In vitro activation of specific helper and suppressor T cells by the type 2 antigen polyvinylpyrrolidone (PVP)

Although type 2 antigens, such as PVP, generally do not activate specific TH, previous studies have established that low doses of PVP (0.0025 microgram) can activate TH in vivo which provide help in primed B cells for PVP-specific IgG responses. Doses of PVP that are optimally immunogenic for IgM antibody production (0.25 to 25 micrograms) preferentially activate PVP-specific TS, which suppress IgG antibody production. In the studies reported here, TH and TS that regulate PVP-specific IgG antibody responses were activated in vitro by culturing normal spleen cells for 4 days with PVP. Induction of the TH and TS is dependent upon the amount of PVP in culture: 10(-4) micrograms PVP activates TH, whereas 10(-2) micrograms PVP preferentially activates TS. TH induced in vitro express Thy-1, L3T4, and I-A determinants and help provided by these TH is similar in magnitude to that provided by TH from mice primed with 0.0025 microgram PVP in vivo. TH can also be activated in vitro if donor mice are treated with Cy before culture of their spleen cells with 10(-2) micrograms PVP. Cy pretreatment prevents TS activation, and TH are then induced in these cultures. The presence of TS does not prevent activation of TH by 10(-2) micrograms PVP, because removal of TS by treatment of T cells with anti-Lyt-2 + complement at the end of culture uncovers TH activity. This TH activity is comparable with that of TH obtained after culture with 10(-4) micrograms PVP. The ability to activate PVP-specific TH and TS in vitro should allow determination of the mechanisms involved in activation of T cells by type 2 antigens and the mechanisms by which TS and TH interact with one another.     

Stimulation of humoral immune responses to a thymic-independent antigen in mice immunosuppressed by a graft-versus-host reaction.

The immunosuppressive effect of the graft-versus-host (GVH) reaction was studied in CBA × A F₁ (CAF₁) mice which had been rendered immunologically unresponsive by the injection of parental A-strain lymphoid cells (GVH mice). When challenged with a single injection of either sheep red blood cells or Escherichia coli lipopolysaccharide (LPS), GVH mice failed to produce a significant number of plaque-forming cells (PFC) or a significant level of antibody against either the thymic-dependent or the thymic-independent antigen. Multiple challenges with SRBC also failed to stimulate a significant humoral immune response to the thymic-dependent antigen. Multiple challenges with LPS, however, resulted in the production of a significant number of LPS-specific PFC and a high titer of anti-LPS hemagglutinating antibodies. These results suggest that GVH-induced suppression of humoral immune responses is directed partly at B-cell activity and partly at the activity of helper T cells.     

Prostaglandin synthesis by lymphoid tissue of mice experiencing a graft-versus-host reaction: relationship to immunosuppression

Studies were carried out on the induction of PGE synthesis during the GVH reaction and its role in GVH-induced immunosuppression. The results demonstrated that spleen, lymph node cells and, to a much lesser degree, thymus cells obtained from adult C57BL/6 × AF₁ mice treated with 50–75 × 10⁶ C57BL/6 lymphoid cells were stimulated to produce PGE during the course of the GVH reaction. The spleen and lymph node PGE production peaked at Day 9 post-GVH induction (30- and 15-fold higher than normal, respectively). Thereafter, it declined to near normal levels by Days 25–30 post-GVH induction. Passage of GVH spleen cells through a rayon column removed macrophages but not mitogen-responsive T and B cells and also removed nearly all of the PGE-producing cells, except during the later course of the GVH reaction. Removal of PGE-producing cells from GVH-immunosuppressed spleen cells significantly reconstituted the mitogen response to PHA and LPS. Treatment of mice experiencing a GVH reaction with indomethacin delayed the onset of suppression of the plaque-forming cell response to sheep erythrocytes. These results suggest that early GVH-induced immunosuppression which may represent an amplified normal regulatory mechanism is mediated by increased macrophage production of PGE which suppresses both B- and T-cell functions, whereas at later stages other immunosuppressive mechanisms become operational.     

Responses in Xenopus to the thymus independent antigen polyvinylpyrrolidone (PVP) and its haptenated derivative, trinitrophenylated PVP (TNP-PVP)

Xenopus laevis (the South African clawed toad) can respond to thymus dependent (TD) and thymus independent (TI) antigens. However, the response to trinitrophenylated Ficoll (TNP-Ficoll), a TI-2 antigen in mammals, is thymus dependent in Xenopus. Polyvinylpyrrolidone (PVP), classed as a TI antigen in mammals, is also a TI antigen in Xenopus, but responses to PVP and TNP-PVP are thymus regulated. As with TNP-Ficoll, capacity to respond to TNP-PVP diminishes during metamorphosis, and tolerance can be induced via the stimulation of TD suppression with trinitrobenzene sulphonic acid. Animals treated with N-methyl-N-nitrosourea and adult-thymectomised Xenopus, which lack certain TD responses, can nevertheless respond to TNP-PVP. Based on this and other information, it is concluded that TNP-PVP should be classed as a TI-2 antigen in Xenopus.     

Superovulation in ewes by a single injection of pFSH dissolved in polyvinylpyrrolidone (PVP): effects of PVP molecular weight, concentration and schedule of treatment

Three experiments were carried out to evaluate induction in ewes of superovulation and embryo production by a single injection of a porcine pituitary extract (pFSH) dissolved in polyvinylpyrrolidone (PVP), investigating the effects of PVP molecular weight and its concentration (Experiment I), time and method of treatment (Experiments II and III). All ewes were synchronized for estrus by vaginal sponges impregnated with fluorogestone acetate (FGA; 30 mg, 9 days) plus PGF(2alpha) (Cloprostenol, 50 microg, 48h before sponge removal - s.r.), and superovulated by 250 IU pFSH. In Experiment I, 60 Gentile di Puglia ewes were subdivided into five experimental groups (n = 12): Group A, the control, received six decreasing intramuscular (i.m.) doses of pFSH, 12 h apart, beginning 48h before s.r.; Groups B and C were given 48 h before s.r. a single i.m. injection of pFSH dissolved in PVP with MW = 10,000, respectively, at concentrations of 15 and 30% w/v; Groups D and E received the same treatments as for B and C using PVP with MW = 40,000. None of the pFSH-PVP treatments were effective in inducing superovulation. In Experiment II, 22 Leccese ewes were subdivided into two groups (n = 11): Group A, control received i.m. four decreasing doses of pFSH, beginning 24 h before s.r., 12h apart; Group B was given a single i.m. injection of pFSH dissolved in PVP (MW = 40,000 at 30% w/v), 24 h before s.r. The pFSH-PVP treatment provided an ovulation rate similar to the control and tended to enhance embryo yield (4.4 versus 2.4, P>0.05). In Experiment III, 60 Leccese ewes were subdivided into six treatment groups (n = 10). Groups A and D served as controls and received i.m. 12 h apart, six doses (from 48 h before s.r.) and four doses (from 24h before s.r.) of pFSH, respectively. Groups B and C were treated by a single injection of pFSH in PVP (MW = 10,000; 30% w/v) 48 h before s.r., respectively by i.m. or subcutaneous (s.c.) administration. Groups E and F received the same treatments as for B and C 24 h before s.r. Intramuscular pFSH-PVP administration 24 h before s.r. provided an ovulation rate (8.1), mean numbers of ova recovered (5.6) and fertilized (4.2) comparable to the six or four dose treatments and significantly higher (P <0.01) compared to the pFSH-PVP treatment carried out i.m. 48 h before s.r.These results show that a single injection of pFSH dissolved in PVP at 30% w/v, performed i.m. 24 h before s.r., is able to induce a superovulatory response comparable to that following multiple injection treatment, regardless of PVP molecular weight.     

Inhibition of the graft-versus-host response by BCGcw-induced suppressor cells or prostaglandin E1

Immunization of C57BL/6 mice with BCGcw stimulated a population of "suppressor cells" which had a decreased capacity to induce the graft-versus-host response. The graft-versus-host response was quantitated using the Simonsen splenomegaly assay. F1 mice (C57BL/6 X CBA) were inoculated intraperitoneally with 1 X 10(8) parental (C57BL/6) or (CBA) spleen cells. The F1 mice were sacrificed 13 days later and the resulting splenomegaly was 3-4 times the normal amount. F1 mice which were injected with parental BCGcw-primed C57BL/6 spleen cells had a 50% inhibition of splenomegaly, whereas BCGcw-primed CBA spleen cells (a strain which does not develop suppressor cells) did not show this inhibition. In vitro results also confirmed that only C57BL/6 mice and not CBA mice developed suppressor cells after BCGcw immunization. A second study showed that X-irradiated (1000 R) BCGcw-primed "suppressor cells" could inhibit splenomegaly caused by the inoculation of normal parental C57BL/6 cells into F1 mice. The mechanism by which BCGcw-primed "suppressor cells" caused this inhibition of splenomegaly was delineated and found to be dependent upon the secretion of prostaglandin (PGE-1). Indomethacin and aspirin, potent inhibitors of prostaglandin synthesis, blocked the activity of C57BL/6 BCGcw "suppressor cells" and splenomegaly resulted. Systemic administration of the prostaglandin (15S)-15-methyl PGE-1 reduced splenomegaly approximately 50% in F1 mice which were injected with C57BL/6 or CBA cells. These results indicated that immunization with BCGcw stimulated a population of "suppressor cells" which could cause a decrease in graft-versus-host response and that the secretion of prostaglandin was responsible for this inhibition.     

Effect of tilorone hydrochloride on graft-versus-host (GvH) reaction in mice.

The effects of tilorone hydrochloride (TH), a powerful interferonogenic agent exhibiting also immune modulatory properties, on GvH reaction was studied, using the popliteal lymph node assay in mice. Administration of TH at ten days 0 or +2 relative to cell transfer to recipient mice led to a significant dose-dependent reduction of GvH reaction, whereas treatment of prospective donor mice at day -4 or -2 induced an enhanced GvH re activity of donor spleen cells. This effect was found not to be due to an altered proportion in the spleen cell inoculum of B and T lymphocytes, which latter are responsible for induction for GvH reaction. However, since normal parental lymphocytes are prepared for an enhanced GvH reactivity by addition of TH-treated macrophages, a stimulatory effect of the latter cells via macrophage-derived mediators, induced by TH, is suggested.     

Leukemia in AKR mice: III. Size distribution of suppressor T-cells in AKR leukemia and neonatal mice

Suppression of in vitro antibody forming potential of normal cells by leukemic cells of AKR and normal neonatal mice have many similarities. In both cases the suppression is by cell contact rather than by the elaboration of soluble suppressive factors and the suppression is sensitive to both x-irradiation and mitomycin C treatment. When the size distribution of suppressing cells in thymus and spleen were compared by velocity sedimentation, both leukemic and neonatal suppressing cells had similar size distribution in each organ. Both large and small cells in the thymus suppress but only large cells (sedimentation velocity >3.5 mm/hr) in the spleen are able to suppress. Leukemic cells in lymph node have a splenic size distribution, viz., only large cells suppress. Both large and small cells of a subcutaneously growing long passage AKR lymphoma are able to suppress. While large cells contain the bulk of cells actively incorporating tritiated thymidine and thus probably in cycle, small but significant amounts of incorporation in small suppressing cells is also seen.     

Interleukin 2 receptor dysfunction in mice undergoing a graft-vs-host reaction

Mice of the (C57BL/10 X B10.A)F1 combination were given a single i.v. inoculation of 3 to 4 X 10(7) B10.A spleen cells, which induces a graft-vs-host (GVH)-associated immune deficiency in F1 mice. Between 1 and 4 wk later, spleen cells from the F1 mice were tested for the expression of IL 2 receptors by flow microfluorometry, using the 7D4 rat monoclonal antibody directed against an epitope murine IL 2 receptor. A reduction in intensity of spleen cell staining with 7D4 was detected as early as 8 days after parental cell inoculation, and no IL 2 receptors were detected by 28 days after initiation of GVH. Furthermore, the loss of IL 2 receptors was correlated with abrogation of proliferative responses to concanavalin A and lipopolysaccharide, of IL 2 production, and of cytotoxic T lymphocyte responses. These observations may be relevant for our understanding of GVH reactions, of immune disorders associated with GVH, and possibly of primary and acquired immunodeficiencies in general.     

"Lymph node" form of graft-versus-host reaction in mice]

Local type of the graft-versus-host reaction was induced in adult hybrid mice (CBA X C57BL) F1 by the transfer of splenic, thymus and bone marrow CBA cells subcutaneously into the right hind footpad.The weight gain of the regional knee lymph node and blast accumulation in it 7 days later were used as indices of the graft-versus-host activity. After the transfer of 5 and 20 x 10(6) splenic cells the absolute weight of the regional lymph node was 8-10 times higher than that of the contralateral control; it was also significantly greater in comparison with controls which received live syngeneic or semiallogeneic dead cells from the same source. Contrary to controls, in case of the live cells a direct dose-effect dependency could be seen. The lymph node weight gain was accompanied by a regular immunoblast accumulation. The effect of the thymus and the bone marrow cells was less pronounced than that of the splenic cells.     

Suppression of IgG memory responses by T cells activated with the type 2 antigen polyvinylpyrrolidone (PVP)

Although type 2 antigens, such as polyvinylpyrrolidone (PVP), generally do not prime for IgG memory responses or activate specific helper T cells (TH), previous studies have established that low doses of PVP (0.0025 microgram) can prime for IgG memory and induce TH in vivo. Doses of PVP that are optimally immunogenic for IgM antibody production (0.25-25 micrograms) do not prime for IgG memory responses and preferentially activate PVP-specific suppressor T cells (TS) which suppress IgG antibody production. The studies reported here further characterize PVP-specific TS and begin to investigate the mode of action of these TS. TS induced with high doses of PVP have a typical suppressor cell surface phenotype in that they are Lyt 2+, I-J+, L3T4-, I-A- T cells. PVP-specific TS are inducible in mice expressing the X-linked immune defect and are Igh restricted in their actions. These TS suppress PVP-specific IgG responses of PVP-HRBC (horse red blood cells)-primed B cells when the TH population is from low-dose PVP-primed mice but not when the TH population is from PVP-HRBC-primed mice. Thus the TS do not apparently directly suppress the B-cell responses but act indirectly to suppress IgG responses by preventing the expression of PVP-specific TH function. The TS induced by 0.25 microgram PVP also prevent the generation of PVP-specific memory B cells apparently by preventing the expression of functional TH which are required for induction of memory B cells. Elimination of TS activation by pretreatment of mice with cyclophosphamide at the time of priming with 0.25 microgram PVP results in the expression of TH function and priming of memory B cells.     

The entry of T and B lymphocytes into rat popliteal lymph nodes undergoing a graft-versus-host reaction

The entry of radiolabeled blood-borne T and B lymphocytes into resting popliteal lymph nodes and popliteal lymph nodes stimulated with semiallogeneic lymphocytes was investigated in rats. Thoracic duct lymphocytes separated into T- and B-lymphocyte populations on nylon-wool columns were radiolabeled with 51chromium and equal numbers of T or B lymphocytes were injected intravenously. While the ratio of T and B lymphocytes in the blood is approximately 3:1 it was found that the ratio of T to B lymphocytes migrating into lymph nodes was approximately 9 T to 1 B lymphocyte in both resting and antigenically stimulated lymph nodes. Since the ratio of T to B lymphocytes in thoracic duct lymph is similar to that of blood, there is a disparity between the number of T cells entering and leaving lymph nodes. These results suggest that some T lymphocytes may return to the blood directly and/or there is increased T lymphocyte death in lymph nodes.     

Synergism of T lymphocyte subsets in the response to Mls-locus coded antigens during graft-versus-host reaction

After transplantation of lymphoid cells into lethally irradiated (semi)allogeneic mice specific anti-host directed effector T cells are generated. This can be demonstrated using a delayed type hypersensitivity (DTH) assay. In H-2 compatible combinations, Mls-locus antigens, but no other minor histocompatibility antigens, can induce the generation of such effector T cells. This paper shows that maximal anti-host DTH responses are obtained when the lymphoid cells transplanted constitute of a mixture of long-lived, recirculating T2 cells and short-lived, sessile T1 cells. It was demonstrated that anti-Mls locus-directed DTH effector T cells are the progeny of T2 cells, and that T1 cells amplify this response. The latter, however, are by themselves incapable of displaying anti-Mls DTH reactivity. The T1 cells were found to be of the Lyt-1+2+ phenotype, and the T2 cells of the Lyt-1+2- phenotype. The same Lyt phenotypes were found for T1 and T2 cells synergizing in the GvH reaction against H-2 alloantigens.