Cytotoxic Antisera Research Articles

Adoptive transfer of immunity against virulent Semliki Forest virus with immune spleen cells from mice infected with avirulent Semliki Forest virus.

This paper describes the minimal requirements to protect mice adoptively against challenge with virulent Semliki Forest virus (SFV). Early immune serum, from donor mice infected with an avirulent strain of SFV, contained mainly neutralizing IgM immunoglobulins. More of these antibodies (1.80 PND50 vs 0.06 PND50) were needed to protect recipient mice against intraperitoneal challenge (10 LD50 of SFV) than against subcutaneous challenge (7 LD50). Adoptive transfer experiments indicated that a minimum of 3 X 10(7) six-day immune spleen cells were also able to protect recipient mice against intraperitoneal challenge with 10 LD50 of SFV. Treatment of these donor cells with cytotoxic antisera and complement revealed both T- and B-lymphocytes were required for optimum adoptive immunity. Surviving recipients of either immune serum or immune spleen cells developed significantly less neutralizing antibodies than control mice. The lower antibody titres in protected mice might be related to either immune serum or immune spleen cell mediated restriction of virus replication; meaning a reduced antigenic stimulus in these mice compared to control mice.

Serologic recognition of a HLA-B44(12) subgroup associated with the 44.2 specificity defined by CML

Several investigators have reported that subdivisions of HLA-B44, which are not defined by available cytotoxic antisera, can be detected by cytotoxic T lymphocytes. The present study was designed to evaluate two sera which appear to recognize a specificity on some HLA-B44 and B45 positive cells and to determine whether the specificity is correlated with one of the B44 subgroups which are defined by cell-mediated lympholysis. Serum BG1359, eluted from a placenta and serum Cutchember (a gift from Dr. Wilma Bias), were tested on a 23-member B44 positive cell panel. The panel was typed independently with CTL Q1/M3 (specificity 44.1) and CTL M3/Q1 (specificity 44.2). Cells classified as 44.2 usually typed positive with serums 1359 and Cutchember, whereas 44.1 cells usually were negative with these sera in the Amos modified cytotoxicity technique. In the 4th American workshop data, serologic reactivity correlated with B44 (r = 0.87 and 0.75) and B45 (r = 0.32 and 0.34) for serums 1359 and Cutchember, respectively. In a North Carolina population study (N = 155), specificity 1359 was detected in 37 of 53 (70%) of B44 positive individuals and in 5 of 6 B45 positive individuals. In three informative families, the 1359 private specificity segregated with B44 haplotypes. The 44.2 subgroup is in positive linkage disequilibrium with C4 and DR7 and possibly with A23, A26, and A29, whereas the 44.1 subgroup seems to be in disequilibrium with A2, Cw5, DR4, and DRw6.

In vitro immune aggression against rabbit aortic smooth muscle cells

Rabbit aortic smooth muscle cells cultivated with certain antisera underwent growth changes and necrosis. These cytotoxic antisera were obtained by immunizing rabbits against rat aorta, human or pig aortic glycoproteins, human serum glycoproteins and E. coli lipopolysaccharide. These different antigens share some biochemical characteristics, and contain four main amino acid residues (Glu, Ala, Asp, Gly) and four sugars (mannose, galactose, glucose, N-acetyl glucosamine). The cytolytic properties of these antisera, however, probably correspond to structural analogies, since although ovalbumin is a glycoprotein, anti-ovalbumin antiserum was not cytotoxic. Antibody cytotoxicity against rabbit arterial smooth muscle cells may depend on the biochemical structure of the antigen used to produce antiserum.

Lack of Rhesus antigen expression by human committed erythroid progenitors

The D antigen of the Rhesus blood group, an erythroid-specific cell surface marker, is expressed by all morphologically recognizable human nucleated red blood cell precursors including, in low density, the pronormoblast. The object of the present study was to determine the expression of the D antigen by committed erythroid progenitors. Under conditions that produced complete inhibition of BFU-E and CFU-E by known cytotoxic antisera, no significant inhibition was produced by anti-D. Use of anti-human IgG (rabbit) to increase sensitivity and trypsinization to reveal cryptic Rh determinants were both without inhibitory effect. Erythroid bursts and colonies grew normally in methylcellulose that contained anti-D. The addition of anti-D to day 7 BFU-E did not inhibit their proliferation to mature bursts at day 14. These results suggest that the D antigen is not expressed by human committed erythroid progenitor cells. The D antigen is therefore an erythroid-specific differentiation marker, rather than an erythroid- lineage-specific antigen. The development of expression of the D antigen during erythropoiesis parallels that of band 3 protein, to which anti-D has been reported to bind. Lack of Rh expression by committed erythroid progenitors is consistent with the rarity of red cell aplasia in Rhesus hemolytic disease of the newborn and in idiopathic and drug-induced autoimmune hemolytic anemia in which the autoantibodies have apparent Rh specificity. These results imply that Rh compatibility is not a contraindication to human bone marrow transplantation.

Lack of Rhesus Antigen Expression by Human Committed Erythroid Progenitors

The D antigen of the Rhesus blood group, an erythroid-specific cell surface marker, is expressed by all morphologically recognizable human nucleated red blood cell precursors including, in low density, the pronormoblast. The object of the present study was to determine the expression of the D antigen by committed erythroid progenitors. Under conditions that produced complete inhibition of BFU-E and CFU-E by known cytotoxic antisera, no significant inhibition was produced by anti-D. Use of anti-human IgG (rabbit) to increase sensitivity and trypsinization to reveal cryptic Rh determinants were both without inhibitory effect. Erythroid bursts and colonies grew normally in methylcellulose that contained anti-D. The addition of anti-D to day 7 BFU-E did not inhibit their proliferation to mature bursts at day 14. These results suggest that the D antigen is not expressed by human committed erythroid progenitor cells. The D antigen is therefore an erythroid-specific differentiation marker, rather than an erythroid-lineage-specific antigen. The development of expression of the D antigen during erythropoiesis parallels that of band 3 protein, to which anti-D has been reported to bind. Lack of Rh expression by committed erythroid progenitors is consistent with the rarity of red cell aplasia in Rhesus hemolytic disease of the newborn and in idiopathic and drug-induced autoimmune hemolytic anemia in which the autoantibodies have apparent Rh specificity. These results imply that Rh compatibility is not a contraindication to human bone marrow transplantation.

CYTOTOXIC ANTIBODIES AGAINST SURFACE IMMUNOGLOBULIN

The reactivity of B lymphocytes, null lymphocytes, T lymphocytes, chronic lymphocytic leukemia (CLL) cells, B and T cultured cell lines, monocytes, and granulocytes was tested against a panel of antisera under cold conditions of 5 C incubation followed by 25 C incubation in complement. The degree of cold cytotoxic activity against these cell types was in the order given above, with the C cells being the most sensitive. Surface immunoglobulin (SIg) staining followed the same distribution sequence, with the B cells containing the most SIg. Human IgM inhibited cold cytotoxic antisera but human IgG or human serum albumin (HSA) did not. IgM did not inhibit HLA antisera. We conclude that at least some cold cytotoxic antibodies react against surface IgM.

Immunoselection of mutants deficient in cell surface glycoproteins encoded by murine erythroleukemia viruses.

We have described a heterogeneously processed glycoprotein with an apparent molecular weight of 55,000 (gp55) that is encoded by the Friend spleen focus-forming virus, an acute erythroleukemia virus [Dresler, S., Ruta, M., Murray, M.J. & Kabat, D. (1976) J. Virol. 30, 564-573]. Several lines of evidence suggest that a small proportion of the gp55 in infected cells is located on the surface membranes. First, different nonproducer cell lines infected with cloned Friend spleen focus-forming virus were efficiently killed in the presence of complement by cytotoxic antisera that react with gp55. Furthermore, a clone of cells selected for resistance to the cytotoxic antibody synthesized an altered intracellular form of gp55. This immunoselection procedure appears to also be useful for isolating glycoprotein mutants of other RNA tumor viruses. Analysis of cell surface proteins labeled with [125I]iodine supported the idea that gp55 occurs on the plasma membranes. However, the cell surface gp55 had more highly processed oligosaccharides than the majority of the gp55, which occurs within the infected cells. In addition, we have found that leukemia cells from mice infected with the erythroleukemic Rauscher virus complex contain a membrane glycoprotein that appears similar to the gp55 encoded by Friend spleen focus-forming virus. The encoding of similar glycoproteins by independently isolated acute erythroleukemia viruses suggests that these glycoproteins may be important in leukemogenesis.

Rapid Identification and Subtyping of Herpes Simplex Virus by Complement-Dependent Cytotoxicity

A simple method for rapid identification and subtyping of herpes simplex virus from clinical specimens is presented. Inoculated cultures demonstrating cytopathic changes were treated with type-specific cytotoxic antisera against herpes simplex virus and complement. Lysis of cells was revealed by staining with trypan blue. The technique allowed identification and subtyping of herpes simplex virus within 2 h after the appearance of CPE.

Phytohemagglutinin-induced differentiation and blastogenesis of precursor T cells from mouse bone marrow.

The cells in mouse bone marrow (BM) capable of responding to phytohemagglutinin (PHA) were shown to be precursor T cells in experiments employing athymic mice, immunofluorescence, and specific lysis of T or B cells with cytotoxic antisera + complement. In contrast, the responses of lymph node (LN) and spleen (Spl) cells to this mitogen were shown by the same techniques to rely upon resident populations of mature T lymphocytes in these peripheral lymphoid organs. Cytolysis of T cells with anti-theta (anti-Thy 1), anti-thymocyte, or anti-brain antisera abolished the PHA responses of LN and Spl, but had no appreciable effect on the BM PHA response. Lysis of B cells with anti-mouse gamma globulin or anti-mouse IgM antisera had no significant effect on either Spl or BM blastogenesis in response to this lectin. Immunofluorescent studies with fluoresceinated anti-brain sera demonstrated acquisition of T-cell surface antigens by BM null lymphocytes during the blastogenic response of this tissue to PHA. The results of these immunofluorescence experiments were reproducible even when marrow obtained from nude mice and pretreated with anti-brain serum plus complement was employed. The implications of these findings with regard to prophylaxis against graft versus host disease in BM transplant recipients are discussed.

Selective depletion of chromosomes in a stable mouse-Chinese hamster hybrid cell line using antisera directed against species-specific cell surface antigens.

A chromosomally-stable mouse-Chinese hamster hybrid cell line was subjected to five rounds of selection with cytotoxic antisera raised in rabbits against either the parental mouse 3T3 cells or the parental Chinese hamster Wg-1 cells. Routine karyological analysis of clones isolated at each stage of serum selection revealed that treatment with either serum resulted in a limited loss of chromosomes (compared to the untreated hybrid cell cultured in parallel) and that the pattern of chromosome loss could not be correlated with the particular antiserum used for selection. However, more detailed analysis with the SSC-formamide C-banding technique, which identifies chromosomes containing a mouse centromere region, demonstrated that while large-scale chromosome loss was not achieved as a result of antiserum selection, the limited loss of chromosomes did, in fact, reflect a specific depletion of chromosomes in response to treatment with cytotoxic antiserum. Specific chromosomal elimination was shown to occur as early asthe first round of antiserum treatment. Antigenic analysis of the serum-selected clones revealed a quantitative decrease in the expression of the species-specific surface antigens selected against, but no qualitative loss of antigens was detected. The results suggest that treatment with cytotoxic antiserum may select for clones that have lost specific chromosomes bearing genes regulating the expression of species-specific surface antigens, rather than for those demonstrating large-scale depletion of chromosomes bearing the corresponding structural genes.

Reactions of Kidney Cells with Cytotoxic Antisera: Possible Evidence for Kidney‐Specific Antigens

HL-A typing of cadaver kidney cell suspension by fluorochromasia cytotoxicity was successful in 76 out of 124 cases. In the 44 cases with confirmed phenotypes, 24 had identical results for lymphocytes and kidney cells. Twenty kidney cells had HL-A antigens not detected on that donor's lymphocytes, most commonly HL-A7 and HL-A8. In eight of these 20 cases, the additional kidney antigens brought the total to more than two per segregant sereis, in disagreement with an earlier report from this laboratory. The discrepancy was traced to a change in the method of complement preparation. The complement was successfully deprived of the resulting non-specific cytotoxicity for kidney cells by absorption with human red blood cells. Prior to absorption, the complement had rendered kidney cells susceptible to the lytic effect of anti-A and B red cell antibodies. Using the absorbed complement, a patient who had hyperacutely rejected two cadaver kidneys provided sera with an antibody reacting with, and absorbed by, the kidney cells but not the lymphocytes of the donors.

Immunologic Induction of Malignant Lymphoma: Identification of Donor and Host Tumors in the Graft-Versus-Host Model 2

Primary lymphoid tumors were induced by inoculation of parental line spleen cells into H-2 incompatible F1 hybrid mice. The genotypes of lymphomas arising in 19 different parent yields F1 combinations were determined with cytotoxic antisera against H-2 specificities of donor and host. Additional chromosome marker studies established the reliability of tumor genotyping by the cytotoxicity test. According to H-2 typing, 12 primary lymphomas were of donor type, 66 of host type, and 14 of intermediate type. As a group, those parent yields F1 combinations in which donor-type lymphomas developed (group 1) showed a higher degree of histocompatibility between donor and host than those combinations in which donor-type lymphomas failed to appear (group 2). Allogeneic inhibition may have suppressed the development of donor-type tumors in the combinations of group 2. The development of malignant lymphomas of donor-type in the graft-versus-host reaction (GNHR) supports the concept of lymphomagenesis as a consequence of persistent stimulation by antigen.

Serological identification of sperm antigens specified by lethalt-alleles in the mouse

Cytotoxic antisera were prepared by immunizing wild-type recipient mice with sperm from donors carrying different recessive lethal alleles at theT locus (T/t0,T/t w 1 ,T/t w 5 , andT/t w 32 ). After removal of sperm autoantibody by absorption with sperm of recipient type, each antiserum reacted only with sperm from males whose genotype contained at least one of the immunizing alleles. Cytotoxicity was high against sperm populations in which both immunizing alleles were represented and was lower when only one was present. Thus, each allele at theT locus which has so far been tested serologically is recognizable as a discrete antigen on the surface of sperm.

HL-A antigens on kidney cells and the use of kidney cells as a target in the detection of donor specific cytotoxic antibodies in renal transplantation.

SummaryKidney cells have been typed for HL‐A antigens using a technique of complement dependent destruction of monolayers of renal epithelial cells in tissue culture by cytotoxic antisera. The degree of concordance between the phenotypes of peripheral blood lymphocytes and kidney cells from the same individual was 80·5%. However, significant discrepancies between the typing of lymphocytes and kidney cells were found for the antigens HL‐A7, 12, 13. Although the majority of the discordant reactions were found with antisera containing more than one antibody, it is felt that the above discrepancies are valid. Positive cross matches between recipient sera and donor kidney cells, together with a negative lymphocytotoxic cross match, were found in four of five cases where the kidney was acutely rejected within three months of transplantation. It is suggested that HL‐A antigens are expressed on both lymphocytes and kidney cells, but either there may be a quantitative difference in their expression on the two cell types, or the monolayer destruction technique may be more sensitive than the lymphocytotoxic technique.

The production of anti HL-A cytotoxic antisera through planned immunization by intravenous injection of small aliquots of whole blood. I. Immunization of unrelated recipients.

Planned immunizations were performed in 20 pairs of unrelated individuals.The immunization procedure, employing weekly injections of small aliquots of whole blood, was shown to be safe and adequate to elicit an anti‐HL‐A response in the great majority of cases.The antibody responses showed two sharply distinguishable patterns: “Restricted”, with the production of monospecific, non cross‐reacting antibody; and “Broad → Restricted”, with the production of an antibody reacting against specificities other than those possessed by the donor, and becoming more and more restricted with time. Both patterns are constantly reproduced upon subsequent stimulations.The pattern of response appears to be determined primarily by some peculiarity of the donor's specificity(ies) or by some characteristic relationship between donor and recipient antigenic constitution, rather than by a special reactivity of the recipient per se.

Alloantigens common to erythrocytes and leucocytes in pigs

SummaryThe presence of pig blood group antigens Ea, Ed, Ef, El and Gb has been demonstrated on pig lymphocytes by the cytotoxic test. The typing of standard blood group reagents in an erythrocyte panel and cytotoxic antisera in a lymphocyte panel (from the same individuals as the erythrocytes) showed that the reactions of some sera with cytotoxic activity corresponded to the distribution of the given erythrocyte antigens.

Histocompatibility in the Rabbi Identification of the Major Locus12

Cytotoxic antisera were produced either by skin graft exchange between related rabbits from the same partially inbred line or by graft exchange between rabbits from different inbreeding lines. By pedigree analysis and correlation with known antisera, these antisera were all shown to detect antigens controlled by a single locus. This locus has been assigned the symbol RL‐A. Skin grafts between siblings incompatible for the RL‐A were all rejected within twelve days, and graft survival time could not be prolonged with relatively mild immunosuppressive treatment with 6‐mercaptopurine. However, a significant number of grafts compatible for the RL‐A locus were maintained for twelve days or longer and graft survival time of compatible grafts could be significantly prolonged with mild immunosuppression, suggesting that the RL‐A locus is the major histocompatibility locus of the rabbit.

Reactions of HL-A antissera in three populations.

Abstract. Cytotoxic antisera against HL‐A antigens were tested in panels of Caucasian, Bantu and Indian donors. A serum that gives reliable results in Caucasians may prove quite unreliable when used in Bantu or Indians, since it may possess extra antibodies against antigens that are very rare in Caucasians but common in the other groups. Every serum that is to be used in a population different from that in which it was standardized must therefore be re‐standardized in the new group before use. Results obtained in previous studies by EDTA agglutination cannot be compared with those obtained by cytotoxicity. The NIH serum Willett (anti‐HL‐A 8) gives identical results by agglutination and by cytotoxicity when tested in Caucasians; but in the Bantu it reacts with 49% by agglutination and only 8% by cytotoxicity. The Bantu evidently possess several unidentified HL‐A antigens.

Reactions of HL-A Antisera in Three Populations

Cytotoxic antisera against HL-A antigens were tested in panels of Caucasian, Bantu and Indian donors. A serum that gives reliable results in Caucasians may prove quite unreliable when used in Bantu or Indians, since it may possess extra antibodies against antigens that are very rare in Caucasians but common in the other groups. Every serum that is to be used in a population different from that in which it was standardized must therefore be re-standardized in the new group before use. Results obtained in previous studies by EDTA agglutination cannot be compared with those obtained by cytotoxicity. The NIH serum Willett (anti-HL-A 8) gives identical results by agglutination and by cytotoxicity when tested in Caucasians; but in the Bantu it reacts with 49% by agglutination and only 8 % by cytotoxicity. The Bantu evidently possess several unidentified HL-A antigens.

Intrarenal complement fixation by cytotxic antibodies.

The reactivity of histocompatibility antigens with serum antibodies and complement was studied in a short term model of allotransplantation. Lewis and DA rat kidneys were perfused with cytotoxic antisera and tissue sections analyzed for adsorption of fluorescent antibodies against gamma-globulin and complement (beta(1C) protein). Both, the presumed cytotoxic antibody and C were localized at similar sites in the endothelial lining and in the walls of the arterial vessels and of the larger veins. The pattern of immune globulin deposition was distinct from that observed in glomerulonephritis. It may thus become helpful in the differential diagnosis between graft rejection and glomerulonephritis. At the sites of the deposits, the endothelial cells appeared enlarged and their nuclei protruded into the lumen. The findings are in line with the hypothesis that an antibody-mediated local activation of serum complement may be involved in the destruction of allografted tissue.