Limiting dilution analysis of T helper cell heterogeneity: a single class of T cell makes both IL 2 and IL 3

We have previously described a limiting dilution assay (LDA) for estimating the frequency, in the spleens of unprimed mice, of alloantigen-responsive cells that, together with their immediate clonal progeny, can produce interleukin 2 (IL 2) in short-term culture. In this paper, we provide further evidence that the limiting cell in these cultures is, in fact, the immediate precursor of the IL 2-producing cell rather than merely a participant in a multicellular cascade that ultimately leads to production of this lymphokine. We also demonstrate the usefulness of the LDA method for estimating the frequency of the limiting cell for production of IL 3. By analyzing the supernatants of these short-term microcultures for both IL 2 and IL 3, we show that essentially all wells that produce IL 3 also produce IL 2. Furthermore, the amount of IL 2 produced in any individual well correlates strongly with the IL 3 production in the same well. Our data suggest that both these lymphokines are produced by a single class of T cell, and further that the regulatory events that control IL 2 generation in allostimulated helper cells act to control IL 3 levels in parallel.     

Generation of alloreactive cytotoxic T lymphocytes: production of T cell and macrophage helper factors in addition to IL 1 and IL 2 by peritoneal cells from mice immunized to Listeria monocytogenes

We investigate the production and biological activity of soluble helper factors produced by peritoneal T cells and macrophage derived from mice primed in vivo with Listeria monocytogenes. Supernatant fluids from co-cultures of these immune T cells and activated macrophages contained Interleukin 1 (IL 1) and Interleukin 2 (IL 2), and had the ability to assist the generation of cytotoxic T lymphocytes (CTL) from a population of nylon wool nonadherent spleen cells sensitized to allogeneic heat-treated thymocytes. The ability to assist CTL development involved T cell and macrophage factors in addition to IL 1 and IL 2. Immune T cells cultured alone produced a factor, devoid of significant IL 2 activity, that assisted CTL development only if adherent cells were present in the responding population. Activated macrophage produced a 38,000 dalton component, distinct from IL 1 on the basis of m.w., that assisted the development of CTL from nylon wool nonadherent splenic cells. Supernatants fluids from co-cultures of immune T cells and allogeneic, nonactivated macrophage contained a CTL helper factor but did not contain IL 1 or IL 2 activities. In contrast, supernatant fluids from co-cultures of immune T cells and syngeneic, nonactivated macrophage contained all 3 activities. This suggests a genetic restriction for the production of IL 1 and IL 2 that does not restrict the production of a CTL helper factor. These results demonstrate that T cell- and macrophage-derived helper factors distinct from IL 1 and IL 2 participate in the development of CTL.     

Antigen-reactive cloned helper T cells. II. Exposure of murine cloned helper T cells to IL 2-containing supernatant induces unresponsiveness to antigenic restimulation and inhibits lymphokine production after antigenic stimulation

Cloned murine helper T lymphocytes (HTL) reactive to alloantigen or to ovalbumin (OVA) become unresponsive to antigenic restimulation after exposure to antigen or to culture supernatant fluids (SF) containing multiple lymphokine activities. Unresponsiveness is manifest by a failure of antigen-stimulated cells to incorporate thymidine or to produce lymphokines after antigenic challenge. Antigen-unresponsive HTL, however, will incorporate thymidine when exposed to an exogenous source of interleukin 2 (IL 2). The duration of unresponsiveness to antigen is correlated with the concentration of IL 2 in SF to which the cloned HTL had been exposed. Chromatographic fractionation of IL 2-containing supernatant from EL-4 thymoma cells (EL-4 SF) yielded a pool of SF that was enriched for IL 2 activity. Exposure of HTL to lymphokines contained in this pool induced unresponsiveness to antigen that was comparable to that observed when HTL were exposed to unfractionated EL-4 SF. Unresponsiveness to antigen also developed after cloned HTL were stimulated with concanavalin A (Con A) or with OVA and syngeneic splenic filler cells. We have used monoclonal antibody (mAb) GK1.5 (anti-L3T4) to investigate the role of lymphokine production in the induction of unresponsiveness. This antibody did not inhibit IL 2-induced thymidine incorporation by cloned HTL, and did not inhibit the induction of unresponsiveness after exposure of cloned HTL to EL-4 SF. In the presence of mAb GK1.5, however, HTL that were stimulated with Con A or OVA did not become unresponsive to antigenic restimulation, an effect that was overcome by the addition of EL-4 SF. These results suggest that HTL become unresponsive to antigen after exposure to IL 2-containing SF, and that stimulation by antigen or Con A can induce the unresponsive state by virtue of stimulating lymphokine production.     

Retrovirus-mediated immunosuppression. II. FeLV-UV alters in vitro murine T lymphocyte behavior by reversibly impairing lymphokine secretion

Murine splenocytes and cloned murine T cells were used to study the in vitro immunosuppressive effects of UV-inactivated feline leukemia virus (FeLV-UV) on lymphokine secretion. FeLV-UV can significantly depress the accumulation of IL 2 in cultures of Con A-stimulated C57BL/6 splenocytes and in cultures containing the alloreactive helper T cell clone B6D/2-2m plus Con A. Inhibition of lymphokine accumulation in these cultures could not be attributed to absorption or inactivation of IL 2 by the FeLV-UV or to the FeLV-UV-induced production of substances which interfere with the IL 2 bioassay. Thus, FeLV-UV appears to block production and/or secretion of IL 2 by a direct inhibitory effect on IL 2-secreting murine T lymphocytes. Additional studies indicate that FeLV-UV impairs IL 2 production only if added very soon after lymphocyte contact with lymphokine-inducing agents and that IL 2 secretion resumes when FeLV-UV is removed from the culture. FeLV-UV also impairs accumulation of MAF (interferon-gamma?) in cultures of Con A-stimulated C57BL/6 splenocytes and in cultures containing the alloreactive cytotoxic T lymphocyte clone B6D/2-7c plus Con A. The latter observation again suggests that FeLV-UV impairs lymphokine secretion by a direct effect on lymphokine-producing T lymphocytes. Furthermore, it suggests that FeLV-UV does not selectively impair production of IL 2 nor does it have selective inhibitory effects on helper T cells. Rather, FeLV-UV appears to have a general inhibitory effect on lymphokine production by T lymphocytes. Finally, concentrations of FeLV-UV which suppress MAF production by the CTL clone have little influence on the cytolysis mediated by the same cloned T cell population. Thus, the immunosuppressive influence of FeLV-UV is selective for phenomena associated with induction of new T lymphocyte functions, such as lymphokine secretion, and spares other immune functions already expressed by the same cells.     

Differential effect of anti-beta 2-microglobulin on IL 2 production and IL 2 receptor expression in the primary mixed lymphocyte culture reaction

The mechanism of inhibition of the proliferative response in primary mixed lymphocyte culture (1 degree MLC) by antibodies to beta 2-microglobulin (beta 2m) was investigated. It is demonstrated that anti-beta 2m antibodies inhibit the production of interleukin 2 (IL 2). In contrast, the expression of IL 2 receptor is not affected by anti-beta 2m. The addition of purified exogenous IL 2 to the antibody-treated 1 degree MLC can completely restore the proliferative response, indicating that anti-beta 2m does not interfere with IL 2 binding to its receptor. Similarly, anti-beta 2m does not interfere with the capacity of IL 2-dependent T cell lines or T cell clones to respond to exogenous IL 2. The inhibition of cell proliferation and IL 2 production by anti-beta 2m is maximal when the antibody is added at the beginning of 1 degree MLC culture, and no effect of anti-beta 2m is seen when added after 3 days of culture. Anti-beta 2m has no effect on mitogen-induced cell proliferation and IL 2 production. Anti-beta 2m acts on the responder cell population, as demonstrated in experiments in which responder cells or stimulator cells are treated separately with the antibody. The expression of HLA-class II antigens (i.e., HLA-DR and DQ (DC) on the T cells activated on 1 degree MLC is not affected by anti-beta 2m. These studies indicate that the HLA-beta 2m class I antigen complex plays a role in T lymphocyte activation via release of IL 2, and suggest the existence of different mechanisms for activation of IL 2 producers and IL 2 responders in 1 degree MLC.     

2'Deoxycoformycin and deoxyadenosine affect IL 2 production and IL 2 receptor expression of human T cells

Congenital deficiency of the enzyme adenosine deaminase (ADA) leads to severe combined immunodeficiency. 2'Deoxycoformycin (dCF), a tightly binding inhibitor of ADA, can induce the metabolic state of ADA deficiency. In vivo, the drug causes specific impairment of lymphocyte function and shows strong immunosuppressive properties. However, to decide whether inhibition of the enzyme ADA offers an attractive approach for immunosuppressive therapy, more information is needed about the immunologic mechanisms affected. In human T cells, we investigated the effect of dCF and deoxyadenosine (AdR) on cell activation, interleukin 2 (IL 2) production, and IL 2 receptor induction after allogeneic and lectin-induced stimulation. After allogeneic stimulation, dCF and AdR affected several events in T cellular immune response. Early events in T cell activation showed to be most sensitive to the drugs. Primary MLC was completely inhibited by concentrations as low as 1 microM dCF and 1 microM AdR. The addition of human recombinant IL 2 (rIL 2) could not abrogate the inhibitory effect of the drugs. Apart from activation of T cells, the drugs interfered with proliferation of activated T cells. Two events in activated T cells were affected: IL 2 production and IL 2 receptor expression. In secondary MLC, IL 2 production was markedly reduced in the presence of 9 microM dCF and 60 microM AdR. These concentrations appeared also to affect IL 2 receptor expression in 12-day primary MLC cells stimulated with rIL 2. Lectin stimulation was also affected by the drugs. In phytohemagglutinin (PHA)-stimulated cultures, 9 microM dCF and 60 microM AdR resulted in inhibition of proliferation and IL 2 receptor expression, whereas IL 2 production was normal. It is concluded that dCF and AdR interfere with several events in T cellular immune response such as cell activation, IL 2 production, and IL 2 receptor expression. According to these results, inhibition of the enzyme ADA seems an attractive approach to immunosuppressive therapy.     

Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins

A panel of antigen-specific mouse helper T cell clones was characterized according to patterns of lymphokine activity production, and two types of T cell were distinguished. Type 1 T helper cells (TH1) produced IL 2, interferon-gamma, GM-CSF, and IL 3 in response to antigen + presenting cells or to Con A, whereas type 2 helper T cells (TH2) produced IL 3, BSF1, and two other activities unique to the TH2 subset, a mast cell growth factor distinct from IL 3 and a T cell growth factor distinct from IL 2. Clones representing each type of T cell were characterized, and the pattern of lymphokine activities was consistent within each set. The secreted proteins induced by Con A were analyzed by biosynthetic labeling and SDS gel electrophoresis, and significant differences were seen between the two groups of T cell line. Both types of T cell grew in response to alternating cycles of antigen stimulation, followed by growth in IL 2-containing medium. Examples of both types of T cell were also specific for or restricted by the I region of the MHC, and the surface marker phenotype of the majority of both types was Ly-1+, Lyt-2-, L3T4+, Both types of helper T cell could provide help for B cells, but the nature of the help differed. TH1 cells were found among examples of T cell clones specific for chicken RBC and mouse alloantigens. TH2 cells were found among clones specific for mouse alloantigens, fowl gamma-globulin, and KLH. The relationship between these two types of T cells and previously described subsets of T helper cells is discussed.     

Antibodies to common leukocyte antigen p220 influence human T cell proliferation by modifying IL 2 receptor expression

The p220 antigen is found on the high m.w. form of the T200 common leukocyte antigen family. Although T200 monoclonal antibodies (MAb) react with all hematopoietic cells, p220 MAb react only with B cells, NK cells, about 50% of CD4+ T helper cells, and about 90% of CD8+ T suppressor cells. The p220 antigen appears to play an important role in T cell activation, because anti-p220 MAb at doses as low as 5 ng/ml accelerate the proliferation kinetics of PHA-stimulated T cells and augment anti-CD3-driven proliferation when IL 2 is in excess. Fab fragments have no effect on PHA-stimulated cells but partially block proliferation in response to anti-CD3. Our data suggest that p220 is functionally related to expression of the IL 2 receptor. Anti-p220 MAb cause an increase in the number of T cells that express the IL 2 receptor early after activation. In addition, T cells begin to turn off expression of p220 after activation, and two-color immunofluorescence shows that by day 3 after activation the cells expressing the most IL 2 receptor have the least p220. The loss of p220 on T cells may reflect a post-thymic differentiation process related to cell activation. Our data are consistent with a model where the p220- T cells in peripheral blood are a more activated population of T cells that have lost p220 and its ability to regulate their IL 2 receptor expression.     

IL 1 induction by murine T cell clones: detection of an IL 1-inducing lymphokine

T cell activation is widely believed to depend on interleukin 1 (IL 1) provided by antigen (Ag)-presenting cells (APC). Because IL 1 is not a constitutive product of APC, we examined the features of its production during the interaction of murine T cell clones and APC. We observed that IL 1 was detectable in supernatants of most myoglobin-specific T cell clones grown with APC and Ag. Two of these T cell clones induced exceptionally high levels of IL 1 in their supernatants, and these same clones demonstrated the unusual restriction to I-Ek, which is a low responder type for sperm whale myoglobin. One of these clones was characterized additionally as to the mechanism of IL 1 induction. This clone rapidly stimulated IL 1 production in the APC population (detectable at 4 hr of co-culture) or in macrophages (M phi) or a M phi-like cell line. IL 1 induction was Ag dependent and H-2 restricted. Induction was radioresistant, both on the part of the T cell and of the IL 1 producer. The IL 1-induction process was attributable to a lymphokine produced by the T cell clone. This lymphokine was distinct from IFN-gamma, TNF and CSF-1 and may account for a principal mechanism of T----APC signalling. The induced IL 1 was the same in size, co-mitogenicity, and pyrogenicity as lipopolysaccharide-induced IL 1.     

Identification of a cord blood T cell subset of CD3+4-8-45R+ suppressing interleukin 2 production in the autologous mixed lymphocyte reaction and the mode of action of exogenous IL2 in the induction of IL2 production

As previously reported, the inability of cord blood T cells to produce IL2 in the autologous mixed lymphocyte reaction (AMLR) could be recovered by the treatment of stimulator non-T cells with interferon-gamma (IFN-gamma) and of the AMLR with exogenous IL2. In the present study, we showed that addition of untreated autologous cord blood T cells to the above-mentioned AMLR abrogated the IL2 production in a dose-dependent manner, suggesting active suppression by the untreated T cells because untreated cord blood T cells did not consume IL2. Suppressor activity was abrogated by the treatment of cord blood T cells with monoclonal anti-CD3 antibody plus complement or with monoclonal anti-CD45R (Leu 18) antibody, but not by the treatment with monoclonal anti-CD4 antibody and/or anti-CD8 antibody plus complement. These data showed that the cord blood suppressor T cells were CD3+4-8-45R+. This suppressor activity also disappeared by culturing with rIL2 for 8 hr. As the frequency of CD45R+ cord blood T cells was comparable to that of CD45R+ adult T cells and was minimally affected by the IL2 treatment, functional modulation of CD45R+ suppressor T cells by IL2 is suggested. Moreover, in spite of the inhibitory effect of anti-CD45R antibody on the suppressor activity, IL2 production was not induced merely by addition of anti-CD45R antibody directly to the responder cells in AMLR. Taken together, these data suggest the requirement of exogenous IL2 for IL2 production in that IL2-producing-precursor T cells themselves should be stimulated by IL2 in addition to the modulation of CD45R+ suppressor T cells by IL2.     

Human B cell-inducing factor(s) for production of IgM, IgG and IgA: independence from IL 2

Human peripheral blood B cells are stimulated into proliferation by killed Staphylococcus aureus bacteria strain Cowan I (Sac). T lymphocytes in the presence of a T cell mitogen induce high numbers of immunoglobulin-secreting cells (ISC) in these Sac-stimulated B cells. The T cells can be largely replaced by a lymphokine factor. We describe here the 11000-fold purification of this B cell-inducing factor (BIF). BIF preparations that are free of IL 2 do not require IL 2 for optimal induction of ISC. This was shown by the lack of effect of IL 2 alone or with suboptimal or optimal concentrations of BIF on the induction of ISC and by the absence of IL 2 production in the purified B cell population which, with other controls, excludes significant T cell contamination. BIF, purified through four fractionation steps and free of IL 2, induces IgM, IgG, and IgA-ISC in approximately the same ratio as unfractionated lymphokine. Because we have not yet attained a pure BIF preparation, the possibility of separate factors for the production of each immunoglobulin isotype cannot be ruled out.     

Estimation by limiting dilution analysis of human IL 2-secreting T cells: detection of IL 2 produced by single lymphokine-secreting T cells

We present here a culture method for the estimation, in human blood, of the number of lymphocytes that can respond to mitogen by producing interleukin 2 (IL 2). T cells are cultured at limiting dilutions with PHA or Con A in the presence of Epstein Barr virus-transformed human lymphoblastoid cells (EB-LCL), and supernatants are tested 3 days later for IL 2 content by a cell proliferation assay. The distribution of negative wells follows the expected Poisson "single-hit" relationship, suggesting that the assay is sensitive to single cells of a single limiting cell type. On average, 16.3% of peripheral blood mononuclear cells can produce IL 2 in such clonal cultures (mean of 12 determinations; SD = 5.6%). Surprisingly, irradiation (up to 2000 rad) of the titrated responder cell population diminishes the estimated frequencies by less than 50%. The ability to detect IL 2 levels in cultures containing only a single, nonproliferating T lymphocyte allows us to estimate the amount of IL 2 generated by an individual effector cell during a 3-day culture interval after mitogen stimulation. The average responding, irradiated T cell generates 0.92 pg of IL 2 (median) within 3 days. The method presented provides a straightforward way to provide independent estimates of responding cell number and of lymphokine production per cell in a variety of clinical situations.     

Demonstration of up-regulated IL 2 receptor expression on an in vitro cloned BCL1 subline

We have established BCL1 CL-3 cells capable of responding to B15-TRF and interleukin 2 (IL 2). This clone has both high affinity and low affinity receptors for IL 2 (IL 2R), but IL 2 by itself did not stimulate either proliferation or immunoglobulin (Ig) secretion. B15-TRF, which possesses both growth and differentiation activity, causes an increase in size of CL-3 cells and renders CL-3 cells responsive to IL 2, including an increased expression of IL 2R (eight-fold to 10-fold) and the differentiation of CL-3 cells into Ig secretion (60 to 80% of cultured cells). CL-3 cells pretreated with B15-TRF for 12 hr become competent to respond to IL 2 by up-regulation of IL 2R within 12 hr. In contrast CL-3 cells pretreated with IL 2 for 12 hr required 24 hr B15-TRF stimulation to result in IL 2R up-regulation. Thus the ordered action of B15-TRF and IL 2 is the most effective operational pathway for the up-regulation of IL 2R. This IL 2-mediated IL 2R up-regulation and induction of Ig synthesis depends upon the concentration of IL 2 in the culture. Both responses seem to be caused by IL 2 molecules bound to high affinity IL 2R. However, the possibility of involvement of low affinity IL 2R can not be vigorously excluded. In fact the level of IL 2 required for a response is far higher than that needed for activated T cell proliferation. This cloned BCL1 subline promises to be a useful tool for studying the regulation and mechanisms of B cell responses.     

IL 2 restores memory B cell activation by antigen-specific T cell clone variants

It has recently been demonstrated that there are at least two separate pathways by which a single keyhole limpet hemocyanin (KLH) reactive T cell clone can induce B cell differentiation. With the use of the high-dose antigen-driven system (10 micrograms/ml trinitrophenyl (TNP)-KLH), a KLH-specific T cell clone was able to induce a primary anti-TNP response in unprimed B cells. In the presence of aliquots of the same T cell clone, a low-dose of antigen (5 X 10(-2) micrograms/ml TNP-KLH) induced an immunoglobulin (Ig)G response in primed B cells. It has also been demonstrated that there are variant subclones of such KLH-specific helper T cell clones that are unable to provide antigen-specific help in the presence of low-dose antigen but maintain the high-dose antigen-driven helper response. This study was undertaken to investigate whether interleukin 2 (IL 2) had some activity in the low-dose, antigen-driven response induced by the T cell clone. With the use of a variant T cell clone (which lost low-dose, antigen-driven helper activity), it was demonstrated that IL 2 was capable of reconstituting the low-dose, antigen-driven helper activity. To investigate whether accessory cells were required in this system, we removed the adherent cell population from the primed spleen cells added to culture. Interestingly, removal of the G10-adherent cells eliminated the low-dose, antigen-driven response induced by IL 2. Additionally by add-back experiments, we were able to demonstrate that the necessary adherent cell population did not require major histocompatibility complex (MHC) restriction for reconstitution of the IL 2-dependent, low-dose, antigen-driven response. Furthermore, 1% concanavalin A (Con A) supernatant (Sn), but not interleukin 1 (IL 1), could replace this adherent cell function. These data suggest that in this system, IL 2 bypasses the MHC-restricted interaction between T cells and antigen-charged adherent cells; B cells can present antigen to cloned helper T cells efficiently for primary responses but need an added factor(s) to induce IgG production; and adherent cells are essential for IgG production in primed B cells, possibly through the release of soluble factor(s) included in Con A Sn.     

IL 2 receptor induction on human T lymphocytes: role for IL 2 and monocytes

In this report we studied the requirements for the activation and proliferation of highly purified human T lymphocytes. Purified T cells incubated for 3 days with PHA neither proliferate nor express IL 2 receptors as detected by FACS analysis with the use of anti-Tac antibodies. However, purified T cells incubated with Con A or anti-T3 moAb do not proliferate, albeit 30 to 35% T cells express Tac epitopes. The addition of IL 2, either natural purified or recombinant, resulted in both the appearance of Tac antigen and the proliferation of PHA-activated T cells. Much to our surprise, IL 2 did not induce proliferation of Tac-positive T cells activated by Con A or soluble anti-T3 unless monocytes were added to the cultures. These data suggested that two classes of IL 2 receptors might exist on T cells, one of which was not functionally involved in T cell proliferation. In keeping with this interpretation, we have been able to demonstrate, using a radiolabeled IL 2 binding assay, that anti-T3 moAb induced almost exclusively IL 2 receptors of low affinity (Kd = 30 to 70 X 10(-9) M) and that additional signals, provided by monocytes, are required for the acquisition of high affinity receptors. IL 2 itself can induce high affinity receptors on PHA-stimulated T cells but not on cells activated by Con A or anti-T3. In this latter case the physical presence of monocytes is required and cannot be substituted by IL 1, thus indicating a previously unreported role for monocytes. It is postulated that the contact of monocytes with T cells induces a switch from an inactive low affinity conformation of the IL 2 receptor to a functional high affinity one.     

Enhancing effect of IFN-gamma on helper T cell activity and IL 2 production

A single injection of young murine immune interferon (IFN-gamma) in young (3 mo) or old (14 to 24 mo) mice 3 days before carrier-priming significantly enhances helper T cell activity of their spleen cells. Maximal enhancement is attained when IFN-gamma is injected once immediately before priming or for 4 consecutive days from the time of priming. Helper activity for anti-TNP antibody response was titrated in vitro by adding graded numbers of spleen cells from HRBC-primed mice of a given age to cultures containing a constant number of spleen cells from 3-mo-old normal mice and TNP-HRBC. When T cell-enriched spleen cells from HRBC-primed young or old mice, uninjected or injected with IFN-gamma, were separated by nylon wool filtration into passed (Thi) and adherent (Th2) cells, the helper activity of both T cell subpopulations was found to be enhanced by IFN-gamma injection. Helper activity of purified Th1 and Th2 cells was also increased by their in vitro preincubation with IFN-gamma. Furthermore, interleukin 2 (IL 2) production by mitogen-activated spleen cells from young and old mice is enhanced by addition of IFN-gamma to cultures. These data altogether indicate that IFN-gamma plays an important role in immunoregulation of helper T cell activity.     

Mechanism of action of Cyclosporin A: inhibition of lymphokine secretion studied with antigen-stimulated T cell hybridomas

We have employed bifunctional T cell hybridomas, which can be stimulated to secrete lymphokine(s) and lyse specific target cells, to analyze the effect of Cyclosporin A (CsA) on T cell helper and effector functions. We report here the effects of CsA on antigen- and lectin-induced lymphokine secretion. We have found that a pharmacologic level of CsA (10 ng/ml) blocks antigen- and lectin-driven interleukin 2 (IL 2) secretion without affecting cell proliferation. In addition, one monoclonal hybridoma that is induced by concanavalin A to secrete colony stimulating factors (CSF) as well as IL 2 is concomitantly blocked by CsA for production of IL 2 and CSF. Because the hybridomas grow constitutively and are devoid of functional IL 2 receptors, they permit analysis of the kinetics of the inhibitory response. We have shown that CsA blocks not only stimulation of lymphokine secretion but also ongoing IL 2 production, probably by interfering with the effective interaction of receptor and antigen. Thus, blocking of IL 2 secretion from preactivated cells by CsA occurs by 1 to 2 hr, the time required to stop IL 2 production by removal of Ag/Lectin stimulator. The results are consistent with a mechanism of action of CsA on T cells that involves a direct interference of CsA with binding of Ag to Ag-receptor and results in blocking of induction and active secretion of multiple lymphokines.     

T cell synergy in the primary MLR: proliferative kinetics, effector cell generation, and IL 2 production

A primary rat MLR was initiated, and on each of 8 consecutive days during the evolving culture, an aliquot of cells was separated into its constitutive helper/inducer (W3/25+) and suppressor/cytotoxic (OX8+) T cell subsets by a monoclonal antibody, Degalan-bead immunoadsorbent column technique. This allowed a detailed kinetic analysis of T cell proliferation, the generation of effector cells, and the production of IL 2 by each subset relative to net whole culture supernatant IL 2 activity. The primary MLR demonstrates an early period of helper/inducer cell proliferation, IL 2 production and accumulation, followed by a period of suppressor/cytotoxic cell (OX8+) proliferation and IL 2 consumption during which there are distinct waves of allospecific suppressor, followed by cytotoxic activity. If fresh T cells of the helper/inducer or suppressor/cytotoxic phenotype were preseparated and then cultured alone with irradiated allogeneic stimulator cells, proliferation was noted in both subsets despite no demonstrable IL 2 activity in cultures of the suppressor/cytotoxic cells. Finally, a suppressed primary MLR exhibited proliferative inhibition of both T cell subsets.     

Antibodies to the L3T4 and Lyt-2 molecules interfere with antigen receptor-driven activation of cloned murine T cells

When L3T4+ cloned murine helper T lymphocytes (HTL) are stimulated with antigen or immobilized anti-T cell receptor (TCR) monoclonal antibodies (mAb) at concentrations which are optimal for proliferation, anti-L3T4 mAb inhibits activation as measured by proliferation and lymphokine production. Under similar conditions, IL 2-independent proliferation of Lyt-2+ cloned murine cytolytic T lymphocytes (CTL) stimulated by anti-TCR mAb is inhibited by anti-Lyt-2 antibodies. Proliferation of cloned HTL and CTL cells stimulated by IL 2 is not affected by the anti-L3T4 and anti-Lyt-2 mAb. The inhibition of TCR-induced activation of the T cell clones is not due to interference with the binding of the anti-TCR mAb. Stimulation of the TCR has been proposed to induce lymphokine secretion and proliferation by T cells through a pathway involving the activation of protein kinase C and the stimulation of an increase in the concentration of intracellular free calcium. However, proliferation of T cells stimulated by PMA (which activates protein kinase C) plus the calcium ionophore A23187 (which increases the concentration of intracellular free calcium) is not affected by mAb reactive with the Lyt-2 or L3T4 structures. If TCR stimulation does indeed activate T cells by activating protein kinase and increasing intracellular free calcium, then our data suggest that anti-L3T4 and anti-Lyt-2 mAb inhibit TCR-driven proliferation at some step before the activation of protein kinase C and the stimulation of a rise in intracellular free calcium concentration. Our results suggest that anti-L3T4 and anti-Lyt-2 mAb interfere with early biochemical processes induced by stimulation of the TCR. In HTL, which proliferate via an autocrine pathway, anti-L3T4 mAb appears to inhibit proliferation by interfering with signaling events involved in lymphokine production. Inhibition of IL 2-independent proliferation of Lyt-2+ cells by anti-Lyt-2 mAb appears to occur by a different mechanism. The precise molecular basis for the interference of each cell type has not yet been characterized.