Two independent pathways of helper activity provided by a single T cell clone

Data presented in this paper demonstrate the existence of two separate pathways by which a single T cell clone can induce B cell differentiation. With the use of high doses of antigen, a T cell clone can induce a primary antibody response in unprimed B cells. With the use of low doses of antigen, the same T cell clone can induce an immunoglobulin (Ig)G response in primed B cells. The primary response is accompanied by T cell proliferation and lymphokine production (interleukin 2, B cell growth factor, B cell differentiation factor for immunoglobulin M, and B cell differentiation factor for immunoglobulin G). The secondary response does not require proliferation and occurs independently of detectable lymphokine production. Variants of the wild type T cell helper clone have been isolated. One variant can provide help to unprimed B cells when high doses of antigen are used. This variant cannot provide help to primed B cells when low doses of antigen are used, nor can it provide help to CBA/N "xid" B cells at any antigen concentration tested. Additional variants have been isolated that proliferate on antigen-pulsed-presenting cells, but fail to secrete detectable lymphokines and do not induce B cell differentiation. These results suggest that a single T cell helper clone has multiple functional activities that can be independently expressed.     

IgM induction in murine B hybridomas with Ia-restricted T cell clones: a monoclonal model for T and B cell interactions in antibody response

The mechanism of MHC-restricted T and B cell interactions in antibody response was studied with IgM-inducible B hybridomas and antigen-specific helper T cell clones. B hybridomas were prepared by fusion between splenic B cells from (CBA/N (H-2k) X BALB/c (H-2d)) F1 (NBF1) male mice and a B lymphoma cell line, M12.4.5. A B hybridoma clone, 1M70, which expressed I-Ad but not I-Ak determinants was chosen in the present study. IgM secretion was induced in 1M70 when it was cocultured with a "resting" KLH-specific and H-2d restricted helper T cell clone in the presence of KLH. A "resting" KLH-specific and H-2k restricted T cell clone did not induce IgM secretion in 1M70 even in the presence of KLH. However, when these KLH-specific T cell clones were activated by KLH and appropriate antigen presenting cells, both H-2d and H-2k restricted T cell clones induced IgM secretion in 1M70 even in the absence of KLH. A monoclonal anti-I-Ad antibody inhibited IgM secretion induced by a "resting" H-2d restricted T cell clone, but not by an "activated" T cell clone. These results indicated that T cell clones recognized antigens in the context of Ia molecules on B hybridomas in a MHC-restricted manner and were activated to produce B cell stimulatory factors which in turn acted on B hybridomas in a non-MHC-restricted manner and induced differentiation of B hybridomas into IgM secreting cells.     

Antigen-specific B cells efficiently present low doses of antigen for induction of T cell proliferation

Previous experiments suggested a role for specific B cells in the induction of antigen (SRBC)-specific T cell proliferation. Two models were proposed: in the first, B cells directly presented antigen to T cells; alternatively, B cells secreted antibody, which opsonized antigen for presentation by macrophages. Experiments to distinguish between these possibilities are presented here. Three lines of evidence support the conclusion that antigen is presented directly by specific B cells. First, nonimmune splenic adherent cells (SAC), which efficiently induced proliferation of appropriately primed T cells to antigens such as OVA and GAT, were unable to induce SRBC-specific proliferation. Secondly, a slope analysis of the logarithmic plot of T cell proliferation vs the number of irradiated B cells suggested that two cells were limiting within the presenting population. The addition of IL 1 or SAC reduced the slope to 1 (although in serum-free conditions, the addition of IL 1, but not SAC, reduced the slope of the line). Specificity of the B cells for the antigen continued to be required in the presence of exogenous IL 1 or SAC. These results suggested that presentation by specific B cells and the amount of IL 1 were the limiting requirements for the induction of SRBC-specific T cell proliferation. The third line of evidence was the demonstration of a restricted interaction between T cells and B cells. The addition of irradiated, allogeneic SRBC-specific B cells to T cell lines and syngeneic SAC failed to support proliferative responses. We further show that a GAT-specific T cell clone was triggered to proliferate by either SAC or B cells, but that antigen-specific B cells were necessary at low doses of antigen. This finding is important in two respects. First, the T cell clone previously has been shown to act as a helper; secondly, when low doses of antigen are used, the requirement for priming of the B cells to the specific antigen is true for a soluble, as well as a particulate, antigen. We propose that at low (physiologic) doses of antigen, presentation to secondary T cells takes place mainly at the surface of antigen-specific B cells. At high doses of antigen,h presentation can also be accomplished by nonspecific cells such as other B cells, macrophages, or dendritic cells.     

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.     

B cell helper factors. II. Synergy among three helper factors in the response of T cell- and macrophage-depleted B cells

The concanavalin A- (Con A) stimulated supernatant of normal spleen cells (normal Con A SN) was shown to contain a set of helper factors sufficient to allow T cell- and macrophage- (M phi) depleted murine splenic B cells to produce a plaque-forming cell response to the antigen sheep red blood cells (SRBC). The activity of normal Con A SN could be reconstituted by a mixture of three helper factor preparations. The first was the interleukin 2- (IL 2) containing Con A SN of the T cell hybridoma, FS6-14.13. The second was a normal Con A SN depleted of IL 2 by extended culture with T cell blasts from which the 30,000 to 50,000 m.w. factors were isolated (interleukin X, IL X). The third was a SN either from the M phi tumor cell line P388D1 or from normal M phi taken from Corynebacterium parvum-immune mice. The combination of all three helper factor preparations was required to equal the activity of normal Con A SN; however, the M phi SN had the least overall effect. The M phi SN and IL 2 had to be added at the initiation of the culture period for a maximal effect, but the IL X preparation was most effective when added 24 hr after the initiation of culture. These results indicate that at least three nonspecific helper factors contribute to the helper activity in normal Con A SN.     

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.     

Induction of antigen-specific antibody responses in primed and unprimed B cells. Functional heterogeneity among Th1 and Th2 T cell clones

CD4+ T cells have been recently divided into two subsets. The functions of these subsets are thought to be distinct: one subset (Th1) is responsible for delayed type hypersensitivity responses and another (Th2) is primarily responsible for induction of antibody synthesis. To more precisely define the roles of both subsets in humoral immune responses, we examined the ability of a panel of nominal antigen specific Th1 and Th2 clones to induce anti-TNP specific antibody synthesis in TNP-primed or unprimed B cells. Four of nine Th1 clones induced little or no antibody synthesis with TNP-primed B cells. However, five other Th1 clones were very effective at inducing IgG anti-TNP plaque-forming cell (PFC) responses in primed B cells. One of these Th1 clones was analysed in detail and found to also provide helper function for unprimed B cells. Cognate B-T cell interaction was required for induction of both primary and secondary responses with this clone, indicating that a Th1 clone could function as a "classical" Th cell. The seven IL-4 producing Th2 clones examined were also heterogeneous in their ability to induce antibody secretion by TNP-primed B cells. Although four of the Th2 clones induced IgG and IgM anti-TNP PFC responses, two Th2 clones induced only IgM and no IgG antibody, and another clone failed to induce any anti-TNP PFC. All Th2 clones failed to induce any anti-TNP PFC. All Th2 clones produced high levels of IL-4, but "helper" Th2 clones produced significantly greater amounts of IL-5 than "non-helper" Th2 clones. These studies indicate that some IL-2- and some IL-4-producing T cell clones can induce TNP-specific antibody in cell clones can induce TNP-specific antibody in primed and unprimed B cells, and that Th1 and Th2 clones are heterogeneous in their ability to induce Ig synthesis. Therefore, although T cell clones can be classified as Th1 or Th2 types according to patterns of IL-2, IFN-gamma, or IL-4 synthesis, the functional capacity to induce antibody synthesis cannot be predicted solely by their ability to secrete these lymphokines.     

A requirement for nonspecific T cell factors in antibody responses to "T cell independent" antigens

Using murine splenic B cell preparations depleted of macrophages and rigorously depleted of T cells, we studied the role of nonspecific helper factors in in vitro antibody responses to T cell-independent (TI) type 1 and type 2 antigens. TNP-lipopolysaccharide, TNP-Brucella abortus, and DNP-liposomes containing lipid A were chosen as examples of TI type 1 antigens. DNP-Ficoll and DNP-liposomes without lipid A were chosen as TI type 2 antigens. Only the type 1 antigens were able to elicit significant, albeit very weak, responses without added helper factors. Both type 1 and 2 antigens required factors present in supernatants from concanavalin A-stimulated spleen cells (Con A SN) to stimulate optimum antibody responses. Interleukin 2- (IL 2) containing supernatant from the T cell hybridoma FS6-14.13 supported suboptimal responses to varying degrees with each TI antigen, in contrast to its lack of effect on responses to sheep red blood cells in the absence of additional factors. This activity of the FS6-14.13 supernatant was removed by absorption with the IL 2-dependent T cell line HT-2, suggesting that IL 2 was the active component. Another factor, IL-X, which is distinct from both IL 1 and IL 2 and is also found in Con A SN, was required in addition to IL 2 to achieve optimal responses with both types of TI antigens. These results clearly establish a role for factors derived from T cells in the activation of B cells by both type 1 and type 2 TI antigens.     

A requirement for physical linkage between determinants recognized by helper molecules and cytotoxic T cell precursors in the induction of cytotoxic T cell responses

It has long been understood that both antibody and delayed-type hypersensitivity responses are induced through collaborative events in which the determinants recognized by the precursor cells must be physically linked to the determinants recognized by the helper. Although it is clear that the generation of memory cytotoxic T lymphocyte precursors (CTLp) involves linked recognition of determinants, the induction of CTL responses has been viewed as being dependent upon interleukin 2 (IL 2), which could be provided by a helper cell, but independent of requirements for antigen bridging. In this work, we have designed a system that lacks exogenous IL 2 by using as our source of help, antigen-specific helper molecules derived from helper T cells. These soluble helper molecules are uncontaminated by IL 2 and unlike a helper cell, are unable to produce IL 2. Helper molecules specific for chicken red blood cells (Crbc) and for a synthetic polypeptide, poly 18, were tested. Thymocyte responders require a source of help to respond to alloantigens intrinsically expressed on the surface of adherent stimulator cells. To analyze the mechanism whereby the helper molecules acted, we used a system involving recognition of haptenic and carrier determinants that were physically linked by virtue of being located on the same cell surface (intra-structural linkage). Adherent stimulator cells were pulsed with Crbc or poly 18 so that the alloantigens recognized by the thymocyte CTLp (intrinsically expressed class I) were either linked or unlinked to the carrier determinants (Crbc or poly 18) presented by the adherent cells and recognized by the helper molecules. Both types of helper molecule were shown to be antigen-specific in crisscross experiments. The helper molecules specific for Crbc were able to induce the thymocyte CTLp only when both hapten and carrier were present on the same stimulator cell surface. Because we were not able to detect a requirement for H-2-restricted recognition of carrier antigen, this inductive event must be viewed as requiring linked associative recognition of determinants, but being noncognate. In contrast, the helper molecules recognizing poly 18 showed a requirement for both physical linkage of determinants and for H-2 restricted recognition, indicating that the mechanism of induction was cognate in nature. Therefore, we have shown that interactions between CTLp and soluble, antigen-specific, helper cell-derived inductive molecules are similar in nature to those of other T cell precursors and of B cells in the stringent requirement for close physical proximity achieved by linked or cognate recognition of determinants across an antigen bridge.     

Characterization of a T4+/Leu-8+ T cell clone that directly helps B cell Ig production by secreting B cell differentiation factor

A human T4+/Leu-8+ T cell clone (YA2) was established by phytohemagglutinin activation and interleukin 2 (IL 2) propagation. Functional characterization of this clone demonstrated that it provided potent help towards Ig production by pokeweed mitogen-stimulated B cells in the presence of small numbers of autologous T cells or by Staphylococcus aureus Cowan I (SAC)-activated B cells in the presence of B cell growth factor (BCGF). YA2 provided no help to resting B cells and minimal help to either unactivated B cells cultured with BCGF or SAC-activated B cells. Supernatant generated from clone YA2 by IL 2 stimulation had significant B cell differentiation activity but no BCGF or IL 2 activity. Thus, YA2 is a T4+/Leu-8+ potent direct helper only to B cells that are activated and proliferating due to its selective secretion of a differentiation factor, and not an activation and growth factor. The availability of phenotypically defined cloned populations of T cells with restricted functional helper activity related to the secretion of selected B cell tropic factors should prove useful in the dissection of the role of individual T cell subsets in the regulation of the human B cell cycle.     

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.     

The regulatory role of sialic acids in the response of class II reactive T cell hybridomas to allogeneic B cells

Two different kinds of alloreactive T cell hybridomas were established in previous experiments. One is reactive and the other is nonreactive to allogeneic I-A region-associated membrane antigen (mIa) on B cells. In the present experiments the difference between these hybridomas were analyzed by using representative clones, B cell mIa-reactive clone CB-11.4, and nonreactive clone HTB-9.3. Unresponsiveness of HTB-9.3 clone to allogeneic B cells could not be due to the inability of B cells in interleukin 1 production or the density of mIa molecules on B cells. HTB-9.3 clone could respond to C57BL/6 mouse B cells treated with neuraminidase (Nase), and Nase-treated HTB-9.3 clone could respond to normal B cells from C57BL/6 mouse, indicating that sialic acid on both B cells and HTB-9.3 clone plays a regulatory role in the alloreactivity of the clone. In response to B cells from C57BL/6 mouse, T cells from C3H/He mouse spleen showed similar reactivity to HTB-9.3 clone; that is, T cells could respond to Nase-treated B cells, and Nase-treated T cells to B cells, and T cells primed with C57BL/6 spleen cells in vitro showed similar reactivity to CB-11.4 clone. These results suggest that HTB-9.3 clone represents virgin T cells and CB-11.4 clone-primed T cells at least in alloreactivity. Anti-L3T4a was shown to block alloreactivities of both T cell hybridomas and splenic T cells against B cells more efficiently than against splenic adherent cells. These results suggest that L3T4a on T cell plays more important role in allogeneic response to B cells than to splenic adherent cells.     

A human helper T cell clone secreting both killer helper factor(s) and T cell-replacing factor(s)

A human helper T cell clone (d4), which showed its helper effect on the differentiation of both T and B cells, was established by MLC reaction of normal T cells against a B lymphoblastoid cell line (CESS) followed by cloning in the presence of IL2 and x-irradiated CESS and autologous non-T cells. d4 cells helped the induction of cytotoxic T cells against UV-treated CESS cells. Antigen-stimulated d4 cells secreted helper factor(s) involved in the induction of cytotoxic T cells (killer helper factor(s), KHF), and KHF activity could be separated into two fractions, one with the m.w. of 15,000 to 20,000 and the other with the m.w. of 45,000 to 50,000. The factor with 15,000 to 20,000 m.w. showed IL 2 activity; the other factor showed gamma-interferon activity without IL 2 activity, suggesting that both IL 2 and gamma-interferon exerted KHF activity. d4 cells or their culture supernatant showed helper activity in the induction of IgG in a B cell line (CESS). The helper activity of the supernatant (TRF) was absorbed with CESS cells but not with IL 2-dependent CTLL, whereas KHF activity was absorbed with IL 2-dependent CTLL but not with CESS cells. The results showed that TRF and KHF were distinct molecules and a single helper T cell clone could secrete helper factors for both B and T cells.     

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.     

Characterization of an antigen-specific suppressive factor derived from a cloned suppressor effector T cell line

A cloned effector-type suppressor T cell line, 3D10, which is known to suppress the antibody response against dinitrophenylated keyhole limpet hemocyanin (KLH), produced a soluble KLH-specific factor (TsF) that can replace the function of parental T cell clones. High activity of TsF was released spontaneously into the culture supernatant when cultured in interleukin 2 (IL 2)-containing medium, requiring no antigenic stimulation. The culture supernatant of 3D10 was also capable of inhibiting the KLH-induced proliferative response of primed T cells in an antigen-specific manner. The direct target of TsF was found to be Lyt-1+2- T cells undergoing an early stage of antigen-specific proliferation. TsF was antigen binding but lacked any other serologic markers such as I-J and immunoglobulin heavy chain-linked allotypic determinants on T cells. No genetic restriction was found in its action on allogeneic T cells. The production of IL 2 in proliferative T cells by antigenic stimulation was not inhibited by TsF. These results indicate that the TsF described here is the legitimate mediator produced by the effector-type suppressor T cell that suppresses the antigen-specific responses of Lyt-1+2- T cells. The m.w. of TsF was approximately 75,000.     

Separable helper factors support B cell proliferation and maturation to Ig secretion

The 24-hr culture supernatant of Con A-activated spleen cells (SN) contains helper factors that enable maturation to high-rate polyclonal Ig secretion and enhance proliferation in cultures of mouse B cells activated with the F(ab')2 fragment of class-specific rabbit antimouse IgM antibody (anti-Ig). When interleukin 2 (IL 2), also called T cell growth factor, is removed from SN by absorption with an IL 2-dependent cell line at either 4 degrees C or 37 degrees C, all the helper activity for anti-Ig-activated B cells is also removed. Partial removal of IL 2 results in partial removal of helper activity for B cells. However, the IL 2-depleted SN appears to contain another helper factor, TRF, that enables anti-Ig-activated B cell cultures to mature to high-rate Ig secretion. This TRF activity is revealed by adding purified human IL 2 or an IL 2-containing supernatant of a cloned, lectin-activated T cell hybridoma line (FS6-14.13) to Il 2-depleted SN, which restores the polyclonal antibody response to anti-Ig. The hybridoma supernatant by itself supports proliferation of anti-Ig-activated B cell cultures, as measured by an increase in cell number, but not maturation to Ig secretion. This proliferative response is likewise IL 2 dependent, although purified IL 2 with anti-Ig is not sufficient. These experiments define separable combinations of factors acting on anti-Ig-activated B cell cultures, one of which (SN) results in both proliferation and maturation to high-rate Ig secretion, whereas the other (hybridoma supernatant) results in proliferation only. IL 2 appears to be an essential component of both combinations, although the target cell for IL 2 action in this system remains to be determined.     

The use of specific helper T cell clones to study the regulation of isotype expression by antigen-stimulated B cell clones

In order to determine the mechanism by which helper T cells regulate the production of the various immunoglobulin (Ig) classes, a number of helper T cell clones specific for keyhole limpet hemocyanin (KLH) were generated. These helper T cell clones then were used in a modified splenic fragment system whereby cloned helper T cells and a source of B cells were limit-diluted into naive, lethally irradiated recipients. The B cell clones that were subsequently stimulated in such an assay system by the addition of the antigen 2,4-dinitrophenol (DNP)-KLH then were tested for the various isotypes produced. The results of these studies indicate that the use of a single helper T cell clone could result in the production of all known Ig isotypes including IgE. Moreover, the use of a single helper T cell clone could result in multiple isotype production by a single B cell clone. However, a comparison of the isotypes secreted by a number of different B cell clones that were stimulated with the same helper T cell clone indicated that a variety of isotypic patterns could be obtained. In addition, it was found that the majority of B cell clones produced in the presence of T cell clones secrete fewer numbers of different isotypes compared with B cell clones generated with a heterogeneous population of T cells. Finally, no evidence could be found for isotype-specific helper T cell clones, although a few of the T cell clones appeared to induce a somewhat restricted isotype pattern in which only two or three different isotypes were observed.     

Antigen-driven T cell clones can proliferate in vivo, eradicate disseminated leukemia, and provide specific immunologic memory

The aim of the current study was to determine the ability of antigen-driven cloned helper cell independent cytotoxic T lymphocytes (HITc) to proliferate and to survive in vivo and to mediate tumor therapy. The HITc clone utilized (denoted 1.B6) was specifically cytolytic to FBL-3, a syngeneic Friend virus-induced murine leukemia. Activation in vitro (48 hr) with FBL-3 induced secretion of interleukin 2 (IL 2), expression of IL 2 receptors (IL 2R), and in vitro proliferation. These cells could be "rested" for several weeks without stimulation, which resulted in reduced expression of IL 2R; however, restimulation with antigen resulted in reinduction of IL 2R and proliferation. The ability of cloned HITc to proliferate and to survive in vivo was examined in cyclophosphamide (CY) pretreated donor mice congenic for the Thy-1 gene. Adoptively transferred cloned HITc could be found in large numbers, and were widely distributed in vivo 1 wk after transfer. In tumor therapy, 1.B6 cells when injected into a site of tumor (i.p.) and used as an adjunct to CY were effective against disseminated FBL-3. In this circumstance, cloned 1.B6 cells could be recovered from cured mice 125 days after transfer and were shown to specifically lyse tumor and proliferate in vitro in response to FBL-3. Thus as an adjunct to CY, tumor-specific cloned HITc are capable of eradicating disseminated leukemia, persisting long-term in vivo, and providing specific immunologic memory.     

T cell regulation of B cell activation. An antigen-mediated tripartite interaction of Ts cells, Th cells, and B cells is required for suppression

To determine the requirements underlying the antigen specificity observed in T cell-mediated immune response suppression, cloned major histocompatibility complex (MHC)-restricted T suppressor (Ts) cells specific for keyhole limpet hemocyanin (KLH) and cloned MHC-restricted T helper (Th) cells specific for fowl gamma-globulin (FGG) were employed to study the regulation of trinitrophenyl (TNP)-specific B cell responses. Neither antigen bridging between Ts cells and Th cells (FGG=KLH) nor bridging between Ts cells and B cells (TNP-KLH) was sufficient to allow suppression; a mixture of FGG=KLH and TNP-KLH was also insufficient for suppression. In contrast, suppression was induced by KLH-specific Ts cells only when suppressor determinants (KLH), helper determinants (FGG), and B cell determinants (TNP) were covalently linked on the same molecule (TMP-FGG)=(TNP-KLH) or TNP-(FGG=KLH)). These findings imply that a tripartite antigen-mediated interaction of Ts cells, Th cells, and responding B cells is necessary for the mediation of this antigen-specific suppression.     

Activation via TCR or IL-2 receptor of a CD8+ suppressor T cell clone: Effect on IL-10 production and on proliferation of the suppressor T cell

In a previous study, we established CD8⁺ suppressor T cell (Ts) clone 13G2 which produced the suppressive lymphokine, interleukin-10 (IL-10). In this study, we examined what physiological activator could induce both production of IL-10 from 13G2 and the proliferation of 13G2. Both the antigenic stimulation mimicked by the anti-CD3 antibody and the T cell growth factor interleukin-2 (IL-2) induced IL-10 production from the 13G2 clone equally well. 13G2 cells proliferated remarkably with IL-2 stimulation, while anti-CD3 only slightly induced proliferation of the clone. 13G2 cells also produced IL-10 in the presence of hydroxyurea which blocked transit of cells from G₁ to S phase. However, cycloheximide blocked the production of IL-10 from the Ts clone. The study demonstrates that both the anti-CD3 antibody and IL-2 induced IL-10 synthesis of the Ts clone equally well, and the proliferative response of Ts cells was induced more by IL-2 than by anti-CD3. IL-2 proved to be a good stimulator for Ts cells to produce suppressive lymphokine and to multiply their population.Abbreviation Ts suppressor T cell - Th helper T cell - Ag antigen - APC antigen presenting cell - IL interleukin - TCR T cell receptor - mAb monoclonal antibody