Regulation of immune response in allogeneic mixed spleen cell cultures. I. Influence of I-region on the generation of suppressor cells

The immune responses of allogeneic mixed spleen cell cultures (MLC) to the T-dependent antigen, SRBC, and to the T-independent antigen, DNP-PAA, were investigated. The immune response to DNP-PAA in MLC with certain strain combinations was always suppressed as compared with the expected PFC response calculated from the PFC responses of the individual strains. This suppression was eliminated by treating the spleen cells with RAMB antiserum plus complement before the incubation of the MLC with DNP-PAA. It can be concluded that the suppression in the PFC response to the T-independent antigen DNP-PAA in MLC is due to the generation of suppressor T-cells. The PFC response to the T-dependent antigen, SRBC, in MLC showed either suppression, no change, or rarely augmenation, suggesting that the allogeneic mixed spleen cell cultures can generate both suppressor and helper T cells and that the balance between helper and suppressor activity regulates the PFC response to a T-dependent antigen. Suppressor activity was also generated in a one-way MLC, but the degree of suppression depended upon which of the two strains was responding. Similar amounts of thymidine were incorporated in the one-way MLR irrespective of which strains was responding. Thus, the extent of proliferation in one-way MLR is not related to the degree of suppressor activity generated. The results further indicate that a difference between two strains in the I-C, S, and G regions of the major histocompatibility complex is required to generate suppressor activitiy that can depress the response to a T-independent antigen, MLC between strains differing in K, I-A, I-B, I-J, I-E, and D regions generate little or no suppressor activity in this system.     

Activation of human B lymphocytes. III. Concanavalin A-induced generation of suppressor cells of the plaque-forming cell response of normal human B lymphocytes.

Con A-induced suppression of the direct PFC response to polyclonal stimulation in human B cells has been described. Two types of experiments are presented. First, Con A was added directly to PWM-stimulated PB or tonsil cells resulting in a dose-dependent suppression of the PFC response, with maximal suppression occurring at a Con A concentration of 10 mug/ml. This suppression is completely removed by the simultaneous addition of alphaMM to the cultures. Secondly, Con A stimulation of tonsil or PB lymphocytes generated a population of cells which when added to autologous lymphocyte cultures induced a marked and reproducible suppression of the PFC response. The generation of suppressor cells is dependent on cell division and is blocked by alpha MM. Once generated the process of suppression is indpendent of the presence of Con A itself and is mediated by an activated lymphocyte population. These studies demonstrate a simple and reproducible model for the generation of a population of suppressor cells capable of inhibiting the direct PFC response to PWM-induced polyclonal activation of normal human B lymphocytes.     

Suppression of cytolytic T-cell activity by staphylococcal enterotoxin B-induced suppressor cells: Role of interleukin 2

We have previously shown that staphylococcal enterotoxin B (SEB) is a potent inducer of suppressor T cells which function to inhibit antibody production in vitro. In the present paper we extend these studies and show that the SEB-induced suppressor cells also inhibit the development of cytotoxic lymphocytes in mixed-lymphocyte reaction (MLR) cultures. Since further analysis also showed that the level of interleukin 2 (IL-2) in cultures of SEB-primed cells was significantly reduced, experiments were carried out to determine the role of IL-2 in the inhibition of cytotoxic cell activity. Attempts to neutralize the suppression by supplementing MLR cocultures with delectinated supernatants from concanavalin A (Con A)-stimulated rat splenocytes were not successful. In addition, MLR cocultures supplemented on Day 0 with 50 units of affinity-purified IL-2 were also suppressed. Further analysis showed that the IL-2 activity in the supplemented MLR cocultures containing suppressor cells were significantly reduced by Day 3. However, repeated supplementation of the MLR cocultures with exogenous IL-2 was successful in achieving (and maintaining) “normal” levels of IL-2. The cytotoxic cell activity in these MLR cocultures remained suppressed. These results suggest that the inhibition of cytotoxic cell activity by SEB-induced suppressor cells is independent of IL-2 levels in the MLR coculture.     

Activation of human B lymphocytes. V. Kinetics and mechanisms of suppression of plaque-forming cell responses by concanavalin A-generated suppressor cells.

The kinetics and mechanisms of suppression of the PWM-induced PFC response of human PB lymphocytes by Con A-activated suppressor cells were investigated. It was necessary that Con A suppressor cells be present early in the process of activation of human B cells toward antibody syntheses, but maximal suppression of the PFC response occurred later in the culture period. In addition, Con A-activated cells, although suppressing the PFC response to PWM greater that 90% of control, did not significantly suppress the blastogenic response to PWM after 3 or 5 days in culture. On the contrary, after 3 days in culture, background tritiated thymidine incorporation as well as tritiated thymidine incorporation to PWM stimulation was increased when Con A suppressor cells are added to fresh autologous peripheral blood lymphocytes. This increased blastogenic response after three days most likely represented an autologous mixed lymphocyte reaction (MLR) or Con A suppressor cells against fresh autologous non-T cells. The induction of autoreactive cells may be one of several modes of suppression of PFC responses by Con A activated suppressor cells.     

MLC-specific suppressor T lymphocytes in man. I. Their induction in vitro by soluble HLA-DR antigens

Human T lymphocytes precultured for 36 hr in the presence of soluble HLA-DR antigens suppress the MLR response of autologous peripheral blood lymphocytes to allogeneic stimulating cells. The suppression is DR antigen-specific in that it appears that the MLR stimulating cell donor and the soluble suppressor-inducing antigen must share DR specificities. The soluble DR antigens were fractionated from the sera of normal donors using QAE-Sephadex chromatography and CNBr-activated Sepharose immunoadsorption. Similarly prepared HLA-A and -B antigens failed to induce suppressive activity. The suppressive activity of DR-antigen cultured T cells is resistant to mitomycin C treatment and, further, the antigen specificity is maintained with or without mitomycin C treatment. The kinetics of suppressor cell induction as well as the kinetics of suppression in the test MLR cultures are presented. The implications of these results are discussed.     

Suppression of the responsiveness of lymphocytes from cancer patients triggered by co-culture with autologous tumor-derived cells

The question as to whether or not cancer patients have "tumor antigen"-induced suppressor T cells is of considerable interest and importance. As an approach to that question, the effect of addition of autologous irradiated tumor-derived cells (TDC) on the mixed lymphocyte response (MLR) of patients' lymphocytes (Ly) and of healthy donor Ly was tested. The rationale for these experiments was based on the fact that circulating antigen-responsive blood lymphocytes can be reactivated in vitro by exposure to the appropriate antigen. Thus, if there are circulating tumor "antigen"-reactive suppressor Ly, exposure to TDC as a source of the antigen should reactivate those cells. Reactivation of suppressor cells might result in diminished responsiveness to other stimuli such as alloantigens in the mixed leukocyte culture. We found that the addition of TDC to Ly cultures produced four distinct patterns of reaction. In 26 of the 74 different patient-tumor assays, the addition of autologous TDC to the patient cultures inhibited MLR, but the addition of the same TDC to cultures of Ly from healthy donors had no effect or increased their responsiveness (Specific Suppression). In 21 cases, the addition of autologous TDC to the patient cultures suppressed the MLR and the addition of the same TDC to control cultures suppressed the response of some but not all the healthy donors (Selective Suppression). In four cases, the addition of TDC to the cultures suppressed the MLR of the patients and all of the control donors (Nonspecific Suppression). In 23 cases, the addition of autologous TDC resulted in no suppression of the patient MLR or of any of the simultaneously tested normal donors (No Suppression). When TDC of patients with noninvasive bladder cancer were added to their own Ly cultures, only four of 11 produced specific or selective suppression compared to 11 of 12 when TDC came from patients with superficially invasive cancer. These data provide indirect evidence to support the hypothesis that human tumors induce circulating suppressor cells that may be reactivated in vitro by co-culture with TDC.     

Comparative study of the proliferative activity of human lymphocytes from different pairs of donors in a mixed culture in vitro

The proliferative response of human lymphocytes was studied in one-way and two-way mixed lymphocyte cultures (MLC). The maximal proliferation was shown to be attained in a two-way MLC containing unequal quantities of the cells from two paired donors. It was also demonstrated in a one-way MLC that the cells of one of the two paired donors are more effective responders. The most powerful proliferation in the MLC of these donors was observed in the cultures containing the excess of more effective cells. Thymosine increased the response of human lymphocytes in the two-way MLC. In vitro cell preincubation reduced the response in the cultures with a high proliferative response in the control. It has been thus demonstrated that lymphocytes from paired donors possess different functional activity in the MLC.     

Ala-1: murine alloantigen of activated lymphocytes. II. T and B effector cells express ala-1.

Ala-1 (activated lymphocyte antigen-1) is a murine alloantigen expressed only on activated peripheral T and B lymphocytes. The presence or absence of Ala-1 on specific functional lymphocyte subsets was determined by treating the relevant cell population with anti-Ala-1 and complement, and assaying for residual functional activity. By this method, Ala-1 was shown to be on in vivo primed killer T cells cytotoxic for allogeneic tumor cells. It was also found on helper T cells generated in vivo to sheep red blood cells, and on IgM and IgG plaque-forming cells (PFC) to sheep red blood cells. In contrast, splenic precursors of helper cells and of IgM PFC to sheep red blood cells were completely resistant to treatment with anti-Ala-1 and complement. These findings indicate that effector cells can be distinguished from their nonactivated precursors by their expression of Ala-1.     

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.     

The differentiation of suppressor cell populations as revealed by studies of the effects of mitogens on the mixed lymphocyte reaction and on the generation of cytotoxic lymphocytes.

The regulation by concanavalin A (Con A) and bacterial lipoloysaccharide (LPS) of the mixed lymphocyte reaction (MLR) and of the generation of cytotoxic lymphocytes (CL) was studied in congenic resistant mice using cortisone resistant thymocytes as the responding cells. LPS enhances the generation of CL selectively when suboptimal numbers of allogeneic cells are present in mixed lymphocyte cultures and also results in the augmentation of the MLR. Mitogenic concentrations of Con A on the other hand suppress the generation of CL regardless of alloantigen dose. The mechanism of suppression cannot be ascribed to the presence of suppressor T cells, since the addition to the cultures of syngeneic cortisone resistant thymocytes activated by Con A does not change the immune response. However, prospective suppressor cells that can be activated by Con A are located in secondary lymphoid organs such as spleen and lymph node. Suppressor activity by those cells is abolished by anti θ plus complement. Con A activated spleen cells suppress the MLR, whereas Con A activated thymocytes amplify the proliferation of responding cells.     

Simple sugars inhibit proliferation of human T lymphocytes in autologous and allogeneic mixed lymphocyte reactions

Several oligo- and monosaccharides were studied for their capacity to modulate lymphocyte proliferation in human allogeneic and autologous mixed lymphocyte reactions (MLR). A defined subset of sugars showed a marked inhibitory effect on lymphocyte proliferative response in the majority of the allogeneic MLR combinations studied. The inhibitory effect disappeared when sugars were added to allogeneic MLR 96 hr after the beginning of culture. These sugars also showed a significant inhibitory power on autologous MLR, performed by using T- and non-T-enriched lymphocytes from the same donor. The reported data suggest that carbohydrate determinants are involved in the proliferative response of human lymphocytes in both autologous and allogeneic MLR.     

Formation of heterophile antibodies by human tonsilar lymphocytes: II. In vitro stimulation with allogeneic cells

Short-term cultures of human tonsilar lymphocytes (HTL), 5 × 10⁶ cells/culture, in medium RPMI 1640 supplemented with human group AB serum were studied for the production of plaque-forming cells (PFC) against sheep (SRBC) and bovine (BRBC) red blood cells following in vitro stimulation by various allogeneic lymphoid cells. Of 55 HTL specimens examined, 48 produced a significant number (50–300/culture) of PFC against SRBC and/or BRBC following the in vitro stimulation. The optimal doses of the stimulator HTL and peripheral blood lymphocytes (PBL) were 10⁷ and 5 × 10⁶/culture, respectively. After the stimulation, PFC appeared in significant numbers on the third day, reached the peak number on the sixth day, and decreased sharply in number thereafter. Removal of E-rosetting cells from both stimulator and responder populations abolished the PFC formation. PFC formation against SRBC was inhibited by solubilized Forssman antigen, while PFC formation against BRBC was inhibited strongly by Hanganutziu-Deicher antigen, hardly by Paul-Bunnell antigen and not at all by Forssman antigen. Supernatants of mixed lymphocyte culture of PBL were shown to enhance PFC formation of HTL cultures stimulated by allogeneic lymphocytes. The results of this study indicated that in vivo primed B cells of the HTL were triggered in vitro by allogeneic stimulation for the heterophile antibody formation. Since these antibodies are apparently directed against Forssman and Hanganutziu-Deicher antigens, the “allo” nature of these antigens as well as their relationship to the previously described heterophile transplantation antigens have to be clarified.     

The plaque-forming cell response of human blood lymphocytes. I. PFC response of lymphocytes to formalin-treated staphylocci.

The plaque-forming cell and proliferative responses of human peripheral blood lymphocytes induced by formalin-treated Staphylococcus aureus of the Cowan strain were studied in vitro. Human blood mononuclear cells were incubated for 6 days with staphylococci in culture medium RPMI 1640 supplemented with 10% human AB serum. The number of anti-sheep erythrocyte plaque-forming cells was determined by the Jerne technique. Lymphocyte proliferation was measured by [³H]thymidine incorporation. Individual lymphocyte donors could be classified as high or low responders to staphylococci. Lymphocyte proliferation appeared necessary for the generation of plaque-forming cells. The plaque-forming cell response was greatly influenced by the source of the human AB serum used in the culture medium. The addition of hydrocortisone to the culture medium augmented the plaque-forming cell response. Human B lymphocytes prepared by passage through a column containing Sepharose 4B conjugated to anti-human F(ab)₂ generated plaque-forming cells when incubated with staphylococci. However, the addition of T lymphocytes to these B-lymphocyte preparations augmented the plaque-forming cell response to staphylococci.     

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.     

Effect of cyclosporin A on human lymphocyte responses in vitro. III. CsA inhibits the production of T lymphocyte growth factors in secondary mixed lymphocyte responses but does not inhibit the response of primed lymphocytes to TCGF

The mechanism whereby Cyclosporin A (CsA) inhibits secondary mixed lymphocyte responses was assessed. CsA added to secondary MLR cultures inhibited proliferation and induction of cytolytic lymphocyte activity. This inhibition was found to be associated with the inhibition of T lymphocyte stimulating growth factor(s) (TCGF) production in the supernatants of secondary MLR cultures. As little as 1.0 micrograms/ml of CsA added to secondary MLR cultures resulted in no measurable TCGF activity. In contrast, moderate doses of CsA (1.0, 2.5 micrograms/ml), which completely inhibited the secondary MLR response to alloantigen, did not inhibit the proliferative and CML response of alloantigen-primed lymphocytes to these stimulating growth factors. Even at high doses of CsA (20 micrograms/ml), substantial levels of proliferation (50% of control response) and CML induction (60% of control response) were observed when the primed cells were exposed to secondary MLR supernatants containing TCGF activity. It was concluded that inhibition of secondary mixed lymphocyte responses by CsA may be due in part to the inhibition of TCGF production rather than the inhibition of the effect of TCGF on mature cytotoxic T lymphocytes.     

3H-uridine incorporation by small lymphocytes of tolerant rats: relationship to T and B lymphocytes.

Tissue tolerance was induced in neonatal rats by the intravenous injection of bone marrow cells from adult allogeneic rat donors. After 6 to 8 weeks, lymphoid cells from rats in which tolerance had been induced were tested for mixed lymphocyte reactivity (MLR), 3H-uridine uptake, and the relationship of uridine incorporation to B and T lymphocytes. Lymph node (LN) and spleen (SPL) cells from the adult inoculated rats showed no reactivity in the MLR or normal lymphocyte transfer reaction (NLTRx), indicating that the animals were tolerant. After in vitro exposure to 3H-uridine, an abundance of small lymphocytes (SL) from these same tolerant rats were heavily labeled, in contrast to nontolerant controls, where relatively few SL were heavily labeled. In order to determine whether the heavily uridine-labeled cells were T cells or B cells, lymphoid cells from the LN and SPL of tolerant animals were exposed to either rabbit anti-AKR brain serum or rabbit anti-rat Ig conjugated with ferritin. The results showed that the heavily uridine-labeled SL of the tolerant rats were mainly Ig-positive cells.     

Cellular requirements for lymphocyte restimulation after a first challenge in vitro.

Human peripheral blood lymphocytes from donors who were sensitized in vivo to bacterial antigens were stimulated by these antigens in vitro. When the cells from these first cultures were challenged with irradiated allogeneic lymphocytes, a proliferative response was obtained, the kinetics of which resembled those of a primary mixed lymphocyte reaction (MLR). On the other hand, the addition, under these conditions, of bacterial antigens never led to any second proliferative response. It was shown that: (1) the addition of irradiated autologous mononuclear cells, together with the bacterial antigens, led to a reconstitution of a proliferative response in second culture; (2) the cells capable of reconstituting the reactivity to tetanus toxoid could also be obtained from donors whose own cells did not respond to that antigen in primary cultures, and (3) the reconstituting activity in the second culture could not be provided by monocytes alone.     

Autologous mixes lymphocyte reaction in human cord blood lymphocytes: decreased generation of helper and cytotoxic T-cell functions and increased proliferative response and induction of suppressor T cells

T lymphocytes from neonates proliferated significantly more than peripheral blood T lymphocytes from adults in autologous mixed lymphocyte reactions (AMLR). AMLR-activated cord, as compared to adult T lymphocytes, exerted significantly less nonspecific cytotoxic activity on PHA-stimulated adult mononuclear cells and Epstein-Barr virus-transformed target cells. The impaired generation of cytotoxicity of cord T cells was not corrected by Interleukin-2. Blood T lymphocytes from adults activated in AMLR synthesized a helper factor that supported PWM-induced proliferation and immunoglobulin production in both adult and cord B lymphocytes. In contrast, cord blood T lymphocytes failed to produce the helper factor for B lymphocytes. T cells from AMLR cultures established with neonatal lymphocytes showed suppressor activity, as assessed in PWM-stimulated immunoglobulin synthesis of adult peripheral-blood mononuclear cells, significantly higher than that exhibited by T cells from AMLR cultures performed with lymphocytes from adults. Finally, neonatal B lymphocytes could be activated to the production of IgM but not IgG by either adult AMLR-derived helper factor plus PWM or by Epstein-Barr virus, whereas adult B cells secreted both IgM and IgG under the same type of stimulation.     

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.     

Formation of Paul-Bunnell antibodies by cultures of lymphocytes from infectious mononucleosis.

Short-term cultures of peripheral blood lymphocytes obtained from 20 infectious mononucleosis patients 2–4 weeks after the onset of the disease were studied for formation of heterophile antibodies. In studying pooled supernatant fluids of lymphocytes from three patients cultured for 3–20 days, lytic antibodies for red blood cells of bovine (BRBC) and sheep (SRBC) origin were demonstrated. These hemolysins were shown to be of IgM nature and Paul-Bunnell specificity. Subsequently, plaque-forming cell (PFC) assays were performed with lymphocyte cultures of 15 patients. Significant numbers (60–750/2 × 10⁷ cells) of PFC secreting antibodies against BRBC were demonstrated in lymphocyte cultures of 12 patients. The number of PFC apparently reached its peak after 5 to 10 days of culturing. No or a very few PFC were observed in the lymphocytes that were not cultured or in lymphocytes cultured for 3 weeks or longer. Lymphocyte cultures prepared in a similar fashion from normal individuals or patients suffering from sore throat and submandibular lymphadenopathy of other than infectious mononucleosis origin did not produce PFC. Production of lytic zones by antibodies to BRBC secreted by PFC was inhibited by preincubation of lymphocytes of infectious mononucleosis patients with solubilized Paul-Bunnell antigen but not with other heterophile antigens, indicating that antibodies involved in the PFC formation are of Paul-Bunnell specificity. An increased number of PFC against BRBC were obtained in two of three lymphocyte cultures after cultivation with BRBC or solubilized Paul-Bunnell antigen.