The role of Ia antigens and Fc receptor-bearing T-cells in the inhibition of macrophage receptors for cytophilic antibody induced by soluble immune complexes

Soluble antigen-antibody complexes composed of 3 M KCl-extracted L1210 antigens and alloantibody to L1210 given to C3H mice caused immunosuppression in the mice. This was reflected in part by the inhibition of cytophilic antibody receptors on macrophages which could be used as a measure of the suppression. Thymocytes or splenic T cells from mice treated with immune complexes could adoptively transfer the suppression to normal syngeneic mice. These cells, which we have termed suppressor inducers, were found to be Ia positive: specifically, I-A⁺, I-J^?. Thus, treatment of the inducers with anti-la or anti-I-A antibodies and complement in vitro abrogated their ability to transfer the suppression to normal mice. In contrast treatment with anti-I-J serum and complement had no effect. Through a similar approach, the cooperating (acceptor) T cells were found to be I-A⁺, I-J^?. Pretreatment of mice with anti-Ia or anti-I-A serum before the administration of antigen-antibody complexes prevented the inhibition of macrophages. This was due at least in part to steric hindrance of adjacent Fc receptors on the FcR⁺ T cells with which the complexes interacted. Early interaction of immune complexes with FcR⁺ T cells was in fact demonstrated directly by the inability of the complexes to induce suppression when FcR⁺ T cells were depleted. The thymocytes or splenic T cells from anti-Ia-pretreated mice failed to transfer the suppression to recipient mice. In contrast, treatment with either anti-Ia or anti-I-A after the immune complexes did not abrogate the generation of suppressor inducers. Treatment of normal recipient mice with anti-Ia serum in vivo before they received the suppressor inducer cells did not prevent cooperation between the two types of cells. By the same token, blocking of Ia antigens of the inducers in vitro with anti-Ia serum (without complement) also did not impair the cooperative interaction. These results indicate that antigen-antibody complexes generate I-A-positive, I-J-negative T-suppressor inducer cells from FcR⁺ naive T cells. These in turn interact with Ia-positive (I-A⁺ and I-J^?) normal thymocytes or spleen T cells. This interaction most likely generates the ultimate suppressor T cells that suppress cytophilic antibody receptors on macrophages in vivo. However, the I-region determined antigens did not appear to be directly involved in the T-T interaction of suppressor inducer and acceptor cells.     

Proliferative response of bone marrow cells to concanavalin A does not require Ia antigen-positive cells

The in vitro proliferative response of murine bone marrow cells to concanavalin A (Con A) and the effect of anti-Ia serum on the response were studied. The incorporation of [3H]thymidine into cells prepared from the bone marrow of C3H/He, ATL, ATH, and C57BL/6 mice increased in the presence of certain doses of Con A. The bone marrow cells of athymic nude mice were also capable of responding to Con A, but cells prepared from the spleens of such mice were not. The addition of anti-Ia serum to the cultures of bone marrow cells did not affect the responses of these cells to Con A, though their proliferative response to bacterial lipopolysaccharide was greatly reduced in the presence of the serum. Moreover, pretreatment of the bone marrow cells with anti-Ia serum or anti-Thy. 1.2 serum and rabbit complement did not abolish the ability of these cells to respond to Con A. These results indicate that there are some Ia negative and Thy. 1.2 negative cell populations in the marrow capable of responding to Con A. Furthermore, the effect of anti-Ia serum on the Con A-induced proliferative response of the spleen cells which had been obtained from gamma-irradiated and syngeneic bone marrow cell-reconstituted mice was examined. The ability of these cells to respond to Con A increased gradually week by week after the reconstitution. The suppressive effect of anti-Ia serum on the response of these cells gradually became much more pronounced after the reconstitution.     

Cellular interaction between cytotoxic and suppressor T cells against syngeneic tumors in the mouse.

Splenic T cells from animals bearing growing syngeneic tumors specifically inhibited the effector process of tumor cell lysis by the cytotoxic T cell which had been activated in vitro by mitomycin C treated homologous tumor. The suppression was strictly specific for the individual tumor by which suppressor cells were generated, whereas in some cases cytotoxic T cells generated by two closely related sarcomas showed a certain degree of crossreactivity. This suggests that suppressor and cytotoxic T cells recognize different antigenic moieties on tumor cells; one unique to the individual tumor and the other shared by related tumor cell lines.The suppressor T cell from tumor bearing animals possessed Ia antigen controlled by a gene in I-J subregion of H-2 major histocompatibility complex. Cytotoxic T cells generated by some but not all syngeneic tumors were also killed by anti-Ia and complement; however, the Ia antigen on such cytotoxic T cells was found to be controlled by a locus in I-A subregion. In general, the cytotoxic T cells generated by newly established tumor cell lines had Ia antigen, whereas some old cell lines, which were capable of growing across the H-2 barrier, activated the Ia negative cytotoxic T cell. These results collectively indicate that the immunological resistance against tumors is dependent on the balance of activations of the cytotoxic and suppressor T cells with different specificities and phenotypic expressions.     

Effect of Anti-Ia Serum In Vitro on Lipopolysaccharide-Induced Proliferative Responses of Murine Bone Marrow and Spleen Cells

The in vitro proliferative response of murine bone marrow cells and spleen cells to bacterial lipopolysaccharide (LPS) and the effect of anti-Ia serum on the response were studied. The incorporation of [³H]thymidine into cells prepared from bone marrow increased in the presence of LPS, but the addition of anti-Ia serum to the cultures reduced the incorporation. Pretreatment of bone marrow cells with anti-Ia serum and complement did not abolish the ability of the cells to respond to LPS, while the same pretreatment destroyed this ability in spleen cells. These results suggest that cultures of Ia-negative bone marrow cells generate Ia-positive cells during the culture period, and the Ia-positive cells are responsive cells to LPS. The proliferative response of 1- or 2-week-old spleen cells was easily suppressed by anti-Ia serum when compared with that of 4-week-old spleen cells. Furthermore, the responses of spleen cells obtained from γ-irradiated and syngeneic bone marrow cell-reconstituted mice were prominently suppressed by anti-Ia serum in comparison with that of normal adult spleen cells. These findings suggest that LPS-responsive lymphocytes in the developmental stage are quite sensitive to anti-Ia serum. The effect of anti-Ia serum on the maturation of bone marrow-derived lymphocytes was discussed.     

Antigen-binding receptors on T cells from long-term MLR. Evidence of binding sites for allogeneic and self-MHC products

Antibody inhibition of radiolabelled stimulator membrane vesicle binding by T blasts activated in the mixed lymphocyte reaction (MLR) was used to identify responder-cell determinants involved in the binding phenomenon. Antisera or monoclonal antibodies against Thy-1, Lyt-1, Lyt-2 and Ly-6 antigens were not inhibitory. However, antibodies against heavy-chain V region (V_H) determinants strongly inhibited vesicle binding by both primary and longterm MLR blasts. Anti-Ia (both alloantisera and monoclonal reagents) caused inhibition of antigen binding by primary MLR blasts only. T blasts from long-term MLR lines were neither Ia-positive, nor susceptible to blocking of antigen binding with anti-Ia. However, these cells were capable of specifically absorbing soluble syngeneic Ia material, with the concomitant appearance of vesiclebinding inhibition with anti-Ia sera. Acquisition of syngeneic Ia by T blasts was effectively blocked with the anti-V_H reagent. Passively bound self-Ia did not interfere with vesicle binding in the absence of anti-Ia. These results strongly suggest the existance of specific self-Ia acceptor sites closely linked to the receptors for stimulator alloantigens on T cells proliferating in MLR. A receptor model based on these findings is briefly discussed.Abbreviations used in this paper B10 C57BL/10 - Con A concanavalin A - FcR Fc receptor - FCS fetal calf serum - H heavy chain - Ia I-region associated antigen - Ig immunoglobulin - LPS lipopolysaccharide - Lyt T-lymphocyte differentiation antigen - MHC major histocompatibility complex - MLR mixed lymphocyte reaction - PM plasma membrane - T thymus derived - Tcr T-cell receptor - V variable region of Ig     

Suppressor factor produced by neonatal mouse spleen cells

Spleen cells from 5- to 6-day- old BDF₁ mice produce a soluble suppressor factor (SF) when cultured for 2–4 days in the presence of 10% fetal calf serum. This suppressor factor inhibits the mixed lymphocyte reactivity of adult mouse spleen cells as well as the in vitro generation of cytotoxic cells. The SF which is not H₂-restricted or antigen specific is most effective when added in the early phase of the culture period. The SF is resistant to heat and uv treatment and appears to consist of a large and small component. It is resistant to treatment with pronase or trypsin. The SF appears to be produced by neonatal spleen cells which are not adherent to plastic or Sephadex G-10 and are insensitive to treatment with anti-Thy 1.2 and complement. Incubation of SF with peritoneal exudate cells reduces suppressor activity.     

Further characterization of the suppressor cells, activated by goat anti-Th-B antibody reagent and responsible for enhanced growth of sarcoma 180 in AKR mice.

In our previous study, thymus cells were shown to be responsible for enhancing the growth of the allogeneic sarcoma 180 (S180) in AKR mice that had been injected with goat anti-Th-B antibody reagent (antiserum raised in goats against Balb/c myeloma MOPC 104E cells and purified). We suggested that the cells producing enhancement are suppressor T cells. We now show that the cells responsible for tumor enhancement are indeed T cells, since they carry the Thy-1 antigen on their surface. Treatment of the cells in vitro with anti-Thy-1 plus complement completely eliminates their ability to enhance tumor growth. The thymocytes responsible for tumor enhancement do not carry the Th-B determinant. Treating thymocytes in vitro with goat anti-Th-B antibody reagent plus complement does not abrogate their tumor-enhancing activity. This suggests that the suppressor T cells involved in tumor enhancement are generated by the interaction of anti-Th-B antibodies with precursor suppressor cells which do carry Th-B. Once generated, the active suppressor cells lose the Th-B antigen. This suggestion is supported by our finding that the thymic precursors of Con A-inducible suppressor cells bear Th-B, since they are killed by anti-Th-B plus complement, whereas active suppressor cells induced by Con A do not carry Th-B, since they are not killed by anti-Th-B plus complement. Neither splenic precursors of Con A-inducible suppressor cells nor the active suppressor cells thus induced carry Th-B since neither is killed by anti-Th-B plus complement. We have also found that there are apparently nonthymic suppressor cell precursors which can also be activated by anti-Th-B, since spleen cells from thymectomized mice bearing S180 and treated with anti-Th-B can transfer the tumor-enhancing effect. We conclude that precursors of suppressor cells carry the Th-B determinant. These precursors differentiate to active suppressor cells when stimulated by anti-Th-B antibodies. This process can take place either outside the thymus or in the thymus. Once differentiated, the mature suppressor cells no longer bear the Th-B marker and migrate from their sites of induction. Such cells can suppress immune mechanisms responsible for allogeneic tumor graft rejection and thus cause tumor enhancement.     

Mechanisms of corticosteroid action on lymphocyte subpopulations. IV. Effects of in vitro hydrocortisone on naturally occuring and mitogen-induced suppressor cells in man.

The effects of in vitro hydrocortisone (OHC) on human peripheral blood (PB) suppressor cell function were investigated. Two types of suppressor cells were studied: (i) the naturally occurring PB suppressor cell seen in 10% of normal people whose lymphocytes do not respond to in vitro PWM stimulation with direct anti-SRBC PFC responses, and (ii) Con A-generated suppressor cells. The addition of OHC to PWM-stimulated cultures from nonresponders reconstituted the PFC response in two of three individuals. The addition of OHC to allogenic cocultures of nonresponder and responder lymphocytes completely inhibited the ability of the naturally occurring suppressor cell of the nonresponder cultures to inhibit the PFC responses of normal responders. Preincubating the nonresponder cultures in 10^?5M OHC for 30 min followed by washing did not inhibit suppressor function, whereas readdition of OHC to cocultures did inhibit nonresponder suppressor cell function. The addition of up to 10^?4M OHC to previously generated Con A-activated suppressor cell-fresh cell cocultures in vitro did not prevent or inhibit mitogen-activated suppressor cell function. However, preincubation of PB cells for 6 hr prior to the addition of Con A prevented the generation of suppressor cells and in two of eight experiments generated a population of cells which were in and of themselves mitogenic for autologous fresh PB. Thus, the function of naturally occurring suppressor cells as well as the induction but not the function of Con A-activated suppressor cells is sensitive to pharmacologic levels of OHC. The effect of OHC on naturally occurring suppressor cell function or on the generation of suppressor cells by Con A did not involve cell lysis, but rather was a reversible phenomenon requiring the continued presence of OHC in culture.     

Characterization of the human blood lymphocytes that produce a histamine-induced suppressor factor (HSF)

The cells which elaborate a soluble suppressor factor in vitro in response to histamine (histamine-induced suppressor factor or HSF) were partially characterized in the present studies. Human blood T- and B-cell populations were purified by affinity chromatography with rabbit anti-human F(Ab′)₂ and examined for their ability to make HSF. Highly purified populations of T cells, but not B cells, produced HSF in response to varying concentrations of histamine (10^?4 to 10^?4M). The HSF-producing cells were characterized further by means of affinity chromatography with columns containing conjugates of insolubilized histamine as well as by rosette formation with IgG (Tγ)- or IgM (Tμ)-coated ox red blood cells. These studies revealed the following: (a) Cells that synthesize HSF are retained on histamine (but not control) columns; (b) cells with histamine receptors comprise approximately 50% of the Tγ subpopulation but are not found in the Tμ subpopulation; (c) cells not retained by histamine columns have a reduced capacity to develop into suppressor cells following stimulation by concanavalin A or specific antigen (compared to unfractionated or control column passed cells). In addition, it was shown that cells synthesizing HSF predominantly express histamine type 2 receptors: (d)4-Methyl histamine (H₂ agonist), but not 2-methyl histamine (H₁ agonist), was capable of inducing HSF production; (e) cimetidine (H₂ antagonist) inhibited HSF production but chlorpheniramine (H₁ antagonist) did not. Taken together, these experiments suggest that T lymphocytes capable of expressing suppressor function following activation by histamine, specific antigen, concanavalin A, or perhaps through their Fc receptors may either be heterogeneous within the same subpopulation or more likely be the same cell with the complement of receptors described above.     

Colony-forming B cells in F1 hybrid and transplanted CBA/N mice.

The conditions for evaluation of suppressor cell regulation of the pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) responses of peripheral blood (PB) B cells in normal individuals using allogeneic cocultures is described. In 14 separate experiments, after preincubation with concanavalin A (Con A) for 2 days, PB cells suppressed the PWM-induced anti-sheep erythrocyte (SRBC) PFC response of fresh allogeneic PB cells to 17% of the expected PFC response (P < 0.05). In addition, control cells incubated for 2 days in the absence of Con A suppressed the PWM- induced PFC response of allogeneic cells in 6 of 14 experiments to the same extent as did the Con A-generated cells (P < 0.01). It was found that unstimulated control cells (without Con A activation) from normal subjects who themselves were nonresponders to PWM stimulation (< 50 PFC/10⁶ cells) usually suppressed the PFC response of allogeneic cells (P < 0.05), while control cells from normal subjects who consistently had a good PFC response to PWM stimulation (> 75 PFC/10⁶ cells) did not suppress the PFC response of allogeneic cells. The spontaneously occurring suppressor cell in nonresponder PB cell suspensions was sensitive to 3000-R irradiation, and the nonresponder state was not associated with a decreased blastogenic response to PWM. Thus, some normal subjects who themselves had a poor PWM-induced PFC response had irradiation-sensitive, spontaneously occurring suppressor cells which were capable of suppressing the PWM-induced PFC response of normal responders. The majority of normal subjects (90%) were good PFC responders to PWM stimulation and did not spontaneously suppress the PFC response of allogeneic cells to PWM, but did have PB cells which were capable of being activated by Con A to suppress.     

Role of the H-2I region in the generation of an antiviral cytotoxic T-cell response in vitro

The participation of H-2I gene products in generating virus-specific proliferative and/or cytotoxic T-lymphocyte (CTL) responses was investigated. Spleen cells from mice infected with vaccinia virus were restimulated secondarily in vitro with syngeneic virus-infected peritoneal exudate cells (PEC) and then restimulated in tertiary cultures with virus-infected PEC from syngeneic and partially histoincompatible strains of mice. Based on the finding that comparable proliferative responses resulted when stimulating the responding cells were histocompatible at the H-2K, I, or D region of the major histocompatibility complex (MHC), the additively enhanced, but not potentiated, proliferation caused by identity at two or three H-2 regions was analyzed. Enhancement of proliferation followed when the H-2K/D components plus virus and the H-2I components plus virus were either on the same, or alternatively on two, stimulating cells. This suggests that H-2K, D, and I plus virus trigger distinct T-cell subsets. A virus-specific CTL response was generated in vitro when spleen cells from virus-primed mice and even unprimed mice were stimulated with cells sharing only H-2K and/or H-2D of the MHC. Identity at the H-2I region did not stimulate a CTL response, nor did it influence the magnitude of the $\text{K}\text{D}$ restricted response. Nevertheless, the presence of anti-Ia antiserum in cultures of syngeneic stimulators and responders inhibited the cytotoxic response to a great extent. Therefore, H-2I region products seem to participate in the generation of virus-specific CTL in vitro.     

Regulatory T cells in pregnancy: V. Allopregnancy-induced T-cell-suppressor factor is H-2 restricted and bears la determinants

Spleen cells of allogeneically pregnant mice at the second week of second to fourth parity contain suppressor T cells capable of suppressing an MLR of maternal responders and releasing a soluble factor. The genetic restrictions of this factor have been studied in a $\text{H}\text{-2}{}^{\mathrm{<mtext>k</mtext>}}\text{H}\text{-2}{}^{\mathrm{<mtext>d</mtext>}}$ system using $\text{k}\text{d}$ or $\text{d}\text{k}$ pregnancies, and responder cells from $\text{k}\text{d}$ recombinants. The factor appeared to be IC subregion restricted. Furthermore, its activity could be removed by passage on insolubilized anti-Ia immune serum. Although factor-releasing suppressor cells are antigen specific the factor itself does not appear so, possibly implicating a mixture of fine specificities.     

Accessory cell function in a Con A response: role of Ia and interleukin 1

Accessory cell (A-cell) function in a Con A response was analyzed. Irradiated P388D1 cells efficiently induced a proliferative response to Con A of T cells purified from spleen cells, whereas paraformaldehyde-fixed P388D1 cells failed to serve as A cells. Although IL-1 containing culture supernatant (SN) of a macrophage hybridoma induced the Con A response of the T-cell preparations, the depletion of Ia+ cells by the treatment with anti-Ia antibody and complement abrogated the response in the presence of IL-1. Fixed P388D1 cells and the hybridoma SN synergized in the reconstitution of the response. A 15,000-Da fraction of the hybridoma SN or human recombinant IL-1 alpha was able to substitute the hybridoma SN for the response. The reconstitution of the response by IL-1 and fixed P388D1 cells was inhibited by the addition of monoclonal anti-Ia antibody. These results indicate that IL-1 or fixed P388D1 cell does not exert a sufficient signal by itself and both of them are required for the reconstitution of a Con A response of highly purified T cells, and that Ia on fixed P388D1 cells play an important role.     

Involvement of suppressor cells in active enhancement of allografted tumors. In vivo (adoptive transfer) and in vitro (MLR) evaluation. Synergy with enhancing antibodies

Specific enhancement of allografted A/J mouse tumor Sa 1 can be transferred not only passively by serum but also adoptively by lymphoid cells from syngeneic donors actively treated for enhancement. This can be done mainly by thymocytes in untreated recipients and by splenocytes in irradiated recipients, the transfer being more efficient in the latter. Results are more striking when the donors' treatment has led to a successful enhancement (AE⁺) demonstrated by an allografted tumor test. The responsible cells, studied in the donors' spleens, were found to be nonadherent to nylon wool, sensitive to specific anti-IJ serum and complement, and sensitive to anti-specific recognition structure serum with complement. Inactive doses of passively transferred serum added to subactive doses of adoptively transferred spleen cells, both from AE⁺ donors, lead to a substantial enhancement of allografted tumors, indicating a synergistic action of enhancing antibodies and suppressor cells. Similar results were obtained in vitro in a MLR system. MLC reactivity of AE⁺ donor cells is impaired. Furthermore, when mitomycin-treated, these cells have a regulatory (suppressor) effect on a MLR of syngeneic spleen cells directed against grafted tumor strain mitomycin-treated spleen cells. This suppressor effect is impaired by pretreating the regulatory cells with anti-IJ serum and complement or with anti-cell recognition structure serum (which has an opposite effect on AE^? cells).     

Human T lymphocytes become glucocorticoid-sensitive upon immune activation

A murine model for Transfer Factor (TF) was used in an attempt to identify the nature of its antigen-specific component. TF was prepared from lymph node cells of CBA/Ca/T6 mice sensitized 30 days previously with 2,4-dinitrofluorobenzene (DNFB). To assay for the specific component of TF, 2 × 10⁷ lymphocyte equivalents were injected intravenously into normal syngeneic recipients. Lymph node cells obtained 18–24 hr later gave a positive response in the macrophage migration inhibition (MMI) test in the presence of the soluble analog of DNFB (sodium 2,4-dinitrobenzenesulfonate). The activity of TF was abrogated by absorption with anti-Ia sera including both an Ia alloantiserum (A.TH anti-A.TL) and a xenogeneic rabbit anti-serum which exclusively recognizes carbohydrate-defined Ia antigens. Analysis by paper chromatography using the technique for purification of carbohydrate-defined Ia antigens revealed that MIF production was obtained exclusively with those fractions known to contain Ia antigenic activity. In addition, pretreatment of TF with insoluble conconavalin A (Con A) which has an affinity for carbohydrate-defined Ia antigens resulted in removal of its activity. Taken together these findings pointed to the presence in TF of I-region gene products. Absorption with antibody directed against the dinitrophenyl determinant abolished the capacity of TF to stimulate macrophage inhibition factor production suggesting that it might also contain antigen fragments possibly in association with Ia. No evidence was, however, obtained for H-2 restriction of the action of TF in vivo since it was found to exert an effect in a variety of strain combinations including A.TH and Balb/c which share no known common I-region specificities. Parallel experiments were carried out with the lymphocyte transformation assay since this is known to be a measure of the nonspecific components in TF. Pretreatment with mouse allo-anti-Ia^k serum directed against both protein-and carbohydrate-defined Ia antigens caused a partial reduction in the proliferative response. In contrast no change in response was observed when the TF was absorbed with insoluble Con A or anti-DNP serum. Furthermore, lymphocyte transformation was obtained with only one of the three paper chromatography fractions positive in the MMI assay as well as two other different fractions. Taken together, these findings permitted a distinction to be made between specific and nonspecific components of TF and indicated that the specificity of TF could be explained in terms of the presence of I-region gene coded products possibly in association with antigen fragments.     

The Lyt phenotype of cells involved in the cytotoxic response to syngeneic tumor and of tumor-specific suppressor cells

Suppressor cells, which specifically suppress the in vitro response of syngeneic spleen cells to the DBA/2 mastocytoma, P815, were identified in the spleens of DBA/2 mice injected intraperitoneally with membrane extracts of the P815 tumor. The Lyt phenotypes of various effector cells were determined. DBA/2 allogeneic killer cells were identified as Lyt-¹²⁺, whereas the syngeneic effector cells were found to be predominantly Lyt-²⁺. The suppressor cell population lost its ability to suppress the in vitro cytotoxic anti-P815 response after treatment with anti-Lyt-1 serum plus complement but not after treatment with anti-Lyt-2 serum, indicating that an Lyt-¹⁺ cell is essential in this suppression.     

Lymphocyte receptors for polyclonal T mitogens: II. High-molecular-weight glycoproteins are the best candidate sites for mitogenic action by concanavalin A and galactose oxidase

Murine lymphocytes oxidized by galactose oxidase were radiolabeled by reduction with NaB³H₄. The labeled cells were incubated with Con A and the Con A-Con A receptor complexes formed in situ on the viable cells were isolated by immuno-precipitation with anti-Con A serum and fixed Staphylococcus aureus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography analysis of the precipitates demonstrated four high-molecular-weight glycoproteins which were oxidized by GO and which bound Con A. These same four glycoproteins were also oxidized and labeled by $\text{IO}{}_4\text{NaB}{}^3\text{H}{}_4$. [³H]Tyrosine biosynthetic labeling identified these four plus several other Con A receptors. Because Con A sterically inhibits GO mitogenic stimulation, these four glycoproteins are likely to represent the necessary sites of oxidative mitogenic action and are good candidates for the targets of Con A mitogenesis.     

Passive transfer of experimental allergic encephalomyelitis in the Lewis rat with activated spleen cells: differential activation with mitogens

It has previously been shown that spleen cell transfer of clinical EAE requires donor cells to be cultured in vitro prior to transfer. Donor cells must be stimulated when cultured, and either Con A or the encephalitogen, guinea pig myelin basic protein (BP), satisfies this stimulation requirement. Following recovery from passive disease, recipients of these in vitro cultured cells will subsequently develop clinical symptoms of EAE sooner than controls when challenged with BP in complete Freund's adjuvant (BP-CFA). In the present study, three T-cell mitogens were evaluated as donor cell stimulants in the required in vitro culture period. Pokeweed mitogen (PWM) as well as Con A stimulated the donor cell population to the degree that clinical EAE could be transferred with 5 × 10⁶ cultured viable cells. Con A at culture levels below 0.25 μg/ml did not yield transfer active cells even though proliferation levels were similar to those found at concentrations of Con A that did yield transfer active cells. Phytohemagglutinin (PHA)-stimulated cultures did not transfer clinical disease even though the degree of lectin induced proliferation ([³H]thymidine uptake as well as recovered cells from culture) was equivalent to the PWM- or Con A-stimulated, transfer positive, cultures. Mixing experiments suggested that the inability of PHA or low doses of Con A to induce transfer active cells was not due to the induction of suppressor cells. Although cells cultured with PHA do not transfer clinical EAE, recipients of these cells as well as recipients of either PWM- or Con A-stimulated donor cells develop an early appearance of disease upon subsequent challenge with BP-CFA. This included cells incubated with a concentration of Con A (0.1 μg/ml) which did not induce cells capable of transferring clinical EAE. These results suggest that PHA and perhaps the low dose of Con A may stimulate the proliferation of the EAE effector cell precursor population without causing the additional differentiation of this precursor population into the effector cell population which is capable of transferring clinical disease. Alternatively, PHA may expand only the helper cell population while effective doses of Con A and PWM would expand both helper and effector cell populations.     

Thymopoietin-induced acquisition of responsiveness to T cell mitogens

A population of murine spleen cells, enriched by flotation in discontinuous bovine serum albumin gradients, was induced to differentiate in vitro by incubation with the purified thymic polypeptide hormone thymopoietin. These cells, normally unresponsive to both T and B cell mitogens, acquired the capacity to respond to the T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) but remained unresponsive to the B cell mitogen lipopolysaccharide from Escherichia coli. The acquisition of responsiveness to mitogens was not impaired by treatment with anti-Thy-1 serum + complement before induction but was prevented by this treatment after induction; thus the cells acquiring the functional capacity to respond to T cell mitogens had also been induced to express the T cell alloantigen Thy-1. Like the expression of T cell alloantigens, the capacity to respond to Con A developed rapidly and reached its maximum within 6 hr. Responses to Con A always greatly exceeded those to PHA. Our data suggest that committed precursor cells, which we believe to be prothymocytes, are induced by thymopoietin to differentiate to cells with an antigenic phenotype and mitogen responsiveness similar to cortical thymocytes.     

Carrageenan-induced decline of natural killer activity: II. Inhibition of cytolysis by adherent non-T Ia-negative suppressor cells activated in vivo

The intravenous injection of 1 to 2 mg of ι-carrageenan (CAR) into (C57BL/6 × C3H)F₁ or BALB/c mice causes a prompt and substantial decline of splenic natural killer (NK) activity against YAC-1 lymphoma targets lasting approximately 1 week in F₁ mice. During this time, NK activity can be enhanced by administration of the interferon inducer polyinosinic-polycytidilic acid. The in vivo effect of CAR requires neither an intact thymus nor unimpaired proliferative capacity of lymphomyeloid cells, according to experiments in congenitally athymic BALB/c.nu/nu mice and in preirradiated (700 rad of γ-rays) F₁ hybrids. The splenic cytotoxic activity lowered in vivo by CAR can be restored in vitro by removing subpopulations of cells that adhere to glass wool or carbonyl iron particles, but not to Sephadex G-10. Thus, the lytic function of mature NK cells is reversibly inhibited in the spleens of CAR-treated animals; differentiation and maturation of NK precursors are not inhibited, as judged by the enhancing effect on NK activity of the interferon inducer. Splenocytes of CAR-treated donors suppress cytotoxic effectors of untreated mice in cell mixing experiments. Athymic and preirradiated animals given CAR are fully competent donors of suppressor cells. Suppressor function is insensitive to irradiation (2000 rad of γ-rays in vitro) and to anti Thy-1 or anti-Ia antibody plus complement. Inhibition of NK cytolysis is not restricted by the major histocompatibility complex and can also be mediated by cell-free supernatants in which suppressor cells were incubated. This model of reversible inhibition of NK activity suggests that activation of thymus-independent suppressor cells is one of the regulatory mechanisms of natural cytotoxic activity in vivo.