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.     

The regulatory role of adenosine-activated T-lymphocyte subset on the immune response in humans: I. Mitogenic response and production of mediators

Human T lymphocytes, rerosetted with sheep erythrocytes in the presence of adenosine, yield two subpopulations: a major one (E_R), still capable of forming E rosettes; and a minor nonrosetting (E_S) one. The two subpopulations differed in their proliferative responses to various mitogens. E_R cells responded well to galactose oxidase (GO), soybean agglutinin (SBA), and phytohemagglutinin (PHA) but responded poorly to concanavalin A (Con A). The response of E_S cells was poor to GO and SBA, intermediate to PHA, and significantly high to Con A. The different response of E_R and E_S subsets to Con A was not greatly affected by adherent cells, but an enhancing effect on the proliferation of E_S cells to Con A was observed when prostaglandin synthesis was inhibited by indomethacin. Addition of E_S cells to E_R cells in a ratio of 1:5 resulted in an enhanced synergistic effect of Con A-induced proliferation. A soluble mitogenic factor released from Con A-activated T cells appeared involved in this enhanced proliferation. This factor (E_SF) was produced only by the minor T-cell subpopulation which is sensitive to adenosine (E_S). The induction of E_SF was not dependent on the addition of adherent cells and required 72 hr of incubation for its production. E_SF was mitogenic to nonactivated and Con A-activated PBL as well as to T, E_R, and E_S subpopulations. Following incubation of E_R cells with E_SF, a suppressor factor (E_RSF) was produced which abolished the mitogenic activity of E_SF. Differences between these factors and a known mediator like Interleukin-2 (IL-2) and suppressor factors are discussed.     

Triiodothyronine affects the phytohemagglutinin to concanavalin A proliferative response ratio in sex-linked dwarf chickens

Euthyroid Cornell K strain and sex-linked dwarf (SLD) strain cockerels (which have abnormally low serum triiodothyronine concentrations) were supplemented with either 0, 0.01, 0.1, or 1.0 ppm of triiodothyronine (T3) in the diet. Peripheral blood lymphocytes (PBL) from these cockerels were obtained by slow-speed centrifugation (slow-spin-prepared PBL). The proliferative response of these PBL to phytohemagglutinin (PHA) and concanavalin A (Con A) was determined when the chicks were 6, 9, and 12 weeks of age. Con A responsiveness was also determined in 12-week-old cockerels using PBL which were separated on Ficoll (Ficoll-prepared PBL). Using slow-spin-prepared PBL, PHA, and Con A responsiveness increased in both strains with increasing levels of T3 supplementation. This enhancing effect of T3 was particularly evident in older cockerels. In 6- and 12-week-old SLD strain cockerels, the PHA:Con A response ratio was significantly (P less than 0.05) lower than in K strain cockerels. At 12 weeks of age the PHA:Con A response ratio of the SLD strain was elevated to K strain control levels by T3 supplementation. Therefore, the lower PHA:Con A response ratio in the SLD strain appears to be partially due to the existing peripheral hypothyroidism in this strain. Using Ficoll-prepared PBL, the effects of T3 on Con A responsiveness differed from those observed when slow-spin-prepared PBL were used. From this study we conclude that T3 supplementation affects mitogen responsiveness and the PHA:Con A response ratio. However, the effects of T3 on mitogen responsiveness depend on the age of the chicken, the level of T3 supplemented, the T cell population stimulated, and the method of lymphocyte enrichment.     

Immunomodulating activity in supernatants from EBV immortalized lymphocytes

Summary Our earlier work has demonstrated that EBV immortalized B lymphocytes are involved in a factor dependent autostimulatory cycle. Soluble growth stimulating activity was released into culture supernatants by these growing B cells. Growth enhancing (GE) media from B lymphocyte lines, immortalized by EBV infection, contained soluble factor(s) which modulated the Con A response of normal human mononuclear cells. Conditioned media from these lines affected the Con A response in a biphasic manner, stimulating the blastogenic response at lower concentrations, while inhibiting at higher concentrations. At stimulatory concentrations, the blastogenic response to Con A began earlier than in controls and was markedly enhanced by day 2. GE media reduced the initial response of purified B cells to pokeweed mitogen. GE media did not support growth of IL-2 dependent cells. GE media from some EBV-carrying B cell lines had measurable IL-1 activity in the mouse thymocyte PHA response. GE media from LPS stimulated B lymphocyte lines produced significant IL-1-like effects on stimulated mouse thymocytes. These results suggested that these B cell lines may produce IL-1-like factors that cooperate in T cell responses. The possibility that such factors may play a role in B lymphocyte transformation by EBV is discussed.Supported by grants from the Concern Foundation and the Brigham Surgical Foundation     

In vitro response of subpopulations of human lymphocytes. II. DNA synthesis induced by anti-immunoglobulin antibodies.

A significant and constant increase in DNA synthesis was observed in human lymphocytes cultured in the presence of purified anti-immunoglobulin antibodies specific for human IgG, IgA, and IgM. This has been found in cultures of lymphocytes isolated from blood, tonsils, spleen, and lymph nodes. The optimal culture conditions for blood and tonsil lymphocytes were determined. As a rule 6-day cultures containing 2 x 10(6) cells/ml and 100 mug/ml of antibody yielded the highest 3H-thymidine uptake. Purified T cell cultures could not be stimulated, whereas a low response could be observed in most of the purified B cell cultures. Optimal culture conditions were the same for the B and total tonsil lymphocytes. However, when the purified B cells were totally depleted of T cells, no response was observed. A T and B cell synergy has been demonstrated by supplementing B cell cultures with purified T cells, whether treated or not with mitomycin. These experiments indicated a permissive and potentiating effect of T cells on the B cell response. Cultures containing mitomycin-treated B cells and purified T cells (mB + T) could be stimulated by a-Ig, thus indicating a T cell proliferation. In keeping with this finding was the observation of an increased response of total lymphocytes supplemented with T cells but not with B cells. Adherent cells are necessary for an optimal response to a-Ig; they enhanced the B cell proliferation observed in (Tm + B) cultures and suppressed the response of T cells in (T + Bm) cultures.     

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.     

Lyt-2+ cells. Requirements for concanavalin A-induced proliferation and interleukin 2 production

The requirements for inducing Lyt-2+ T cell proliferation in response to concanavalin A (Con A) were examined. Purified Lyt-2+ or L3T4+ spleen cells of C57BL/6 origin were stimulated with Con A and syngeneic macrophages (MO) in the presence of monoclonal antibodies to T cell markers or to polymorphic determinants on major histocompatibility complex molecules, and assessed for the ability to proliferate and to produce interleukin (IL) 2. alpha I-Ab failed to inhibit the Con A response of Lyt-2+ cells at dilutions that significantly inhibited the response of L3T4+ cells. In contrast, alphaKb/Db or alpha Lyt-2.2 specifically inhibited the response of Lyt-2+ cells, but not L3T4+ cells. The ability of alpha Kb/Db and of alpha Lyt-2.2 to inhibit the response of Lyt-2+ cells was dependent upon the concentration of Con A. These data demonstrate that optimal triggering of T cell subsets to proliferate and to produce IL-2 in response to Con A requires interactions with the appropriate restricting major histocompatibility complex molecule. The role of accessory cells in Lyt-2+ Con A-induced proliferation and IL-2 production was also investigated. Purified Lyt-2+ cells and purified L3T4+ cells failed to respond to Con A in the absence of MO. IL-1 reconstituted the response when MO were limiting, but failed to restore the response of either Lyt-2+ or L3T4+ cells when T cells were rigorously purified to remove all MO. These results demonstrate that triggering Lyt-2+ T cells, like L3T4+ T cells, requires accessory cells, and that this does not merely reflect a requirement for IL-1 production. Thus, Con A-induced proliferation and IL-2 production by Lyt-2+ T cells requires intimate contact with accessory cells and interactions dependent upon the class I-restricting element.     

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.     

Subpopulations of human T lymphocytes. VII. Cellular basis of concanavalin A-induced T cell-mediated suppression of immunoglobulin production by B lymphocytes from normal humans.

Purified peripheral blood T lymphocytes from normal subjects were pretreated with varying concentrations of concanavalin A (Con A) for a period of 18 or 48 hr. Following treatment, these T lymphocytes were examined for the proportions of Tμ and Tγ cells and their regulatory effect on immunoglobulin production by normal allogeneic B cells in presence of pokeweed mitogen. A significant decrease in the proportions of Tμ cells and increase in Tγ cells were observed when concentration of Con A, 40 μg/ml, was used to treat purified T cells for either 18 or 48 hr. Significant suppression of in vitro immunoglobulin synthesis was observed at a similar concentration in mixing experiments. The mechanism of Con A-induced T cell-mediated suppression of immunoglobulin secretion by allogeneic B cells is discussed.     

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.     

The anti-T cell monoclonal antibody 9.3 (anti-CD28) provides a helper signal and bypasses the need for accessory cells in T cell activation with immobilized anti-CD3 and mitogens

CD28 is an antigen of 44 kDa which is expressed on the membrane of the majority of human T cells. The present study examines the functional effects of an anti-CD28 monoclonal antibody (mAb 9.3) on T cell activation induced with immobilized anti-CD3 mAb OKT3 or with mitogens, in the absence of accessory cells. To this end, we used blood resting T cells that were completely depleted of accessory cells (monocytes, B cells, and natural killer cells), and consequently did not respond to recombinant interleukin-2 (rIL-2), to immobilized OKT3, to PHA, or to Con A. Addition of mAb 9.3 to the cultures enhanced IL-2 receptor expression (Tac antigen) on PHA- or immobilized OKT3-stimulated T cells and induced IL-2 receptors on Con A-stimulated T cells. Moreover, addition of mAb 9.3 to cultures of T cells stimulated with PHA, Con A, or immobilized OKT3 resulted in IL-2 production. Soluble mAb 9.3 was a sufficient helper signal for T cell proliferation in response to PHA or immobilized OKT3. Crosslinking of mAb 9.3 by culture on anti-mouse IgG-coated plates enhanced the helper effect and was an essential requirement for the induction of T cell proliferation in response to Con A. No other anti-T cell mAb (anti-CD2, -CD4, -CD5, -CD7, -CD8) was found to provide a complete accessory signal for PHA or Con A stimulation of purified T cells. T cell proliferation induced by the combination of PHA and mAb 9.3 was strongly inhibited by the anti-IL-2 receptor mAb anti-Tac. In conclusion, mAb 9.3 can provide a signal bypassing monocyte requirement in T cell activation with immobilized OKT3, PHA, and Con A, resulting in an autocrine IL-2-dependent pathway of proliferation.     

Comparison of mitogenic responses of young and old rhesus monkey T cells to lectins and interleukins 2 and 4

Purified T cells from rhesus monkeys, like human T cells, do not show a significant mitogenic response to lectins or PMA, but when combined with PMA or accessory cells, PHA and Con A induce a vigorous mitogenic response. This response is strongly impaired in purified T cells from old rhesus monkeys compared to young T cells, from 56 to 72%, and parallels results obtained with T cell preparations containing accessory cells. Likewise, purified T cells do not respond to interleukin 2 (IL-2) or IL-4, but in the presence of PMA, a significant mitogenic response occurs in the young but not the old T cells. This response is augmented by accessory cells, but is still very deficient in the old T cells. These results show that the IL-2 independent activation of T cells triggered by IL-4, like the conventional IL-2 activation, is age impaired. The deficient response to IL-2 implies an age-related deficiency in IL-2 receptor as well in aged rhesus T cells, and may account for the less effective response of the old cells to calcium ionophore (+PMA) activation. The use of purified T cells in these studies obviate the influence of accessory cells, and thus simplify interpretation.     

Structure and biological function of human IgD: XV. Biological potential of IgD-bearing cells from cord blood,and normal human adults

Specifically purified intact IgG anti-human δ or F(ab′)₂ anti-δ stimulated the in vitro incorporation of [³H]thymidine by cord blood and adult human peripheral blood lymphocytes. The magnitude of stimulation was unrelated to the increased frequency of IgD-bearing cells observed in cord blood over that seen in adult blood. Purified T cells lacked the capacity to undergo mitogenesis when incubated with anti-δ antibody. Of special interest was the lack of anti-δ enhancement of phytohemagglutinin (PHA) responsiveness of cord blood lymphocytes that is often observed when adult peripheral blood lymphocytes are treated with anti-δ, then PHA. This observation could be interpreted as indicating: (a) that the anti-δ-activated neonatal B cells, or their lymphokines, were preferentially activating a population of suppressor cells, which in turn prevented the PHA-augmentation usually seen with adult cells, (b) that the neonatal B cells do not possess the T cell-augmenting capacity or, (c) that the neonate lacks a subpopulation of T cells that can be acted upon by the B-cell or B-cell factors.     

Modification of immune competence by parasitic infections. I. Responses to mitogens and antigens in mice treated with Trichinella spiralis extract

Spleen cells from mice pretreated with a Trichinella spiralis extract (TsE-mice) showed severe depression of the response to lipopolysaccharide (LPS) and to concanavalin A (Con A), slight depression to phytohemagglutinin (PHA) and normal response to tuberculin purified protein derivative (PPD) as compared to saline-pretreated controls. Mice pretreated with bovine serum albumin (BSA-mice) revealed greatly reduced responses to LPS, somewhat reduced response to Con A, and normal responses to PHA and to PPD. Only TsE-mice showed significant reduction in the number of rosette-forming cells and of direct and indirect plaque-forming cells (DPFC and IPFC). BSA-mice exhibited some reduction of the DPFC only. Direct hemagglutinating (HA) titers were equivalent in the 3 groups after immunization with sheep erythrocytes but facilitated HA titers were depressed in TsE-mice. The total number and the number of viable cells were similar in the spleens of all animals. TsE treatment causes a reduction in the number of T1 lymphocytes and an inhibition of the late differentiation of B cells in the spleen. Suppressor T-cells apparently play a major but not exclusive role in T. spiralis-induced nonspecific immunodepression.     

Identification of a factor(s) from cloned murine natural suppressor cells that inhibits IL-2 secretion during antigen-driven T cell activation.

Crude supernatants were obtained from cloned murine natural suppressor (NS) cells activated in vitro with phorbol-myristate-acetate (PMA) and calcium ionophore (A23187). Supernatants suppressed IL-2 production in the mixed leukocyte reaction (MLR) with BALB/c spleen cells, but no reduction was observed in the response to PHA, Con A, or anti-CD3 antibody. Suppressive activity was partially purified by DEAE ion exchange chromatography, and inhibited the antigen-presenting function of the macrophage line 1G18-LA in an assay system with the ovalbumin-specific T cell hybridoma, 3DO-18.3. In addition, the antigen-presenting function of the A20 B cell line was inhibited in an assay with a sperm whale myoglobin (SpWMb)-specific T cell hybridoma (8.2.1d.H1a). Results with blocking antibodies suggest that this factor appears to be a unique cytokine.     

Properties and applications of monoclonal antibodies directed against determinants of they Thy-1 locus.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.     

Cytotoxic activity of lymphocytes. VII. cellular origin of alpha-lymphotoxin.

To detect the cellular origins of alpha-lymphotoxin (alpha-LT), we cultured various subpopulations of human blood lymphocytes separated by erythrocyte-rosetting techniques with various mitogens. T cell-enriched subpopulations responded to PHA by increased 3H-thymidine uptake into DNA and large amounts of alpha-LT production. SPL and Con A-Sepharose stimulated DNA synthesis in T cell-enriched cultures if the macrophage content was greater than 1.5%; however, alpha-LT production was not induced by these two mitogens even when reconstituted with 10% macrophages. B and/or null cell-enriched populations severely depleted of T cells (less than 0.7% did not respond to PHA, SPL, or Con A-Sepharose. However, reconstitution to 5 or more percent in E-RFC allowed all three mitogens to stimulate DNA synthesis and alpha-LT production. The LT made by all cell populations 5 and 7 days after stimulation were equally neutralized by a heterologous antiserum to alpha-LT. These results show that human T and B and/or null cells, when appropriately stimulated, can produce alpha-LT.     

Differential requirement for accessory cells in polyclonal T-cell activation

Highly purified rat Ia-negative (OX-6-) and Ia-positive (OX-6+) T cells were employed to examine the requirement for accessory cells (AC) and/or soluble factors in the activation of resting T cells with Con A, PHA, sodium periodate, or antigen. A variety of cells were employed as AC, including Ia-positive and Ia-negative macrophages (M phi), gamma-irradiated (2000 rad) or non-irradiated OX-6+ T cells, and several Ia-negative adenovirus-transformed rat embryo fibroblast cell lines. Our results suggested that for the expression of IL-2 receptors (IL-2R) and proliferation of OX-6- T cells in response to Con A, PHA, or antigen, there was an obligatory requirement for the presence of AC which could not be overcome by the addition of IL-1 and/or IL-2. Activation of OX-6- T cells with antigen required the presence of Ia+ AC, while activation with mitogens could be initiated with Ia- AC. M phi were efficient in AC function in all responses tested, while the AC function of OX-6+ T cells (TAPC) proved discriminatory under different conditions. The optimal response to PHA required much higher concentrations of TAPC as AC than for the Con A response. TAPC failed to stimulate sodium periodate-treated T cells under any conditions tested. Furthermore, when TAPC were employed as AC, their antigen-presenting ability was radiosensitive, while their AC function for Con A and PHA was radioresistant. These results suggest that molecules involved in T cell-AC interactions may differ, depending on the source of AC and/or type of the proliferative stimulus provided to T cells. This data has been discussed in the context of T-cell activation.     

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.