Two types of murine helper T cell clone. II. Delayed-type hypersensitivity is mediated by TH1 clones

We have previously shown that at least two types of Lyt-1+, Lyt-2-, L3T4+ helper T cell clones can be distinguished in vitro by different patterns of lymphokine secretion and by different forms of B cell help. Evidence is presented here to show that one type of helper T cell clone (TH1) causes delayed-type hypersensitivity (DTH) when injected with the appropriate antigen into the footpads of naive mice. The antigen-specific, major histocompatability complex (MHC)-restricted footpad swelling reaction peaked at approximately 24 hr. Footpad swelling was induced by all TH1 clones tested so far, including clones specific for soluble, particulate, or allogeneic antigens. In contrast, local transfer of TH2 cells and antigen did not produce a DTH reaction, even when supplemented with syngeneic spleen accessory cells. Similarly, local transfer of an alloreactive cytotoxic T lymphocyte clone into appropriate recipients did not produce DTH. The requirements for the DTH reaction induced by TH1 cells were investigated further by using TH1 clones with dual specificity for both foreign antigens and M1s antigens. Although these clones responded in vitro to either antigen + syngeneic presenting cells, or M1s disparate spleen cells, they responded in vivo only to antigen + MHC and did not cause footpad swelling in an M1s-disparate mouse in the absence of antigen. Moreover, in vitro preactivation of TH1 or TH2 cells with the lectin concanavalin A was insufficient to induce DTH reactions upon subsequent injection into footpads. From these results, we conclude that the lack of DTH given by TH2 clones in vivo could be due to the inability of the TH2 cells to produce the correct mediators of DTH, or to a lack of stimulation of TH2 clones in the footpad environment.     

Interleukin 2 inhibits antigen-stimulated lymphokine synthesis in helper T cells by inhibiting calcium-dependent signalling

Certain L3T4+, Lyt-2- cloned murine helper T lymphocytes (HTL), when cultured with a high concentration of interleukin 2 (IL 2), become temporarily unresponsive to antigenic stimulation, as indicated by failure to proliferate and by reduced secretion of lymphokines when challenged with antigen. Exposure of cloned HTL to IL 2 also renders these cells less responsive to concanavalin A (Con A). Here we demonstrate that antigen-unresponsive HTL also accumulate reduced levels of lymphokine mRNA, thus indicating a pretranslational block of the response to antigen. However, HTL which had been pretreated with IL 2 and were unresponsive to antigen responded strongly to antigen + A23187 or to A23187 + PMA but failed to respond to antigen + PMA. With HTL made unresponsive to antigen or to Con A by exposure to IL 2, increases in intracellular calcium ion levels stimulated by Con A also were reduced. Thus, for mouse HTL clones, the IL 2-induced state of unresponsiveness to antigen or Con A appears to reflect an inability of such HTL to increase intracellular free calcium to a level sufficient for activation of lymphokine genes.     

Antiproliferative effect of IFN-gamma in immune regulation. III. Differential selection of TH1 and TH2 murine helper T lymphocyte clones using recombinant IL-2 and recombinant IFN-gamma

Supernatants collected after primary or secondary stimulation of spleen cells contain different arrays of lymphokines. Primary supernatants from spleen cells stimulated with Con A or allogeneic spleen cells (MLC-SF) contain IL-2 but little IL-4 or IGN-gamma; in contrast, secondary MLC-SF contains IL-2 as well as substantial IL-4 and IFN-gamma. Our laboratory previously had always used secondary MLC-SF for cloning T cells, and had routinely obtained TH1 helper T lymphocyte clones. In the present study, when primary Con A-SF was used as source of growth factors, TH2 and not TH1 clones were preferentially derived. Considering the possibility that IFN-gamma may be one important factor in determining whether TH1 or TH2 clones are preferentially obtained, clone derivation was then performed either in the presence of rIL-2 or rIL-2 plus rIFN-gamma. The majority of clones derived using rIL-2 alone were TH2 cells, whereas the majority of clones derived using rIL-2 plus rIFN-gamma were TH1 cells. Using either procedure, some clones were obtained that produced IL-2, IL-4, and IFN-gamma. These data are consistent with our previous observations that IFN-gamma inhibits the proliferation of TH2 but not TH1 clones, and suggest that the presence of IFN-gamma during an immune response would result in the preferential expansion of helper T lymphocytes of the TH1 phenotype. Our procedure for the differential selection of TH1 and TH2 clones reactive with the same Ag should be useful for designing in vitro systems for studying the function of these cell subsets in specific immune responses.     

Functional analysis in vitro and in vivo of Mycobacterium bovis strain BCG-specific T cell clones

Several cloned T cell lines specific for PPD and BCG were obtained. All clones were able to secrete lymphokine, i.e., MAF/interferon, upon antigenic stimulation. The surface phenotype of all these different clones was Thy-1.2+, L3T4+, Lyt-2-, suggesting that these lines belonged to the helper/inducer T cell subset. The T cell clones displayed various degrees of helper activity as tested in a secondary antibody response in vitro. The capacity of these clones to elicit DTH reactions in the presence of antigen and their ability to inhibit mycobacterial growth in vivo were tested by transferring locally the different clones to normal mice. The clones which exhibited little or no helper activity were able to elicit DTH responses, whereas the clone with strong helper activity did not. Both types of functionally defined clones had the capacity to inhibit the growth of intracellular mycobacteria in vivo.     

Functional analysis of antigen-nonspecific T-cell suppression. I. Effect of mitogen-induced T suppressor cells on helper-T-cell clones

The effect of mitogen-induced nonspecific suppressor T cells (Ts)2 on T-helper-cell activity was investigated using isolated clones of murine T-helper cells as targets. TNP-self-reactive Thy1+, Ly1+ T-cell clones were isolated after continuous culture of T cells derived from picryl chloride-sensitized mice and were characterized by their ability to proliferate in an antigen-specific and MHC-restricted manner. In addition, selected T-cell clones were found to produce both interleukin-2 (Il-2) and T-cell replacing factor (TRF), lymphokines characteristic of helper T cells. Concanavalin A (Con A)-induced Ts cells inhibited the antigen-specific proliferation of these helper-T cell clones in a noncytotoxic manner even in the presence of exogenous Il-2. This implied that failure to proliferate was not merely due to an inability of these clones to produce Il-2. The kinetics of suppression also suggested that early T-cell activation signals were not affected. Furthermore, coculture experiments indicated that while proliferation could be severely inhibited, the actual secretion of lymphokines such as Il-2 and TRF by the T-helper clones was not. Our data suggest that nonspecific Ts modulation of proliferation versus helper factor production are under separate control in cloned T-cell populations, with lymphokine secretion remaining intact in the presence of Con A-induced Ts cells.     

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.     

Heterogeneity in direct cytotoxic function of L3T4 T cells. TH1 clones express higher cytotoxic activity to antigen-presenting cells than TH2 clones

In the process of generating culture supernatant from T cell clones, with anti-CD3 antibodies and the B lymphoma A20 as APC, a striking difference in the stimulation of TH1 and TH2 clones was observed, i.e., TH2 clones produced higher levels of lymphokines than TH1 clones. This prompted us to test the hypothesis that differential killing of APC (thus the removal of stimuli) by T cells led to differential T cell activation. By studying a panel of five TH1 and seven TH2 clones, it was demonstrated that TH1 clones mediated significantly higher levels of cytotoxicity toward A20 cells in the presence of soluble anti-CD3 antibody (as opposed to immobilized anti-CD3). Although T cell clones could, when activated with immobilized anti-CD3, produce lymphokines cytotoxic to A20 cells, experiments in which lymphokine production was blocked indicated that T cell clones, in the presence of soluble anti-CD3, mediated killing of A20 through direct cytotoxicity. A higher level of cytotoxicity, by TH1 compared with TH2 clones, was not restricted to anti-CD3 or a particular target cell type, because it also occurred with Con A- or Ag-dependent killing (a monocyte-macrophage cell line), and LPS blasts. Furthermore, the higher cytotoxic activity of TH1 clones compared with TH2 clones was independent of the stage of T cell activation and was unlikely a result of the length of in vitro culture. High levels of killing of APC led to low levels of T cell activation, the significance of which may be as a negative feedback mechanism in the immune response. Other biologic relevancies of higher cytotoxic activity in TH1 vs TH2 cells were also discussed.     

Measles virus-specific murine T cell clones: characterization of fine specificity and function

Measles virus (MV)-specific murine helper T cell clones (Thy-1.2+, CD4+, CD8-) were generated from mice immunized with MV-infected mouse brain homogenate by limiting dilution and in vitro stimulation of spleen cells with UV-inactivated MV Ag. The protein specificity of 7 out of 37 stable T cell clones, which displayed MHC-restricted MV Ag recognition, could be assessed by using purified MV proteins. Two fusion (F) protein-specific, two hemagglutinin-specific, and three nucleoprotein- or matrix protein-specific clones were shown to be established. The F protein-specific T cell clones together with a panel of previously generated F protein-specific T cell clones were characterized for their fine specificity by using beta-galactosidase fusion products, which contained different parts of the F protein. It was shown that at least two epitopes on the major part of the F protein (amino acid 2-513) can be recognized by mouse T cells. Functional characterization of three T cell clones showed that they were able to assist MV-specific B cells and bystander B cells for antibody production. Furthermore, they were shown to produce the lymphokines IL-2 and IFN-gamma. It was also shown that these T cell clones induced a MV-specific delayed type hypersensitivity response. These observations suggest that all of the T cell clones characterized belong to the TH1 helper subset.     

Cloned T cells with "co-helper" activity. I. Biologic activities of the clone

Clones of sheep erythrocyte-(SRBC) specific helper T cells with the surface phenotype Thy-1+, Ly-1+, Ly-2- have been derived that grow in vitro in the absence of exogenous antigen or added growth factors. The IL 2-independent clone, 101.6 has been shown to produce a supernatant factor that augments the primary anti-SRBC but not anti-burro RBC responses of whole spleen cells or Ly-1 T plus B cell cultures. The supernatant does not help B cells directly. This augmenting activity is terminated "co-helper" because the enhancement requires the presence of normal Ly-1 T cells. The supernatant of 101.6 was not shown to contain IL 2; co-helper activity was distinguishable from IL 2 activity by absorption with SRBC but not with Con A blasts, and we observed that co-helper activity does not act on spleen cells that differ at the major histocompatibility complex.     

The murine IL 2 receptor. III. Cellular requirements for the induction of IL 2 receptor expression on T cell subpopulations

The accessory cell requirements for the induction of the IL 2 receptor by the lectin Con A on murine T cell subsets were directly assayed with anti-IL 2 receptor monoclonal antibodies. Substantial levels of IL 2 receptor expression were induced on T lymphocytes of the MHC class I-restricted, suppressor/cytotoxic phenotype (L3T4-, Ly-2+) in the presence and absence of accessory cells. In contrast, high levels of IL 2 receptor expression could only be induced on T cells of the MHC class II-restricted, helper/inducer phenotype (L3T4+, LY-2-) in the presence, but not in the absence, of accessory cells. Ia- cells such as the P388D1 macrophage line or cultured fibroblasts (DAP X 3) were as efficient as the Ia+ B cell hybridoma LB in providing accessory cell function for the L3T4+, Ly-2- subset. PMA, but not purified human IL 1, could substitute for accessory cells for both IL 2 receptor expression and IL 2 secretion by the L3T4+, Ly-2- subset. These data suggest that IL 2 receptor induction on the L3T4+, Ly-2- subset is complex, possibly requiring a T cell-accessory cell interaction, whereas the lectin may directly trigger IL 2 receptor expression on L3T4-, Ly-2+ T cells.     

CD4+ subset regulation in viral infection. Preferential activation of Th2 cells during progression of retrovirus-induced immunodeficiency in mice.

Progressive lymphoproliferation and increasingly severe immunodeficiency are prominent features of a syndrome, designated mouse AIDS, which develops in susceptible strains of mice infected with the mixture of murine leukemia viruses, termed LP-BM5. Development of splenomegaly and lymphadenopathy, caused primarily by increases in B cell immunoblasts, requires the presence of CD4+ T cells and is assumed to be mediated by lymphokines produced by these cells inasmuch as progression of disease is markedly inhibited by treatment of infected mice with cyclosporin A. Studies of spleen cells from infected mice revealed spontaneous production of cytokines (IFN-gamma, IL-2, IL-4, IL-5, and IL-10) characteristic of Th0 (or a mixture of Th1 and Th2) T helper cells at 1 wk after infection. At later times, IFN-gamma and IL-2, characteristic products of Th1 helper clones, were expressed poorly, either spontaneously or after stimulation of cells with Con A. In contrast, IL-4, IL-5, IL-6, and IL-10, cytokines typically synthesized by Th2 cells, were produced in response to Con A or spontaneously through 18 wk post-infection. Increased serum IgE levels and enhanced IL-10 mRNA expression were consistent with expression of Th2 cytokines at biologically significant levels in vivo. Selective depletion of T cell subsets before stimulation with Con A showed that CD4+ T cells were the primary source of IL-2, IL-4, IL-10, and, to a lesser extent, IFN-gamma in spleens and lymph nodes of normal or infected mice. These results suggest that persistent activation of CD4+ T cells with the lymphokine profile of Th2 helper clones is responsible for chronic B cell stimulation, down-regulation of Th1 cytokines, and impaired CD8+ T cell function in mouse AIDS. This provides the first demonstration that, like many parasitic infections, viruses encoding potent antigenic stimuli can markedly affect the balance of Th subset expression.     

Biological and biochemical characterization of bovine interleukin 2. Studies with cloned bovine T cells

Bovine peripheral blood lymphocytes stimulated with the T cell mitogen concanavalin A (Con A) secrete a lymphokine with biological properties similar to T cell growth factor (TCGF) or interleukin 2 (IL 2) from other species. The material supports proliferation of Con A-derived T cell blasts, limiting dilution cloning of T cell blasts, and continuous growth of T cell clones for over 6 mo in vitro. A quantitative microassay with the use of TCGF-dependent, Con A-unresponsive cloned T cells was used to determine the biological activity during purification of IL 2. A single peak of activity with an apparent m.w. of 25,000 to 28,000 was recovered after gel filtration. This material eluted from DEAE-Sephacryl between 135 and 165 mM NaCl. After isoelectric focusing, high pressure liquid chromatography, and gel electrophoresis under reducing conditions, peak IL 2 activity was associated with proteins having m.w. of 20,000 and 23,000.     

Evidence implicating utilization of different T cell receptor-associated signaling pathways by TH1 and TH2 clones

We have reported recently that high concentrations of anti-CD3 mAb inhibited IL-2-dependent proliferation of TH1 but not TH2 clones. The selective inhibitory effect on TH1 clones suggested that the two helper T lymphocyte subsets might utilize different TCR-associated signal transduction mechanisms. In the present study, we demonstrate that this distinction was not due to a gross difference in the level of TCR expression by TH1 and TH2 clones. Inhibition of TH1 proliferation by anti-CD3 mAb appeared to depend on calcium for maximal effect, suggesting that a substantial elevation of intracellular free calcium concentration ([Ca2+]i) might not occur after ligation of the TCR complex of TH2 clones. Calcium ionophore inhibited IL-2-dependent proliferation of both subsets, suggesting that receptor/ligand systems which stimulate elevated [Ca2+]i would be expected to inhibit proliferation. Although elevated [Ca2+]i and generation of inositol phosphates were readily detected in TH1 clones, these second messengers were not detected following stimulation of TH2 clones via the TCR complex. In addition, lymphokine production by TH1 clones was more sensitive to inhibition by cholera toxin, 8-bromoadenosine 3':5'-cyclic monophosphate, and cyclosporin A than was lymphokine production by TH2 clones. Collectively, these results suggest that TH1 and TH2 clones utilize distinct TCR-associated signal transduction mechanisms for lymphokine gene expression. The difference in signaling mechanisms suggests a potential pharmacologic target for intervention in situations where inappropriate activation of TH1 or TH2 cells occurs in vivo.     

Functional diversity of T lymphocytes due to secretion of different cytokine patterns.

Different helper T cell subsets secrete different patterns of cytokines when stimulated by antigen. The TH1 and TH2 subsets differ in the secretion of at least eight cytokines, and three or more other cytokine secretion patterns also exist among both mouse and human T cell clones. Several properties of strong immune responses suggest that at least the TH1 and TH2 phenotypes can be present in vivo. As cytokines are major determinants of the functions of the T cells that produce them, these patterns lead to different properties of the T cell subsets. TH1 cells mediate several functions connected with cytotoxicity and local inflammatory reactions, and so these T cells are particularly effective at combating viruses and intracellular bacteria and parasites. TH2 cells are much more effective at stimulating B cells to produce antibody, and so should be more effective against free-living bacteria, and in inducing protective humoral immunity. Antibody and delayed inflammatory reactions are often mutually exclusive during immune responses, and this can be at least partially explained by cross-inhibition of TH1 and TH2 cells. A newly discovered cytokine, IL10, has been implicated as one of the cross-regulatory cytokines, as this TH2 product inhibits cytokine synthesis by TH1 cells.     

Distinct signals are required for proliferation and lymphokine gene expression in murine T cell clones

Experiments were performed to assess the capacity of lectin (Con A), ionomycin, phorbol ester (PMA), and recombinant IL 2 to mediate proliferation as well as the expression of cell surface IL 2 receptors, two lymphokine genes, IL 2 and IFN-gamma, and the c-myc proto-oncogene in cloned T cell populations. Stimulation of T cell clones with recombinant IL 2 resulted in proliferation and sustained expression of the c-myc cellular proto-oncogene, but did not induce the expression of mRNA for the lymphokines IFN-gamma and IL 2. In contrast, stimulation of cloned T cells with lectin alone induced significant IFN-gamma and IL 2 mRNA expression, up-regulation of the number of cell surface IL 2 receptors, and transient c-myc expression. Ionomycin alone was not a sufficient signal for lymphokine mRNA induction. The phorbol ester PMA alone induced neither proliferation nor lymphokine gene expression but potentiated lectin and ionomycin-mediated signals. We also performed experiments to examine whether the T cell response to extracellular stimuli was a function of the activation state of the cell. Reexposure of 48-hr antigen-activated cloned cells to identical stimuli revealed several differences. Low but significant levels of IFN-gamma mRNA were now also reinduced in activated clones cells in response to IL 2 or PMA alone. Activated cells were refractory to reinduction of IL 2 mRNA by any stimulus, which may reflect a physiologic mechanism to limit clonal expansion after antigenic stimulation. This could be partially reversed by restimulation with lectin in the presence of cycloheximide, suggesting a role for a labile protein repressor in the down-regulation of IL 2 mRNA expression. PMA alone induced an IL 2-independent proliferative response. We demonstrate that distinct signals are required for lymphokine gene expression vs cellular proliferation in cloned T lymphocyte populations, and that the capacity of extracellular stimuli to reinduce expression of lymphokine genes or to mediate cell proliferation is altered by prior activation.     

In vitro induction and expression of interleukin 2 receptor in a clonal T helper cell differentiation model

Induction and expression of interleukin 2 (IL 2) receptor have been studied using a poly( Glu60 Ala30 Tyr10 ) (GAT)-specific T cell clone of mouse origin. This clone (52-3) has been characterized and it exhibits functional properties of T helper (TH) cells: it leads to a specific anti-DNP response in the presence of DNP-GAT and DNP-primed B cells and it secretes biological activities which can induce polyclonal B cell proliferation and IgM secretion. In vitro this clone mimics the activation stages of normal T lymphocytes and can be obtained under two states of differentiation. depending on the antigen-specific signal provided by antigen-presenting cells (APC). The expression of IL 2 receptor by this clone has been studied by (i) its response to IL 2, (ii) its capacity to absorb IL 2 bioactivity, and (iii) its reactivity with monoclonal antibody 7D4 specific for mouse IL 2 receptor. All the results indicate that the unstimulated state does not express the IL 2 receptor while the activated state does. Clone 52-3 has been compared with clone 14-1.6 that derives from a TH cell line and expresses the IL 2 receptor constitutively. 52-3 offers a good experimental model for studying in vitro, in a clonal TH cell population, the detailed mechanism of IL 2 receptor induction.     

Establishment of continuous cultures of thy1.2+, Lyt1+, 2-T cells with purified interleukin 3

IL-3 is a recently described lymphokine that induces the expression of the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH) in an early T-cell precursor. We demonstrate that purified IL-3 an be used to establish continuous cultures of a discrete subpopulation of T cells with virtually 100% efficiency from normal, unstimulated splenic lymphocyte populations. Once established over a period of approximately 4-6 weeks, the cultures can be readily cloned either in soft agar or by limiting dilution. All of the established lines are Lyt1+, 2- la-, lg-, Tdt-, 20 alpha SDH+, a phenotype characteristic of helper T cells; they are therefore distinct from continuous cultures of T cells established with IL-2. although initiation of these cell lines was absolutely dependent on IL-3, once established all of the cell lines were independent of exogenously added lymphokines for their growth in vitro. However, all of the cells lines were found to constitutively produce IL-3 at high levels. None of the cell lines constitutively produced lL-2, but could be readily induced to produce this lymphokine by treatment with phorbol-myristic acetate. The ability to produce lL-3 and lL-2 is a further indication that all the cell lines are helper T cells. The possible mechanisms by which lL-3 allows the specific differentiation and/or amplification of T cells of helper phenotype in tissue culture are discussed.     

Gangliosides: differentiation markers for murine T helper lymphocyte subpopulations TH1 and TH2.

On the basis of the pattern of lymphokines they secrete, murine T helper clones can be divided into two subsets, TH1 and TH2. This concept of two different T helper effector cells helps to explain the diversity of immune reactions occurring in different parts of the body. The in vivo localization of T helper subtypes is of great interest, but up to now no biochemical or surface markers were available to distinguish between them. We analyzed the glycolipids from altogether 12 murine TH1 and TH2 cell lines or clones. A comparison of the gangliosides by thin-layer chromatography showed differences between the TH1 and TH2 cells. Binding studies with specific antibodies to asialo backbone structures after degradation by neuraminidases showed that the main gangliosides from these lymphocytes shared a common GgOse4 backbone and thus differed only in their degree or position of sialylation. Two disialogangliosides appeared to be characteristic. They were isolated from the D10.G4.1 TH2 cell clone and identified by fast atom bombardment mass spectrometry as IVNeuAc,IINeuAc-GgOse4Cer (GD1a) and IVNeuAc,IIINeuAc-GgOse4Cer (GD1 alpha), respectively. GD1a was characteristically only detected in TH2 cells, whereas GD1 alpha was preferably, but not exclusively, expressed by TH1 lymphocytes. Although GD1a was also found in the lung, heart, kidney, and spleen, its expression within the murine immune cells under investigation was unique to TH2 lymphocytes. Scarcely any GD1a was found in thymocytes, B cells, or CD8 positive (cytolytic) T cells, but significant expression was seen in CD4 positive (helper) T cells which include the TH2 subpopulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of CTL responses to alloantigens by a Db-specific T helper clone

A T cell helper clone was derived 2 yr ago from a mixed lymphocyte culture. This clone, referred to as clone 9, was propagated in interleukin 2 (IL 2)-containing medium in the presence of irradiated stimulator and irradiated syngeneic spleen cells. Clone 9 was of H-2d origin and was found to be Thy-1+ and Lyt-1-2-. Clone 9, as well as supernatant factor(s) derived from it, were able to enhance the primary cytotoxic responses of Db responder cells to alloantigens. Furthermore, clone 9 cells or its factor(s) were only active when added during the first 24 hr of a 5-day culture period. When a low stimulator cell dose (10(4) cells per 0.2 ml culture) was used, it was possible to demonstrate that clone 9 also required a source of irradiated allogeneic splenic accessory cells to exert its helper action. Under these conditions, clone 9 or its factor(s) could also synergize with IL 2-containing medium in mounting cytotoxic responses to alloantigens. Synergy between IL 2-containing medium and clone 9 or its factor(s) was observed only when Db responder cells were used. The helper activity in clone 9 supernatant was also specifically absorbed out by Con A-stimulated Db spleen cell blasts. Preincubation with clone 9 supernatant for 1 hr at room temperature also led to enhanced cytotoxic responses of Db responder cells to alloantigens. Clone 9 supernatant was also found to be devoid of detectable IL 2 activity. Thus, clone 9 or its helper factor(s) appear to exert its helper activity by an early interaction with Db cytotoxic T lymphocyte precursors (CTL-P).     

Immunoglobulin-dependent helper T cells: studies in the MOPC-315 system suggest a novel surface antigen phenotype

We have previously provided evidence that the SRBC-immune helper T (TH) cells which enhance MOPC-315 plasmacytoma cell secretory differentiation in vivo (THd cells) differ in specificity, accessory cell requirements, and Qa-1 expression from the SRBC-immune TH cells which enhance MOPC-315 cell growth in vivo (THg cells). Indeed, like other immunoglobulin-dependent TH cells, THd cells in the 315 system do not develop in anti-IgM-treated, B cell-deficient mice, whereas THg cell development is unaffected. In this report, we provide evidence for other differences in the expression of surface antigens by these two TH cell populations. We find that, like most Lyt-1+, 2- T cells, the THg cells can be eliminated by monoclonal anti-L3T4 antibody and complement treatment, whereas such treatment had no effect on adoptive transfer of SRBC-immune THd cell activity. Similarly, THg cell activity was eliminated from SRBC-immune T cells by treatment with monoclonal anti-T cell receptor beta-chain allotope antibody plus anti-rat IgG and complement, whereas THd cell activity remained intact. Both helper cell activities were deleted by either anti-Lyt-1.2 or anti-Thy-1.2 antibody and complement treatment. Interestingly, the THd cell activity was abrogated by treating SRBC-immune T cells with monoclonal anti-B220 or monoclonal anti-p50 antibodies (RA3-3A1/6.1 and RA3-2C2/1, respectively) and complement, whereas THg cell activity was unaffected. Additional controls indicated that the THd effects did not arise by virtue of a two-cell interaction between a Thy-1+, B220- cell and a Thy-1-, B220+ cell, and it is therefore proposed that the THd effect arises from a single population of cells that exhibit a unique phenotype (Thy-1+, Ly-1+, 2-, L3T4-, B220+). The proposal is further supported by studies conducted with a T cell clone which promotes MOPC-315 cell secretory differentiation in vitro and which exhibits this surface antigen phenotype. The serologic differences between these two TH cell populations stress even further the likelihood that B cell growth and differentiation enhancement are mediated by distinct T cell subsets in this system, and raise the possibility that immunoglobulin-dependent TH cells in other systems will routinely exhibit a unique surface antigen profile. These data also imply that immunoglobulin-dependent TH cells (such as the THd cells) may not express antigen receptors that are identical to those expressed by MHC-restricted helper cells (such as our THg cells).