Characterization of a T-cell-derived mast cell costimulatory activity (MCA) that acts synergistically with interleukin 3 and interleukin 4 on the growth of murine mast cells.

The proliferation of mucosal mast cells (MMC) depends on the presence of interleukin 3 (IL 3) and can be further enhanced by interleukin 4 (IL 4). The supernatant of a TH2 cell clone (ST2/K.9) stimulated by concanavalin A was found to contain a factor, provisionally termed mast cell costimulatory activity (MCA), that substantially enhances the proliferation of MMC promoted by a combination of IL 3 and IL 4. In comparison to other lymphokines MCA is rather resistant to tryptic digestion but is very sensitive to pH values lower than 6.0 and to organic solvents. Chromatographic fractionation of MCA revealed that activity is associated with protein(s) or glycoprotein(s) of 35 to 40 kDa. Partial…

Co-development of naive CD4+ cells towards T helper Type 1 or T helper type 2 cells induced by a combination of IL.-12 and IL-4

Abstract Cytokines were found to play a key role in Th cell differentiation. Among them IL-12 was shown to be a potent differentiation factor for Th1 cells, whereas IL-4 is the only known cytokine that promotes the development of Th2 cells. Upon addition of comparable amounts of IL-4 and IL-12 to a primary culture of naive CD4 + T cells activated by immobilized anti-CD3 mAb, it was found that the Th1-inducing capacity of IL-12 is dominated by the Th2-promoting effect of IL-4. However, high amounts of IL-12 (10,000 U/ml) in combination with low amounts of IL-4 (100 U/ml) led to the development of a Th cell population that, upon rechallenge, showed a substantial secondary IFN-γ (Th1 cytokine)…

Specificity of H-2-linked Ir gene control in mice: recognition of the core structure A--L in defined sequence analogues of (T,G,)-A--L.

For further characterization of the processes involved in Ir gene control, the specificity of antibodies and the cross-reaction on the level of helper T cells was studied for a series of polypeptide antigens related to poly-L(Tyr,Glu)-poly-DL-Ala–poly-LLys[(T,G)-A–L] but carrying more defined side chains. Helper cell specificity was assayed in an in vitro secondary anti-dinitrophenyl (DNP) response by cross-stimulation of primed T cells with the various polypeptide carriers. It was established that these polypeptides, although showing the same response pattern, were recognized as distinct entities in the immune response by B and T cells. If this common pattern is due to the effect of the sa…

Components of an antigen-/T cell receptor-independent pathway of lymphokine production

The general way to induce the synthesis of lymphokines by T cells is the stimulation through the T cell receptor (TcR) complex which results in an increase of intracellular [Ca2+] and in the activation of a tyrosine kinase as well as of protein kinase C. Lymphokine production induced via the TcR is inhibited by the immunosuppressive drug cyclosporin A (CsA). However, an alternative pathway of lymphokine production exists. Several T lymphocyte clones can synthesize interferon-gamma (IFN-gamma), granulocyte-monocyte colony-stimulating factor, and small amounts of interleukin (IL3) when stimulated with syngeneic or allogeneic accessory cells (AC) plus IL2. In contrast to the TcR pathway the al…

Epitope specificity and Ia restriction of T cell responses to insulin in a system of complementing Ir genes: analysis with primed lymph node T cells and a long-term cultured T cell line.

The antibody response of (H-2b X H-2k)F1 mice to pig insulin (PI) has previously been shown to be under the control of H-2-linked, complementing Ir genes. In addition, this response was reported to depend on the genetic background of the parental strains (Keck, K., Eur. J. Immunol. 1977. 7: 811). Here it is demonstrated that the secondary in vitro response of proliferating T cells shows the same dependence on H-2-linked Ir genes yet an influence of the background genes could not be detected. The complementing genes were mapped to the Kb, I-Ab and Kk, I-Ak regions. For restimulation of F1 T cells by PI, the Ir genes of both parental chromosomes have to be expressed in the same antigen-presen…

Requirements for the growth of TH1 lymphocyte clones.

Besides the signal generated in a T lymphocyte after triggering the T cell receptor (TcR), most lymphocytes need a "second signal" to become fully activated. The necessity and nature of the "second signal" differs between different types of T cells. At the level of CD4-positive T helper lymphocytes interleukin 1 (IL 1) serves as "second signal" for those of the TH2 subtype (IL4, 5, 6 producer) but not for those of the TH1 subtype (IL 2, IFN-gamma producer). This correlates with the absence of the IL 1 receptor at the surface of TH1 clones. We report herein the further purification of T cell stimulating factor (TSF), a soluble mediator involved in the proliferation of TH1 lymphocytes. A prep…

Freshly isolated mouse 4F7+ splenic dendritic cells process and present exogenous antigens to T cells.

The antibody 4F7 was reported to recognize an epitope expressed on dendritic cells (DC) from various tissues. To study the ability of splenic 4F7+ dendritic cells to process antigen for presentation to CD4+ T cells, DC were enriched using a separation procedure avoiding overnight culture which could lead to an altered phenotype. These DC were used as antigen-presenting cells (APC) in stimulation cultures of major histocompatibility complex class II-restricted T cells. It was found that they induce antigen-dependent lymphokine production by T cells and therefore could present exogenous antigens. These processing takes place intracellularly, because fixation abrogates presentation to T cells.…

Specificity and Restriction of T Cells in a System of Complementing Ir Genes

The phenomenon of Ir gene control of immune responsiveness is intimately related to the fact that antigen-specific stimulation of T cells with the Lyt-1 surface phenotype requires the simultaneous recognition of both external antigen and Ia antigen on the surface of antigen-presenting cells. There is increasing evidence indicating that Ia antigens encoded for by the I-A and I-E/C subregions of the H-2 complex represent the products of Ir genes.

The efficient bovine insulin presentation capacity of bone marrow-derived macrophages activated by granulocyte-macrophage colony-stimulating factor correlates with a high level of intracellular reducing thiols

Bone marrow-derived macrophages (BMM phi) were shown before to function as antigen-presenting cells. We show here, that the antigen presentation capacity of BMM phi depends on the nature of the antigen and is differently regulated by the lymphokines interferon-gamma (IFN-gamma) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). When bovine insulin (BI) was employed as antigen, only BMM phi treated with GM-CSF (GM-CSF-M phi) were efficient presenters, but when presentation of the antigens ovalbumin and conalbumin was tested, IFN-gamma-pulsed BMM phi (IFN-gamma-M phi) proved superior to GM-CSF-M phi. The lack of efficient BI presentation function of IFN-gamma-M phi was only obviou…

Characterization of a T cell-derived lymphokine that acts synergistically with IL 3 on the growth of murine mast cells and is identical with IL4

Abstract A mast cell-like cell line (SN-1) was established with the aid of growth factor(s) present in the supernatant of a Con A-stimulated L3T4 + T cell line. In analogy to other mast cell lines, IL 3 was identified as a growth factor for SN-1 cells. In addition, a second lymphokine produced by the T cells synergistically enhanced the IL 3-induced growth. This factor, originally termed mast cell growth enhancing factor (MaGEF), could be separated from IL 2, IL 3, a CSF-like activity and was purified to homogeneity. The N-terminal amino acid sequence (8 residues) and the functional properties of this lymphokine proved to be identical with those reported for BSF-1 (IL 4). Unless applied at …

Tcgfiii/p40 is produced by naive murine cd4+ t cells but is not a general t cell growth factor*

Several antigen-specific T cell lines were found to secrete a lymphokine upon activation by antigen or lectin that was provisionally termed T cell growth factor III (TCGF III) because it induced the proliferation of a CD4+ T cell clone independently from IL2 and IL4. Amino acid sequence analysis (and the functional properties of TCGF III) revealed that TCGF III was identical with a recently identified lymphokine termed P40. TCGF III/P40 was not only produced by long-term cultured T cell lines but also upon stimulation of freshly isolated Mlsa-reactive T cells. In addition, naive CD4+ T cells secreted TCGF III/P40 upon activation by lectin or allo-major histocompatibility complex structures.…

Presentation of insulin and insulin A chain peptides to mouse T cells: involvement of cysteine residues.

The requirements for insulin presentation and recognition by A alpha b A beta b- and A alpha b A beta k-restricted mouse T cells were studied using a variety of derivatives of the insulin A chain. It was found that A chain peptides with irreversibly blocked Cys residues are non-stimulatory for the T cells. This suggests that at least one of the Cys residues is essential for recognition. On the other hand, all A chain peptides containing Cys residues modified in a way reversible by reaction with thiols are stimulatory yet differ in antigenic potency. All these A chain derivatives including a 14 amino acid fragment require uptake by antigen presenting cells (APC) for efficient presentation. D…

Development of T cell clones reactive to two defined restriction elements in conjunction with two defined epitopes of antigen

A previously described pig insulin (PI)-specific T cell line of (B10 X B10.BR)F1 origin was assayed for its reactivity with species variants of insulin in the presence of antigen-presenting cells (APC) of various H-2 haplotypes. In addition to its reactivity with PI and bovine insulin (BI) in the context of syngeneic F1 (H-2b X k)-APC, a weak cross-reactivity was observed with parental B10 (H-2b)-APC and BI but not PI. The cross-reactive cells could be selected out by several restimulations with the combination of BI and B10-APC. From the resulting, strongly cross-reactive T cell line several interleukin 2-dependent sublines were developed which did not require antigen-specific restimulatio…

Lymphokine profile and activation pattern of two unrelated antigen- or idiotype-specific T suppressor cell clones.

Two T suppressor (Ts) clones of different specificity have been analyzed for their lymphokine spectrum. BVI/5 is an I-Ek-restricted bovine serum albumin (BSA)-specific Ts cell clone from a CBA/J mouse tolerized by low doses of BSA. It affects directly or indirectly the function of BSA-specific T helper (Th) cells. The Ts cell clone 178-4 from a BALB/c mouse is I-Ed restricted and recognizes the public J558 Id on B cells. It prevents alpha(1----3)dextran B 1355S (Dex)-specific IgG antibody production and drives Dex-specific J558 idiotype-bearing B cells into an anergic B IgG memory cell state. Both Ts cell clones thus cause specific suppression, yet in different experimental systems using di…

Cloned T helper cells reverting to a resting state develop increasing sensitivity in their antigen-mediated interaction with accessory cells.

A cloned murine T cell line, KIII5, specific for the polypeptide poly-L(Tyr,Glu)-poly-D,L-Ala--poly-L-Lys [(T,G)-A--L] was compared at different stages after antigenic stimulation with respect to the conditions required for the reinduction of growth by varying concentrations of antigen presented on different types of accessory cells (AC). We show that the dose of antigen necessary for inducing half maximal proliferation in the presence of splenic AC shifts to considerably lower concentrations when the T cell blasts revert to a resting state (100 micrograms/ml on day 7 to 10 micrograms/ml on day 21-35). During the same time period the expression of interleukin 2 (IL2) receptor and the reacti…

Perpetual proliferation of LYT-1 cells requires repetitive signals for IL-2 receptor induction by antigen-presenting cells.

Abstract T cell lines with specificity for bovine insulin and ovalbumin were maintained by serial stimulation with antigen presented on irradiated syngeneic spleen cells, alternating 3 days later with subculture in IL-2 containing medium (CM). When the cultures were repetitively split in CM, with concomitant dilution of antigen-presenting cells, a gradual loss of proliferative capacity of the cells in the presence of CM was observed. Absorption studies revealed a 20-fold reduction of IL-2 receptors on the surface of T blasts assayed 12 days after antigenic stimulation as compared with day 5 blasts. This decrement in the number of IL-2 acceptor sites reflected an actual decrease in cell surf…

Interleukin 12 and its Receptor

Processing without proteolytic cleavage is required for recognition of insulin by T cells.

Beef insulin as well as a chymotryptic A-chain fragment [BI-A1-14(SSO3-)3] need uptake by antigen-presenting cells (APC) for efficient presentation in combination with major histocompatibility complex class II molecules to insulin-specific T cells. This could be shown by the inability of aldehyde-fixed APC to present these antigens to T cells. Furthermore, presentation of the insulin fragment as well as presentation of ovalbumin (OVA) was inhibited by treatment of APC with chloroquine, cerulenin or tunicamycin. This was not the case for a processing-independent OVA peptide. Treatment of APC during antigen pulsing with various protease inhibitors, active on all classes of proteases, did not …

Xid-defective male (CBA/N x C57BL/6)F1 accessory cells present bovine insulin to long-term cultured F1-restricted T-cells

The reactivity of H-2b-restricted murine T cells towards bovine insulin was reported to depend on the expression of Ia. W39, a private specificity of I-Ab, on antigen-presenting cells. Cells of male (CBA/N X B6)F1 mice carrying the mutation xid on the X chromosome lack IA. W39 on the cell surface. These cells are unable to present bovine insulin to primed T cells derived from female (CBA/N X B6)F1 mice. We show here that spleen cells of male (CBA/N X B6)F1 hybrids served perfectly as accessory cells for the insulin-dependent induction of a proliferative response of long-term cultured T cells with (B10 X B10.BR)F1 genotype, restricted to recognizing insulin in the context of F1-unique I-A de…

An antigen-independent physiological activation pathway for L3T4+ T lymphocytes.

The data presented in this report describe an antigen-independent activation pathway leading to reinduction of proliferation of class II major histocompatibility complex (MHC)-restricted murine T cell lines that after previous antigen-specific stimulation reverted to a resting state. Antigen-independent proliferation and interleukin 2 (IL2)-receptor expression occur in the presence of splenic accessory cells, exogenous IL2 and a soluble factor(s) provisionally termed T cell-stimulating factor(s) (TSF). Each of these components is essential for inducing growth. TSF is found in the supernatant of an autoreactive T cell line upon stimulation with syngeneic accessory cells. Neither TSF nor acce…

Processing requirements for the recognition of insulin fragments by murine T cells.

In this study we investigated aspects of antigen processing using insulin and insulin A chain-derived fragments as model antigens in Ab alpha Ak beta-restricted T-cell stimulation. Similarly to other proteins, the immunodominant region of insulin recognized by these T cells is limited in size. It is located on the insulin A chain and encompasses a portion of the molecule that is represented faithfully by peptide A1-14(SSO3-)3. Efficient presentation of intact insulin and its entire A chain is dependent on uptake and processing by APC. Whereas peptides stemming from various globular proteins are known to be presented to T cells by APC without requiring processing, this is not the case with A…

The Immunostimulatory Function of IL-12 in T-Helper Cell Development and Its Regulation by TGF-β, IFN-γ and IL-4

T cell factor (interleukin 2) allows in vivo induction of T helper cells against heterologous erythrocytes in athymic (nu/nu) mice.

Mice carrying the nude mutation (nu/nu) lack a functioning thymus and do not contain detectable levels of immunocompetent T cells. We now report that nu/nu mice do have lymphocytes which can be activated in vivo by heterologous erythrocytes and a Lyt-1 T cell-derived factor (interleukin 2) to generate T helper cells. Thus, a lymphokine is described which is able to restore in vivo T helper cell immunocompetence of nu/nu mice. The data may suggest that nu/nu mice contain a low number of T lymphocytes influenced by the cystic remnant of the nu/nu thymus anlage. Alternatively, the data imply that interleukin 2 circumvents the requirement of a thymus during ontogeny of T lymphocytes.

Involvement of soluble mediator(s) different from interleukin (IL)1 in the antigen-induced IL 2 receptor expression and proliferation of L3T4+ (CD4+) T lymphocytes

Proliferation of T lymphocytes (T cells) requires the interaction of interleukin 2 (IL 2) with the high affinity form of the IL 2 receptor (IL 2R). IL 2 production as well as IL 2R expression are generally induced simultaneously in T cells by the recognition of specific antigen displayed on the surface of syngeneic antigen-presenting cells. The experiments described herein show that the expression of IL 2R has different requirements than the production of IL 2 (and other lymphokines). Stimulation of antigen-specific L3T4+ T cell lines with antigen-pulsed spleen cells (SC) treated with ultraviolet (UV) light results in efficient IL 2 production but only minimal proliferation due to reduced I…

Co-activation of naive CD4+ T cells and bone marrow-derived mast cells results in the development of Th2 cells

Activation of naive dense CD4+ T cells by plate-bound anti-CD3 antibodies favors the development of Th1 cells which, upon re-stimulation, produce significant amounts of IFN-gamma but no IL-4. However, co-activation of such naive T cells in the presence of IgE [anti-dinitrophenyl (DNP)]-loaded bone marrow-derived mast cells (BMMC) on plates coated with anti-CD3 antibodies and DNP-BSA led to the development of IL-4-producing Th2 cells. The same result could be observed if irradiated (800 rad) BMMC were applied as co-stimulators. Moreover, BMMC could be replaced by the supernatant of IgE-activated BMMC suggesting that a soluble mediator, presumably IL-4, was responsible for this effect. This a…

Requirements of Exogenous Protein Antigens for Presentation to CD4+ T lymphocytes By MHC Class II-Positive APC

The antigen-specific activation of CD4-positive T helper cells depends on the recognition of a complex of MHC class II molecules and an antigen-derived peptide on the surface of antigen-presenting cells (APC). For most antigens generation of this MHC/peptide complex requires the uptake of the respective antigen by APC, followed by intracellular processing. The latter leads to suitable peptides of the antigen which are able to bind to MHC class ll-molecules. Subsequently the resulting complexes are transported to the cell surface. Evidence supporting this concept came mainly from the finding that agents such as chloroquine1, interfering with the function of endosomes and lysosomes, can block…

Induction of anamnestic T cell proliferation by antigen-pulsed, bone marrow-derived macrophages.

Bone marrow-derived macrophages (BMM phi) were grown in a liquid culture system in the presence of L cell-conditioned medium as a source of colony-stimulating factor. After a 4-h pulse with antigen, cultured irradiated BMM phi were capable of presenting the antigen to primed T cells as assessed in a T cell proliferation assay. Proliferation was optimal when BMM phi were used between days 5 and 8 of bone marrow cell culture. T cells of Lyt1 and Lyt123 phenotype had to be present at the start of the culture period to yield an optimal response. Conventional antisera and monoclonal antibodies directed against the H-2 I region and the I-A subregion, respectively, proved inhibitory in this system…

Mast cell growth-enhancing activity (MEA) is structurally related and functionally identical to the novel mouse T cell growth factor P40/TCGFIII (interleukin 9).

We have previously shown that certain bone marrow-derived mast cell (BMMC) lines proliferate in response to a mast cell growth-enhancing activity (MEA) that is distinct from interleukin (IL) 3 and IL 4. Here we provide evidence that MEA is identical with the recently cloned mouse T cell growth factor P40. The evidence is as follows: (a) recombinant P40 displayed all the biological activities ascribed to MEA: it supported the growth of MEA-sensitive BMMC lines, it induced IL 6 secretion by these cells, and it enhanced survival of primary mast cell cultures; (b) highly purified MEA stimulated the growth of P40-dependent cell lines; (c) a rabbit monospecific antiserum directed against P40 spec…

NF-ATp plays a prominent role in the transcriptional induction of Th2-type lymphokines

Establishment of different T cell sublines using either interleukin 2 or interleukin 4 as growth factors

Purified protein derivative reactive T cell lines were established under identical conditions with the exception that different lymphokines, namely interleukin (IL) 2 and IL 4 were employed as growth factors. IL 2 favored the development of T cell lines (LNC.2) which upon activation by concanavalin A (Con A) secreted predominantly lymphokines characteristic of TH1 cells. By contrast, T cell lines established with the aid of IL 4 as growth factor (LNC.4) produced mainly lymphokines representative of TH2 cells. Apart from their pattern of lymphokine secretion LNC.2 and LNC.4 T cells were found to differ in their proliferative response to lymphokines and Con A. LNC.2 T cells proliferated only …

Characterization of the Adjuvant Effect of IL-12 and Efficacy of IL-12 Inhibitors in Type II Collagen-Induced Arthritis

A destructive joint disease can be induced in susceptible DBA/1 mice by immunization with type II collagen emulsified with oil and either killed Mycobacterium tuberculosis or IL-12 as adjuvant. Cellular and humoral anti-collagen immune mechanisms appear to be involved in the pathogenesis of arthritis. We have characterized the adjuvant effect or IL-12 in more detail and addressed the question whether mycobacteria might act via the induction of endogenous IL-12. Injections of IL-12 into collagen-immunized DBA/1 mice promoted the development of IFN-gamma-producing CD4+ T cells and strongly upregulated the production of complement-fixing IgG2a and IgG2b antibodies resulting in severe arthritis…

Characterization of lymphokine-mediated activation of macrophages for antigen presentation: studies with long-term cultured bone marrow-derived macrophages and cloned T cells.

In cultures of bone marrow (BM) supplemented with L cell-derived colony-stimulating factor a pure population of macrophages (M phi) differentiates, which can be further propagated with a doubling time of 3.8 days. "Young" BMM phi obtained on day 8 of culture were shown to act as antigen-presenting cells inducing the antigen-specific proliferation of the cloned T cell line ST2/K.9, whereas "old" M phi had lost this ability. However, at any time tested (up to 132 days) the presentation function of old BMM phi could be completely restored by pulsing the cells with lymphokines (LK). A duration of 11 hr for the LK-pulse was sufficient to trigger the M phi to exert an optimal presentation functio…

Processing and Presentation of Protein and Parasite-Derived Antigens by 4F7+ Dendritic Cells

The dendritic cell (DC), a trace component of splenocytes is the principal cell type required for a primary mixed lymphocyte reaction.1 Splenic DC are described as antigen presenting cells (APC) capable to generate immunogenic fragments of intact protein antigens for presentation to MHC class II restricted T cells,2 in contrast to former findings.3 Langerhans cells (LC) as potent APC are members of the dendritic cell lineage forming a system of potent APC, first identified by Steinman and Conn.4 Importanly Schuler and Steinman have subsequently presented experimental evidence suggesting that LC represent immature DC.3 During infectious diseases caused by parasites of the genus Leishmania LC…

Differential expression of mRNA encoding interleukin-12 p35 and p40 subunitsin situ

Interleukin-12 (IL-12) is a heterodimeric cytokine that plays an important role in the regulation of the immune response. For biological activity the expression of both subunits of IL-12, p35 and p40, is required. Moreover, in the mouse the p40 chain of IL-12 specifically inhibits the effects of the IL-12 heterodimer. In the present study we have analyzed by in situ hybridization the expression of the p35 and p40 mRNA in the spleens of BALB/c and mutant (SCID, nude, beige) mice, unstimulated and after in vivo stimulation with lipopolysaccharide (LPS) and with staphylococcal enterotoxin B (SEB). In unstimulated spleens of BALB/c mice p35 and p40 mRNA were only detectable in a few strongly st…