Fowl Gamma Globulin Research Articles

CD8+ T cells reduce in vitro interferon-gamma production in Theiler's murine encephalomyelitis virus-induced demyelinating disease model.

In the Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease model for multiple sclerosis, regulatory CD8+ T cells prevent demyelinating disease and reduce in vivo interferon (IFN)-gamma production by anti-TMEV CD4+ blast cells in BALB/c mice. We describe here that regulatory CD8+ T cells reduce in vitro IFN-gamma production by lymph node cells from both TMEV and fowl gamma globulin immunized mice without affecting interleukin (IL)-4, IL-10, tumour growth factor-beta or tumour necrosis factor-alpha production.

Selective inactivation of lymphocytes after psoralen/ultraviolet light (PUVA) treatment without affecting systemic immune responses.

Psoralens, together with ultraviolet light A (PUVA), are used for the treatment of several proliferative diseases. It has been suggested that the effectiveness of this treatment is due to induction of a specific immune response by PUVA-treated lymphocytes that down-regulates untreated cells of the same specificity. The present studies show that the clinically used psoralen analogue 8-methoxypsoralen (8-MOP), as well as 4,8-dimethyl-5'-(pyridiniummethyl)psoralen bromide (4NQ), a derivative that does not cross the cell membrane, when activated by UVA light, render lymphocytes unresponsive to mitogenic, antigenic, or phorbol myristate acetate + ionomycin-induced stimulation. This state of unresponsiveness was not reversed by exogenous recombinant interleukin-2. Treatment of lymphocytes with 4NQ and UVA light had no effect on the viability or the homing pattern of the cells to spleen or liver, whereas 8-MOP induced toxicity in about 50% of cells after 3 days in culture. Using an adoptive transfer mouse model, we found that antigen-specific lymphocytes treated with PUVA (8-MOP or 4NQ) had no effect on the ability of these mice to respond to a subsequent challenge with the same or an irrelevant antigen. This was tested by measuring the T cell proliferative responses of lymph node cells from mice primed with keyhole limpet hemocyanin (KLH) or fowl gamma globulin (FGG) before or after adoptive transfer of large numbers of KLH- or FGG-specific PUVA-treated lymph node cells. No effects of antigen-primed PUVA-treated cells on antigen-primed untreated cells were observed in vitro in mixed lymphocyte cultures responding to the relevant antigen. These results suggest that, in our model system, PUVA induces cell inactivation but does not affect systemic T cell responses.

Analysis of T hybridomas prepared from a T cell clone with three specificities. Recognition of self + X and allo-H-2 determinants segregates from recognition of Mlsa determinants.

To see k information on T cell recognition of Mlsa determinants, hybridomas were prepared from a well-characterized F23.2+ (V beta 8.2+) T cell clone specific for three different ligands, i.e., 1) Mlsa determinants, 2) fowl gamma-globulin (F gamma G) plus self-H-2 (H-2d), and 3) allo-H-2, e.g., H-2p, determinants. Fusion of the clone to the BW5147 thymoma line produced a triple-reactive T hybridoma which generated two types of spontaneous variants. One type of variant (type I) lost Mlsa reactivity but retained reactivity to both F gamma G/H-2d and allo-H-2p. These variants, which were generated at high frequency, stained strongly with a mAb, A1.57, with idiotypic specificity for the TCR molecules of the parental clone. The second type of variant (type II) reacted to Mlsa determinants but showed no reactivity to F gamma G/H-2d or to allo-H-2p. These variants failed to express the A1.57 idiotypic determinants of the parent clone, but were F23.2+ (V beta 8.2+); nonequilibrium pH gradient electrophoresis analysis suggested that these hybrids expressed a mixed TCR heterodimer composed of the parental clone beta-chain and the BW5147 alpha-chain. Three aspects of the data are very difficult to accommodate with the view that Mlsa, F gamma G, and allo-H-2 determinants are all recognized via a common TCR molecule: 1) the independent (and frequent) segregation of Mlsa reactivity from F gamma G/H-2d and allo-H-2p reactivity, 2) the retention of Mlsa reactivity by the type II variants despite loss of the parental clone alpha-chain, and 3) the loss of Mlsa reactivity by the type I variants despite high expression of the A1.57+ TcR molecules derived from the parental clone. The data support a model in which Mlsa determinants are recognized by a separate T cell structure, which we envisage as a monomorphic accessory molecule unrelated to the TCR. Since the type II hybridoma variants invariably retained quantitatively normal TcR expression, the triggering phase of anti-Mlsa responses appears to be TCR dependent. The model we favor is that anti-Mlsa/Mlsa interaction increases TCR binding with Ia epitopes to above the threshold required for cell triggering. A key feature of this model is that Mlsa and Ia determinants are recognized as separate structures rather than as a complex.

Downregulation of T cell responses by antibodies to the T cell receptor.

We have derived a T cell clone that recognizes and responds to three different types of antigen: self + X (fowl gamma globulin + H-2d), allo-H-2p,b, and minor lymphocyte-stimulating (Mlsa,d) determinants. Anti-TcR mAb and their F(ab')2 and Fab fragments were tested for their capacity to block the response of this clone. When responses were assayed on day 4 or later, addition of KJ16 or F23.1 mAb caused a marked inhibition of the response to each of the three antigens recognized by the clone. Responses measured at earlier time points however were unaffected or enhanced. This finding suggested that the inhibitory effects of anti-TcR mAb that followed the phase of enhancement might have reflected downregulation of the cells rather than simple blockade of TcR. In support of this possibility it was found that addition of anti-TcR mAb caused marked inhibition of the response of the clone to IL-2, i.e., a response that is not known to involve the TcR.

Different T cell requirements for specific memory induction in normal and xid B cells.

The immune response to TTGG-A--L, a defined-sequence, branched-chain polypeptide, is regulated by MHC-linked Ir genes. TTGG-A--L specific B cells can be demonstrated in both normal and immune-defective low responder strains by activation to specific antibody secretion after immunization with TTGGAA-F gamma G, a conjugate of the hexapeptide TTGGAA and the immunogenic carrier fowl gamma-globulin. It is shown that immunization with TTGG-A--L induces specific memory B cells with equal efficiency in normal low and high responder strains but not in immune-defective low responder strains. We conclude that memory induction in xid B cells in contrast to normal B cells is dependent on MHC-restricted, carrier-specific helper T cells. Other observations also suggest a more stringent requirement for MHC-restricted, carrier-specific helper T cells in the induction of TTGGAA-specific antibody secretion by xid as compared to normal B cells. Both normal and immune-defective H-2k/b hybrids between the mutant CBA/N strain and TTGG-A--L high responder BALB.B are responders to TTGG-A--L. In contrast, normal but not immune-defective H-2k/d hybrids with responder BALB/c are responders to TTGG-A--L. This identifies H-2d as a TTGG-A--L high responder haplotype for normal B cells but a low responder haplotype for xid B cells, whereas H-2b is a high responder haplotype for both normal and xid B cells. This must reflect a quantitative or qualitative difference in Ir gene-mediated cellular interactions required for induction of antibody secretion in normal and xid B cells.

TTGG-A-L-specific memory B cells induced in low responder strains.

The immune response to TTGG-A--L, a defined-sequence, branched-chain polypeptide, is regulated by MHC-linked Ir genes. TTGG-A--L-specific B cells can be demonstrated in low responder strains by activation to specific antibody secretion after immunization with TTGGAA-F gamma G, a conjugate of the hexapeptide TTGGAA and the immunogenic carrier fowl gamma-globulin. It is shown that immunization with TTGG-A--L induces specific memory B cells with equal efficiency in low and high responder strains. This finding demonstrates that memory formation in a B cell subpopulation represented by TTGG-A--L-specific precursors is independent of carrier-specific, MHC-restricted helper T cells. This conclusion is further supported by the demonstration in an adoptive transfer model that immunization with TTGG-A--L induces equivalent levels of TTGG-A--L-specific memory B cells in T cell-deficient nude mice and their normal heterozygous littermates.

Bystander help in primary immune responses in vivo.

We evaluated the requirement for hapten-carrier linkage in the primary, T cell-dependent antibody response in vivo. Mice immunized with mixtures containing nonimmunogenic and immunogenic proteins developed antibody that was specific for determinants present on the nonimmunogenic carrier. Therefore, hapten-carrier linkage was not necessary for the generation of primary antibody responses. The magnitude of the bystander response was a function of the immunogenicity of the coimmunogen and the quantity of determinant-specific B cells available for activation. Interestingly, the kinetics of the bystander response, in contrast to the cognate response, were not accelerated in the presence of primed Th cells. Adoptive recipients reconstituted with primed Th cells developed accelerated cognate but not bystander antibody response, as compared with unprimed recipients. This phenomenon may reflect a regulatory mechanism invoked to limit the potentially harmful effects of nonspecific help. It was observed that while animals are tolerant to immunization with mouse (self) hemoglobin, immunization with a mixture containing mouse hemoglobin plus fowl gamma globulin resulted in the production of hemoglobin-binding autoantibodies. Thus bystander help induced by coimmunization may serve as a model for the induction of autoantibodies during normal immune responses in vivo.

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.

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.

Genetic control of immune response to myoglobin. Ir gene function in genetic restriction between T and B lymphocytes.

We studied the genetic restrictions on the interaction between T cells, B cells, and antigen-presenting cells (APC) involved in the H-2-linked Ir gene control of the in vitro secondary antibody response to sperm whale myoglobin (Mb) in mice. The B cells in this study were specific for Mb itself, rather than for a hapten unrelated to the Ir gene control, as in many previous studies. Low responder mice immunized in vivo with Mb bound to an immunogenic carrier, fowl gamma globulin (F gamma G), produced B cells competent to secrete anti-Mb antibodies in vitro if they received F gamma G-specific T cell help. However, (high-responder X low responder) F1 T cells from Mb-immune mice did not help these primed low responder (H-2k or H-2b) B cells in vitro, even in the presence of various numbers of F1 APC that were demonstrated to be component to reconstitute the response of spleen cells depleted by APC. Similar results were obtained with B6 leads to B6D2F1 radiation bone marrow chimeras. Genotypic low responder (H-2b) T cells from these mice helped Mb-primed B6D2F1B cells plus APC, but did not help syngeneic chimeric H-2b B cells, even in the presence of F1 APC. In contrast, we could not detect any Ir restriction on APC function during these in vitro secondary responses. Moreover, in the preceding paper, we found that low responder mice neonatally tolerized to higher responder H-2 had competent Mb-specific helper T cells capable of helping high responder but not low responder B cells and APC. Therefore, although function Mb-specific T cells and B cells both exist in low responder mice, the Ir gene defect is a manifestation of the failure of syngeneic collaboration between these two cell types. This genetic restriction on the interaction between T cells and B cells is consistent with the additional new finding that Lyb-5-negative B cells are a major participant in ths vitro secondary response because it is this Lyb-5-negative subpopulation of B cells that have recently been shown to require genetically restricted help. The Ir gene defect behaves operationally as a failure of low responder B cells to receive help from any source of Mb-specific T cells either high responder, low responder, or F1. The possible additional role of T cell-APC interactions, either during primary immunization in vivo or in the secondary culture is discussed.

Genetic control of the immune response to myoglobins. Both low and high responder T cells tolerant to the other major histocompatibility complex help high but not low responder B cells.

We sought to examine the role of immune response (Ir) genes in helper T cells. To eliminate allogeneic effects, we used neonatally tolerized mice. The results bear not only on the mechanism of Ir genes, but also on the development of the T cell repertoire. B 10.BR (H-2(k)) or C57BL/10 (H-2(b)) mice, which were low responders to myoglobin (Mb), were neonatally tolerized to high responder H-2(d) alloantigens, and B10.D2 mice, which were high responders to Mb, were neonatally tolerized to low responder H-2(k) or H-2(b) alloantigens. Spleen cells from these tolerized mice did not show any reactivity in mixed-lymphocyte reaction or cell-mediated lympholysis against alloantigens used in tolerization. Mb-immune F(1) B cells were helped comparably by Mb-immune tolerized low or high responder T cells. Thus, low responder T cells functioned equivalently to high responder T cells. The failure of nonimmune T cells from tolerized low responder mice to help F(1) B cells and antigen-presenting cells (APC) indicated that collaboration between B10.BR or C57BL/10 T cells and F(1) B cells was not caused by a positive allogeneic effect. Spleen cells from tolerized mice were contaminated with 2-4 percent chimeric F(1) cells, as judged by fluorescence-activated cell sorter analysis, and no F(1) alloantigens were detectable in the thymus. However, removal of chimeric F(1) T cells from the tolerized cell population by treatment with anti-H-2 and complement did not change the helper activity of tolerized low responder T cells. These data indicated that helper activity in the T cell population from low responder mice was not due to F(1) cells. Also, the level of contamination was not sufficient to quantitatively account for the help. In examining the genetic restriction of these tolerized T cells, we found that T cells from tolerized low responder B10.BR or C57BL/10 mice helped F(1) or high responder B10.D2 B cells and APC but not syngeneic B10.BR or C57BL/10 B cells and APC, which were immunized with Mb-coupled fowl gamma globulin instead of Mb to prime low responder B cells with Mb. On the other hand, high responder B 10.D2 tolerized T cells helped syngeneic B 10.D2 B cells but not allogeneic low responder B10.BR B cells. These data indicated that clones of helper T cells specific for Mb exist in low responder mice, and these are not phenotypically different from those in high responder mice, in that both help high responder and F(1) but not low responder B cells and APC. These data are discussed in terms of the mechanism for Ir gene control, and the mechanism of T cell repertoire development- whether intra- or extrathymically-in neonatally tolerized mice.

T cells cooperating in the induction of delayed-type hypersensitivity act via the linked recognition of antigenic determinants.

Culture conditions have been established that allow the induction of delayed-type hypersensitivity (DTH) precursor cells present in a cell population derived from unsensitized spleen cells only when antigen-specific, radioresistant, Thy-1-bearing helper cells are added. This specific cellular cooperation acts via the linked recognition of two determinants on the antigen; thus, cells primed to the protein antigen fowl gamma globulin (FGG) will only allow the induction of DTH reactivity against the second antigen, burro erythrocyte (BRBC), when the conjugate FGG-BRBC is present in the cultures. The requirement for physical linkage between the two antigens has been demonstrated by the observation that DTH to BRBC is induced when the conjugate FGG-BRBC is present and not when BRBC and FGG are given an uncoupled molecules.

Selective inhibition of the induction phase of delayed type hypersensitivity in mice by niridazole

Niridazole, a nitrothiazole derivative, used in the treatment of schistosomiasis, inhibits cell-mediated immunity in mice. The drug depressed delayed type hypersensitivity (DTH) 3 3 Abbreviations used: CMI, cell-mediated immunity; DTH, delayed type hypersensitivity; FGG, fowl gamma globulin; KLH, keyhole limpet haemocyanin; POL, polymerized bacteria flagellin; NIP, 4-hydroxy-3-iodo-5-nitrophenylacetic acid; PFC, plaque-forming cells. when administered at the time of sensitization, and lymph node cells from niridazole-treated mice were found to be less efficient than cells from untreated controls in transferring DTH, or in providing helper function in a plaque-forming cell assay. In contrast, the drug was unable to prevent passive sensitization when given to the recipient of sensitized cells. These studies suggest that niridazole inhibits cell-mediated immune responses primarily by interfering with the afferent arm of T-cell sensitization.

Genetic control of T helper cell function in the clawed toad Xenopus laevis.

The genetic control of the collaboration between Xenopus T and B cells has been analyzed in vivo using cells from five strains of major histocompatibility complex-defined Xenopus. When carrier (fowl gamma-globulin)-primed T cells and hapten (dinitrophenylated keyhole limpet hemocyanin)-primed B cells differed by minor histocompatibility antigens or by only one haplotype of the major histocompatibility complex, the collaboration was efficient in the sense that large numbers of plaques, low-molecular weight antibodies and high-affinity IgM antibodies could be recorded in the cultures challenged with dinitrophenylated fowl gamma-globulin. However, when T and B cells differed at both alleles of the major histocompatibility complex, lower numbers of plaques were obtained, no low-molecular weight anti-hapten antibodies could be detected, and the IgM antibodies that were sometimes synthesized were of low affinity. This suggests that the major histocompatibility complex, or a gene linked with it, affects the collaboration between Xenopus T and B cells in a way perhaps similar to that described in mammals.

Immune suppression in vivo with antigen-modified syngeneic cells. V. Interacting T cell subpopulations in the suppressor pathway.

Intravenous administration of syngeneic C57BL/6 cells coupled with the palmitoyl derivative of the protein antigen fowl gamma-globulin (p-FgG) results in a state of FgG carrier-specific nonresponsiveness, mediated in part by suppressor T cells. At least two populations of cells are involved in generating a suppressor pathway in this system, one induced in recipients of p-FgG coupled cells and the other contributed by normal recipients of cells from suppressed donors. Induction of T cells capable of transferring suppression to normal recipients is sensitive to 1) low doses of cyclophosphamide, 2) adult thymectomy, and 3) in vivo treatment with anti-I-J antisera. This T cell subset bears I-J determinants and is functionally sensitive to irradiation. The induction of the second population required for immune suppression is sensitive to 1) low doses of cyclophosphamide and 2) adult thymectomy. In addition, compatibility of genes at the Igh locus appears not to be essential for cellular interaction between these populations. The evidence that interacting T cell subpopulations are involved in a suppressor pathway induced with protein-modified syngeneic cells is discussed relative to other systems in which T-T cell interactions are postulated.

Cell collaboration in a marine teleost. A demonstration of specificity in the carrier effect

The possible role of helper cells for the antibody response was studied in a marine teleost, the winter flounder ( Pseudopleuronectes americanus). The fish were injected with either fowl gamma-globulin (FGG), bovine gamma-globulin (BGG), or bovine serum albumin (BSA). Three weeks later they were immunized with a hapten conjugate, either NIP-FGG or NIP-BGG, and bled after another 3 weeks. A specific carrier priming effect was observed: animals immunized with NIP-FGG produced 10 times higher anti-NIP titres when they had been primed by FGG than when primed with BGG or BSA. The specific priming effect also held true for animals immunized with NIP-BGG; BGG priming was associated with 10 times higher anti-NIP titres than FGG priming. Sera of unimmunized winter flounders had higher natural anti-NIP titres than the sera of other animals tested, and immunization by NIP-FGG or NIP-BGG without carrier priming did not increase the background titre within 3 weeks. An 8-fold increase was observed in carrier-primed animals (average of 3 experiments) and a 100-fold increase in one group that was primed with whole hapten—carrier conjugate (a true secondary response, one experiment).

Idiotypic profile of the response to phosphorylcholine induced in the absence of the homologous antigen.

In the absence of the homologous antigen, an anti-phosphorylcholine (PC) plaque-forming cell (PFC) response has been induced in vitro which is restricted to the TEPC 15 idiotype (T15). Anti-T15 antibodies were used to focus either the keyhole limpet hemocyanin (KLH) or the fowl gamma-globulin (FGG) carrier molecules on the membrane of B cells carrying the T15 idiotype on their immunoglobulin receptors; thereafter, these cells were allowed to cooperate in vitro for 5 days with T cells primed to the appropriate carrier molecule. A response, virtually 100% T15+, could be induced both in normal BALB/c and in T15 neonatally suppressed mice which had lost the T15+ clonal dominance. The magnitude of this response is comparable to that obtained in the presence of the PC antigen. The role of the membrane-bound immunoglobulin receptor (sIg) in the expression of different anti-PC clones was also investigated. We have focused either the KLH or the FGG carrier molecules on the membrane of B cells via anti-H-2 antibodies and then cultured these cells for 5 days with the appropriate carrier-specific T cells. Under these conditions, B cells are activated in the absence of interaction at the sIg. The idiotypic profile of the anti-PC PFC obtained with the anti-H-2-mediated activation was then compared with the profile of the anti-PC response obtained in the presence of PC antigen. Since similar idiotypic profiles were obtained in both cases, it can be excluded that sIg plays a direct role in favoring the expression of T15+ over T15- anti-PC clones.

Regulation by the H-2 gene complex of delayed type hypersensitivity

Identity at the major histocompatibility complex (MHC) was essential for successful transfer of delayed type hypersensitivity (DTH) in mice. The regions of the MHC involved differed according to the antigen used for sensitization. In the case of fowl gamma globulin (FGG), identity atI-A was necessary, whereas with dinitrofluorobenzene (DNFB), identity at theK, I, orD region was sufficient. These different genetic constraints probably reflect differences in the mechanisms by which antigens are presented to T lymphocytes. Cells from sensitized (CBA×C57BL)F1 mice transferred DTH to FGG into parental-strain mice, but transfer was more effective in C57BL than in CBA with the same cell dose. This phenomenon is governed by the MHC, since there was better transfer intoH-2 b than intoH-2 k mice, regardless of their backgrounds. It may reflect the activity of an Ir gene-dependent process. Cells of one genotype (e.g., CBA), sensitized in chimeric mice derived from two MHC-incompatible strains (CBA↔C57BL), transferred DTH to both strains. These results do not support the notion that the genetic constraint observed in DTH transfer may be a result of the necessity for sensitized T and stimulator cells to match an identical MHC-coded cell interaction molecule. Rather, they favor the hypothesis that T cells recognize antigen, not as a naked determinant, but in close association with products of genes of the MHC.

SPECIFIC SUPPRESSION OF THE IMMUNE RESPONSE BY T CELLS

Publisher Summary This chapter presents two groups of experiments designed to study the mechanism of T cell tolerance. In the first group, tolerance was induced in CBA mice to the soluble protein antigen fowl gammaglobulin (FγG) by an injection of 15 mg of deaggregated material per mouse. A tolerant state was detectable 6–10 days after innoculation. It was stable on adoptive transfer into irradiated mice and predominantly affected IgG production. Specificity was established by demonstrating an unimpaired response of the same recipients of tolerant cells to an unrelated antigen, donkey red cells or horse red cells. Trace-labeling studies with 125 I-labeled deaggregated FγG excluded transfer of significant amounts of non-cell -associated tolerogen. Tolerance induction to protein antigens at the level of the T cell was manifested by a suppressor effect. The suppressor effect was exerted directly by T cells. Suppression abrogated antibody production, not by a direct effect on AFCP (hapten sensitive cells) but via nontolerant carrier reactive (T) cells of appropriate specificity.

Serum containing endotoxin-induced tumour necrosis factor substitutes for helper T cells.

WHEREAS immunocompetent precursor B cells recognise antigenic determinants by way of specific surface receptors (an interaction which presumably initiates cell division1,2), the binding of antigen to surface receptors alone does not result in the differentiation of B cells to antibody-secreting cells. To accomplish this differentiation step a second signal is needed. This signal may be provided by T cells in the form of a soluble factor which they produce in vitro after contact with antigen3–5. Besides T cells, activated peritoneal macrophages have also been shown to produce a factor which allows B cells from athymic Nu/Nu mice to produce antibody against fowl gammaglobulin in vitro. Production of this factor is increased by addition of bacterial lipopolysaccharide (LPS) to the culture medium6. LPS itself has been shown to provide the second signal (antigen being the first) for antibody production by B cells7, but it is not clear whether the effect on antigen-reactive B cells is direct, or mediated by other cells. We report here that injection of LPS, in certain conditions in the mouse, induces the release of factors into the serum which enable B cells to produce antibody to heterologous red-cell antigen, in vitro in the absence of helper T cells. The factor is released in large quantity when LPS is injected into mice that have been primed with BCG. It is hardly, if at all, detectable in the serum of mice injected with LPS but not pretreated with BCG. Our investigation was prompted by the discovery that serum from BCG-infected mice given LPS causes acute necrosis of established tumour grafts in syngeneic mice (tumour necrosis serum, TNS)8. After partial purification, tumour-necrotising activity was shown to reside in a glycoprotein fraction (termed tumour necrosis factor, TNF) with a molecular weight of about 150,000 that migrates with the α-2-globulins9.