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.     

Generation of cytotoxic T lymphocytes from thymocyte precursors to trinitrophenyl-modified self antigens. I. Requirement of both killer-helper factor(s) and interleukin 2 for CTL generation from a subpopulation of thymocytes

The requirement for signals in the induction of cytotoxic T lymphocytes (CTL) from thymocyte precursors has been investigated. Either unfractionated or peanut agglutinin-binding (PNA+) C3H/He thymocytes were stimulated with mitomycin C(MMC)-treated, 2,4,6-trinitrophenyl(TNP)-modified syngeneic spleen cells in the presence of a variety of lymphokine preparations. Cellfree supernatant (CFS) from purified protein derivatives(PPD-CFS) stimulated Mycobacterium tuberculosis (Tbc)-primed cells, or partially purified interleukin 2 (IL 2) mediated strong cytotoxic responses in unfractionated thymocytes, whereas only PPD-CFS at final concentrations beyond 30% was active for CTL generation in PNA+ thymocytes. Neither IL 2 at concentrations of below 60 U/ml nor a low concentration of PPD-CFS (at final below 10%) had such a capacity. The addition of monoclonal anti-IL 2 receptor antibody completely blocked CTL generation induced by PPD-CFS in PNA+ thymocytes. In contrast, anti-immune interferon (IFN-gamma) antibody showed a marginal effect. PPD-CFS (10%) and IL 2 (10 U/ml) could synergistically trigger PNA+ thymocytes to induce CTL generation. These results suggested that both IL 2 and "helper" factors other than IL 2 are required for CTL generation from PNA+ thymocytes. We refer to these kinds of helper factors as killer helper factors (KHF). Partially purified IL 2-free KHF show two peaks of activities at apparent m.w. 14,000 to 34,000 and 44,000 to 90,000, and are heterogeneous with respect to isoelectric point, which is between 4.5 and 5.1. Cultures that received TNP-modified syngeneic cells and KHF on day 0 and IL 2 on day 2 generated potent CTL responses, whereas the addition of IL 2 on day 0 followed by the addition of KHF on day 2 to the culture was ineffective, suggesting that KHF is required in the early phase of the culture to achieve optimal CTL responses.     

Antigen-specific helper factors present in the supernatant of concanavalin A-induced spleen cell cultures

The supernatants from cultures of concanavalin A-induced spleen cells contained both antigen-specific and nonspecific (Interleukin 2) helper factors (Hf). The antigen-specific factor could be isolated from the supernatant by adsorption onto and elution from antigen-Sepharose immunoadsorbents. Specific Hf was produced in cultures of either immune or nonimmune spleen cells although in the latter case the quantity of Hf was significantly less. The specific Hf did not manifest the thymocyte stimulatory property of 112.     

T cell-derived glucosteroid response-modifying factor (GRMFT): a unique lymphokine made by normal T lymphocytes and a T cell hybridoma

Mouse spleen cells and a murine T cell hybridoma, FS6 14.13.1, produce a glucosteroid response-modifying factor (GRMFT) after stimulation with concanavalin A. GRMFT blocks glucosteroid suppression of helper T cell function and the growth of granulocyte/macrophage progenitor cells in vitro. IL 1 also protects helper T cells and myeloid precursors from glucosteroid suppression. This suggests that GRMFT and IL 1 act congruently to ensure that an effective immune response is generated when endogenous glucosteroid levels are elevated. To understand the role of GRMFT in normal immune responses and in disease states characterized by imbalances in the immune system, we began to purify and characterize GRMFT. GRMFT appears to be distinct from other well-characterized T cell-derived factors. GRMFT is larger than IL 2 as determined by gel exclusion chromatography and is completely separated from IL 2 by isoelectric focusing. Furthermore, purified IL 2 does not have GRMFT activity. Purified IL 3 also lacks GRMFT activity, and conditions that inactivate immune interferon have no effect on GRMFT. Thus, GRMFT is different from IL 2, IL 3, and immune interferon. GRMFT also lacks activity in the thymocyte co-mitogenic assay and is therefore different from IL 1. Finally, FS6 14.13.1 reportedly does not produce TRF or CSF, which suggests that GRMFT is different from these molecules as well.     

Induction of the differentiation of memory T killer cells with factors released from macrophage-like cell lines

Macrophage-like cell lines J774.1 and WEHI-3 as well as peritoneal exudate macrophages have been demonstrated to produce factors which induce the differentiation of memory cells into specific T killer cells in the absence of an added antigen. LPS stimulation was required for J774.1 cells and peritoneal macrophages to produce the factors but not for WEHI-3 cells. Interferon seemed to be one of the responsible factors. However, macrophages seem to produce other active factors; one has a molecular weight (MW) of more than 80,000 and lacks thymocyte mitogenic activity; another, with a weak thymocyte mitogenic activity, has a MW of 38,000 to 44,000. The low MW thymocyte mitogenic factor (interleukin-1) showed weak T killer cell differentiating activity.     

Epidermal cell (keratinocyte)-derived thymocyte-activating factor (ETAF)

In order to determine whether keratinocytes play a role in the modulation of the immune response, we investigated the murine keratinocyte cell line Pam 212. In culture these cells generate a substance with a biologic activity that greatly enhances phytohemagglutinin-induced thymocyte proliferation. We have, therefore, called this substance epidermal cell thymocyte-activating factor (ETAF). This keratinocyte-derived supernatant activity is mainly produced at the onset of the logarithmic growth phase and is directly mitogenic for murine thymocytes. Although ETAF by itself exhibits no T cell growth factor activity, ETAF enhances Interleukin 2 production by mitogen-stimulated murine spleen cells. Murine ETAF is not genetically restricted and lacks species specificity since it decreases lectin-induced proliferation of human peripheral blood lymphocytes (as well as murine spleen cells) and also enhances the production of human Interleukin 2. The factor has a m.w. between 15,000 and 25,000 as determined by gel filtration and elutes as a single peak from anion exchange chromatography columns. The activity is maintained mainly at alkaline pH and is rapidly destroyed at temperatures above 60 degrees C. These observations suggest that epidermal cells may interact with the immune system by elaborating nonspecific factors that modulate lymphocyte proliferation and augment lymphokine production.     

Lymphokines: their role in lymphocyte responses. Properties of interleukin 1

Interleukin 1, or IL 1, otherwise known as lymphocyte-activating factor, is a macrophage-derived 12,000- to 15,000-dalton polypeptide. Isoelectric focusing of human IL 1 reveals three peaks at pI's of 5.2, 6.0 and 6.9 respectively. IL 1 can be depleted of lymphocyte-derived IL 2 by SP-Sephadex chromatography. IL 1 augments the mitogenic response of PNA- Lyt 1+ thymocytes, and promotes thymocyte helper functions and B cell antibody production. IL 1 induces stable E rosette formation and the production of lymphokines such as T cell growth factor (IL 2) by peripheral T lymphocytes. Others have shown that IL 1 or closely related factors also stimulate hypothalamic cells to induce fever; induce in vitro fibroblast growth, prostaglandin, and collagenase production; and stimulate hepatocytes to produce acute phase proteins such as serum amyloid A. Murine epidermal cells also produce a 15,000-dalton factor that is mitogenic for thymocytes and may be similar to IL 1. We have recently hybridized spleen cells from mice sensitized with partially purified human IL 1 with a myeloma cell line. Clones have been isolated that produce supernatants that partially inhibit the thymocyte proliferative response to IL 1 but not the T cell growth factor activity of IL 2. Should these hybridoma products prove to be monoclonal anti-IL 1 antibodies, they will facilitate the further purification and characterization of IL 1.     

Stimulation of macrophage H2O2 release by resident thymocytes: effect of a soluble factor distinct from interferon-gamma

Activated T cells are known to stimulate macrophage oxidative metabolism and antimicrobial activity through release of interferon-gamma (IFN-gamma). In contrast, the role of nonactivated T cells in regulating macrophage effector functions is less well defined. We have previously reported that a low molecular weight soluble factor derived from resident (nonactivated) thymocytes enhances macrophage receptor-mediated phagocytosis. In the present study, we examined the capacity of resident murine thymocytes to stimulate the respiratory burst and microbicidal activity of peritoneal macrophages. Macrophages cultured for 1-2 days with cell-free thymocyte supernatant (TS) released two to three times more H2O2 in response to PMA or opsonized zymosan than did control macrophages. The H2O2-stimulating factor in TS was distinguished from IFN-gamma by its heat stability (100 degrees C, 20 min), approximate MW of 2400 Da (gel filtration high-pressure liquid chromatography), and absence of interferon activity in both antiviral and enzyme-linked immunosorbent assays. TS-treated macrophages, however, did not exhibit a greater capacity to kill or inhibit the intracellular growth of Toxoplasma gondii, indicating that the thymocyte factor did not fully activate macrophage microbicidal mechanisms. These data suggest that thymocytes can increase the respiratory burst capacity of macrophages in the absence of antigen-specific immune responses.     

Biochemical relationship of thymocyte mitogenic factor and factors enhancing humoral and cell-mediated immune responses.

Supernatants from concanavalin A-induced mouse spleen cells contain soluble factors which: 1) augment the anti-sheep erythrocyte hemolytic plaque-forming cell response of nude mouse spleen cells, 2) facilitate the generation of antigen-specific cytotoxic cells by alloantigen-stimulated thymocytes, and 3) enhance the mitogenic response of thymocytes in the presence or absence of phytohemagglutinin. The uninduced control spleen cell supernatants contained variable but significantly less biologic activity within the difference assay systems. The principle enhancing activity (apparent m.w. 35,000 to 38,000) in each of the assay systems was found to be inseparable after a multiple step purification scheme involving ammonium sulfate precipitation, two-cycle gel filtration, hydroxylapatite chromatography, and hydrophobic chromatography on Phenyl Sepharose. These results indicate that either a single factor (designated thymocyte mitogenic factor) augments both humoral and cell-mediated immune responses or, alternatively, the different biologic activities are mediated by different factors that share a common core structure.     

Regulation of the production of immune interferon and cytotoxic T lymphocytes by interleukin 2

The activation of alloantigen-specific cytotoxic T lymphocyte precursors is dependent upon the presence of both macrophages and helper T cells or regulatory molecules derived from these facilitative cells. Three biochemically distinct helper factors have been identified: interleukin 1 (macrophage-derived), Interleukin 2 (T cell derived), and immune interferon. All 3 factors are found in supernatants of mixed lymphocyte cultures (MLC), however, the removal of macrophages from these cultures completely ablates the production of these factors as well as the induction of cytotoxic T lymphocytes (CTL). The addition of IL 2 to these macrophage-depleted MLC restores the ability of responder T cells to: 1) bypass the requirement for macrophage soluble function, 2) produce immune interferon, and 3) generate CTL. The kinetics and dose response of immune interferon production in response to IL 2 correlates with the generation of CTL. The production of immune interferon as well as the generation of CTL requires T cells, alloantigen, and IL2. Furthermore, the induction of CTL by IL2 was neutralized by the addition of anti-immune interferon. These data suggest that: 1) the regulation of immune interferon production is based on a T to T cell interaction mediated by IL 2, and 2) immune interferon production may be required for IL 2 induction of CTL. These findings are consistent with the hypothesis that the induction of CTL involves a linear cell-factor interaction in which IL 1 (macrophage-derived) stimulates T cells to produce IL 2, which in turn stimulates other T cells to produce immune interferon and become cytotoxic.     

Regulation of lymphocyte proliferation and differentiation by macrophages.

In this paper, we consider the role of a macrophage secretory product in promoting thymocyte differentiation, as well as a macrophage-immune T cell interaction that results in augmented secretion of lymphostimulatory factors. When cultured with the thymocyte-differentiating factor (TDF), thymocytes show a physiological increase in H-2D and K, decreased sensitivity to lysis with anti-TL and complement, and acquisition of responsiveness in the mixed lymphocyte culture. Development of the mature phenotype requires 2 to 3 days of culture and, once attained, is stable. The induced antigenic changes do not require cell division. The activity demonstrated by TDF, which is not attributable to interferon and cannot be replaced by 2-mercaptoethanol, is also displayed by normal thymic macrophages themselves. Enhanced secretion of TDF and of a distinct mitogenic protein follows the interaction of macrophages and immune T cells. This interaction is shown to require physical contact of the two cell types and is regulated by products of the I-A region of the major histocompatibility complex.     

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.     

In vivo activity of affinity-purified helper factor from antigen-specific helper clone

Supernatant from culture of a virally transformed OVA-specific helper T clone (C-41) was examined for the presence of soluble helper factor. Inoculation of helper clone supernatant into DNP-KLH-primed mice enhanced the IgG anti-DNP response when given with DNP-OVA. The C-41 supernatant did not trigger the DNP-primed B cells in mice when injected with hapten (DNP) coupled to an unrelated carrier (BSA). The carrier-dependent helper activity of C-41 supernatant in vivo demonstrates the presence of an antigen-specific T helper factor in the media of the cultured helper clone. Extensive immunization of F1(C57BL X BALB/c) mice with the helper clone resulted in the production of anti C-41 antibodies. Monoclonal antibodies prepared from the immunized mice were screened for specificity of binding to other transformed T lines and clones, some specific to OVA. Monoclonal antibodies that stained the C-41 cells exclusively were considered clone-specific. Supernatants of the helper clone were passed over columns of anti-clone-specific antibodies. The eluates from three antibodies were active as antigen-specific helper factor, i.e., they elevated the IgG anti-DNP response in vivo in a linked recognition fashion in the presence of DNP-OVA. The affinity-purified factor was inactive when injected with DNP-BSA or DNP-BSA + OVA. Thus, we describe the antigen-specific immune function of a clone-produced helper factor in normal mice.     

Helper factors for hapten-self cytotoxic T cells occur in NZB culture supernatants despite deficient AMLR

The ability of helper T cells from NZB mice to produce non-interleukin 2 (IL-2) lymphokines in the autologous mixed lymphocyte reaction (AMLR) was examined. Factors present in normal AMLR culture have been previously reported to mediate the development of a cytotoxic T-cell response to trinitrophenyl (TNP)-modified syngeneic thymocytes. Young NZB mice, like the normal strains, were able to produce the helper factors in the AMLR and to utilize these mediators in the cytotoxic induction system. Old autoimmune NZB mice demonstrated a poor proliferative response in the AMLR and were unable to activate hapten-specific cytotoxic cells in the presence of AMLR culture supernatant from either young or old mice. This was not due to a lack of cytotoxic precursors, nor was it a normal consequence of aging, but may be related to decreased IL-2 production by helper T cells. Interestingly, supernatant from AMLR proliferation deficient old NZB mice contained normal amounts of the AMLR helper factor. These data suggest that AMLR helper factor production is not directly related to the proliferative response and that two different helper-T-cell subpopulations may be responsible for these activities. The production of these mediators in mice which cannot utilize them raise questions about their role in autoimmunity.     

Functional analysis of human T cell subsets defined by monoclonal antibodies. III. Regulation of helper factor production by T cell subsets

In the present report we extended our previous studies demonstrating that obligatory T-T interactions are important in regulating human immune responses in vitro. Functionally distinct human T cell subsets were isolated by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Evidence was obtained that during allogeneic interactions, OKT4+, but not OKT8+, responder T cells are required to generate helper factor(s) capable of polyclonally activating human B cells independent of additional T cell help. Importantly, the alloantigen-induced helper factor(s) production and/or release was found to be suppressed by addition of graded numbers of radiosensitive OKT8+ cells. On the other hand, no evidence was obtained that supernatant derived from alloactivated OKT8+ cells could counterbalance the helper activity generated in the presence of supernatant from alloactivated OKT4+ cells. Furthermore, OKT8+ cells, known to suppress PWM-driven B cell differentiation in the presence of OKT4+ cells, do not suppress B cell differentiation induced by preformed helper factor even in the presence of OKT4+ cells. These data further underscore the importance of functional T-T interactions in immunoregulation in vitro and support the idea that the target of suppression of B cell differentiation, induced either by alloantigen-triggered helper factor or PWM, are OKT4+ cells and not B cells themselves.     

The necessity for T cell help for human tonsil B cell responses to pokeweed mitogens: induction of DNA synthesis, immunoglobulin, and specific antibody production with a T cell helper factor produced with pokeweed mitogen.

Human B lymphocytes obtained from tonsils do not proliferate when stimulated with pokeweed mitogen. A soluble factor produced from T cells cultured with pokeweed mitogen stimulates B cells to synthesize DNA and differentiate into immunoglobulin producing cells. This PWM produced supernatant induced a PFC response to SRBC. The T cell supernatant activity is produced within 12 hr of stimulation in the presence of serum and without a requirement for T cell division. Optimal stimulation of B cells occurred at 7 to 9 days of culture. This helper factor activity eluted postalbumin from a column of Sephadex G-200. Insolubilized pokeweed mitogen was not mitogenic for B cells. The continuous presence of the lectin in culture was not required for B cell proliferation or for immunoglobulin synthesis.     

Antigen-specific helper factor production by immortalized clone of OVA-specific T cells. I. In vitro activity

Immortalized clones of virally transformed OVA-specific T cells produce antigen-specific helper factor upon stimulation in vitro. The helper factor activate DNP-primed B cells to multiply and synthesize IgG anti-DNP antibodies. The trigger of the helper clone is antigen specific and the B cell-stimulating hapten must be coupled to the specific T cell carrier in order to transfer the help signal from the activated T clone to the B lymphocytes. Activation of the helper clone is performed by antigen-pulsed macrophages and cannot be achieved by the free soluble antigen. However, cell-free supernatant of the antigen-pulsed macrophages can stimulate the helper cells. Thus the antigenic determinant must be presented to the helper cell in the form of macrophage-processed antigen. These requirements for antigenic stimulation and the activity of the secreted helper factor demonstrate that the immortalized helper clone preserved the cellular components which control the antigen-specific immune function of the normal T lymphocyte.     

Helper factor production in murine secondary syngeneic mixed leukocyte reactions

Culture supernatants of murine thymocytes or spleen cells responding in a secondary syngeneic mixed leukocyte reaction (SMLR) were studied for their biologic effects on cell-mediated immune responses in vitro. Such supernatants contained helper factor(s) that facilitated the development of alloantigen-specific cytotoxic T lymphocyte (CTL) responses from thymocyte precursors. Thymocytes, but not spleen cells, required activation by allogeneic effect factor (AEF) in primary culture in order to proliferate and produce biologically active mediator(s) during a secondary SMLR. The same culture supernatants possessed, in some instances, weak T cell growth factor (TCGF; IL 2) activity. However, TCGF activity could be dissociated from helper factor(s) active in the CTL induction assay because some culture supernatants that had potent helper activity were devoid of TCGF activity. This lack of TCGF activity was not due to a lower degree of sensitivity of the TCGF assay or to the presence of a selective TCGF inhibitor in the SMLR-derived supernatants, indicating that the helper factor(s) studied is distinct from TCGF. Production of immunoregulatory lymphokines during the SMLR may serve as a physiologically relevant model for studying the role of T cell-derived lymphokines in immunoregulation.     

Generation of cytotoxic T lymphocytes from thymocyte precursors to trinitrophenyl-modified self antigens. II. Establishment of a T cell hybrid clone constitutively producing killer-helper factor(s) (KHF) and functional analysis of released KHF

T cell hybridoma lines were constructed by fusion of Mycobacterium tuberculosis-primed and boosted BALB/c T cells with the AKR-derived T lymphoma cell line BW5147. Certain of the hybridomas prepared in this manner secreted constitutively into their culture supernatants biologically active molecules that displayed precursors of cytotoxic T cell activating properties characteristic of killer-helper factor (KHF). Cell surface analysis revealed that the hybridomas were indeed somatic cell hybrids between the two respective partner cells used for fusion. KHF properties of these hybridoma supernatants were verified by their capacity to stimulate peanut agglutinin-binding (PNA+) C3H/He thymocytes to respond in vitro to 2,4,6-trinitrophenyl(TNP)-modified syngeneic stimulator cells in conjunction with suboptimal doses (10 U/ml) of interleukin 2 (IL 2) for the generation of H-2-restricted, TNP-reactive cytotoxic T cells. The biologically active molecules secreted by a T cell hybrid clone (2Y4) were, like conventional KHF, distinct from IL 1, IL 2, or immune interferon (IFN-gamma). The partially purified KHF derived from 2Y4 cells shows activity at apparent m.w. range of 34,000 to 60,000 on gel permeation, and is relatively homogeneous with respect to isoelectric point, which was approximately 4.5 to 4.7. The partially purified 2Y4-KHF is able to augment proliferation of as well as the expression of IL 2 receptors on PNA+ thymocytes in conjunction with IL 2. Finally, addition of 2Y4-KHF on day 0, followed by the addition of IL 2 on day 2 for 7 days of culture was effective in generating potent CTL responses, whereas addition of IL 2 on day 0, followed by the addition of 2Y4-KHF on day 2 to the culture was ineffective.     

Bone marrow-thymus axis in senescence

This presentation offers a brief review of the bone marrow-thymus axis in senescence, a putative model for thymocyte differentiation, and recent results of our work on the status of pre-thymic stem cells in aged mice. The data presented here provide further evidence for a thymus endocrine influence on the bone marrow stem cells, specifically lymphocyte precursors. It has been postulated that the thymic hormones may act on lymphocyte precursors in the bone marrow and that the loss of thymic factors during senescence may be a contributing factor to the decreased cellular immune function. This study used Haar's in vitro model to investigate the bone marrow-thymus axis in aged mice. Erythroid-depleted bone-marrow cells from 3-month- and 24-month-old CBA (Thy 1.2) mice were placed in the upper half of a blind-well chamber with thymus supernatant in the lower half. Experimental cells were treated with thymus supernatant for 1 hr prior to migration. This study confirmed that pre-thymic stem cells in aged bone marrow are deficient in their ability to migrate to the thymus supernatant. It also revealed that treatment of the old bone marrow with thymus supernatant, made from neonatal thymus cultures, could dramatically improve the thymus migrating ability of the aged bone-marrow stem cells.