Regulatory role of a monomorphic determinant of HLA Class I antigens in T cell proliferation

The role of HLA Class I antigens in T cell proliferation was investigated by using the anti-HLA Class I monoclonal antibodies (MoAb) CR10-215, CR10-325, and CR11-115. MoAb CR10-215 and CR11-115 recognize the same (or spatially close) monomorphic determinant, which is distinct and spatially distant from that reacting with MoAb CR10-325. Addition of MoAb CR10-215 and CR11-115 to cultures of peripheral blood mononuclear cells stimulated with MoAb OKT3, MoAb Pan T2, PHA, or PPD inhibited cell proliferation. The blocking is specific in that the anti-HLA Class I MoAb CR10-325 and the Pan T MoAb Pan T1 had no effect on the proliferation. The inhibitory activity of MoAb CR10-215 and CR11-115 does not reflect i) toxic effects, ii) induction of suppressor cells and factors, iii) blocking of the binding of mitogens to lymphocytes, iv) inhibition of the production of interleukin 1 (IL 1) and interleukin 2 (IL 2), or v) function of IL 2 receptor. Anti-HLA Class I MoAb were able to inhibit the proliferation of purified, Tac-, T cells. The inhibited cells did not express Tac antigen, as assayed by direct immunofluorescence, with MoAb anti-Tac, but released a normal amount of IL 2 in culture medium. These results indicate that monomorphic determinants of the HLA Class I complex are involved in the regulation of T cell proliferation. The effect appears to occur at the level of IL 2 receptor expression.     

Induction of monocyte procoagulant activity with OKT3 antibody

OKT3 monoclonal antibody (MoAb), a mouse MoAb against cluster of differentiation 3 (CD3) molecule, induced a large amount of procoagulant activity (PCA) in human peripheral blood mononuclear cells (PBM). The PCA-inducing capability in OKT3 MoAb was abolished by absorption with T lymphocytes or Sepharose-conjugated antibody to mouse IgG. Most of the PCA in PBM was associated with monocytes. There was a dose-dependent increase in PCA when increasing numbers of T cells were added to the monocytes in the presence of OKT3 MoAb. OKT3 MoAb did not induce PCA in either T cells or monocytes alone. T cells pulsed with OKT3 MoAb only in the presence of monocytes could induce PCA in monocytes. Culture supernatants (CS) from PBM stimulated with OKT3 MoAb did not enhance PCA in monocytes; however, it did induce PCA in the human monocyte-like cell line (U937) which differs in some properties from monocytes; this activity could be abolished by the MoAb against human interferon-gamma (IFN-gamma). Nevertheless, neither human IFN-gamma nor interleukin 1 or 2 had significant direct effect in inducing PCA in U937 cells; CS from either monocytes or T cells alone stimulated with OKT3 MoAb did not induce PCA in U937 cells. This apparent discrepancy suggests that there may be factors in CS that induce PCA in U937 cells only in the presence of IFN-gamma. The PCA induced in monocytes or U937 cells was tissue factor-like because of the dependence on coagulation factors V, VII, and X. These observations suggest that OKT3 MoAb is a potent T cell-dependent monocyte PCA inducer and stimulates T cells only in the presence of monocytes. The direct cellular interaction between monocytes and stimulated T cells appears to be necessary to elicit monocyte PCA with OKT3 MoAb stimulation. Thus, monocytes may play a dual role, not only as effector cells, but also as cells that collaborate with T cells after OKT3 MoAb stimulation so as to produce PCA.     

Lack of a role of monocytes in the inhibition by monoclonal antibodies to monomorphic and polymorphic determinants of HLA class I antigens of PHA-P-induced peripheral blood mononuclear cell proliferation

This study aimed at characterizing the mechanism(s) underlying the regulatory role of distinct determinants of HLA Class I antigens in PHA-P-induced T cell proliferation and the involvement of monocytes in this phenomenon. The anti-HLA-A2,A28 monoclonal antibodies (MoAb) CR11-351, the MoAb Q6/64 to a determinant restricted to the gene products of the I antigens HLA-B locus, and the MoAb CR10-215 and W6/32 to distinct monomorphic determinants of HLA Class I antigens were found to inhibit PHA-P-induced peripheral blood mononuclear cell (PBMC) proliferation in a dose-dependent fashion. The inhibition is specific and reflects neither inhibition of PHA-P binding to cells nor a toxic effect of the anti-HLA Class I MoAb. The latter differed in the concentration required to induce inhibition, in the influence of the concentration of PHA-P used as mitogen, in the differential effect on the donors used as a source of PBMC, and/or in the requirement of the Fc portion to induce inhibition. At variance with the information in the literature, the inhibitory effect of anti-HLA Class I MoAb on PHA-P-induced PBMC proliferation neither reflected their interaction with accessory cells nor was mediated by suppressor factors released by monocytes stimulated with PHA-P in the presence of anti-HLA Class I MoAb. Therefore, the regulatory role of HLA Class I antigens in T cell proliferation is not likely to be mediated by monocytes and/or factors released from them, but may reflect an involvement of these molecules in T cell activation pathways.     

The role of three distinct Ia-like antigen molecules in human T cell proliferative responses: effect of monoclonal anti-Ia-like antibodies

Murine monoclonal antibodies (MoAb) to three distinct Ia-like molecules were studied for their inhibitory effects on antigen- and alloantigen-induced T cell proliferations. The MoAb were classified into three groups according to the molecules they recognized. Both the group I MoAb reacting with DR molecules and the group III MoAb were capable of inhibiting T cell proliferative responses to PPD- and HSV-Ag-pulsed APC, autologous B-LCL, and alloantigens. On th other hand, the group II MoAb, which reacted with a determinant on the molecule carrying MB1 determinants, was only capable of inhibiting T cell responses to alloantigens. These results suggest that the structure of the molecules correlates with the functional repertoire of the human Ia-like antigens.     

Antigenic profile and functional characterization of human peritoneal macrophages

The antigenic profile and the functional properties of human peritoneal macrophages have been analyzed by using a panel of monoclonal antibodies (MoAb) and functional assays. All peritoneal macrophages were stained by the anti-class I HLA MoAb Q6/64. Between 40 and 100% of the cells were stained by the anti-HLA-DR + DP MoAb Q2/80, Q5/6, and Q5/13; approximately 80% of the cells were stained by the anti-HLA-DQ MoAb BT3.4, and about 95% were stained by the anti-macrophage MoAb OKM1. Peritoneal macrophages were not stained by the anti-dendritic cells MoAb Ki-M4 or by MoAb to T cell subsets, although all of the MoAb were reactive with the appropriate substrates. More than 60% of the cells expressed Fc receptors and C3 receptors, and displayed phagocytic activity. Peritoneal macrophages were effective in stimulating autologous and allogeneic lymphocytes and in presenting soluble antigens to T cells. These reactions were blocked by the anti-HLA-DR + DP MoAb Q5/13, but were not affected by the anti-dendritic cells MoAb Ki-M4 or by the anti-class I HLA MoAb Q6/64. These results suggest that human peritoneal macrophage preparations, without detectable contamination with dendritic cells, can induce proliferation of autologous and allogeneic T cells, and that class II HLA antigens play a significant role in these phenomena.     

Immunological and biochemical characterization of an epitope of the transferrin receptor involved in the production of interferon-gamma and B-cell growth factor

We have investigated the effect of a newly developed monoclonal antibody, MoAb NDA9, on human lymphocyte function. This MoAb inhibits the capacity of peripheral blood lymphocytes to display blastogenic responses and to produce immunoglobulins when stimulated in vitro with PWM or with soluble antigens. The inhibitory effect seems to result from the decreased ability of T lymphocytes to produce B cell growth factors (BCGF) in the presence of MoAb NDA9. This antibody also blocks the capacity of polyclonal or monoclonal populations of activated human T cells to produce immune interferon (gamma) but has no direct effect on B cell activation and growth in T-cell-independent systems. Immunochemical studies of the antigen recognized by MoAb NDA9 showed that it is an epitope of the transferrin receptor molecule which is distinct from that recognized by the MoAb OKT9.     

Regulation of helper T cell clone proliferation via the CD2 molecule

We have investigated the requirements for CD2-induced proliferation of a CD4+, CD8-, CD3+, CD2+ antigen-specific, class II-restricted proliferating cloned cell line. A combination pair of two monoclonal antibodies (MoAb) recognizing, respectively, TII1 and D66 epitopes on the CD2 molecule was used as a stimulus. The regulatory function of accessory cells and various interleukins in this proliferation was determined. The results show that although this clone was able to proliferate in the absence of accessory cells (AC) or interleukin 1 (IL-1) when stimulated by these MoAb, AC constantly enhanced the response to these MoAb. AC acted by increasing high-affinity IL-2 receptor expression. On the contrary they did not play any role in IL-2 production. This regulation of IL-2 receptor expression by AC was specific of adherent cells, did not involve Fc receptors, was impaired when AC were metabolically inactivated and did not require T cell-AC interaction via LFA1, CD4, or HLA molecules. The AC function was not abrogated by anti-IL-1 antibodies and could not be replaced by exogenous IL-1. These results were compared to previously described AC effects on resting T-cell proliferation when stimulated with the same pair of anti-CD2 MoAb. Clear differences in activation requirements in resting and activated T cells via CD2 molecules were found.     

In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.     

Monoclonal antibodies that inhibit activation and proliferation of lymphocytes. II. Requisite role of the monoclonal antibody-defined antigen systems in activation and proliferation of human and rat lymphocytes

A murine monoclonal antibody (MoAb) B3 to rat cells and MoAb HBJ127 and HBJ98 to human cells were found previously to recognize the homologous antigen systems (gp130 in the rat and gp125 in the human) which are predominantly distributed on the cell surface of proliferating cells of the respective species, and the expression of the antigen systems in lymphocytes were indicated previously to correlate closely with the activation and proliferation of the lymphocytes. In this respect, the in vitro effects of these MoAb on the nucleic acid synthesis, cell cycles, or proliferation of stimulated rat and human lymphocytes were examined by use of T cell-enriched and B cell-enriched cell populations. The addition of B3 MoAb to cultures diminished Con A-induced or allogeneic mixed lymphocyte culture-induced rat T cell proliferation and lipopolysaccharide-induced rat B cell proliferation, whereas B31 MoAb, which is unreactive with the gp130 antigen, did not inhibit these lymphocyte responses. Similarly, both HBJ127 and HBJ98 MoAb could inhibit the human lymphocyte proliferation in vitro, although HBJ127 MoAb showed about eight times greater inhibitory activity than did HBJ98 MoAb; HBJ127 MoAb almost completely inhibited the DNA synthesis of the Con A-stimulated lymphocytes at concentrations higher than 13 micrograms/ml. The flow cytometric analysis of the cellular nucleic acid contents with acridine orange-stained cells showed that when B3 MoAb and Con A were simultaneously added to unstimulated rat T cells, progression of the cell cycle was blocked at the G0 to G1 transition. In this culture condition, the appearance of the B3-defined antigen was arrested in a moderate level, as determined with fluorescein-stained cells. On the addition of B3 MoAb to the culture of the T cells after 24-hr Con A stimulation, the MoAb also strongly inhibited the cellular DNA synthesis, but it did not arrest the cell cycle at a certain phase and did not modulate the corresponding antigen. These data suggest that the B3 MoAb-defined antigen on the rat lymphocytes and the HBJ127/HBJ98 MoAb-defined antigen on the human lymphocytes may play some requisite roles not only in lymphocyte activation but also in the subsequent progression through the cell cycle to proliferate.     

A novel monoclonal antibody, 1B3.1, binds to a new epitope of the VLA-1 molecule

A monoclonal antibody, 1B3.1, was raised against a cloned IL-2-dependent T cell line that expresses the T gamma delta T cell receptor. MoAb 1B3.1 reacted with long-term cultured T cell lines of both T gamma delta and T alpha beta lineage, and with in vivo-stimulated T cells, derived from synovial fluid, but not with resting or short-term activated T cells, B cells, or macrophages. Immunoprecipitation of the 1B3.1 target antigens showed that 1B3.1 recognizes a 200/110 kDa molecule that is identical to the VLA-1 heterodimer precipitated by MoAb TS2/7. 1B3.1, however, binds to an epitope of VLA-1 that is distinct from the TS2/7 binding site. This new MoAb could be useful in further studies of the functions of VLA-1, and of the cells that express this molecule.     

Production and characterization of monoclonal antibodies against the excretory-secretory antigen of the liver fluke (Opisthorchis viverrini).

Monoclonal antibodies (MoAb) were produced against a major soluble metabolic product (excretory-secretory, ES) of Opisthorchis viverrini. The latter was obtained in a form of spent culture medium in which the adult flukes had been maintained in vitro. The MoAb produced were exclusively associated with either IgG or IgM isotypes. When screened against a panel of parasite antigens by indirect ELISA, these MoAb exhibited three patterns of reactivity. Approximately 50% of the MoAb were highly specific for O. viverrini and another 25% cross-reacted only with Clonorchis sinensis. The remaining 25% cross-reacted extensively with other parasites. Results from radioimmunoprecipitation and immunoblotting experiments showed all MoAb to react with the 89-kDa glycoprotein. By indirect immunofluorescence, these MoAb reacted almost exclusively with the tegumental surface, tegumental cells, cecum and developing miracidium. Different lines of evidence suggest that these MoAb reacted with different epitopes on the same 89-kDa polypeptide carrier.     

Production of a species specific monoclonal antibody against a 56-kDa Mycobacterium marinum antigen which is potentially useful in culture identification

A monoclonal antibody (MoAb) designated 1H11 has been produced against a 56-kDa antigen from Mycobacterium marinum. This MoAb has been shown to be species specific by both enzyme-linked immunosorbent assay and Western blot. The relationship of this 56 kDa to other known mycobacterial antigens is currently under investigation. MoAb 1H11 recognised bacilli taken from cultures by both immunofluorescence and immunoperoxidase staining. This MoAb 1H11 may provide a simple method for rapid identification of M. marinum from cultures of clinical isolates.     

Immunoconjugates of doxorubicin and murine antihuman breast carcinoma monoclonal antibodies prepared via an N-hydroxysuccinimide active ester intermediate of cis-aconityl-doxorubicin: preparation and in vitro cytotoxicity

Doxorubicin (DXR) conjugated to murine monoclonal antibodies (MoAb) raised against human breast tumor cells demonstrated a MoAb-specific, molar ratio-dependent in vitro cytotoxicity. These conjugates were prepared on a scale sufficient to allow for subsequent clinical trials (1 to 3 g of MoAb per conjugation reaction). The conjugation reaction proceeded via an N-hydroxysuccinimide (NHS) active ester intermediate of cis-aconityl-DXR (CA-DXR), resulting in a cis-aconitate acid-sensitive linker between the DXR and MoAb. Molar ratios of DXR to MoAb ranged from 40 to 45. The immunoreactivity of conjugated MoAb was only slightly decreased from naked MoAb. When immunoconjugates were incubated with MoAb-reactive tumor cells for 3 hours, specific cell-killing was observed. If the exposure time was lengthened to 18 hours, however, nonspecific killing resulted. Incubation of the immunoconjugate with the nonspecific adsorbant Amberlite XAD-2 caused an average 30% decrease in the DXR-to-MoAb molar ratio, suggesting a population of drug that is tightly but noncovalently associated with MoAb.     

The monoclonal antibody CR11-351 discriminates HLA-A2 variants identified by T cells

Serologic and immunochemical assays have shown that the monoclonal antibody (MoAb) CR11-351 recognizes a determinant expressed by HLA-A2 and A28 alloantigens. The MoAb CR11-351 blocks the cytotoxicity of some, but not all, anti-HLA-A2 and anti-HLA-A28 alloantisera tested. These findings suggest that each allospecificity consists of several determinants, only some of which are spatially close to the determinant defined by the MoAb CR11-351. The binding of the MoAb CR11-351 to HLA-A2 lymphoid cells is not effected by their precoating with the HLA-A, B-specific MoAb CR10-214, Q6/64, and 6/31 but is enhanced by at least 20% by the MoAb CR10-131, CR10-402 and by the beta 2-m-specific MoAb NAMB-1. The MoAb CR11-351 did not react with one of four HLA-A2 variants which are indistinguishable with conventional anti-HLA-A2 sera, but are not recognized by "normal" HLA-A2-restricted cytotoxic T cells and possess structurally distinct HLA-A2 heavy chains. Therefore the MoAb CR11-351 provides the first evidence of a serologically detectable difference between the four HLA-A2 variants and "normal" HLA-A2 antigens.     

Malignant human B cells express two populations of p24 surface antigens

Monoclonal antibodies (MoAb) to a leukemia-associated p24 cell surface antigen are currently being used to purge bone marrow of malignant cells before autologous transplantation for acute lymphoblastic leukemia (ALL). Their use as potential diagnostic reagents for hematologic disorders is also under investigation. It has been assumed throughout these investigations that the p24-specific MoAb produced by different laboratories all identify the same antigen. Our present studies indicate that at least two populations of p24 antigens, having different chemical properties and cellular distributions, exist on malignant B cells. For example, eight MoAb raised to ALL cells (ALL-MoAb) identify a p24 antigen on these cells but do not react with the Burkitt's lymphoma cell line Ramos. In contrast, six MoAb raised to Ramos (Ramos-MoAb) identify a p24 antigen on both Ramos and ALL cells. Quantitative binding of both sets of MoAb to ALL cells is comparable. The ALL-MoAb react with platelets, granulocytes, and activated but not resting T lymphocytes, whereas the Ramos-MoAb react with both resting and activated T lymphocytes but not with platelets or granulocytes. The ALL-MoAb react with 11 of 34 human hematopoietic cell lines tested; the Ramos-MoAb react with all 34. Both sets of MoAb react with most of the nonhematopoietic human cell lines tested. Reciprocal exhaustive radioimmune precipitation experiments performed with an ALL cell line indicate that the antigenic determinants recognized by these two sets of MoAb are present on different molecules. Similarly, proteolytic digests of iodinated antigens identified by these two sets of MoAb on ALL cells confirm the unique chemical identities of these molecules and suggest that they reflect the products of different genetic loci. The presence of the antigen identified by the Ramos-MoAb on every cell population tested except granulocytes suggests that it may serve an important cellular function. The existence of two populations of p24 antigens on at least some hematopoietic cells indicates the need for caution when comparing the results of studies of these antigens by groups employing different MoAb.     

Analysis of the interaction between a human high molecular weight melanoma-associated antigen and the monoclonal antibodies to three distinct antigenic determinants

The restricted tissue distribution and the limited heterogeneity that appear in melanoma lesions of the high M.W. melanoma-associated antigen (HMW-MAA) suggest that this antigen may be an appropriate marker for radioimaging, and a useful target for immunotherapy in patients with melanoma. Therefore, in this study we analyzed other characteristics that are important in the selection of reagents for radioimaging and immunotherapy purposes. The affinities of the monoclonal antibodies (MoAB) 149.53, 225.28S, and 763.74T to distinct determinants of the HMW-MAA were found to be at least 1 X 10(8) mol/L. Furthermore, the effects of the concentrations of unlabeled MoAb on the dissociation rates suggest that the binding of MoAb 149.53 and 225.28S to melanoma cells (Colo 38) is preferentially bivalent, whereas that of MoAb 763.74T is preferentially univalent. These results suggest that the latter MoAb is the reagent of choice for assays that make use of soluble HMW-MAA, whereas the former two are the reagents of choice for assays with membrane-bound HMW-MAA, such as imaging with radiolabeled MoAb. The density of the HMW-MAA on cultured Colo 38 melanoma cells appears to be in the range of approximately 5 X 10(6) molecules/cell. The HMW-MAA was not susceptible to MoAb-mediated modulation under a variety of experimental conditions that included various concentrations of modulating MoAb, different incubation times, the use of an anti-mouse Ig antiserum, and the relaxation of equilibrium by diluting cells in MoAb-free medium. These results indicate that the HMW-MAA and the available corresponding MoAb meet the criteria to be reagents for radioimaging and immunotherapy in patients with melanoma.     

Altered cell surface antigen expression in bladder carcinoma detected by a new hemagglutinating monoclonal antibody

A hemagglutinating monoclonal IgM antibody (MoAb145) was produced against a high incidence red blood cell membrane antigen. By the specific red cell adherence test, the antibody also reacted with human bladder epithelium; in addition, expression of the MoAb145 antigen was lost in some cases of transitional cell carcinoma of the bladder, in a manner similar to the ABH blood group. Hemagglutination studies with a panel of erythrocytes lacking specific high incidence red blood cell membrane antigens indicated that MoAb145 did not recognize ABH specificity but rather a determinant absent from rare MN variant erythrocytes, including En(a-) erythrocytes, which lack glycophorin-alpha. Failure of MoAb145 to stain, by indirect immunofluorescence, the erythroleukemia cell line K562, which expresses glycophorin-alpha and the MN blood group, and failure to inhibit MoAb145 hemagglutination with an erythrocyte sialoglycoprotein fraction that contained MN blood group activity suggests that MoAb145 does not recognize either glycophorin-alpha or the MN blood group, but rather another membrane determinant, which is altered in En(a-) erythrocytes. This study demonstrates a new epitope detected by MoAb145 that is shared between human erythrocyte membranes and bladder epithelia, and is affected by neoplastic transformation in transitional cell carcinoma of the bladder.     

Augmentation of antileukemia lytic activity by OKT3 monoclonal antibody: synergism of OKT3 and interleukin-2

We have shown that short-term incubation (45 min) of peripheral blood lymphocytes of normal donors with OKT3 monoclonal antibody (MoAb), directed against T-cell-associated antigen CD3, resulted in an acquisition of lytic activity against fresh leukemic cells. Induction of such antileukemia activity was specific for OKT3, since Leu-1 MoAb (directed against another T cell surface molecule, CD5) did not induce a lytic effect. The OKT3-generated antileukemia effect was displayed against various types of leukemia including chronic myelogenous leukemia and acute myelogenous leukemia of various histological subtypes (M1, M2, M5). We furthermore demonstrated that OKT3 MoAb substantially enhanced leukemia killing by interleukin-2 (IL-2)-activated killer cells obtained from peripheral blood of patients with leukemia. Of most importance was the observation that the combined treatment of effector cells with IL-2 and OKT3 MoAb resulted in the highest levels of lysis of both autologous and allogeneic fresh leukemic cells that have been observed in leukemic patients to date. Of importance was to note that OKT3 treatment was effective in induction of cytotoxic activity also in patients whose effector cells were unresponsive to stimulation with IL-2 alone. All of these observations suggest that IL-2-activated and OKT3-MoAb-treated effector cells may represent the most aggressive population of antileukemia-directed killer cells and may play a significant role in the treatment of human leukemia.     

Analysis of the human CD36 leucocyte differentiation antigen by means of the monoclonal antibody NL07.

The murine monoclonal antibody (MoAb) NL07 was generated by immunization with human platelet extracts. NL07 MoAb recognized a molecule expressed by human platelets, monocytes, and endothelial cells, as well as by the myelomonocytic line U937 and by some melanoma cells or lines. Normal endothelial cells and the melanoma cells express the NL07 epitope only while adhering to a substrate. SDS-polyacrylamide gel electrophoresis and two-dimensional gel analysis indicate that the molecule recognized by NL07 MoAb on platelets is a single chain structure featuring a molecular weight of 85 kDa under reducing conditions, with an acidic isoelectric point ranging from 5.2 to 5.5. The specific phenotype distribution and the biochemical structure indicate that NL07 MoAb recognizes the platelet GPIV (CD36) molecule, a surface glycoprotein with a functional role of thrombospondin receptor. The results of competition tests with OKM5 MoAb (specific for the CD36 molecule) confirm the molecular specificity and epitope coincidence. Furthermore, upon binding to the platelets, NL07 MoAb is able to transmit via CD36 an activation signal which is followed by a potent aggregation. On the contrary, there is lack of evidence concerning the ability of the CD36 molecule of transmitting signal(s) on the U937 cells.     

Rat monoclonal antibody distribution in mice: An epitope inside the lung vascular space mediates very efficient localization

MoAb to an epitope on lung endothelial cells accumulates rapidly in the lung resulting in localization ratios of over 100. MoAb to a macrophage antigen found in spleen and lung has maximum localization ratios of 24 and 5, respectively, while MoAb specific for a human tumor grown in nude mice has a maximum ratio of about 4. Epitope concentrations in target organs (300–600 ng/mg protein) are comparable in all three systems, indicating that the MoAb to endothelium is efficient in localization to the lung.