Immunodeviation by passive antibody, an expression of selective immunodepression. II. Action of guinea pig IgG1 and IgG2 anticarrier antibodies.

The effect of passively administered IgG1 and IgG2 anticarrier antibodies on the IgG1 and IgG2 antihapten response has been studied. Guinea pigs were immunized with dinitrophenylated bovine gamma globulin mixed with purified IgG1 or IgG2 antihuman gamma globulin antibodies, i.e., antibodies directed against a limited range of the carrier determinants. Humoral IgG1 and IgG2 anti-DNP antibody contents were assayed at weekly intervals and 4 to 10 days after a booster injection of antigen in saline given on the 12th wk. The main finding was the sustained suppressive effect of passive IgG1 anti-carrier antibodies on the active IgG1 antihapten response. This result is compared with the enhancing effect of passive IgG1 antihapten antibodies and is discussed in the light of T cell-B cell and hapten-carrier relationships, leading to the proposal of a regulatory function of the Fc portion of the IgG1 anticarrier antibody, combined with the antigen, on the T cell.

Immune status of mice tolerant of living cells. II. Continuous presence and nature of facilitation-enhancing antibodies in tolerant animals.

CBA mice were rendered highly tolerant to A/Jax cells by neonatal intravenous injections of (CBA x A)F(1) spleen cells. The high degree of tolerance was ascertained by the absence of circulating antibodies detected in the sera by the usual tests and by the perfect state of A skin grafts during all the experiments. Tolerant sera (sera from tolerant animals) were studied at three periods of tolerance: before skin test grafting, from 2 to 11 wk after grafting, and at time of sacrifice at almost 6 months of age. The tolerant sera were shown to have specific facilitation-enhancing properties promoting the take and growth of A/Jax sarcoma (SaI and /Sa 15091a grafted on normal CBA mice. These properties were present throughout the duration of the experiments, showing that they were not the result of a beginning interruption of tolerance. The tolerant sera, although lacking the usual serological properties (hemagglutination, hemolysis, cytotoxicity, passive cutaneous anaphylaxis) had, however, specific synergistic hemagglutinating properties (increasing the hemagglutinating titer of a reference immune serum). Antibodies giving direct specific hemagglutination could be extracted from spleens of 20% of highly tolerant mice. The tolerant sera were also found to contain more IgG1 and more IgA than normal sera while they contained normal quantities of the complement-fixing immunoglobulins IgG2 and IgM. Fractionation of tolerant sera on DEAE chromatography column confirmed the data concerning immunoglobulin classes and demonstrated direct specific serological activities undetected in unfractionated sera: a weak hemolysis in the most cationic fractions and a weak hemagglutination in the middle fractions. Synergistic hemagglutination, detected in unfractionated serum, was localized in fast anionic fractions containing high IgA concentration, along with facilitation-enhancing activity, thus confirming a link suggested previously between these three properties. The relation between immunological tolerance and facilitating antibodies was discussed in the light of the fact that antibodies, possibly of a particular class continuously present at low dose in the sera of highly tolerant animals, are able to transfer (at least partly) this state of tolerance provided a sensitive test system is utilized.

Evidence for absence of toxicity of T101 immunotoxin on human hematopoietic progenitor cells prior to bone marrow transplantation.

T101-ricin A-chain immunotoxin is a hybrid molecule made up of the T101 monoclonal antibody bound to the A-chain of ricin. It specifically destroys cells expressing the cell surface T65 antigen. We have designed a preclinical study to evaluate its possible use for the in vitro treatment of T-cell hematological cancers prior to autologous bone marrow transplantation. The data presented here show that conditions previously defined to produce high tumor cell killing, i.e., a 20-hr incubation at 37 degrees in the presence of T101-ricin A-chain immunotoxin up to 10(-7) M in a 10 mM ammonium chloride solution, do not affect the in vitro proliferative capacity of human hematopoietic stem cells studied by means of semisolid medium cultures (granulocyte-macrophage progenitors, burst-forming units-erythrocyte) and continuous liquid cultures (pre-granulocyte-macrophage progenitors). Therefore, autologous bone marrow transplantation with T101-ricin A-chain immunotoxin-treated graft should be feasible.

A rapid and reliable in vivo method for anti-GPLA (class I and class II antigens) antibody titration and GPLA typing.

A reverse Arthus reaction (RAR) may be successfully used in guinea pigs to detect histocompatibility alloantigens of the GPLA type. Epidermal cells carry class I GPLA antigens, and Langerhans cells also bear class II alloantigens. It is therefore possible to elicit an RAR by intradermal injection of relevant alloimmune sera in the skin of guinea pigs of known GPLA haplotype. RAR is detected by increased vascular permeability due to IgG1 antibody and hemorrhage due to IgG2 antibody. Compared with an in vitro protein A-rosetting method RAR is easier and quicker. It proved more sensitive for class II antigens in which Langerhans cells are the target for anti-class II antibody and rather less sensitive for class I antigens. RAR is a convenient method for following the course of GPLA alloimmunization, allowing titration of antibodies against both classes of antigen. It may also be used to type guinea pigs of unknown GPLA haplotype.

Classical and alloimmune anaphylactic degranulation of isolated single mast cells.

Viable mast cells, directly isolated by micromanipulation from a mouse peritoneal cell suspension, were deposited on the bottom of microtiter-plate wells and submitted to histamine release. Conventional antigen-induced anaphylactic degranulation as well as direct allogeneic anaphylactic degranulation were strongly inhibited when these mast cells were settled on normal tissue culture plastic surfaces. Nevertheless, normal degranulation could be recovered by pretreatment of the experimental surface with a multipositive charged molecule (poly-L-lysine). Under these conditions, we demonstrate that the degranulation of one isolated mast cell is possible and consequently, as regards the direct allogeneic anaphylactic degranulation, confirm the "self-triggering mechanism" in which the recognition of histocompatibility antigens on the membrane of the mast cell itself is the trigger to the secretory response. The technique of monocellular degranulation described in this paper provides a new tool which leads us to think that the problem of detection of anaphylactic antibody-secreting cells can be solved.

Immune status of mice tolerant of living cells. III. Presence and evolution of cells cytotoxic to the tolerated strain.

Spleen cells from CBA mice neonatally rendered highly tolerant to A/Jax (keeping a skin graft in perfect shape for more than 1 year and without detectable hemagglutinating or cytotoxic antibodies) contain cells cytotoxic for YAC 222 (A/Jax) in Cr release assay. The degree of the cytotoxicity depends on the age of the mouse, following a curve lower than, but parallel to, the one followed by the cytotoxicity of cells from CBA rendered immune by injecting them with A/Jax cells 1 week previously. The maximum of the cytotoxicity curve is reached during the 9th and 10th weeks. Normal CBA cells themselves are moderately cytotoxic to YAC 222. This "natural" cytotoxicity, significantly less intense and presumably directed against Moloney virus-related determinants, does not follow the same time pattern. The cytotoxic indices from both immune and tolerant cell populations are significantly reduced by CBA and anti-A/Jax immune serum. The tested sera of the tolerant mice did not contain hemagglutinating or in vitro-blocking antibodies in the Cr release assay (only the sera from unsuccessfully treated mice, having rejected their skin grafts, had some degree of blocking activity). However (and in agreement with previous experiments), these sera often contained synergistic hemagglutinins and in vivo enhancing properties of A/Jax tumors (Sal) grafted on CBA recipients.

Passive allograft enhancement by subclasses of polyclonal antibodies directed toward restricted regions of the major histocompatibility complex.

Two parameters of enhancing major histocompatibility complex (MHC) antibodies, previously separately studied, namely, Ig class and antigen specificity, have been treated simultaneously. In the experimental model used, Sa 1 tumor cells, indigenous of A/J (H-2a) were grafted on CBA (H-2k) or C57BL/Ks (H-2d) mice. Immune sera specific for the H-2 K/D- or H-2 I coded antigens of the A/J haplotype (anti-Kk, or IAk, IBk, IJk, IEk, or ICd, Sd, Gd, or Dd) and their immunoglobulin fractions (separated on protein A-Sepharose columns) were injected either i.v. or locally as mixture with the challenging Sa 1 cells. Within the limits of the studied system, the following results were obtained: (1) Sa 1 cells do possess Iak antigens at their surface detected by C-dependent cytotoxicity; no ICd, Sd, or Gd products were detected. (2) The bulk of enhancing activity is concentrated in IgG1 anti-K/D antibodies (anti-Dd when Sa 1 was grafted on CBA mice and anti-Kk, on C57BL/Ks). (3) Anti-Iak antibodies have some activity on Sa 1 cells grafted on C57BL/Ks mice. This activity is significant for IgG1 anti-Iak and suggestive for IgG2 of the same specificity. (4) No enhancing activity was detected in the other antibodies: IgG2 anti-Dd, IgG2 anti-Kk, IgG1, or IgG2 anti-ICd, Sd, Gd as well as in fractions containing IgM and IgA antibodies directed against any studied portion of the MHC products. This results are discussed in terms of the mechanisms involved in enhancement.

Ia versus K/D antigens in immunological enhancement of tumor allografts.

The respective role of anti-H-2 K/D and anti-H-2 Ia antibodies in allotransplanted tumor enhancement was tested in vivo on two experimental tumors. Sa I A/4 (H-2a, i.e., H-2k/d) was enhanced in CBA (H-2k) and C57BL/Ks (H-2d) strains with anti-A/J immune sera prepared in CBA and C57BL/Ks, respectively. EL 4, C57BL/6 (H-2b) lymphoma, was enhanced in DBA/2 (H-2d) and BALB/c (H-2d) with immune sera prepared in DBA/2 and BALB/c. Anti-K/D antibodies were obtrained by elution from glutaraldehyde-treated RBC previously incubated with corresponding alloimmune sera prepared in mice immunized with spleen cells, thymocytes, or two consecutive skin grafts syngeneic to the RBC. The residual complement-dependent serocytotoxicity for target lymphocytes observed after complete hemagglutinin absorption on corresponding RBC was attributed to anti-Ia antibodies. RBC eluates (anti-K/D) were found to be enhancing for both experimental tumors and for all studied sera. After RBC absorption, the sera lost all enhancing activity when they were prepared by immunization with spleen or thymus cells, but remained enhancing in some sera prepared by immunization with skin grafts. Both types of antibodies (anti-K/D and anti-Ia) therefore appear able to enhance allografts. These results are compatible with the in vitro correlates of the two phases of the transplantation reaction: initiation phase (mixed lymphocyte reaction) inhibitable by anti-Ia and effector phase (cell-mediated cytotoxicity) inhibitable by anti-K/D.

Ultrastructural localization of guinea pig spermatozoal autoantigens on germinal cells by immunoperoxidase techniques.

Three guinea pig spermatozoal autoantigens S, P and T, each one able to induce autoimmune aspermatogenic orchiepididymitis and autoantibodies, were ultrastructurally localized in male germinal cells by immunoperoxidase techniques. Both living and prefixed sectioned cell preparations were treated and examined. Fab antibody fragments were used to study intracellular antigens (whole antibodies were inefficient). Water-soluble S and P autoantigens were found in acrosomal structures in the same sites: proacrosomal and acrosomal granules of the young spermatids, on the head caps of spermatids and acrosomal cap of spermatozoa, along the inner and outer acrosomal membranes and in the outer zone of the acrosomal matrix of the same cells. S was never found in the inner zone of spermatid or spermatozoa acrosomes, while P was present in this inner zone, but only of young spermatids. Water-insoluble T autoantigen was found on the plasmalemma and outer acrosomal membranes of spermatids and spermatozoa, inside the spermatid cytoplasm and, sometimes, on the inner acrosomal membrane of young spermatids. The specificity of the immunological localization for each antigen was confirmed by testing with specific antisera following absorption with homologous and heterologous antigens. No other testicular cell type (including Sertoli cells per se) was found to bear S, P or T autoantigens. When use was made of autoimmune sera obtained through autologous whole spermatozoa, the observed staining was an additive combination of what was observed when using the preceding three immune sera, anti-S, anti-P and anti-T.

Alloantibody bipolar bridging; a new mechanism of cell surface activation.

Bipolar bridging of cellular membrane receptors and epitopes by alloantibodies (Fab bridging the MHC antigens and Fc the Fc receptors) has been shown on a murine mast cell model to be a way of cell signaling and activation. In order to test a possible general significance of this phenomenon, another model was studied, namely guinea pig neutrophils. It was found l(1) that neutrophils from S2, S13 and BIO-AD strains both express class I (B) and class II (Ia) antigens on their surface, as detected by a Prot.A-SRBC rosetting method, after cell incubation with related alloantibodies; (2) that Fc receptors for IgG (Fc gamma R) were specific for IgG2 subclass, as determined by the same rosetting method after binding of preformed immune complexes (IgG1, IgG2 and F(ab')2 anti-DNP-DNP25 BSA); and (3) that specific alloantibodies of IgG2 subclass were able to specifically activate the neutrophil oxidative metabolism as shown by superoxide anion (O2-) release, detected by the luminol-dependent chemiluminescence method. Neither the IgG1 nor F(ab')2 portion were able to trigger O2- release. This demonstrates a second situation of a cell membrane activation through alloantibody bipolar bridging.

Resistance of female guinea pig fertility to efficient iso-immunization with spermatozoa autoantigens.

Virgin female guinea pigs received two courses of immunization with S, P or T spermatozoa autoantigens and Freund's complete adjuvant and were mated from 1 up to 18 weeks after the end of each course. The immunizations were efficient as judged by the titers of circulating antibodies to S, P or T, the existence of antibodies to the corresponding immunizing antigen in cervico-vaginal secretions and by cutaneous reactions of delayed hypersensitivity. In spite of this successful immunization, the fertility rate was 100% after the first course and only slightly decreased after the second one. The only significant events were a delay in the time of fertilization and a high rate of intrauterine death (as already observed following anti-S immunization). The absence of fertility impairment was not due to a lack of a relevant antigen in the injected preparations since immunizing female guinea pigs with either epididymal spermatozoa or crude water-soluble extract also did not decrease the fertility. The mechanisms responsible for such a resistance remain to be elucidated; they may involve spermatozoa coating substances, enhancing antibodies or sperm immunosuppressive factors.

Maternal alloimmune reactions towards the murine conceptus and graft-versus-host reaction (GVHR). I. Priming for anti-paternal GVHR by gestation.

In the H-2 compatible (but minor loci-incompatible) BALB/c-DBA/2 strain combination (both H-2d), intravenous injection of 1.3 X 10(7) BALB/c spleen cells from virgin females into DBA/2 newborn mice less than 18 h old does not result in a significant lethal graft-versus-host reaction (GVHR). A strong GVHR (79% lethal) is induced if the BALB/c donors have been preimmunized to DBA/2. Spleen cells from BALB/c mice pregnant by DBA/2 males are also able to induce a significant, but weaker, GVHR (16% lethal) indicating a cellular priming to paternal antigens by gestation. A significant difference exists between anti-DBA/2 GVH reactivity of spleen cells from primiparous (22% lethal) and multiparous (9% lethal) allopregnant BALB/c mice, indicating that the allogeneic boosters of successive allogestations act more on the target-protective side of immunity than on the target-aggressive one. Sera from allopregnant mice (BALB/c X DBA/2) inhibit the GVHR induced by their own cells, while sera from isopregnant ones (BALB/c X BALB/c) have no effect. Thymectomy performed at 6-wk of age, six weeks before gestation did not significantly modify the maternal reactivity. A similar priming by allogestation in the same strain combination was found for local GVHR (induced in adult F1 hybrids) resulting in higher (+132%, P less than 0.005) stimulation indices and seen to be specific for the paternal strain, the indices induced by the same cells being lower (-35%, P less than 0.05) compared to that induced by cells from virgin BALB/c, when injected into irrelevant F1 hybrids (BALB/c X CBA).

Immunological relationships between murine surrogate mothers and transplanted embryos, as studied by local GVH reactivity.

C57BL/Ks (H-2d) female mice were transplanted with early (stage 2) embryos of the A/J (H-2a) strain. Spleens from mice exhibiting successful pregnancies were tested at days 16 to 19 of gestation in a local graft versus host (LGVH) assay using (C57BL/Ks X A/J)F1 recipients and proved to be significantly more reactive than virgin controls or mice carrying transplanted syngeneic fetuses. This increased reactivity was specific for the transplanted embryo's strain. Other controls included donors with semi-allogeneic (F1) transplanted fetuses and females naturally pregnant by allogeneic males which did not give reactions significantly different from virgin control spleen cells. Para-aortic lymph node cells (PALN) obtained from the same A/J embryo-transplanted females showed a strong T suppressive activity both on their own spleen cell (SC) reaction as well as on the reaction obtained with virgin SC. This suppressive activity also appeared to be embryo-strain specific. Serological tests revealed the presence of mast cell-degranulating (anaphylactic) antibodies but not of hemagglutinating or complement-fixing cytotoxic activities. The A/J offspring obtained after embryo transfer to C57BL/Ks females presented at the age of two months significantly lower LGVH reactivity against the surrogate mother's strain. The differences in the responsiveness of the mice transplanted with allogeneic embryos compared with those with conventional pregnancies are discussed.

Antagonistic maternal immune reactions (rejection and facilitation) to the embryo in the urodele amphibian Salamandra salamandra lin.

In vitro assays have been employed to demonstrate that pregnant salamanders mount an immune reaction against their embryos. Maternal spleen cells kill up to 85% of dissociated embryonic epidermal cells during a 48 h incubation period. The degree of killing depends upon the ratio of maternal to embryonic cells and on the number of embryos borne by the mother. The cytotoxicity shows considerable specificity for the embryos of a given mother although a weak degree of killing can occur with embryos from other mothers, presumably due to some form of cross-reactivity. The effect is inhibited by the addition of maternal serum to the cultures. The degree of protection is also a function of the number of embryos borne by the mother. Pre-incubation experiments indicate that the maternal serum has a protective action on the embryonic cells which is largely specific for the female's own embryos (and suggested to be antibody in nature) and an inhibitory action on the maternal spleen cells which occurs also with spleen cells of other females (and suggested to be either an immune complex or a nonimmunological substances). An increase in beta protein peaks is seen following electrophoresis of sera from pregnant (and also allografted) salamanders. These findings indicate that the pregnant salamander mounts a double immune reaction against her embryos, an aggressive (rejection) reaction and a protective (facilitation) reaction.

Allogeneic reactivity of maternal lymphoid cells during the course of gestation. Modifications and sex differences in a local GVH assay.

The immune reactivity of lymphoid cells from pregnant mice was studied during the course of pregnancy in primiparous and multiparous animals either " isopregnant " (male and female of same strain) or " allopregnant " (male and female differing at H-2), using a local GVH assay (CBA lymphoid cells injected into (CBA X A/J)F1 recipients). The findings were as follows: The lymphoid cell number in the para-aortic lymph nodes ( PALN ) was increased at all stages of gestation. The peak occurred in the 2nd week in primiparity and as early as 60 h after fertilization in multiparity. PALN cell alloreactivity was weak at the beginning and higher than normal in the third week of pregnancy. Spleen cell alloreactivity was increased in the second week and decreased in the third week in primiparous compared with multiparous animals. Anti-paternal alloreactivity exhibited by spleen cells of allogestation was decreased (as compared to cells of isogestation ) especially in primiparous mice, particularly in the third week. At this time, the anti-paternal alloreactivity of PALN cells was increased. The influence of the recipient's sex on GVHR intensity was reversed when the cells were obtained from a pregnant donor, becoming stronger in male compared with female hybrids.

Maternal alloimmune reactions towards the murine conceptus and graft-versus-host reaction (GVHR). II. Inhibition of priming by placental extracts.

Gestation can induce a priming for a GVHR towards paternal strain antigens, although this priming is significantly lower than the one induced by experimental immunization. A role has been sought for placental substances in decreasing this priming through immunomodulation. BALB/c (H-2d) spleen cells do not usually induce a systemic, lethal GVHR in DBA/2 (H-2d) newborn mice except when the donors are preimmunized with DBA/2 cells. Placental extracts (as well as RPMI medium or liver extracts used as controls) were added to DBA/2 cells injected into BALB/c mice used as cell donors for GVH induction. The latter's spleen cells, harvested on day 6 after immunization, were used for systemic and local GVHR. In the systemic assay (lethal effect on DBA/2 newborn mice injected i.v. with BALB/c spleen cells) a significant protection was observed. In the local assay (popliteal lymph node assay in F1 hybrids injected with BALB/c spleen cells into the foot-pad) a highly significant inhibition of priming was detected in recipients injected with spleen cells from placental extract-treated donors. The stimulation index was even lower than that obtained with unprimed BALB/c spleen cells. The same type of local GVHR in (CBA/Ca X A/J) F1 hybrids injected with CBA cells led to similar results. In both situations (systemic and local GVHR) the observed inhibition was found to be specific to the priming cell strain. These results support the working hypothesis that placental substances are able to modify the systemic response of an organism towards both H-2 and non-H-2 alloantigens.

In vitro immunomodulatory effects of placental substances on preparatory MLR and resulting modifications of lymphocyte reactivities.

The immunomodulatory effects of murine placental extracts (PE) were studied in vitro using mixed lymphocyte culture (MLC) and resulting cell-mediated lympholysis (CML). The results showed that preparative cultures in the presence of PE syngeneic to the responding cells led to a low secondary MLR response with a concomitant generation of suppressor cells. At the efferent phase, cells from the same preparative culture showed a weaker cytotoxic activity than controls cultured in the absence of extract. Furthermore, the induction of regulatory cells able to inhibit CTL in vitro activity was also observed. The active substances can be found in the 30% ammonium sulphate precipitate as well as in some gel filtration fractions showing several main bands from 115 to 43 kDa in SDS-PAGE.

Deviation of humoral and cellular alloimmune reactions by placental extracts.

Modifications of the alloimmune response at both the humoral and the cellular levels by placental extracts (PE) syngeneic to the recipient were studied in the mouse using two different H-2 strain combinations. CBA (H-2k) or C57BL/Ks (H-2d), immunized with A/J (H-2a) spleen cells. The tests included in vivo tumor allograft evolution (accelerated rejection or enhancement reactions), and in vitro analysis of the involved immune agents, both cellular and humoral, using mixed lymphocyte reactions (MLR) and biological activity studies of serum samples. Animals from the recipient strains exhibited a delayed rejection of A/J tumor Sa 1 allografts if preimmunization was carried out with 10(6) A/J spleen cells combined with PE syngeneic to the recipients, as compared to controls immunized with A/J cells only or supplemented with isogeneic liver extracts (LE). The serological analysis revealed that PE treatment did not modify the overall hemagglutinating antibody production but resulted simultaneously in both a decreased production of cytotoxic complement fixing antibodies and an increase of specific anaphylactic mast cell degranulating antibodies, as compared to controls. The sera from PE-treated donors also demonstrated enhancing activity following passive transfer to isogeneic recipients. MLR regulatory activity was exhibited by spleen cells from PE- and immunogen-treated mice although the same or stronger activity was obtained from mice immunized without the addition of PE. However, in vivo transfer of these cells to syngeneic recipients showed that PE treatment erased the accelerated rejection caused by allogeneic immunization in the absence of PE and could even cause some degree of allografted tumor enhancement. The cells responsible for this inhibitory effect were mainly IJ+ lymphocytes, since their elimination with a relevant anti-IJ serum and complement restored a secondary type rejection pattern. These results show that PE present during the onset of immunization can promote the activation of regulatory agents such as enhancing antibodies and suppressor cells favoring allograft survival.

Modification of immune responsiveness to tumour grafts (enhancement or inhibition) induced in mice by allogeneic embryos.

Immune reactivity of the female recipients, made artificially pregnant, of fully allogeneic embryos was investigated. Allogeneic blastocysts were transferred into hormonally synchronized foster mothers in two strain combinations A (H-2a)--CBA (H-2k) and C57BL/6 (H-2b)--DBA/2 (H-2d). Immunogenicity was tested by growth of tumours Sa 1 (syngeneic with A strain recipients) and EL 4 (syngeneic with C57BL/6 strain recipients). Allogeneic blastocysts implanted in the host's uterus are able to elicit a stronger immune response than in semi-syngeneic pregnancies. The immune reaction was manifested by enhanced growth of tumour Sa 1 and accelerated rejection of EL 4.

[Immunotoxins]. / Les immunotoxines.

Immunotoxins are conjugates between antibodies especially directed against cancer cells and a subunit of a powerful toxin. We used the A-chain of ricin. These conjugates are specifically cytotoxic when used at very low concentrations in vitro and can destroy more than 99.99% of clonogenic cells. The efficacy of immunotoxins was also demonstrated in vivo but is inferior to its in vitro potency. For this reason the first use of immunotoxins in man can be the cleaning up of bone marrow from leukemic cells in the near future.