T-cell Helper Factor Research Articles

In vitro T-cell functions specific to an anti-DNA idiotype and serological markers in patients with systemic lupus erythematosus (SLE)

The human monoclonal autoantibody 16/6 is a common anti-DNA idiotype found to have clinical relevance in patients with systemic lupus erythematosus (SLE). Therefore the ability of peripheral blood T cells of SLE patients and healthy controls to proliferate and to produce helper T-cell factors following stimulation with this idiotype was tested. It was found that T cells of 75% of healthy donors proliferated to the 16/6 idiotype, whereas only 22% of SLE patients responded to this idiotype by proliferation. On the other hand, the capability to produce T-cell helper factors specific to the 16/6 idiotype was found in a higher percentage of SLE patients (48%) as compared to healthy controls (31%). The low frequency of proliferative responses in SLE patients might be due either to the chronic exposure to the 16/6 idiotype or to the production of antiidiotype antibodies against the 16/6 idiotype, which interfere with the response to the latter stimulator.

MODE OF ACTION OF ANTIGEN‐SPECIFIC T‐CELL HELPER FACTORS SECRETED BY A T‐CELL LINE AND CLONES

Antigen-specific T-cell helper factors were secreted from a (T,G)-A--L specific T-cell line and clones. The factors were released upon antigenic stimulation and could be induced by a low or a high dose of antigen. The factors secreted upon low-dose stimulation possessed the antigenic specificity of the secreting cells, while the high dose-induced factors had a broader antigenic specificity and could react with the closely related polypeptide (Phe,G)-A--L, even when the cells were restricted to (T,G)-A--L. Both the low dose- as well as the high dose-induced factors could not trigger antibody production in the presence of a non-relevant antigen, and did not collaborate with B cells immunized with a non-related antigen for the production of antibodies. The helper factors, like their secreting cells, were H-2-restricted in the collaboration with B cells. In contrast to the helper cells, however, they did not require accessory cells for triggering the B cells in the process of antibody production. Some preparations of helper factors were found to be inactive. The helper activity could be restored by IL-2. Thus, IL-2 is an additional essential factor required for the antigen-specific collaboration of B cells and T-cell helper factors.

The isolation of immunogenic molecular entities from immunogenic and nonimmunogenic tumor homogenates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

YAC, a Moloney-virus-induced tumor of A-strain mice, is a nonimmunogenic tumor. Mice injected with the inactivated neoplastic cells and challenged with viable tumor cells did not survive longer than mice that received the challenge dose alone. The homogenate of this nonimmunogenic tumor was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the gel slices containing isolated molecular entities were injected into various groups of mice. The mice were challenged with low doses of viable tumor cells (10-30 cells) and their survival time was recorded. Small but significant numbers of mice injected with apparent 80-90 K SDS-PAGE-isolated molecular entity rejected the tumor or survived longer than the control groups of mice. Spleen cells from mice injected with 80-90 K molecular entity inhibited the YAC tumor cotransferred with them to naive recipients (Winn assay). Spleen cells from mice injected with monoclonal antibody against nonspecific T-cell helper factor and immunized with 80-90 K SDS-PAGE-isolated molecular entity failed to inhibit the tumor growth in naive recipients, indicating that helper T cells are involved in induction of the antitumor resistance. Nylon-wool-passed splenocytes from mice injected with 80-90 K inhibited tumor growth in some of the recipient mice. Spleen cells from these mice treated with anti-Thy-1 and complement also inhibited the tumor growth in some of the recipients, suggesting that the effector cells were both T and non-T cells. C57BL/6 mice immunized with apparent 20 K SDS-PAGE-isolated molecular entity of RBL5 tumor also induced in vivo resistance to the syngeneic viable RBL5 cells, but not to the syngeneic B16 melanoma cells, indicating the specificity of the protective effect. The practical and theoretical implications of these findings are discussed.

An insight into the T-cell recognition systemusing antigen-specific functional T-cell lines

Summary For a better understanding of the molecular nature of the antigen-specific T-cell recognition system, continuous T-cell lines specific to the synthetic polypeptide (T,G)-A-L, have been established from murine and human activated T cells. These lines are constitutive secretors of antigen-specific T-cell replacing helper factors. Lines prepared from (T,G)-A-L activated C3H.SW (high-responder) T cells were cloned and characterized. The cells and their secreted products were identical in their fine antigenic specificity, as tested using synthetic polypeptides closely related to (T,G)-A-L. The secreted T-cell helper factor was shown to possess MHC determinants as well as V-region determinants or, more specifically, idiotypic determinants. Using the fluorescence-activated cell sorter (FACSII), we demonstrated the expression of cross-reactive idiotypic markers on the monoclonal helper T cells. Gel analysis of the twice affinity-purified eluate of a (T,G)-A-Lcolumn revealed the existence of iodinated bands with a molecular weight of 15 kd and 17 kd, in addition to a diffuse band of high molecular weight. The specific helper activity of the factors was associated with a 65–75% ammonium sulfate precipitate. Gel electrophoresis experiments indicated that both a high (less than 67 kd) and a low (15–17 kd) molecular weight fraction contained the biological activity of the factor. The two fractions were also shown to be synthesized by the T-cell lines, as indicated by internal labelling experiments using 35 S-methionine. A (T,G)-A-L-specific long-term T-cell line of human origin which constitutively secreted a helper factor, was established from antigen-activated T cells of a donor previously determined to be a responder to (T,G)-A-L. In addition to the antigenic specificity of the factor, it was found to possess V H determinants in addition to the relevant HLA-DR determinants. The latter finding was confirmed by the demonstration that the factor reacted with monoclonal anti-Ia antibodies. Thus, the molecular characterization of the active products of the antigen-specific T-cell lines of murine and human origin will hopefully lead to elucidation of the T-cell receptor.

In Vitro Effects of Cyclosporin A on Human B‐Cell Responses

The in vitro effects of cyclosporin A (CsA) on T-cell-dependent and T-cell-independent mitogen responses of human B cells were studied. T-cell-dependent, pokeweed mitogen (PWM)-induced B-cell proliferation and B-cell differentiation to Ig-secreting cells were significantly inhibited by CsA, when purified B cells were cultured with T-cell helper factor containing supernatants instead of T cells. This indicates that the inhibitory effect of CsA on T-cell-dependent, PWM-induced B-cell proliferation and differentiation is not exclusively due to direct effects on helper T cells. B-cell proliferations induced by anti-IgM antibodies and by Staphylococcus aureus bacteria were also found to be sensitive to CsA. Since both types of reactions are T-cell-independent, the concept that responses of human B cells can also be affected by CsA in ways that seem to be independent of the well-documented direct effects of CsA on T cells is further supported. This seems not to be a general phenomenon, however. Epstein-Barr-virus-induced activation of human B cells, as reported previously and also observed by us, is completely insensitive to CsA. It seems, therefore, that certain B-cell activation mechanisms are sensitive to CsA while others remain unaltered. The difference between these two reaction patterns cannot be exclusively explained by a T-cell dependence or T-cell independence of these responses. CsA effects on certain functional B-cell subsets or interference with accessory cell mechanisms might be responsible.

Induction of proliferation and differentiation of neoplastic B cells by anti-immunoglobulin and T-cell factors.

Sepharose-bound anti-immunoglobulins, which are potent mitogens for normal adult B cells, are not mitogenic for tumor cells freshly isolated from mice carrying the B-cell leukemia BCL1. However, after 4 or more days of in vitro cultivation, BCL1 cells can be stimulated to divide by either anti-mu or anti-delta antibodies. These results suggest that in vitro cultivation of BCL1 cells results in their differentiation into more mature cells which can be triggered to proliferate by their interaction with anti-Ig antibodies. Addition of T-cell helper factors to anti-Ia treated BCL1 cells results in their differentiation into Ig-secreting cells. These results indicate that surface Ig molecules on BCL1 cells are capable of delivering an activation signal to the cells but that the cells require a second signal from T cells for induction of Ig secretion.

Shared idiotypic determinants on antibodies and T-cell-derived suppressor factor specific for the random terpolymer L-glutamic acid60-L-alanine30-L-tyrosine10.

T-cell derived suppressor factors (TsF) specific for the random copolymers L-glutamic acid60-L-alanine30-Ltyrosine10 and L-glutamic acid60-L-alanine40, referred to as GAT and GA, respectively, were prepared and partially purified on the approprate antigen immunoadsorbents. GAT-TsF obtained from nonresponder DBA/1 (H-2q) and SJL (H-2s) mice were passed over immunoadsorbents prepared from normal guinea pig serum (NGPS) or guinea pig anti-idiotype antiserum (anti-CGAT) specific for a common cross-reactive idiotype found on most anti-GAT antibodies in all mouse strains tested. Both the directly suppressive activity of the GAT-TsF and the ability of GAT-TsF to induce new suppressor T cells (Ts2) in vitro were adsorbed to and fully recoverable from the guinea pig anti-CGAT-Sepharose immunoadsorbent, while the TsF passed through the control NGPS-Sepharose without appreciable binding. The SJL GAT-TsF specifically eluted from anti-CGAT-immunoadsrobents was shown to still posses I-J determinants. These data provide evidence suggesting a sharing of V region structures between B-cell antibody and T-cell suppressor factor specific for an antigen (GAT) under Ir gene control, in agreement with earlier studies on T and B-cell alloreceptors, T-cell helper factors, and T and B-cell receptors for conventional antigens.

Antigen specific T-cell helper factor cross reacts idiotypically with antibodies of the same specificity

THE recognition system of bone marrow-derived (B) cells and their products is based on the interaction of the active site of immunoglobulins with the specific antigenic determinants. However, the molecular nature of the recognition component of thymus-derived (T) lymphocytes is not yet established. Studies on antigen specific T-cell helper or suppressor factors1–5 and isolation of the antigen receptor directly from T cells, or from serum6,7 are examples of approaches to elucidate this problem. The synthetic polypeptide poly(Tyr,Glu)-poly(DLAla)--poly(Lys) [abbreviated (T,G)-A--L] has been extensively used in studies on the molecular basis of antigenicity8 and genetic regulation of immune responsiveness9–11. Studies on the antibody response to (T, G)-A--L have revealed the involvement of several cell types and a T-cell replacing soluble factor which are antigen specific and genetically controlled12–14. An important feature of the T-cell factors is their antigen specificity. The specificity of a T-cell factor produced to (T, G)-A--L was demonstrated by the removal of its activity on specific antigen-Sepharose columns15 and also by demonstrating that the in vivo helper effect was specific to the antigen used in the T cell education1,16. It is very likely that the specific T-cell factor represents the molecular recognition component of T cells—the T-cell receptor. If this is the case, the molecular identification and characterisation of the T-cell factor may answer one of the most controversial questions in immunology. It has been suggested that antigen-specific molecules of B and T cells share idiotypic dterminants17,18. It was, therefore, of interest to find out whether the (T, G)-A--L specific factor will share idiotypic determinants with antibodies of the same specificity. The results of the present study demonstrate that B-cell products (antibodies) and T-cell products (factors) possess cross-reacting idiotypic determinants. This strongly suggests the presence of similar variable regions in both antigen binding entities, which are probably coded by the same variable region genes. Cross-reactive idiotypic determinants are also present on (T, G)-A--L specific factors obtained from low responder C3H/DiSn T cells confirming previous reports on the activity of these factors.

Antigen-specific helper factor in man.

Supernates of tetanus toxoid (TT) antigen-stimulated human T cells were studied for the presence of an antigen-specific T-cell helper factor (ASF). Supernates were circulated over an immunosorbent column consisting of insolubilized TT antigen. The material which bound to the column was eluted with 3 M NaCNS and was shown to contain a factor which in the presence of TT-induced specific IgG anti-TT antibody synthesis in autologous B cells without causing readily detectable proliferation. ASF activity was partially inhibited by antisera directed against the B-cell alloantigens of the ASF donor. Immunosorbent columns containing such antisera removed ASF activity. Immunosorbent columns containing antisera to human immunoglobulin heavy chain determinants did not remove ASF activity; whereas immunosorbent columns containing rabbit idiotypic antiserum directed against anti-TT antibodies completely removed ASF activity. ASF was destroyed by treatment with proteolytic enzymes; its molecular weight was estimated by Sephadex G-100 gel column chromatography to be between 25,000 and 75,000 daltons.

The specificity of T-cell helper factor in man

Supernates of human T cells stimulated with TT antigen contain a factor that induces mitogenesis and immunoglobulin synthesis in autologous as well as allogeneic B cells. A fraction of the IgG produced has specificity against TT. The T-cell-derived LMF-TT eluted after albumin on Sephadex G 200 and did not contain immunoglobulin heavy chain determinants. LMF-TT was active on B cells from TT immune as well as TT- nonimmune individuals but in the latter instance the IgG secreted had no specificity against TT. B cells incubated with LMF-TT in the presence of a second antigen (DT) made IgG with specifity to that antigen provided the B-cell donor was immune to that second antigen. LMF-TT-containing supernates were depleted of TT antigen by Sephadex G 200 chromatography followed by passage over anti-TT immunosorbent columns. The antigen-free supernates were able to induce mitogenesis and IgG synthesis in B cells but the IgG produced failed to exhibit specificity against TT unless the TT antigen was readded to the B-cell cultures. The optimal concentration of LMF-TT (50 percent) inducing B-cell mitogenesis was different from the optimal concentration (20 percent) causing IgG synthesis by B cells. At low LMF concentrations (less than or equal 10 percent) addition of a second antigen to which the cell donor was immune caused an increase in the degree of B-cell mitogenesis. Submitogenic concentrations of LMF-TT (less than or equal to 5 percent) were still capable of inducingimmunoglobulin synthesis in B cells At these low concentrations of LMF-TT the proportion of anti-TT IgG over total IgG increased sharply. B cells from TT immune donors were separated on TT immunosorbent columns. Cells that bound to the column were more sensitive to the mitogenic and IgG synthetic effects of LMF-TT than unfractionated B cells. Thus, LMF is a nonspecific human T-cell helper factor which behaves like a polyclonal B-cell activator. However, in the presence of specific antigen (TT) the antigen-specific B cell is preferentially triggered by LMF. The experimental design of the present study does not rule out the additional presence of an antigen-specific helper factor in the supernates of TT-stimulated human T cells.

Antigen-specific helper factors in rabbit lack both V and C region Ig determinants.

THE nature and genetic origin of the T lymphocyte antigen-recognition system is an intriguing and compelling immunological problem. T cells differ from B cells in not synthesising classical antibody and in not expressing a readily detectable immunoglobulin (Ig) as their surface receptor for antigen1. It has recently been found, however, that in some cases T cells and serum antibody carry the same idiotypic determinants2–4, inferring identity between the binding sites, that is the variable regions, of the T cell antigen receptor and immunoglobulin. It has also recently been discovered that T cells can produce antigen-specific molecules or factors which carry determinants of the major histocompatibility complex (MHC) in addition to a binding site for antigen5–8. The existence of the specific T cell factors raises the possibility of variable (V) region genes separate from those contributing to antibody and B cell receptors, and perhaps located in the MHC9. In order to resolve these possibilities we have attempted to produce antigen-specific T cell factors in the rabbit. The rabbit is the ideal animal for such a study, since it uniquely carries allotypic markers in the V region of its immunoglobulins (see reviews in refs 10 and 11). The sharing of such a V-region allotypic marker by both Ig and the specific T cell factor would establish a common genetic basis for antigen recognition in the two molecules. The results below show, however, that the Ig V region allotypes determined by the a locus and indeed other Ig markers are not present on the specific T-cell helper factor in the rabbit and imply the existence of separate V genes for antibody and T cell factors.