Induction of IgG in young nude mice by lipid A or thymus grafts.

Postnatal serum concentrations of IgG2a of paternal allotype, measured in congenitally thymusless nude mice, increase with kinetics and titers comparable to their normal congeneic counterparts. Lipid A, the mitogenic part of LPS, stimulates IgG synthesis in nude mice when it is given 7 days after birth. IgG concentrations at 15 days of age are 6- to 8-fold higher than in untreated control nudes; this is considerably lower, however, than in normal mice, which show up to 45-fold higher IgG2ab levels after lipid A treatment. A thymus graft from nearly congeneic donors of the same age, transplanted at 4 days after birth, also stimulates long-lasting IgG synthesis in the nude recipients. If the grafted nudes are injected with lipid A 3 days later, IgG synthesis is further stimulated 8- to 16-fold. The data are discussed in relation to the thymus dependency of IgG production and the conditions for lipid A stimulation.

Induction of IgG by lipid A in the newborn mouse.

IgG of paternal allotype first becomes detectable in the serum of (BALB/c x C57BL/6)F(1) mice between day 12 and 14 after birth and reaches adult levels at an age of 5 wk. Since in mice there is a transfer of maternal IgG molecules through the placenta and via milk, F(1) heterozygous at the allotype locus were used and the concentrations of IgG with paternal allotype were measured. This was done by a sensitive method capable of detecting IgG concentrations as low as 5 x 10(-4) of normal adult serum levels. It is based on the quantitative inhibition of allotype-specific facilitation of hemolysis. When lipid A or Salmonella bacteria were injected into neonatal mice, a stimulation of IgG synthesis was observed. Thus IgG levels were enhanced 10-30-fold compared to the nontreated mice. No increase in IgG levels was obtained in adult mice after treatment with lipid A. Whether the newborns were injected at birth, on day 2, 4, or 7, IgG was first demonstrable in the treated mice at an age of 6-11 days. The increase in IgG levels was not paralleled by a demonstrable antibody activity against lipid A, SRBC, and LPS. Thus the bulk of newly induced IgG is probably a statistical distribution of different specificities.

Human testis cytosol and ovarian follicular fluid contain high amounts of interleukin-1-like factor(s).

Human testicular cytosol and ovarian follicular fluid were analyzed for the presence of interleukin-1 (IL-1)-like factors. Both the follicular fluid and testis cytosol preparations exhibited significant IL-1-like activity as determined by the murine thymocyte proliferation bioassay. The dose-response lines obtained with the gonadal preparations were parallel to each other and to those obtained with monocyte-derived IL-1 and the activity of the gonadal IL-1 could be neutralized by specific IL-1 antibodies. After gel chromatography of human follicular fluid (hFF) and human testis cytosol (hTC) proteins, IL-1 activity was found in the molecular weight region between 30 and 50 kilodaltons (kDa). Chromatofocusing of IL-1 from hFF and hTC revealed that the major part of IL-1 in both cases exhibited similar charge properties (pI less than 6.0). However, two extra peaks (pI 7.0 and greater than 9.0, respectively) were observed in hFF preparations. After isoelectrofocusing (IEF), IL-1 activity of hFF was also found in two different pH regions; a broad area of activity was localized between pH 5.5 and 7.0, while a sharp peak was observed with an approximate pI value of 9.5. Re-chromatofocusing or IEF of alkaline IL-1-like activity resulted in a heterogeneous profile of IL-1-like activity suggesting that the alkaline material may represent either a precursor or an aggregated form of the acidic IL-1. None of the IL-1 peaks obtained from hFF or hTC exhibited IL-2 activity as assessed in a specific IL-2 bioassay. The results of the present study indicate that both gonads may produce high amounts of IL-1-like factor(s) which might play a regulatory role in normal gonadal function.

Cross-reactivity patterns of IgM and IgG anti-lipopolysaccharide antibodies in individual mice.

A sensitive method has been developed which permits comparative analysis of IgM and IgG antibody specificity against lipopolysaccharide (LPS) antigen. It is based on hemolysis of LPS-coated red blood cells and on its inhibition by homologous and heterologous LPS. By appropriate use of anti-immunoglobulin sera, indirect (facilitated) lysis due to IgG antibodies is obtained, whereas IgM gives direct lysis and is 2-mercaptoethanol-sensitive. IgG can be analyzed either by facilitation with a rabbit anti-mouse Ig or with anti-allotype sera. By use of anti-allotype sera in F1 hybrids, both parental antibody types can be studied separately. Antibodies of either class from individual mice may display different cross-reactivity patterns. Furthermore, for IgM and IgG within a given serum, both similarities and differences have been found. Some of the cross-reactivity patterns have been followed over one year. With few exceptions, individual patterns remained constant throughout this period.

Inheritance of antibody specificity: the IgM anti-lipopolysaccharide response in mice.

Mice of different genotypes were immunized with Salmonella anatum. The cross-reactivity patterns of their IgM anti-S. anatum lipopolysaccharide (LPSAN) antibodies were characterized by their relative avidity toward heterologous LPS. When the LPS from S.cholera suis (LPSCHS) was used as the heterologous LPS, clear differences between mouse strains were found. DBA/2 and DBA/1 showed cross-reacting IgM, whereas C57BL/10, C57BL/6 BABL/c-Igb and B10. D2 had mainly noncross-reacting IgM. In C3H and C57BL/6-Iga, individual mice express either the cross-reacting or the noncross-reacting antibodies. The IgM antibodies from individual mice were further characterized for their cross-reactivity toward the LPS from S. strasbourg (LPSSTR) and S illinois (LPSILL). Only individual patterns with no correlation to the cross-reactivity pattern with LPSCHS were found.This shows that more than one antibody type is characterized by cross-reactivity.(B10.D2 X DBA/1)F1 mice showed a biphasic distribution of cross-reactivity. Of the F1 X DBA backcross mice 21% had IgM antibodies which showed no cross-reaction with LPSCHS. This still is in agreement with one locus controlling this phenotype. This locus segregates independently from Ig allotype since no correlation was found between allotype and cross-reactivity pattern in F1 X DBA backcross mice.

Analysis of T-T lymphocytes and T-accessory cell interactions using cloned T cells: MHC I restricted cloned T cells activate MHC II restricted T helper cells.

We evaluated the role of molecules of the major histocompatibility complex (MHC) involved in the cellular interactions of two T-cell clones by testing the effect of monoclonal antibodies on the responses of the clones in vitro. The two T-cell clones used in the study are specific for minor histocompatibility antigens and restricted to the H-2Kk. In the absence of exogenous IL-2 the clones require the presence of Ia+, Thy-1- accessory cells and of Thy-1+, Lyt-1+2- cells in the irradiated spleen cell suspension used as stimulator. It is also necessary that both the accessory cells and the T cells in the stimulator cell populations are recognized specifically by the clones. Monoclonal antibodies specific for the H-2K product inhibited the lytic effector function of the cytolytic clone. These antibodies when added to cultures of stimulator cells and clones inhibited also the proliferation of this clone and of a nonlytic clone. When antigen recognition was measured by the increase in sensitivity of the clones to IL-2 while confronted with uv-irradiated stimulator cells, both clones were blocked efficiently by anti-H-2K antibodies. Thus, these results suggest that the interaction of monoclonal antibodies with the restricting H-2K molecule is sufficient to block the recognition signal, a prerequisite for proliferation. In contrast, monoclonal antibodies specific for A alpha A beta and/or E alpha E beta had no effect on cytolysis or on restricted recognition. However, they inhibited the proliferative responses as efficiently as the H-2K specific antibodies. Inhibition by class II-specific antibodies was not abolished when stimulator cell populations were depleted of Lyt-2+ cells. The blocking effect, however, was reversed by the addition of IL-2. No inhibition was obtained with antibody specific for E alpha E beta when B10.A(4R) spleen cells, which do not express E alpha E beta, or when B10.A(4R) accessory cells, which were reconstituted with (BALB/c X B10.A(4R] F1 T cells, were used as stimulators. Stimulator cells heterozygous for H-2 could be inhibited by antibodies to the parental haplotype not encoded in the clones (H-2Kd). These and previous results suggest that H-2K-restricted minor histocompatibility antigen-specific recognition transmits an activating signal to the clones and to the stimulator cells. The clones probably are induced to express more IL-2 receptors. The stimulator T cells seem to interact through A alpha A beta and E alpha E beta molecules with syngeneic accessory cells.(ABSTRACT TRUNCATED AT 400 WORDS)

T cell lines and variant subclones with differing susceptibility to tumor promoters.

The effect of tumor-promoting phorbol esters on cloned cytotoxic T cell (CTL) lines, H-2 restricted and specific for minor histocompatibility antigens, was studied. We found that two of the three CTL lines tested can be induced by 12-O-tetradecanoylphorbol 13 acetate (TPA or PMA) to synthesize DNA. One of the lines did not respond. TPA concentrations in the range of 1 nmol were active, and induction of DNA synthesis was independent from cell densities. Testing of other phorbol esters showed correlations with their tumor-promoting activity, because TPA and phorbol 12,13-didecanoate were active, whereas phorbol and 4 alpha phorbol 12,13-didecanoate had no effect. Interestingly, two subclones isolated from one of the lines responding to TPA behaved differently: one of the subclones synthesized DNA when incubated with TPA, whereas the other remained completely negative. This difference was reflected also in the dissimilarity of morphologic alterations (observed by scanning electron microscopy) occurring in the responding cells. The nonresponsiveness, in terms of DNA synthesis, by some of the CTL lines is not due to a lack of interaction between TPA and the cells. When incubated simultaneously with TPA and T cell growth factor (IL 2), all lines responded with greater DNA synthesis than when incubated with IL 2 or TPA alone, thus demonstrating a synergistic action between TPA and IL 2. We have also done experiments to test whether TPA induces the synthesis and secretion of a factor leading to proliferation of the cells. From bystander experiments it appears that the action of TPA is not mediated by a secreted product but is a direct one.

T cell clones interacting with accessory and T helper cells for a proliferative response.

The requirement for cell interactions in T cell activation has been studied with two continuously in vitro growing T cell clones. These clones are specific for minor histocompatibility antigens, are H-2K restricted, and one clone is functionally a cytolytic T lymphocyte. Both can proliferate when interleukin 2 is added to the cultures, but for continuous growth they require irradiated spleen cells carrying the specific minor histocompatibility antigen and the restricting H-2. In this study we show that for proliferation the clones require at least two cell populations in the stimulator spleen, one is a splenic-adherent cell (SAC), the other a T cell. The SAC are plastic adherent, Thy-1-, Ia+. The T cells are nylon wool nonadherent, Thy-1+, Lyt-1+2- and Ia-. Cell mixing experiments of stimulator cells (all were done with H-2-syngeneic cells), depleted of either SAC or T cells confirm the requirement for a specific interaction between these two cell types and the T clone. Neither SAC, syngeneic with the T clone when mixed with T cells of the stimulator type, nor T cells syngeneic with the clone added to stimulator SAC, can induce an optimal proliferative response. Such a response is obtained only if both cell types, SAC and T cells, are of the stimulating genotype. This suggests that, in addition to an interaction of clonal T cells with SAC, a specific recognition at the T cell level between T stimulator and T clone is necessary. The interaction of the T clones with stimulator SAC and T cells leads to an activation, mediated by antigen recognition, of all three cell populations. Since we also show that each of the stimulator cell types are impaired by ultraviolet light irradiation, we conclude that factor production by SAC and T helpers is the final prerequisite for clonal expansion.

Antigen-dependent, H-2-restricted cytolytic and noncytolytic T cell lines with specificity for minor histocompatibility antigens.

Several cloned T cell lines were isolated from primed mixed lymphocyte cultures immunized against minor histocompatibility antigens. These lines were selected with irradiated stimulator cells as antigen and require restimulation at intervals to keep growing. They are responsive, as measured by proliferation, to interleukin 2 (T cell growth factor) but cannot be grown in it continuously. These T cell lines have either the H-2d or H-2k haplotype. They all show exquisite H-2 restriction and minor histocompatibility antigen specificity. We did not observe any alloreactivity on 8 different H-2 haplotypes. For the H-2k T cell lines, the restriction element could be mapped to either the K or D end of the H-2 complex. No I-A-restricted cell line was found. It is of interest that all these T cell lines need the presence of T cells in the irradiated stimulator cell population. This suggests a more complex interaction between irradiated stimulators and responder T cells than just H-2K- or D-restricted antigen interaction. This recognition, though necessary, does not seem sufficient to induce the T cell clones to proliferate.