Enhancement of DNA synthesis and cAMP content of mouse thymocytes by mediator(s) derived from adherent cells.

Supernatants of adherent mouse peritoneal exudate cells or human mononuclear cells were used as the source of lymphocyte activation factor (LAF). LAF was found to potentiate the effect of mitogens such as PHA and Con A on DNA synthesis by mouse thymocytes. However, LAF also was capable of reducing vigorous thymosyte reactions to Con A. Thus, LAF usually enhanced the effect of PHA on DNA synthesis by BALB/c thymocytes to a relatively greater degree than that of Con A. This change in the ratio of Con A to PHA response of thymocytes suggests that LAF can serve as a regulator of thymocyte DNA synthesis. Moreover, in the presence of LAF, allogeneic thymocytes developed the ability to have bidirectional mixed thymocyte reactions. Exposure to LAF not only improved the ability of parental thymocytes to act as responder cells, but, in addition, led to increased stimulatory activity of F1 thymocytes, presumably by promoting the differentiation of stimulator cells. These indications that LAF affected differentiation were investigated further by studying its effect on the cAMP content of thymocytes. LAF stimulated significant immediate but transient elevations of intracellular cAMP and adenylate cyclase activity in thymocyte membranes. In contrast, the mitogens themselves failed to elevate or to influence the effect of LAF on the content of intracellular cAMP of thymocytes. Furthermore, the potentiating effect of LAF on mitogen-induced thymocyte DNA synthesis at times was enhanced by exogenous cGMP, carbachol, or imidazole. These findings suggest that LAF, through its stimulation of cAMP levels in thymocytes may in turn promote thymocytes to differentiate sufficiently to become competent to proliferative in response to mitogens.     

Effect of T-cell mitogens and T-lymphocyte deficiency on splenic colony formation

A study was made of the influence of T-cell mitogens (Con A and PHA) on the colony formation and differentiation of hemopoietic stem cells from normal and thymectomized mice, as well as of the relationship between the colony formation and the dose of injected thymocytes. The incubation of bone marrow cells with Con A and PHA was shown to inhibit the growth of spleen colonies. This inhibition is reduced by thymocytes within the dose intervals of 0.25-2.0 X 10(7) cells/mouse. Administration of these agents serially has led to the potentiation of inhibition effect and to the inability of thymocytes to reverse it. Con A and PHA exert no effect on the differentiation of stem cells. Incubation of the bone marrow cells from thymectomized mice with Con A is much less effective in the depression of colony formation, if compared with the treatment by intact bone marrow preparations. A reversed picture was observed using antiserum to mouse brain (RAMBS). It is proposed that regulation of stem cells is governed by different subpopulations of thymocytes.     

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.     

Effects of cyclophosphamide and cortisone on the virus-immune response characteristics of thymocytes and the early reconstitution profiles of P → F1 chimeras

Thymocytes from normal adult mice that have been treated 1 or 2 days previously with a low dose of cyclophosphamide (30 or 40 mg/kg), hydrocortisone acetate (2.5 mg), or hydrocortisone succinate (5.0 mg) show an enhanced potential to generate vaccinia-immune cytotoxic T lymphocytes (CTL) when stimulated for 6 days in irradiated, virus-infected recipients. However, the capacity of thymocytes (though not of spleen cells) to develop such CTL activity is lost within 8 days of treatment with the depot drug, hydrocortisone acetate. In contrast, thymocytes from mice treated 8 days previously with hydrocortisone succinate show response patterns equivalent to those found for normal adults, with the increased response characteristic of recent corticosteroid treatment being recalled by giving a second dose 2 days prior to sampling. “Cortisone resistance” is thus both a function of the form of drug given, and the time of sampling. A large dose of cyclophosphamide (150 mg/kg) completely eliminates thymocyte responder potential within 2 days of treatment. This protocol, and the administration of 2.5 mg of hydrocortisone acetate (but not hydrocortisone succinate), serves to remove the radiation-resistant host cells which initially (14 days after bone marrow reconstitution) dominate the CTL response patterns of adoptively transferred thymocytes from P1 → (P1 × P2)F1 bone marrow chimeras. A smaller dose of cyclophosphamide (100 mg/kg) delays the emergence of the radiation-resistant host component in both thymus and lymph node, and skews the emerging CTL activity toward the donor.     

Separation of polypeptides which induce a mitogen response of thymocytes from the culture supernatant of a thymic epithelial cell line

To examine thymic hormonal factors, four polypeptide fractions (estimated molecular weight: I, 10 K; II, 7 K; III, 3 K; IV, 2.5 K) were separated from the culture supernatant of a rat thymic epithelial cell line by high-pressure liquid chromatography (HPLC) with a gel-filtration column. The effects of the fractions on response to mitogens of three small-lymphocyte subsets were studied. All fractions enhanced response to concanavalin A (Con A) of the lighter subset containing mainly immature thymocytes, but only fractions II and IV increased response to phytohemagglutinin (PHA) of the heavier subset containing relatively mature thymocytes. When fraction IV was subfractionated by reversed-phase HPLC, the polypeptides that enhanced response to Con A and PHA were separated into hydrophobic and hydrophilic subfractions, respectively. Fraction I was subfractionated by a similar method, and the inducing activity of Con A response was found in a relatively hydrophobic subfraction. These data suggested that the cell line secretes several kinds of bioactive polypeptides that affect the thymocytes at different stage of maturation.     

Differences in the pattern of proliferative response with age in thymocytes undergoing spontaneous and induced proliferation

The proliferative capacity of thymocytes from C3H/HeJ mice decrease as the animals attain maturity. The proliferative response of thymocytes from 24- to 28-week-old mice to stimulation with concanavalin A (Con A) is only 20% of that observed at 4 weeks of age. The decreased proliferative capacity of thymocytes in response to Con A stimulation observed between 4 and 24 weeks of age closely correlates to the drop in thymic weight and cellularity observed during this period. In contrast, the spontaneous proliferative capacity of thymocytes, as well as proliferation of thymocytes in response to stimulation with phorbol myristate acetate (PMA) and ionomycin, drops only slightly during this period, as proliferation under these condition in thymocytes from 24- to 28-week-old mice is approximately 65-70% of that observed in 4-week-old animals. We have previously shown that cytoplasmic extracts from proliferating lymphoid cells contain a factor, termed the activator of DNA replication (ADR), which is capable of inducing DNA synthesis in isolated, quiescent nuclei. We show in this study that the decreased proliferative capacity of thymocytes during whole organism maturation and thymic involution is associated with decreased endogenous levels of ADR, while nuclear sensitivity of thymocyte to ADR was retained during these process. The diminution of ADR activity during thymic involution was quantitatively greater than the loss in proliferative capacity.     

Defective thymic T cell activation by concanavalin A and anti-CD3 in autoimmune nonobese diabetic mice. Evidence for thymic T cell anergy that correlates with the onset of insulitis.

In nonobese diabetic (NOD) mice, T cells play a major role in mediating autoimmunity against pancreatic islet beta-cells. We and others previously reported that age-related alterations in the thymic and peripheral T cell repertoire and function occur in prediabetic NOD mice. To study the mechanism responsible for these T cell alterations, we examined whether a defect exists in the thymus of NOD mice at the level of TCR-mediated signaling after activation by Con A and anti-CD3. We found that thymocytes from NOD mice respond weakly to Con A- and anti-CD3-induced proliferation, compared with thymocytes from control BALB/c, BALB.B, (BALB.B x BALB.K)F1, C57BL/6, and nonobese non-diabetic mice. This defect correlates with the onset of insulitis, because it can be detected at 7 to 8 weeks of age, whereas younger mice displayed a normal T cell responsiveness. Thymic T cells from (NOD x BALB/c)F1 mice, which are insulitis- and diabetes-free, exhibit an intermediate stage of unresponsiveness. This T cell defect is not due to a difference in the level of CD3 and IL-2R expression by NOD and BALB/c thymocytes, and both NOD CD4+ CD8- and CD4- CD8+ mature thymic T cells respond poorly to Con A. BALB/c but not NOD thymic T cells respond to Con A in the presence of either BALB/c or NOD thymic APC, suggesting that the thymic T cell defect in NOD mice is intrinsic to NOD thymic T cells and is not due to an inability of NOD APC to provide a costimulatory signal. The defect can be partially reversed by the addition of rIL-2 to NOD thymocytes. To determine whether a defect in signal transduction mediates this NOD thymic T cell unresponsiveness, we tested whether these cells elevate their intracellular free Ca2+ ion concentration in response to Con A. An equivalent Con A-induced increase in Ca2+ ion concentration in both NOD and BALB/c thymocytes was observed, suggesting a normal coupling between the CD3 complex and phospholipase C in NOD thymocytes. In contrast to their low proliferative response to Con A or anti-CD3, NOD thymocytes respond normally (i.e., as do BALB/c thymocytes) to the combinations of PMA plus the Ca2+ ionophore ionomycin and PMA plus Con A but weakly to Con A plus ionomycin. Our data suggest that the age-related NOD thymocyte unresponsiveness to Con A and anti-CD3 results from a defect in the signaling pathway of T cell activation that occurs upstream of protein kinase C activation.     

Thymocyte-stimulating factor from peripheral blood cells.

Human peripheral blood leukocytes (HPBL) produce a thymocyte-stimulating factor (TSF-HPBL) that enhances the phytohemagglutinin (PHA) and concanavalin A (Con A) responsiveness of murine thymocytes. This activity is considerably specific for thymocytes. TSF-HPBL is not mitogenic by itself. Experiments with cell cultures pretreated with carbonyl iron particles showed that phagocytic cells are not involved in the production of mouse and rat TSF but are involved in the production of TSF-HPBL. The dose-response profile to PHA of murine thymocytes cultured in the presence of TSF-containing supernatants is similar to that of mature, immunocompetent spleen cells. TSF-HPBL, however, does not enhance the PHA responsiveness of murine thymocytes at low (<0.25 μg/microwell) concentrations of mitogen. TSF enhances the PHA and Con A responsiveness of the high-density subpopulations of thymocytes isolated on a Ficoll-Hypaque gradient. In general, the enhancing effect of TSF-HPBL on these subpopulations of thymocytes is smaller than that exerted by TSF. While supernatants containing TSF confer to thymocytes the ability to participate in a mixed lymphocyte reaction, this effect is not exerted by supernatants containing TSF-HPBL. A factor enhancing the PHA and Con A responsiveness of murine thymocytes is also produced by murine peripheral blood leukocytes (TSF-MPBL). This factor, similarly to TSF-HPBL, is produced by phagocytic cells and does not confer to murine thymocytes the ability to participate in a mixed lymphocyte reaction. Human T-cell lines do not enhance the PHA or Con A responsiveness of murine thymocytes. TSF-HPBL has a molecular weight of about 30,000 daltons, as measured by Sephadex filtration. Its half-time of inactivation as 56 °C is 162 ± 8 min.     

Interaction of levamisole and mercaptans during thymocyte proliferation induced by concanavalin A.

Levamisole enhances[³H]thymidine uptake of murine thymocytes stimulated by concanavalin A (Con A). The proliferative response of thymocytes to Con A can also be enhanced by addition of mercaptans. Six different mercaptans were examined for this effect; three of them, 2-mercaptoethanol, cysteamine, and l-cysteine, stimulated the Con A response. Addition of levamisole to an optimal stimulatory dose of 2-mercaptoethanol or cysteamine resulted in complete inhibition of cell proliferation. Three other mercaptans, penicillamine, d-cysteine, and glutathione, failed to enhance the Con A response and, in fact, were mildly inhibitory. Levamisole gave only slightly less than normal stimulation in the presence of these mercaptans. In the absence of Con A neither levamisole nor the mercaptans stimulated cell proliferation. Oxidized 2-mercaptoethanol reacted analogously to reduced 2-mercaptoethanol both in the presence and absence of levamisole. We have interpreted these results as suggesting that the effect of levamisole is dependent upon the state of activation of the lymphocyte.     

Thymic lymphocytes. III. Cooperative phenomenon in the proliferation of thymocytes under Con A stimulation

In the present paper, the response of thymocytes to Con A is analyzed in terms of a cooperative phenomenon between medullary thymocytes, cortical thymocytes, thymic accessory cells, and interleukin 2. Medullary thymocytes respond spontaneously to Con A and produce IL-2. The addition of exogenously produced IL-2 enhances their proliferation. Small numbers of cortical (PNA+) thymocytes do not respond to Con A, even in the presence of IL-2-containing supernatant. By increasing the number of PNA+ cells per well, sensitivity to Con A and IL-2 appears. This response may be linked either to the increase in a minor PNA+-responding population and/or to the enhanced contamination by medullary thymocytes and macrophages in non-responding PNA+ thymocyte population. In this hypothesis, either the contaminating cells respond by themselves and/or cooperate with PNA+ cells to induce their proliferation. Coculture of non-responding low numbers of PNA+ thymocytes with Con A- and IL-2-containing supernatant in the presence of PNA- cells containing thymic medullary thymocytes and macrophages always produces a higher response than that of each individual population. These results show that a cooperative phenomenon occurs in the cocultures of PNA+ and PNA- thymic cells. We can show using PNA+ and PNA- thymocytes with different Thy 1 alleles, that indeed both PNA+ and populations participate PNA-thymocytes with different Thy 1 alleles, that indeed both PNA+ and PNA- populations participate in the generation of proliferating cells. We can demonstrate, by lysis experiments with monoclonal antibodies and complement that at the end of coculture, most of the proliferating cells are Lyt 1+, and part are Lyt 2+ or L3T4+. We discuss the fact that the phenotype of the cells after activation does not allow us to deduce the phenotype of their precursors. Lysis of Ia+ cells prior to coculture, reduces the level of the proliferative response but does not modify the percentage of cooperation produced by the coculture. Cooperation with medullary mature thymocytes or the presence of active Ia- accessory cells possibly able to convert to Ia expression during coculture experiments may account for these results.     

T and B lymphocyte populations of the spleen and thymus in normal and immunosuppressed BALB/c mice.

The antituinor agent 1,3 bis (2-chloroethyl)-1-nitrosourea (BCNU) has been studied in order to determine its effect on thymic and splenic T and B lymphocytes in normal and immunosuppressed BALB/c mice. Utilizing indirect immunofluorescence and lymphocyte proliferation studies we detected an initial reduction of splenic T and B cells as a result of the administration of an optimal dose, 30 mg/kg, of BCNU. The population dynamics of the thymic lymphocytes are totally different in their mitogenic reactivity than that of the splenic lymphocytes. An initial decline in the PHA and LPS-sensitive splenic lymphocytes of BCNU-treated mice was temporary. However, there was no return to normal levels detected for the Con A-sensitive splenic lymphocytes. On the other hand, the PHA-sensitive thymic lymphocytes of BCNU-treated mice not only failed to repopulate but were totally depleted by the tenth day.     

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.     

Beta-endorphin modulation of mitogen-stimulated calcium uptake by rat thymocytes

Lymphocytes stimulated by mitogens or antigens exhibit an enhanced calcium uptake early in the proliferation or activation response. Modulation of this calcium uptake results in alterations of proliferation and immunocompetence. beta-endorphin and other opioids affect several parameters of lymphocyte competence. Limited data are available concerning the mechanism(s) of these effects. This study examines whether a possible opioid mechanism is the modification of the early calcium influx into stimulated lymphocytes. The time course of both concanavalin A (Con A) and phytohemagglutinin (PHA)-stimulated 45Ca2+ uptake into thymocytes was characterized to determine the optimal time for testing the effects of opioids. beta-Endorphin 1-31 significantly enhanced Con A-stimulated 45Ca2+ uptake into rat thymocytes. This peptide had no significant effect on PHA-stimulated 45Ca2+ uptake or on basal thymocyte 45Ca2+ flux. The beta h-endorphin stimulatory effect was titratable in the range of 0.1 nM to 10 microM. Naloxone did not reverse the enhancement. Met-enkephalinamide and other opioid agonists did not duplicate the stimulatory effect. Thus, the beta h-endorphin 1-31 enhancement of Con A-stimulated 45Ca2+ uptake by rat thymocytes does not operate via classical opioid receptor mechanisms. beta h-endorphin 1-31 appears to be acting on a subset of T cells that are responsive to Con A but not to PHA.     

Muramyl dipeptide induced augmentation of the proliferative response of thymocytes to phytohemagglutinin

N-Acetylmuramyl-L-alanyl-D-isoglutamine (MDP) augmented the proliferative response of thymocytes to phytohemagglutinin (PHA). The augmenting effect of MDP disappeared by passage of glass-nonadherent thymocytes through Sephadex G-10 (G-10) column or by removal of low density cells by the Ficoll-Conray gradient centrifugation. The diminished augmenting effects of MDP on the proliferative response of glass-nonadherent-G-10 nonadherent thymocytes was restored by the addition of the G-10 adherent cells. When G-10 adherent cell fraction was extensively depleted of macrophages by glass adherence and EA-rosetting, it was found that neither the macrophage-depleted G-10 adherent cell fraction nor the macrophage fraction supported by itself the proliferative response of G-10 nonadherent thymocytes. However, addition of macrophage fraction together with the macrophage-depleted G-10 adherent cells did support the proliferation of G-10 nonadherent thymocytes. It was further shown that peritoneal exudate macrophages could be substituted for thymic macrophage fraction. These results suggested that both the G-10 adherent-glass nonadherent cells and macrophages were essential for the MDP-induced augmentation of the proliferative response of thymocytes to PHA and these cells exerted different accessory roles in this response.     

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.     

Effect of hydrocortisone on peripheral blood phagocytic cells under beta-adrenoreceptors blockade

The effect of hydrocortisone (50 mg/kg body wt i.p.) under beta-adrenergic receptors blockade (four subcutaneous injections of propranolol in single dose of 5 mg/kg body wt with 3 h interval) on phagocytic activity and oxygen dependent microbicidal activity in NBT-test of peripheral blood phagocytic cells in male Wistar rats was investigated. It was established that hydrocortisone stimulated neutrophil phagocytic activity through 6, 24 and 48 h after hormone injection and decreased oxygen-dependent microbicidal activity of phagocytic cells in NBT-test. Hydrocortisone in vitro (500 ng/ml) decreased neutrophil phagocytic activity that indicated on realization of stimulating effect of hydrocortisone in vivo through complex of other indirect mechanisms. Administration of hydrocortisone led to depression of eosinophil phagocytosis and lesser decrease in monocyte phagocytic activity. Hydrocortisone effects were significantly modified under blockade of beta-adrenoceptors that indicated on its mediation by endogenous catecholamines through modulation of beta-adrenoceptor expression.     

In vitro proliferation of blood,spleen and thymus leukocytes from the turtle Mauremys caspica

Cell-mediated immune responses is commonly evaluated by cell proliferation assays. Mitogens are known to induce a vigorous proliferative response in lymphoid cells from mammals but relatively fewer studies have investigated mitogen-mediated lymphoproliferation in non-mammalian animals. In the present work, we incubated spleen, thymus and blood leukocytes with phytohaemagglutinin (PHA), concanavalin A (Con A), lipopolysaccharide (LPS) and pokeweed mitogen (PWM), by different times of incubation (96 and 120 h) and at different concentrations. Our results show that the optimal mitogen concentrations inducing proliferation on leukocytes from Mauremys caspica were 20 microg/ml PHA, 1 microg/ml Con A, 12.5 microg/ml LPS and 1/150 dilution PWM. The optimal time of incubation was dependent on the type of leukocytes (peripheral blood leukocytes, splenic leukocytes or thymic cells) and the mitogen utilized.     

Effects of costimulator on immune responses in vitro.

We recently described a factor, costimulator, that is required for the concanavalin A-induced proliferation of CBA mouse thymocytes in vitro (see Reference 1). Using the costimulator dependence of mouse thymocytes as an assay, we have now determined that spleen cells from congenitally athymic (nude) BALB/c mice do not produce costimulator in response to Con A, and spleen cells depleted of Thy 1-positive cells do not respond to it in the presence of Con A. Thus, costimulator both requires thymus-derived (Thy 1+ lymphocytes for its production and has an effect on this type of cell. (However, the costimulator-producing and responsive cells may be different.) Purified costimulator preparations are a source of the required second component for the stimulation of adult, CBA/J thymic lymphocytes by PHA, normally a poor mitogen for these cells. They also enhance the level of DNA synthesis in a mixed leukocyte reaction, and the specific generation of cytotoxic lymphocytes to allogeneic tumor cells in vitro. Costimulator is not H-2 restricted in its effects, and it is produced in mixed leukocyte reactions. Finally, it has been possible to grow normal, primary thymic lymphocytes in culture for about 20 days by adding partially purified costimulator to the cultures.     

Selection of CD4+CD8+ thymocytes by complex formation with medulla-derived epithelial cells

The role of lymphostromal complexes in T-cell differentiation is far from elucidated, mainly because a clear association of a particular stromal cell type with a distinct thymocyte subset has never been identified. Using an in vitro system, detecting the adherence of thymocytes to a thymic medullary epithelial cell line (E-5), we showed that the phenotype of these thymocytes was that of cortical type: Thy-1hi, LFA-1+, PNAhi, CD4+CD8+, MEL-14-/lo, IL-2R-, CD3-/lo, and TcR V beta 8-/lo. They were enriched in cells in G2/M at the time of complex formation, showed a higher basal proliferation in culture, and did not respond to PHA, IL-2 and only marginally to Con A. These data show that complex formation with mouse thymic medullary epithelium selects for CD4+CD8+ thymocytes, as shown by the marked decrease in CD4+CD8-/CD4-CD8+ thymocytes, and the incapacity of CD4-CD8- thymocytes to adhere.     

Differential suppressive effects of antiserum to thymus ribosomal fraction on mitogen responsiveness of thymus cells

The effect of antiserum to thymus ribosomal fraction (ATRS) on the responsiveness of thymus cells to mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) was investigated. ATRS preferentially suppressed the responsiveness of thymus cells to Con A whereas their PHA responsiveness remained unchanged. In contrast, thymus cells from mice treated with a single injection of cortisone acetate showed significant increases in thymidine uptake when stimulated by both PHA and Con A.