Immunoglobulins in NZB-BL mice. I. Serum immunoglobulin levels and immunoglobulin class of erythrocyte autoantibody.

Determination of serum immunoglobulin levels in NZB and normal mice has indicated that an elevation of the gammaM-globulin level occurs in NZB mice. This can be demonstrated in young NZB mice well before other autoimmune manifestations are observed. A slight elevation of gammaM-globulin was also observed in NZB hybrid mice. Erythrocyte autoantibodies were analyzed by the direct Coombs' test with specific antiimmunoglobulin reagents. Autoantibodies in NZB mice can be of all immunoglobulin classes, although predominantly of gammaG(1)-class. In (NZB x NZC)F(1) hybrid mice, although Coombs' positivity develops around the same age as in NZB mice, there is a greater restriction of the autoantibodies into gammaM- and gammaG(1)-classes. Preliminary attempts to quantitate immunoglobulins coating NZB red cells have shown that there are approximately 400 molecules of gammaM and 5000 molecules of gammaG-globulin per NZB mouse red cell.

Immunoglobulin isoantigens (allotypes) in the mouse. II. Allotypic analysis of three gammaG2-myeloma proteins from (NZB x BALB/c)F1 hybrids and of normal gammaG2-globulins.

Further analysis of the isoantigens (allotypes) of 2 classes of normal mouse immunoglobulins, gammaG(2a) and gammaG(2b), has shown a minimum of 10 specificities for the Ig-1 locus (controlling gammaG(2a)-antigens) and 3 specificities for the Ig-3 locus (controlling gammaG(2b)-antigens). Three gammaG(2)-myeloma proteins of plasma cell tumors induced in (NZB x BALB/c)F(1) mice have been analyzed for the isoantigens they carry. NZB mice are genotypically Ig-1(e) Ig-3(e), while BALB/c are Ig-1(a) Ig-3(a). Two of the myeloma proteins are gammaG(2a)-globulins. One of these, GPC-7, carries all the isoantigenic specificities of the Ig-1(e) allele while the other, GPC-8, carries all the isoantigenic specificities of the Ig-1(a) allele. Thus only one of the parental alleles of the mouse in which the tumor arose is expressed in each of these myeloma proteins. The third myeloma protein GPC-5, also carries the antigens of only one parental strain (NZB). However GPC-5, a gammaG(2b)-globulin, carries only one of the Ig-3 specificities normally associated with gammaG(2b)-globulins of NZB. Most remarkably it also carries one Ig-1 specificity normally associated with gammaG(2a)-globulins of NZB. This is the first analyzed mouse myeloma shown (a) to express some but not all the antigenic specificities normally associated with an allele and (b) to carry antigenic specificities controlled by two distinct immunoglobulin loci. The implications of these findings are discussed in relation to the genetic control of immunoglobulins.

Transplantation behavior of A.TH and A.TL T-cell lymphomas in congenic resistant and hybrid strains.

Seven spontaneously arising T-cell lymphomas originating in A.TH or A.TL mice, which are congenic for the immune response gene (I) chromosomal segment were described. When transplanted into partner strains which were incompatible at the I region, the tumors were rapidly rejected. Rejection was proposed to be due to the presence of antigens controlled by I-region genes.

Defects in hematopoietic differentiation in NZB and NZC mice.

Hematopoietic stem cell activity in inbred NZB and NZC mice has been determined by transplantation and endogenous spleen colony assays. Whereas NZB mice show normal colony-forming unit (CFU) activity in the transplantation assay, they show markedly elevated endogenous CFU. NZC mice also show this markedly elevated endogenous CFU activity, but in the transplantation assay show only about 5-10% of normal CFU counts. When NZC stem cells are tested for CFU activity in irradiated recipients of the H-2(d) type, almost normal colony numbers occur. NZB stem cells however also cannot form colonies in NZC mice. These results suggest that NZC mice have a defect in the micro-environment of the spleen which renders them incapable of allowing transplanted CFU to form colonies. Genetic analysis of both the NZC defect as a CFU recipient, and the elevated endogenous count in NZB and NZC, shows that both are controlled by single recessive genes which are not linked to either coat color, agouti, H-2 or Ig loci. Of even more relevance is the finding that these hematopoietic abnormalities are not linked to the genes involved in controlling autoantibody formation to red cells in the NZB mice. These mice therefore appear to show two distinct hematopoietic abnormalities, the analysis of which may be of considerable value in understanding the detailed events of hematopoietic stem cell differentiation.

Tolerance and immunity to maternally derived incompatible IgG 2a -globulin in mice.

Progeny mice were confronted with maternal gamma-globulin of a different allotype by either back-cross mating, intercross mating, or by foster nursing. In all cases, many mice subsequently produced alloantibodies directed against the incompatible maternal type of IgG(2a)-globulin. In one series of experiments, immunologic tolerance to the maternally derived gamma-globulin was demonstrated to exist in the period before formation of spontaneous antibody. The state of tolerance was then lost, unless maintenance injections of foreign gamma-globulin were given. These studies demonstrate in a natural situation that maternally derived foreign proteins can first induce a state of immunological tolerance which is followed, after disappearance of the antigen, by a state of immunity. As such, this parallels the experimental induction of tolerance to foreign proteins by neonatal injections.

Surface immunoglobulins on thymus and thymus-derived lymphoid cells.

Lymphoid cells from thymus, thoracic duct lymph (TDL), and thoracic duct lymph in irradiated animals reconstituted with allogeneic thymus cells (TTDL) were labeled with radioiodinated anti-immunoglobulins using radioautographic techniques. Thymus and TTDL were labeled (14.4 and 37.0%, respectively) with anti-light chain protein after prolonged exposures (30-60 days). No labeling was observed on thymus and TTDL with anti-polyheavy chain globulin. In contrast 18.5-19.0% of TDL labeled on short exposure (6 days) with anti-polyheavy chain and anti-light chain materials. It is proposed that the difference between the labeling observed on short exposures versus long exposures can be related to the difference in surface density of immunoglobulins between nonthymus-derived (B) and thymus-derived (T) cells. The distribution of labeled cells in the thymus was preferentially among the larger cells (greater than 10 micro diameter). The TTDL population was mostly composed of a larger, blast-like population and the distribution of label was independent of size. As the thymus and TTDL preparations contain almost exclusively T cells, this represents a direct demonstration of surface immunoglobulin light chains on T lymphoid cells.

A receptor for antibody on B lymphocytes. II. Immunochemical and electron microscopy characteristics.

Binding of antibody to the surface of B lymphocytes was shown to involve the Fc piece of the immunoglobulin molecule. This property was not shared equally by all immunoglobulin classes as revealed by direct binding and inhibition studies. Total IgG globulin was found to label cells more heavily than IgM, and IgG1-containing fractions more heavily than IgG2 fractions lacking IgG1. The ability of various purified myeloma proteins to inhibit attachment of antibody to B cells was examined. Pretreatment of B cells with excess IgG(2a), IgA, or light chain proteins failed to inhibit, whereas IgG1 proteins and to a lesser extent Ig(2b) and IgM proteins at the same concentrations did so. At lower protein concentrations, IgG1 myeloma protein alone retained the capacity to inhibit binding. The conclusion was reached that the receptor on B cells for antibody has a marked predilection for the IgG1 class. Although IgM and IgG(2b) antibody may bind, they do so with lower avidity and probably in insignificant amounts if IgG1 antibody is present in excess. No evidence was found to implicate complement in the binding process. For example, heat-inactivated sera at high dilution retained the ability to label B cells, while the use of purified low molecular weight anticomplementary factor, a potent inhibitor of C'3, did not interfere with the formation of the bond between antibody and cell surface. The failure of anti-mouse immunoglobulin F(ab)'(2) fragments to prevent access of antibody to B cells implied that the antibody-binding receptor and antigen-binding (immunoglobulin) receptor are discrete entities on the B cell membrane. Despite this, a marked similarity between their surface distribution was observed on electron microscopy. The antibody-binding receptor was shown to be a marker for mature B cells. It did not appear to be present on hematopoietic precursor stem cells and was lost during differentiation to antibody-forming cells.

Concomitant induction of the cell surface expression of Ia determinants and accessory cell function by a murine macrophage tumor cell line.

This study demonstrates that an uncharacterized soluble factor produced in concanavalin A-induced rat spleen cell suspensions has the capacity to induce the increased expression of cell surface H-2K and H-2D molecules and the expression of I-region gene products on murine monocyte-macrophage lineage tumors that are not Ia positive in the absence of the factor. In parallel with induction of serologically defined Ia specificities, Ia-induced WEHI-3 macrophage tumor cells are capable of providing accessory cell function in stimulating IL-2 production by T-T hybridomas that are activated in a major histocompatibility complex-restricted, antigen-dependent fashion. The uninduced Ia-negative WEHI-3 tumor cells do not trigger a comparable response in this assay system.

Quantitative features of a sandwich radioimmunolabeling technique for lymphocyte surface receptors.

The present study was designed to devise and characterize an indirect or sandwich radioimmunolabeling technique for the study of lymphocyte surface receptors of immunoglobulin nature. Mouse lymphocytes from various sources were treated by the method of Shortman et al. to remove debris and damaged cells. This was an important preliminary step, as without it, little meaning could be attached to bulk scintillation counting of labeled cell suspensions, in view of the marked tendency of dead or damaged cells to adsorb protein nonspecifically. Next, cells were reacted at 0 degrees C for 30 min with graded dilutions of unlabeled rabbit antisera against defined mouse Ig chains. After washing, the cells were reacted with a sheep anti-rabbit globulin reagent labeled with (125)I, again at graded concentrations. After further washing, lymphocyte labeling was quantitated by both bulk scintillation counting and radioautography. Conditions were defined in which nonthymus-derived cells (B cells) but not thymus-derived cells (T cells) could be labeled. Most B cells displayed kappa- and micro-chains on their surface, but some also displayed alpha- and gamma(2)-chains, though in smaller amounts. When the concentration of both the first and the second reagents were raised considerably, conditions were defined under which virtually all T cells could be labeled by polyvalent antiglobulin sera, anti-kappa sera, or, with more difficulty, by anti-micro sera. A large series of control experiments confirmed the serologic specificity of this labeling. It was shown that under equivalent conditions, B cells bind 100-400 times more antiglobulin than do T cells. The theoretical implications of the results are briefly discussed. It is argued that the sandwich approach offers certain technical advantages over direct labeling procedures for further analyses of T cell receptors and for studies of receptor metabolism.

Expression of Lyt-1 antigen on certain murine B cell lymphomas.

Although the Lyt-1 antigen has previously been considered a T cell-specific marker, recent evidence suggests that a population of Thy-1-, Lyt-1+ cells exists in normal lymphoid tissues. In this study, we have observed that the WEHI-55, WEHI-259, and CH5 B cell lymphomas express high levels of the Lyt-1 antigen, as detected by monoclonal antibodies using the fluorescence activated cell sorter. Three other B cell lymphomas of the 11 examined also gave weak but detectable reactions with the anti Lyt-1 monoclonal antibody. Except for the expression of the Lyt-1 antigen, these lymphomas are typical of cells in the B cell lineage with respect to surface phenotype. The Lyt-1 glycoprotein immunoprecipitated from metabolically labeled WEHI-55 cells is similar in structure to the Lyt-1 glycoprotein on thymocytes. These findings are similar to recent reports that B-type human lymphocytic leukemia cells express the putative human homologue of Lyt-1, the Leu-1 antigen.

Growth of B-lymphocyte colonies in vitro.

In semisolid agar cultures containing mercaptoethanol, cells from the spleen, lymph nodes, marrow, peritoneal cavity, thoracic duct, and blood of normal mice generated clusters and colonies of up to 3,000 cells. Colony numbers and growth were markedly enhanced by the addition of sheep red cells. The frequency of colony-forming cells in the spleen or lymph nodes was 0.5-2.0%, and cluster forming cells were approximately five times more numerous. The mononuclear cells comprising these colonies had the electronmicroscopic morphology of immature lymphoid and plasma cells. The majority of the cells possessed Fc receptors, 61-69% reacted with anti-mu-serum and 4-11% with anti-gamma2-serum. Analysis of single cells from individual colonies indicated a higher frequency of the cells with membrane immunoglobulin and a clonal pattern of anti-mu or anti-gamma-reactivity. The clonal nature of colonies was supported by an analysis of NIP-binding cells in colonies grown from CBA spleen cells enriched for NIP-binding cells. Mass-harvested colony cells synthesized immunoglobulin in short-term liquid cultures. It is concluded that the colonies are clones of functionally active B-lymphoid cells.

Cell-to-cell interaction in the immune response. VII. Requirement for differentiation of thymus-derived cells.

Experiments were designed to test the possibility that thymus-derived (T) cells cooperate with nonthymus derived (B) cells in antibody responses by acting as passive carriers of antigen. Thoracic duct lymphocytes (TDL) from fowl gammaG-tolerant mice were incubated in vitro with fowl anti-mouse lymphocyte globulin (FALG), which was shown not to be immunosuppressive in mice. On transfer into adult thymectomized, irradiated, and marrow protected (TxBM) hosts together with a control antigen, horse RBC, a response to horse RBC but not to fowl gammaG was obtained. By contrast, TxBM recipients of nontolerant, FALG-coated TDL responded to both antigens and the antibody-forming cells were shown to be derived from the host, not from the injected TDL. These findings suggested that, under the conditions of the experiment, triggering of unprimed B cells in the spleens of TxBM hosts was not achieved with antigen-coated tolerant lymphocytes. Another model utilized the ability of B cells to bind antibody-antigen complexes. Spleen cells from TxBM mice, incubated in vitro with anti-fowl gammaG-fowl gammaG.NIP, were injected with or without normal TDL (a source of T cells) into irradiated hosts. Only mice given both cell types could produce an anti-NIP antibody response. In a further experiment, spleen cells from HGG.NIP-primed mice were injected together with NIP-coated B cells (prepared as above) into irradiated hosts. A substantial anti-NIP antibody response occurred. If, however, the T cells in the spleens of HGG.NIP-primed mice were eliminated by treatment with anti-theta serum and complement, the NIP response was abolished. It was concluded that antigen-coated B cells could not substitute for T cells either in the primary or secondary response. Treatment of T cells from unprimed or primed mice with mitomycin C impaired their capacity to collaborate with B cells on transfer into irradiated hosts. Taken together these findings suggest that before collaboration can take place T cells must be activated by antigen to differentiate and in so doing may produce some factor essential for triggering of B cells.

Tumor immunity to murine plasma cell tumors. V. Genetic control of the in vitro cytotoxic T-cell response to plasma cell tumor-associated antigens of NZB mice.

Cytotoxic T-lymphocytes (Tc) were induced in vitro to plasmacytoma tumor-associated antigens (TAA) by coculture of irradiated cells of plasma cell tumors (PCT) from NZB mice and viable nonimmune or PCT-immune spleen cells from NZB. (NZB X C57BL)F1, (NZB X B10.D2)F1, and (NZB X B10,BR)F1 mice. When nonimmune spleen cells were used, major histocompatibility complex (MHC)-linked genetic control of te primary in vitro induction of Tc to NZB PCT was demostrated for a shared TAA expressed on NZB and BALB/c PCT. Evidence was also obtained for a non-MHC-linked genetic control of the primary in vitro induction of Tc to a second TAA that ws expressed on both PCT and T-lymphomas. When the spleen cells were obtained from mice preimmunized to an NZB PCT, a secondary in vitro Tc response was observed, and a PCT-specific and strain-specific TAA or NZB mice was identified. In addition, results with the F1 hybrids also indicated an MHC-linked genetic control of the in vitro Tc response to this strain-specific TAA.

Effect of Corynebacterium parvum on tumor growth in normal and athymic (nude) mice.

The effect of systemic or local injection of Corynebacterium parvum at the tumor site on the growth of various murine tumors was studied in intact and congenitally athymic BALB/c mice. Systemic injection of C. parvum usually had a marked antitumor effect in both types of mouse. Two lymphomas, which regressed spontaneously in untreated intact mice but not in athymic mice, grew progressively in intact mice given systemic C. parvum, though their growth was inhibited in similarly treated athymic mice. Local injection into the site of the tumor markedly inhibited tumor growth in intact mice but was without effect in athymic mice. C. parvum was believed to exert its antitumor effects by two different mechanisms, only one of which was T-cell dependent. The mechanism not dependent on T-cells was particularly activated by systemic C. parvum injection.

Tumor immunity to murine plasma cell tumors. III. Detection of common and unique tumor-associated antigens on BALB/c, C3H, and NZB plasmacytomas by in vivo and in vitro induction of tumor-immune responses.

Tumor-associated antigens (TAA) were demonstrated on plasmacytomas derived from BALB/c, NZB, and C3H mouse strains, by in vivo and in vitro techniques. By immunizing the appropriate F1 hybrid mice with these tumors, it was possible to show that all the plasmacytomas expressed cross-reactive tumor-associated transplantation antigens. When cytotoxic lymphocytes (CL) were induced in vitro by the coculturing of syngeneic or F1 hybrid spleen cells with irradiated plasmacytoma cells, "shared" and "unique" plasmacytoma TAA were demonstrable. This was accomplished by inducing CL in vitro against one syngeneic plasmacytoma and assaying for lytic activity on a range of 51Cr-labeled BALB/c, NZB and C3H plasmacytoma cells in vitro. In a second in vitro assay, unlabeled plasmacytoma cells were tested for their ability to inhibit the lysis of a particular 51Cr-labeled plasmacytoma, with the use of CL induced in vitro against it. The possibility that these TAA were "self" antigens was excluded by demonstrating in the inhibition assay that they were not present on T lymphomas and spleen cells of the same strain, and that CL "autosensitized" in vitro could not significantly lyse 51Cr-labeled plasmacytoma cells in vitro. From both in vivo and in vitro studies of immunity to these tumors, it was concluded that any one plasmacytoma line possesses multiple TAA of both shared and unique types.

In vitro induction of tumor-specific immunity. II. Activation of cytotoxic lymphocytes to murine oncofetal antigens.

The presence of oncofetal antigens (OFA) on a wide variety of murine tumor cells was demonstrated to a totally in vitro system of cellular immunity. Nonimmune spleen lymphocytes were cocultivated with irradiated syngeneic fetal liver cells and, at various times after initiation of culture, were tested for the presence of cytotoxic lymphocytes (CL) by 51Cr-release assay with labeled tumor target cells. Significant cytotoxic activity was regularly detected after such culture, whereas only minor levels appeared in control cultures of spleen lymphocytes with irradiated syngeneic spleen cells. Specificity of the reaction was assessed by inhibition tests in which nonlabeled cells were admixed to the CL and 51Cr-labeled tumor targets. Fetal liver cells gave significant inhibition; however, no inhibition was found with adult spleen cells. Various tumor types gave inhibition, and fibrosarcomas were more effective than plasmacytomas or lymphomas. The results suggested that all tumor types tested possess such OFA, as well as their unique or virus-associated, tumor-associated transplantation antigens, and that the in vitro system permits a more active response to the tumor-associated OFA than that observed in in vivo studies.

Heterogeneity of HLA-A,B, Ia-like, and melanoma-associated antigen expression by human melanoma cell lines analyzed with monoclonal antibodies and flow cytometry.

With the use of monoclonal antibodies, indirect immunofluorescence, and flow cytometry, human melanoma cell lines (Colo 38, M-16, and M-21) were examined for the quantitative level of expression of HLA-A,B antigens, Ia-like antigens, and two human melanoma-associated antigens (MAA) referred to as 280K and 94K MAAs. Each of the melanoma cell lines examined showed tremendous heterogeneity with regard to antigen expression. A detailed study of the heterogeneity of the four antigens listed revealed that variation in cell size could, in part, account for the large differences in antigen expression observed. Cell surface density of HLA-A,B antigens, Ia-like antigens, and the 280K and 94K MAAs remained relatively constant over a wide range of cell sizes that were examined, with the exception that small melanoma cells showed a slightly lower mean surface density of MAA expression than did large cells. A novel method was used to detect the expression of the two MAAs as a function of the Colo 38 cell cycle using dual-laser beam flow cytometry. The results of these experiments show that both the 280K MAA and the 94K MAA are differentially expressed during various stages of the cell cycle, with each antigen being maximally detected during G2 + M.

Evidence for common and distinct determinants of colon carcinoembryonic antigen, colon carcinoma antigen-III, and molecules with carcinoembryonic antigen activity isolated from breast and ovarian cancer.

This study was designed to answer the question, do molecules with carcinoembryonic antigen (CEA) activity from colon, breast, and ovarian cancer differ? Extracts of two breast and three ovarian cancers with CEA activity were compared to three colon cancer CEA preparations and to the related antigen, colon carcinoma antigen-III, in terms of lectin- and antiserum-binding properties. With the use of Farr-type radioimmunoassays with the lectins, concanavalin A and wheat germ agglutinin, the iodinated colon CEA and CEA-like preparations from breast and ovarian cancer all showed distinctly different patterns of binding. Specificity of binding was confirmed by inhibition studies with the relevant monosaccharides. Similarly, with antisera prepared against colon CEA, colon carcinoma antigen-III, or breast CEA, it was shown that, although all preparations shared some antigens, unique antigenic determinants were also present on all preparations. These data are consistent with the concept of a series of closely related CEA and CEA-like molecules with distinct characteristics for each tissue source of CEA.

A human natural killer cell-associated antigen defined by monoclonal antibody anti-Leu (NKP-15): functional and two-color flow cytometry analysis.

A monoclonal antibody, anti-Leu 11a (NPK-15), was generated against human large granular lymphocytes (LGL). Anti-Leu 11a reacted with the majority of Percoll gradient-enriched LGL cells, a subpopulation of peripheral blood lymphocytes (PBL) approx (approximately 10-20%), and most granulocytes, but not with a significant number of monocytes, T lymphocytes, or erythrocytes. Cell sorting experiments demonstrated that the Leu 11a+ population encompassed essentially all functional natural killer (NK) cells in the peripheral blood. Two-color flow cytometry analysis of PBL populations stained with anti-Leu 11a and anti-Leu 7 revealed the existence of four distinct populations: Leu 11a-, 7+; Leu 11a+, 7-; Leu 11a+, 7+; and Leu 11a-, 7-. The Leu 11a+ population did not appear to include cells marked with the T cell-associated antigens Leu 1, Leu 2, or Leu 3. The existence of a cell surface antigen common to granulocytes and NK cells, which is capable of distinguishing subpopulations of Leu 7+ cells, provides a useful probe to analyze the nature of the NK lineage.

Characterization of subsets of bone marrow-derived macrophages by flow cytometry analysis.

Normal C3H bone marrow cells were grown 7 days in medium containing L cell-derived colony stimulating factor-1 (CSF-1). During the first 4 days of culture, erythroid and granulocytic cells decreased while macrophages increased exponentially with a doubling time of about 31 hr. Only 0.3% of all cells in the initial bone marrow suspension formed discrete colonies of mononuclear phagocytes, but by day 6 60% of the nonadherent cells were capable of forming macrophage colonies, representing a 200-fold enrichment of the original progenitor population. Using flow cytometry, mononuclear phagocytes obtained after 4 days of culture were separated into two distinct phenotypes based on their autofluorescence. Nonadherent cells were a discrete population of small cells exhibiting low autofluorescence, and the adherent cells were a broad heterogeneous population of large cells exhibiting high autofluorescence. A panel of currently available rat monoclonal antibodies (MABs) against murine hematopoietic cells were used to determine whether unique subsets of macrophages could be resolved. The MABs RA 31B6 and H-11 stained virtually all the nonadherent cells but not adherent cells. The MABs E-2 and 11-4.1 (anti-H-2Kk) stained almost all the adherent cells and demonstrated no significant staining of nonadherent cells. Nearly all the nonadherent and adherent cells were stained by the MABs DNL 4.4 and MAC-1. Additionally, the data suggest that the epitopes for MAC-2 and MAC-3 and gamma 2a Fc receptors develop late in nonadherent progenitor cells as they mature into adherent macrophages.