Stimulation of human peripheral blood lymphocytes by periodate, galactose oxidase, soybean agglutinin, and peanut agglutinin: differential effects of adherent cells.

Blastogenic responses of normal human peripheral lymphocytes to three distinct groups of mitogens were studied: Group I--phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM); Group II--soybean agglutinin (SBA) and peanut agglutinin (PNA); and Group III--galactose oxidase (GO) and sodium periodate (IO4-). SBA was mitogenic for human cells, and this effect was enhanced by treating the cells with neuraminidase (NA). PNA was mitogenic only after cells had been treated with NA. GO was effective before and activity was increased after lymphocytes were treated with NA. Responses to Group II and III mitogens were more variable than were those to Group I mitogens. Studies with purified T and B cells indicated that SBA and PNA were T cell mitogens, whereas IO4- and GO failed to stimulate either T or B cells. Adding macrophages back to this system indicated that they were both T cell mitogens with strict macrophage requirements. T cell responses to SBA and PNA were enhanced over responses to unfractionated cells to a degree that could not be explained simply by enrichment of the cultures with T cells. Removal of adherent cells from unfractionated cell suspensions again revealed a marked enhancement of responses to SBA and PNA, a consistent decrease in responses to IO4-, and a variable decrease in responses to GO. Similar results were found with 14C-leucine and 3H-uridine incorporation, as well as 3H-thymidine for the assessment of bastogenic response. Mechanisms responsible for these differential effects of macrophage depletion on lymphocyte responses to different groups of mitogens are yet to be determined. Either different mitogens require different lymphocyte to macrophage ratios for optimal stimulation, or some mitogens (i.e., SBA and PNA) form inhibitory complexees in the lymphocyte-macrophage mixture. In any case, variability in response to mitogenic agents in normal as well as pathologic states may be dependent on adherent cell populations, rather than on the lymphocytes themselves.     

Activation of human T lymphocytes by 12-O-tetradecanoylphorbol-13-acetate: role of accessory cells and interaction with lectins and allogeneic cells

Stimulatory effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on human T lymphocytes have been investigated. TPA was found to stimulate highly purified T cells (obtained by a three-step isolation procedure involving plastic adherence, nylon wool passage and Ig-anti-Ig column passage) in the absence of accessory cells (stimulation index of 5 to 10), whereas phytohemaglutinin (PHA) and concanavalin A (Con A) did not. This response was, however, increased by the addition of autologous adherent cells. Addition of TPA, but not adherent cells, induced T-cell proliferation in response to the nonmitogenic lectin, wheat germ agglutinin (WGA), while both adherent cells and TPA restored T-cell proliferation to mitogenic lectins such as PHA and Con A. Furthermore, TPA greatly increased the mixed-lymphocyte response of purified T cells to otherwise nonstimulating allogeneic cells such as T lymphocytes or tumor cells from some patients with chronic lymphocytic leukemia. These results suggest that TPA can directly act on human T cells to render them reactive to a variety of stimuli.     

In vitro response of subpopulations of human tonsil lymphocytes. I. Cellular collaboration in the proliferative response to PHA and Con A.

Human tonsil lymphocytes have been separated into three subpopulations of cells: purified B cells and two subsets of purified T cells (F1 and F2). B cells were obtained by rosetting with neuraminidase treated SRBC. F1 and F2 were separated by filtration on a nylon wool column using different speeds of elution. Purified B cells contained less than 5% T cells, the T cells preparations contained less than 5% B cells for F1 and 10 to 15% for F2, respectively. A significant contamination in cells not identified by any B or T marker was observed in purified B cells and in F1. Adherent cells enhanced the response of each lymphochte population to PHA and Con A. This explained the paradoxically low responsiveness of the purified T cells. Purified B cells did not respond to these mitogens in different culture conditions. However, a small B cell response was observed when they were cultured in the presence of mitomycin-treated T cells. Striking was the enhancing effect of B cells on the T cell response to PHA and Con A. This enhancing effect was observed even when B cells were treated with mitomycin or depleted in adherent cells. The comparison of the F1 and F2 response suggested that they contained distinct types of T cells.     

Potentiation of the T lymphocyte response to mitogens. V. Inhibition of the response to concanavalin A by supraoptimal doses or by other lectins and its aversion by LAF.

The enhanced thymidine incorporation in murine lymphocytes induced by Concanavalin A (Con A) was markedly inhibited in the presence of other lectins, which are poorly mitogenic (phytohemagglutinin {PHA} or pokeweed mitogen), or non-mitogenic (soybean agglutinin {SBA}). The level of inhibition was found to be inversely proportional to the mitogenic effect of the lectins. Our results did not support the notions that the lectins inhibit the lymphocyte responses by competing with Con A, or by activating suppressor cells. Rather, the data suggest that the lectins cause cytotoxic or cytostatic effects. The effects of the inhibitory lectins were found to resemble those of supraoptimal doses of Con A. In particular, both effects were partly averted by the lymphocyte activating factor (LAF). The mitogenic effect of LAF was not inhibited by the non-mitogenic lectin, SBA, whereas the poor responses to PHA or to moderately supraoptimal doses of Con A were markedly potentiated by this factor. It is thus suggested that LAF activity counteracts the inhibitory processes provoked by the lectins.     

Effect of T- and B-lymphocyte mitogens on interactions between lymphocytes and macrophages.

The mitogenic response of murine T cells ² to Con A, S-Con A and PHA was found to be macrophage-dependent. Optimal mitogenic responses were obtained when macrophage-depleted T-cell populations were reconstituted with 5% normal peritoneal macro-phages. Studies were carried out to investigate the effect of T- and B-cell mitogens on in vitro physical interactions between murine lymphocytes and macrophages. This was done by determining the number of T- or B cells binding to macrophages in the absence and in the presence of T- and B cell mitogens, and comparing the results of these experiments with the induction of lymphocyte proliferation. Con A increased the binding of T cells to macrophages when used in mitogenic doses (1–5 μg/ml). Dose response experiments showed that the same dose of Con A which produced maximal mitogenic stimulation also induced the greatest number of T cells to bind to macrophages. Nonmitogenic doses of Con A (20–50 μg/ml) did not enhance the binding of T cells, while identical doses of S-Con A both induced T cell mitogenesis and increased the number of T cells bound to macrophages. Similar results were obtained with PHA. None of the B-cell mitogens tested (LPS, EPO 127 and LAgl) increased the binding of either T or B cells to macrophages. PWM, which is mitogenic for both T and B cells, increased the binding of T cells to macrophages, but not that of B cells. In brief, the four T-cell mitogens tested (Con A, S-Con A, PHA, and PWM) induced specific physical interactions between T cells and macrophages, while none of the B-cell mitogens had any effect on the physical interactions between either B or T cells and macrophages when used in mitogenic doses.     

Some requirements for the response of separated T-cell sub-populations to the mitogens phytohaemagglutinin and concanavalin A.

The proliferative response of various separated populations of mouse spleen and thymus lymphocytes to the mitogen phytohaemagglutinin (PHA) was not a direct function of the level of responsive T cells, but was governed by other regulatory effects. These included a stimulation by adherent macrophages, an inhibition by a separate population of adherent cells and an adherent cell independent restriction of proliferation at high cell concentration. In contrast, the proliferative response to Concanavalin A (Con A) was more closely related to the level of responsive T cells. All density and electrophoretically isolated sub-sets of splenic T cells appeared capable of a proliferative response to PHA and Con A, although under some conditions the PHA responsiveness of certain fractions was suppressed. In the thymus, the minor low theta sub-population appeared capable of response to both mitogens, and accounted for all the activity of the unfractioned thymus cells. No response to either mitogen could be obtained from the major, high theta thymocyte population.     

Author index for volume 49

Using mouse thymocytes, mitogen-induced [³H]thymidine incorporation was compared with a recently developed flow-cytometric technique, based on acridine orange staining of cells, which differentiates the G₀ and G₁ phase of thymocytes. PHA induces a transient but considerable G₀-G₁ shift without any substantial proliferation. On the other hand, crude supernatants derived from Con A-stimulated human peripheral blood mononuclear cells induce only a minor G₀-G₁ shift and no proliferation. However, PHA in the presence of this supernatant induced an increased [³H]thymidine uptake in thymocytes and a shift from G₁ to S. These results support the current hypothesis that a factor present in Con A-activated supernatants in conjunction with PHA stimulation indeed facilitates the entrance of G₁ cells into the S phase. The flow-cytometric technique might be used in the study of the interaction of endogenous mediators with exogenous mitogenic agents in activating lymphocytes to proceed through the initial G₀-G₁ phases of the cell cycle.     

Rabbit T cells with and without Fc receptors

Earlier, indirect evidence for rabbit subpopulations differing in Fc receptors and in response to mitogen has been directly tested. T cells were purified from spleen suspension by removal of adherent cells, followed by removal of Ig-bearing cells on petri dishes coated with antibody, directed against the light chain allotype of Ig receptors. The purified cells were further fractionated by formation of EA rosettes and separation on Ficoll-Hypaque. T cells which lacked Fc receptors had a larger response when stimulated with Con A or PHA than did T cells which possessed Fc receptors. Both subpopulations responded more when irradiated nonadherent B cells were added to the mixture, but the extent of help was the same for both cell populations. T cells which contained both Fc receptor-bearing cells and cells which lacked the receptor had a response which was intermediate between that of the two separated subpopulations.     

Modulation of T-cell functions: I. Effect of 2-mercaptoethanol and macrophages on T-cell proliferation

The in vitro effects of 2-mercaptoethanol (2-ME), macrophages (MØ), and concanavalin A (Con A) on the proliferation of normal spleen cells (NSC), MØ-depleted spleen cells (DSC), T cells, T-cell subpopulations, and B cells were assessed by [³H]thymidine incorporation. 2-ME alone was consistently shown not to be mitogenic for purified T cells; however, 2-ME enhanced the early (Days 1 and 2) Con A (2 μg/ml)-induced response of NSC, DSC, and T-cell preparations, but depressed the late response (Days 4 and 5). 2-ME alone was mitogenic for purified B-cells, as reported previously; and the 2-ME-induced B-cell response was inhibited by Con A. Preincubation of T cells with 2-ME was sufficient for enhanced Con A responsiveness; however, if 2-ME was added 24 hr after the initiation of culture, no alteration of the Con A-induced response was observed. Ly-2,3⁺ T cells were unresponsive to Con A (0.3–20 μg/ml), but the addition of 2-ME or peritoneal cells enhanced the Con A responsiveness of Ly-2,3⁺ T cells over 200-fold. Ly-1⁺ T cells responded with a similar doseresponse and kinetic profile as unselected T cells. Although Ly-1⁺ T cells responded to Con A, unlike Ly-2, 3⁺ T cells, extensive removal of MØ significantly reduced the Con A-induced responsiveness of the Ly-1⁺ T cells. The reactivities of Ly-1⁺ and Ly-2,3⁺ DSC could be reconstituted by the addition of MØ or 2-ME; however, the kinetic response of Ly-1⁺ T cells peaked on Day 2–3, and Ly-2,3⁺ T cells had a delayed response which peaked on Day 4–5. The results indicated that (i) 2-ME and/or MØ accelerate the response kinetics of T-cells to Con A; (ii) T-cell subpopulations have differential requirements for MØ and/or 2-ME in the response to Con A; (iii) T-cell subpopulations exhibit differential dose responsiveness to Con A; and (iiii) 2-ME alters Con A responsiveness by a direct effect on T cells.     

Independent analysis of T helper and T killer cell function in young and old NZB mice.

With age, NZB mice lose their ability to develop a cytotoxic response after alloimmunization in vitro. This decline is shown to coincide with a diminution of T-helper cell activity as assessed by proliferation in mixed lymphocyte culture or in response to PHA. When cytotoxic T cell precursors are activated with the polyclonal activator Con A, there is no reduction in the number of cytotoxic effector T cells that develop. No autoreactive cytotoxic cells are seen in Con A-activated cultures. These findings are related to previous work on cell-mediated immunity in NZB and B/W mice.     

Biological expressions of lymphocyte activation. IV. Concanavalin A-activated suppressor cells in mouse mixed lymphocyte reactions.

Thymus-derived lymphocytes (T cells) from mouse spleen, activated in vitro or in vivo with concanavalin A (Con A), suppress proliferative responses of syngenic lymphocytes in mixed lymphocyte reactions (MLR). Replication in vitro was not required for expression of suppressor activity by Con A-activated cells and was blocked in MLR by treating suppressor cells with mitomycin C or irradiation. Kinetics of MLR responses and viability of cultures were not altered by addition of activated suppressor cells. The data are consistent with a direct inhibitory effect of suppressor T cells on antigen-induced DNA replication. These observations extend a model previously described for regulation of antibody synthesis by Con A-activated T cells to control of cell-mediated immune responses. This model should be particularly useful in further definition of regulatory T cell subpopulations, and in investigation of interactions and relationships between such populations.     

Inability of newborns' or pregnant women's monocytes to suppress pokeweed mitogen-induced responses

Although an excess of human adult blood adherent cells inhibits the pokeweed mitogen- (PWM) induced normal adult lymphocyte proliferation and B cell maturation into immunoglobulin-containing cells (ICC), adherent cells collected from newborn infants or pregnant women at time of delivery were unable to exert a similar suppressor activity. After activation by Concanavalin A (Con A), newborns' and pregnant women's adherent cells acquired a suppressor activity comparable to that of control adult adherent cells. The adherent suppressor cell was shown to be radioresistant (3000 rad), indicating its probable monocytic origin. Both monocyte-suppressor activities (MSA) observed in adulthood (spontaneously) and in the neonatal period (after activation) were dependent on prostaglandin E2 (PGE2) secretion, because they were abolished by indomethacin or a specific anti-PGE2 antiserum. Expression of MSA appeared to be under a negative regulation exerted by naturally occurring T suppressor lymphocytes present in the blood of newborns or pregnant women, because incubation of adult monocytes or Con A-activated newborn monocytes with newborns' or pregnant women's T lymphocytes resulted in a dramatic decrease of their MSA. These results strongly suggest that the lack of MSA in the neonatal period and in late pregnancy is a consequence of activation of T suppressor lymphocytes.     

Identification and characterization of a B cell growth inhibitory factor (BIF) on BCGF-dependent B cell proliferation

The culture supernatants of Con A-activated human peripheral blood mononuclear cells (PBM) contained at least two regulatory factors upon B cell proliferation. One was B cell growth factor (BCGF), which activated antigen-stimulated B cells to proliferation and clonal expansion, and the other was its inhibitory factor, arbitrarily named B cell growth inhibitory factor (BIF). This BIF inhibited the effect of BCGF on anti-mu-stimulated B cells or the monoclonal mature B cell line (CLL-T.H.) obtained from the peripheral blood lymphocytes of B cell-type chronic lymphocytic leukemia patients, which were activated only with BCGF and without adding other proliferating stimuli (e.g., anti-mu). BIF activity was detected in the 24 hr culture supernatants of Con A-activated human PBM in FCS containing medium and also in serum-free RPMI 1640 medium. This substance with BIF activity could not be derived from FCS. Con A-induced BIF (m.w. of 80,000 and an isoelectric point of pH 5.4) was analyzed by Sephadex G-200 gel filtration and chromatofocusing. BIF was stable at pH 2.0 and at 56 degrees C for 30 min. Partially purified BIF had no effect on cell viability and almost no interferon activity (less than 1 IU/ml). BIF with high titer had a slight but significant inhibition on TCGF-dependent T cell growth and on PHA or Con A responses, but the extent of these inhibitions was far less than that of BCGF-dependent B cell growth. Absorption of BIF with Con A blasts made its inhibition on T cell growth even less. On the other hand, BIF activity could not be absorbed with Con A blasts but was almost absorbed with large numbers of CLL-T.H. cells. BIF had almost no inhibitory effect on the proliferation of a mouse fibroblast cell line (NIH 3T3), a mouse myeloma cell line (NS-1), human lymphoid cell lines (MOLT-4, HSB-2, and Daudi), or a human myeloid cell line (K-562). BIF-producing cells were estimated to be T cells and were identified as T8+ T cells. On the other hand, Con A-induced BCGF was demonstrated to be produced predominantly by T4+ T cells. These results show that human B cell proliferation is regulated by interaction between T4+ and T8+ cells via soluble factors, namely BCGF and BIF, respectively.     

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.     

Proliferation of murine thymic lymphocytes in vitro is mediated by the concanavalin A-induced release of a lymphokine (costimulator).

Mitogen-induced proliferation of lymphocytes may in theory result directly from the interaction of mitogen with the cells, or indirectly as a result of the mitogen-stimulated release of lymphokines. In the case of murine thymic lymphocytes exposed to concanavalin A (Con A) in tissue culture, we have determined that mitogenesis depends upon a lymphokine. Interaction of the thymic lymphocytes with lectin is necessary, but not sufficient, for mitogenesis. A lymphokine, or costimulator for mitogenesis, is released by normal spleen or thymus cells during the first 16 hr of their exposure to Con A, and in the presence of a phytomitogen it stimulates thymic mitogenesis. Under conditions of low costimulator levels, no mitogenesis follows the interaction of Con A with cells. The response of adult CBA/J mouse thymocytes to phytohemagglutinin (PHA) is very low, compared to their response to Con A. When costimulator is added to PHA, the cells respond as well as they do to Con A. Costimulator does not act through Con A-binding sites on thymus cells. Its production is dependent on both cells carrying omega surface antigen (T lymphocytes) and adherent cells of the macrophage-monocyte series. The adherent population, but not the T cells, may be heavily irradiated without affecting production of costimulator. Costimulator is not a mitogen on its own.     

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.     

Developmental changes in humoral suppressor activity released from concanavalin A-stimulated human lymphocytes on B cell differentiation

Cell-free culture supernatants (Con A-activated supernatants) were obtained by incubating peripheral blood lymphocytes (PBL) from cord blood, healthy children of various ages, and healthy adults with mitogenic doses of concanavalin A (Con A) for 48 hr. It is well known that human T lymphocytes are activated by Con A to manifest suppressor function in vitro. One mechanism whereby these suppressor cells act has been shown to be by the secretion of a soluble suppressor factor. The present study has investigated the Con A-inducible suppressor cell function in cord blood, children of various ages, and adults by comparing the ability of each Con A-activated supernatant to inhibit the generation of immunoglobulin-producing cells (Ig-PC) in pokeweed mitogen- (PWM) stimulated cultures of adult PBL. Con A-activated supernatants from adults could markedly suppress the generation of Ig-PC by allogeneic as well as autologous PBL in response to PWM. Such suppression appeared to be equally effective on the generation of IG-PC of 3 major classes, IgG, IgM, and IgA. On the contrary, Con A-activated supernatants from cord blood and newborn infants showed only a negligible suppression on PWM-induced adult B cell differentiation. But the suppressor activity found in Con A-activated supernatants gradually increased with advancing age, and reached approximately to the adult level at 4 yr of age or later. The results suggest that human T lymphocytes may be relatively deficient in their Con A-induced suppressor cell function in the early period of life.     

Specific triggering by concanavalin A of a secondary T killer cell-mediated anti-tumor immune response.

Spleen cells from C57 B1/6 mice which rejected a Moloney sarcoma virus (MSV)-induced tumor elicited a strong cytolytic reaction against MSV-associated antigens when incubated for 7 days with a mitogenic dose (5 μg/ml) of concanavalin A (Con A). The cytolytic activity evaluated in a chromium release test was shown to be mediated by T lymphocytes, by anti-Thy 1–2 serum treatment, and independent of remaining Con A, by treatment with d-[α-methyl]mannopyranoside. A similar reactivity was obtained with phytohemagglutinin (PHA) at mitogenic doses but not with the B-cell mitogen lipopolysaccharide (LPS). This “secondary-like” response was regenerated up to 50 days post-MSV inoculation but decreased regularly. The cytolytic activity had an antigenic pattern indistinguishable from that of the relevant tumor cell reactivation and was directed by the same H-2 restriction.     

The role of the 2H4 molecule in the generation of suppressor function in Con A-activated T cells

The molecular basis for the suppression generated in a concanavalin A (Con A)-activated T cell culture remains unknown. In this study, we have attempted to determine whether the 2H4 and 4B4 molecules on Con A-activated T cells play some role in the generation of suppression by such cells. We have shown that Con A-activated suppressor cells belong to the 2H4+ subset of T cells but not the 4B4+ (2H4-) subset. Con A-activated T cells exerted their optimal suppressor function on day 2 in culture, a time at which the expression of 2H4 on such cells was maximal and 4B4 was minimal. Furthermore, the stimulation of T cells with the higher concentration of Con A generated the stronger suppressor function. At the same time, both 2H4 expression and density were increased and 4B4 expression and density were decreased on such Con A-activated T cells. More importantly, the treatment of Con A-activated T cells with anti-2H4 antibody but not with anti-4B4, anti-TQ1, or anti-T4 antibodies can block the suppressor function of such cells. Taken together, the above results strongly suggest that the 2H4 molecule itself may be involved in the generation of suppressor function in Con A-activated T cells. The 2H4 antigen on such cells was shown to be comprised of 220,000 and 200,000 m.w. glycoproteins. Thus this study indicates that the 220,000 and 200,000 m.w. structure of the 2H4 molecule may itself play a crucial role in the generation of suppressor signals of Con A-activated cells.     

Immune interferon induction by T-cell mitogens involves different T-cell subpopulations.

Recent studies of mitogen-induced DNA synthesis in cells from various lymphoid tissues indicate that certain mitogens may selectively activate specific T-cell subpopulations. Staphylococcal enterotoxin A (SEA) optimally stimulates thymocytes and spleen cells (presumed T1 cells) and phytohemagglutinin P (PHA) optimally stimulates lymph node and spleen cells (presumed T2 cells); concanavalin A (Con A) stimulates cells from all these sources well. To determine further the specificity of mitogens for T-cell subpopulations, immune interferon (IIF) production was studied in spleen cells from mice treated in vivo with cortisone acetate (CA), preferentially a T1-cell inactivator, and antithymocyte serum (ATS), preferentially a T2-cell inactivator. The effect of administering gallic acid (3,4,5 trihydroxy-benzoic acid) (GA) in vivo was also studied, since in vitro studies showed GA to be capable of blocking IIF production. Results indicate that SEA induces IIF primarily in T1 cells, PHA induces IIF primarily in T2 cells, and Con A induces IIF in both T1 and T2 cells. Furthermore, GA was shown to mimic the ability of CA to inactivate T1 cells selectively in vivo. The data indicate that more than one type of T-cell subpopulation is capable of producing IIF.