Rosemary L. Sparrow

Hu Ly-m5: A unique antigen physically associated with HLA molecules

A new human cell surface antigen (Hu Ly-m5) detected by a murine monoclonal antibody (E4.3) is described. The tissue distribution of the Hu Ly-m5 antigen is similar to the HLA antigens (with which it was initially confused) but it is not present on all bone marrow cells nor the U266 myeloma, and is expressed on the HLA-negative K562 cell line. Nevertheless, the Hu Ly-m5 antigen has some affinity for HLA molecules as the two entities cocap and the Hu Ly-m5 antigen copurifies with the HLA antigens on an anti-beta 2-microglobulin immunoabsorbent column; however, the antigen complexes did not withstand the procedures used for coprecipitation. Despite their similarities, the Hu Ly-m5 and HLA antigens are distinct molecular entities--Hu Ly-m5 consists of two bands of apparent molecular weight 69 and 60 K while HLA is comprised of the 43 and 12 K bands of the HLA heavy chain and beta 2-microglobulin, respectively. The function of the Hu Ly-m5 antigen is unknown, but no involvement in the cytotoxic T-lymphocyte response to influenza virus-infected cells could be demonstrated. The two properties described (apparent molecular weight and physical association with the HLA antigens) suggests that the Hu Ly-m5 antigen may be a viral-encoded protein.

Description of a mouse monoclonal anti-HLA-B27 antibody HLA-ABC-m3

The production and characterization of a new anti-HLA-B27 monoclonal antibody HLA-ABC-m3 is described. This cytotoxic IgG2a antibody binds protein A and is able to precipitate cell surface molecules of 43,000 and 12,000 daltons corresponding to the HLA heavy chain and beta 2-microglobulin. Population testing revealed that the HLA-ABC-m3 antibody reacted with the peripheral blood lymphocytes of 47/47 individuals conventionally typed as HLA-B27+ and with 5/105 HLA-B27- individuals. These five extra reactions were with individuals expressing the cross-reactive HLA-B7 alloantigen, although the affinity of the monoclonal antibody for B27 heterozygous individuals (approx 10(9) M-1) was tenfold greater than with B7 individuals (approx 10(8) M-1). In addition, HLA-ABC-m3 reactivity segregated with HLA-B27 in two families. This monoclonal antibody should be of value in the investigation of the role of HLA-B27 in disease.

Murine megakaryocyte colony stimulating factor: its relationship to interleukin 3.

A protocol for the partial purification of murine megakaryocyte colony stimulating factor from WEHI-3 cell conditioned medium is described. The procedure involving separation by anion exchange chromatography and molecular sieving on Sephadex G100, gave a 260-fold purification over starting material, as determined by in-vitro megakaryocyte colony formation. Fifty percent of maximum colony numbers were obtained at a dose of 1 microgram protein/ml. Electrophoretic analysis of the partially purified preparation followed by silver nitrate staining revealed four major protein bands with apparent molecular weights of 67 Kd, 43 Kd, 38 Kd and 28 Kd. All megakaryocyte colony stimulating activity and interleukin 3 activity was found in the 22-25 Kd fraction from a preparative SDS-polyacrylamide gel using non-reducing conditions, a molecular weight range that coincided with the 28-Kd molecule obtained when reducing conditions were employed.

Haemopoietic growth factors stimulating murine megakaryocytopoiesis: Interleukin-3 is immunologically distinct from megakaryocyte-potentiator

The biological and immunological properties of stimulators of in vitro murine megakaryocytopoiesis were studied using a heterologous anti-interleukin 3 (IL-3) serum. All megakaryocyte colony development was inhibited with the antiserum using three sources of IL-3, including WEHI-3 cell conditioned medium (WEHI-3CM), pokeweed mitogen-spleen conditioned medium (PWM-SCM) and recombinant IL-3. The data indicate that IL-3 is an absolute requirement for murine megakaryocyte colony development in this system. By comparison the antiserum abolished all myeloid colony growth stimulated by WEHI-3CM, but not PWM-SCM. The in vitro development of single megakaryocytes stimulated by a second putative growth factor, megakaryocyte-potentiator, was not inhibited by the antibody. The antiserum precipitated a 26 kd molecular weight protein from a radioiodinated sample of IL-3. No crossreactivity by the antiserum with other colony-stimulating factors (CSF) including CSF-1 and granulocyte-macrophage CSF was observed. The data indicates that IL-3 and megakaryocyte-potentiator are immunologically unrelated and provides further support that the two factors are separate molecules.

Cross-reactivity of a normal human cell surface antigen with primate retrovirus glycoproteins

Normal human cells express a human-specific antigen, HuLy-m5 (defined by the E4.3 monoclonal antibody), cross-reactive with determinants of the primate retroviruses, MPMV(Mason Pfizer monkey virus) and GALV (gibbon ape leukemia virus). Purified virus preparations of MPMV and GALV absorbed E4.3 antibody activity while antisera to these retroviruses blocked the binding of E4.3 antibody to human target cells. Sequential immunoprecipitation and two-dimensional gel analysis both indicated that the anti-primate retrovirus sera recognize the same molecular entity (a two-chain glycoprotein of Mr60 and 69Kd) as does the E4.3 antibody. These results suggest that normal human cells express primate retroviral proteins (most probably viral envelope glycoprotein, gp69) at the cell surface.

Hu Ly-M3--a human leukocyte antigen.

A monoclonal antibody, 5–4.8, was produced against human peripheral blood lymphocytes and it appears to be leukocyte-specific in that it reacts with a common determinant (called Hu Ly-m3) present on the peripheral blood T, B and null lymphocytes of 40 individuals. The antibody also reacts with thymocytes, spleen cells, and bone marrow cells (30%) and weakly with granulocytes and platelets—but not with heart, liver, or kidney. Affinity to lentil-lectin and molecular weight analysis demonstrated that Hu Ly-m3 is a glycoprotein consisting of a single chain of 47,000 daltons which is not HLA because it is not present on all cells; because it is present on the surface of the phenotypically HLA-Daudi cell line; and because soluble HLA antigens did not inhibit the binding of the 5—4.8 antibody.

Human 'Ia' antigen populations defined by monoclonal antibodies.

The relationships between the antigens recognized by four monoclonal anti‐human ‘Ia’‐like antibodies were investigated using sequential immunoprecipitation and capping techniques. Two of the antibodies were ‘monomorphic’ and have previously been shown to recognize epitopes in which carbohydrate residues are involved, whereas the two ‘polymorphic’ antibodies recognized protein‐defined epitopes—one of these epitopes being present on MB+DR‐ molecules. In the absence of an indisputable anti‐DR monoclonal antibody, it was not possible to conclusively verify which ‘Ia’‐encoded antigens were detected by the anti‐‘Ia’‐like monoclonal antibodies. Nevertheless, several firm conclusions could be drawn: (a) so‐called ‘monomorphic’ antibodies do not necessarily react with all ‘Ia’ molecules encoded by a single locus—from the results using the two monomorphic antibodies, B5.1 and 3F1.1, described herein, two populations of antigens being B5.1+3F1.1+ and B5.1+3F1.1‐ were identified; (b) cross‐reactivity of a polymorphic determinant expressed on antigenically‐separable ‘Ia’ molecules was noted—using the two polymorphic antibodies, 26.1 and F5C9, molecules which were 26.1+F5C9+ and 26.1‐F5C9+ were identified; and (c) the data clearly point to the existence of at least two loci coding for ‘Ia’‐like antigens (one of which may or may not be the HLA‐DR locus). Given that polymorphisms can now include protein‐ and carbohydrate‐defined epitopes, that cross‐reactions occur and that the definition of DR itself by monoclonal antibodies is not clear, the complexity of the human ‘Ia’ antgens is apparent.

Human class II antigens. Implication of carbohydrate in the determinants recognized by several antihuman class II monoclonal antibodies.

Several monoclonal antibodies (McAbs) to human class II antigens are described; three of these recognize monomorphic epitopes, and two are polymorphic. The biochemical nature of the reactive epitopes was investigated by determining the binding ability of the McAbs to protease- or glycosidase-treated antigen preparations. Both of the polymorphic McAbs recognized protein-defined epitopes. However, for one of the antibodies F5C9, carbohydrate residues were also implicated. The extent of carbohydrate involvement varied with class II antigens of different DR specificity, suggesting that the proximity of the F5C9 epitope to carbohydrate side chains can vary. One of the three monomorphic McAbs recognized a protein-defined epitope, while the other two antibodies detected epitopes in which carbohydrates were involved. These results indicate that both protein and carbohydrate residues of the human class II antigens can influence the epitopes recognized by murine McAbs. This could explain some of the apparent complexities of the anti-human class II McAbs.

An in situ immunoperoxidase staining procedure for human cell colonies grown in semi-solid agar culture

An adaptation of the immunoperoxidase staining procedure is described which enables the detection of antigens expressed by human hemopoietic cells grown in semi-solid agar culture. Positive staining was achieved using several different monoclonal antibodies recognizing an array of cell surface antigens on developing myeloid cells.