-presenting cells, the induced Ta was able to augment the antibody response of (A x B) F1 B cells in the presence of Th cells from F1----A but not from F1----B radiation bone marrow chimeras. This indicates that the induction of Ta in an F1 T cell population is dependent on the H-2 haplotype of antigen-presenting cells during in vitro cultivation. The restriction specificity of the established Ta is, however, not directed to the class II antigen itself but to the restriction specificity of Th cells that recognize class II antigen. In support of this is the fact that the elimination of A-restricted Th cells during cultivation by treatment with anti-I-J mAb, which is known to react with H-2-restricted Th cells, resulted in failure of induction of Ta cells having the augmenting activity for the A-restricted response.     

In vitro effects of 4-hydroperoxycyclophosphamide on concanavalin A-induced human suppressor T cells

Summary Treatment in vitro of human peripheral blood lymphocytes (PBL) with ConA induced the generation of suppressor cells which inhibited T cell blastogenic response to ConA and of allogeneic response in the mixed lymphocyte reaction (MLR). Treatment of PBL with 4-hydroperoxycyclophosphamide (4-HPCy) before incubation with ConA markedly decreased the generation of suppressor cells by ConA. The effect of 4-HPCy on generation of suppressor cells was more pronounced in the test of ConA stimulation than in the MLR. Treatment with 4-HPCy had no effect on suppressor cells already induced as shown by incubation of PBL with 4-HPCy after incubation with ConA.     

In vitro T cell-mediated killing of Pseudomonas aeruginosa. III. The role of suppressor T cells in nonresponder mice

T lymphocytes from immune BALB/c mice can adoptively transfer protection against infection with the extracellular Gram-negative bacterium Pseudomonas aeruginosa to nonimmune recipients, and in vitro, immune T cells are able to kill these bacteria. Earlier studies indicated that this killing is mediated by a bactericidal lymphokine. The current studies demonstrate that T cells from immunized CB.20 mice, a strain congenic with BALB/c, fail to kill Pseudomonas aeruginosa in vitro. This nonresponsiveness is attributable to the activity of suppressor T cells of the Lyt-1-, 2,3+, I-J+ phenotype. CB.20 mice are known to differ from BALB/c mice only at a single locus, which includes the Igh-1 allotype CH genes. These results suggest a critical role for this locus or closely linked genes in the control of T cell killing of this extracellular bacterium.     

In vitro growth of murine T cells. IV. Use of T-cell growth factor to clone lymphoid cells

Murine bone marrow cells can suppress the in vitro primary antibody response of normal spleen cells without apparent cytotoxicity. The bone marrow cells suppress the response to both T-dependent (SRBC) and T-independent (DNP-Ficoll) antigens. When bone marrow cells are fractionated on a sucrose density gradient, the suppressive activity is found in the residue rather than the lymphocyte fraction. The suppressive activity is either unaffected or enhanced by treatment with anti-T- and anti-B-cell serums. Pretreatment of mice with phenylhydrazine which reduces the number of pre-B cells did not reduce the suppressive activity of their bone marrow cells. Suppressive activity is abolished by irradiation of the marrow cells in vitro with 1000 R prior to assay. The activity is present in the marrow of thymus deficient (nude) mice, infant mice, and mice which have been made polycythemic by transfusion. Furthermore, the suppressor cell can phagocytize iron carbonyl particles, is slightly adherent to plastic and Sephadex G-10, and can bind to EA monolayers. We conclude that the suppressor cell is not a mature lymphocyte or granulocyte nor a member of the erythrocytic series, but is likely to be an immature cell possibly of the myeloid series. We speculate on the physiologic role of this cell.     

Regulation of the primary in vitro response to TNP-polymerized ovalbumin by T suppressor cells induced by ovalbumin feeding

Spleen cells from DBA/2 mice that received a single feeding of 20 mg of ovalbumin (OVA) 7 days previously were specifically hyporesponsive to primary in vitro challenge with the thymic-dependent antigen TNP-polymerized ovalbumin (TNP-POL-OVA). The tolerance observed in spleen cells from OVA-fed animals was dependent upon OVA-specific T suppressor cells, because splenic T cells from OVA-fed mice suppressed the primary response to TNP-POL-OVA of cultures containing normal T and B cells. The tolerance and suppression was OVA specific, because spleen cells from OVA-fed animals responded well to other antigens (including TNP on another carrier), and splenic T cells from OVA-fed mice did not affect the response of normal T and B cells to sheep erythrocytes. These data confirm the existence of T suppressor cells after OVA feeding and provide a direct means of assaying their activity in a primary in vitro response.     

Induction of suppressor T cells and tolerant B cells in vitro by DNP-IgG

Intravenous injection at proper time of irradiated reticulum cell sarcoma cells into SJL mice immunized with dinitrophenylated (DNP) keyhole limpet hemocyanin inhibits the production of anti-DNP IgG₁ and IgG₂ antibodies.