Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells

Anti-CD20 monoclonal antibodies have been successfully employed in the clinical treatment of non-Hodgkin's lymphomas in both unmodified and radiolabeled forms. Previous publications have demonstrated that the antitumor effects of unmodified anti-CD20 mAb are mediated by several mechanisms including antibody-dependent cellular cytotoxicity, complement-mediated cell lysis, and induction of apoptosis by CD20 cross-linking. In this report, we demonstrate induction of apoptosis by three anti-CD20 monoclonal antibodies [1F5, anti-B1, and C2B8 (Rituximab)]. The magnitude of apoptosis induction was greater with the chimeric Rituximab antibody than with the murine 1F5 and anti-B1 antibodies. Apoptosis could be enhanced with any of the antibodies by cross-linking with secondary antibodies (or Fc-receptor-bearing accessory cells). The signaling events involved in anti-CD20-induced apoptosis were investigated, including activation of protein tyrosine kinases, increases in intracellular Ca²⁺ concentrations, caspase activation, and cleavage of caspase substrates. Our results indicate that anti-CD20-induced apoptosis can be attenuated by PP1, an inhibitor of protein tyrosine kinases Lck and Fyn, chelators of extracellular or intracellular Ca²⁺, and inhibitors of caspases, suggesting that anti-CD20-induced apoptosis may involve modulation of these signaling molecules. We also demonstrated that varying the expression of Bcl-2 did not affect the magnitude of anti-B1-induced apoptosis, possibly because of the sequestering effects of other Bcl-2 family members, such as Bad. These studies identify several of the signal-transduction events involved in the apoptosis of malignant B cells that transpire following ligation of CD20 by anti-CD20 antibodies in the presence of Fc-receptor-expressing cells or secondary goat anti-(mouse Ig) antibodies and which may contribute to the tumor regressions observed in mouse models and clinical trials. Received: 27 May 1999 / Accepted: 1 October 1999     

Characterization of a neutralization-sensitive epitope on the Am 105 surface protein of Anaplasma marginale

Purified immunoglobulin from each of two hybridoma cell lines (ANA 15D2 and ANA 22B1) significantly neutralized the infectivity of 10⁸Anaplasma marginale initial bodies for cattle. Both cell lines produce antibody to the same Am 105 epitope as they inhibited the binding of each other to Am 105 in a competition radioimmunoassay. Complete digestion of Am 105 with proteinase K, pronase, or trypsin prevented monoclonal antibody binding indicating that the epitope was protein in nature rather than surface polysaccharide. In addition, evidence that the neutralization-sensitive epitope was not membrane-protein-bound polysaccharide included: [1] ³⁵S-methionine, but not ³H-glucosamine, was metabolically incorporated into Am 105 during short-term in vitro culture; [2] Am 105 was surface radiolabeled using ¹²⁵I in a lactoperoxidase mediated reaction, but not labeled using a galactose oxidase-NaB[³H]₄ mediated reaction with or without neuraminidase pretreatment; and [3] Am 105 did not bind to concanavalin A, Helix pomatia lectin, peanut lectin, soybean lectin, or wheat germ lectin.     

Specific suppression of the in vitro parent anti-hybrid reaction: II. Parameters influencing suppressor cell induction in the F1 hybrid

In the accompanying paper (K. Kosmatopoulos et al. Cell. Immunol.104, 319–334, 1987) we have reported that the spleens of B6D2F1 hybrids pretreated with B6 spleen cells 7 days earlier contain a cell which specifically suppresses the in vitro proliferative and cytotoxic B6 anti-B6D2F1 responses. The results we present here concern the in vivo conditions under which this suppressor cell can be induced. Suppressor cell activity appears early after the injection of B6 spleen cells (day +1), increases on Day 7, and disappears by Day 30; it is always detectable after the injection of 5 × 10⁷ B6 spleen cells and never after the injection of 1.25 × 10⁷ cells, the intermediate dose of 2.5 × 10⁷ cells being followed by variable results. This variability is attributable to the age of B6 donor and B6D2F1 recipient mice, and suppression is never observed when 2.5 × 10⁷ spleen cells from 6-week-old B6 mice are injected into 6-week-old B6D2F1 hybrids. The suppressor cell is induced by the injection of B6 spleen cells of the Thy-1⁺ Ly-1⁻2⁺ phenotype, even if they are irradiated at 1000 R just before their injection. Lymph node cells from B6 mice induce the suppressor cell, whereas thymocytes do not. Irradiation of B6D2F1 hybrids at 600 or 950 R does not prevent the induction of suppressor cell, nor does thymectomy. Moreover, in the thymectomized or 600 R-irradiated B6D2F1 animals suppression can be induced even by the injection of only 1.25 × 10⁷ B6 spleen cells. This phenomenon of specific suppression is not limited to the B6-B6D2F1 genetic combination since it has been observed in all parent-hybrid combinations tested to date.     

In vivo and in vitro effects of monoclonal antibody to Ly antigens on immunity to infection

Injection of CBA mice with Brucella abortus strain 19 leads to chronic infection during which both cell-mediated immunity (delayed hypersensitivity and macrophage activation) and antibody production occur. Protection was efficiently transferred to naive mice using spleen cells from mice infected 5 or 12 weeks earlier. Selective lysis in vitro of these cells by antibody to cell surface antigens showed that Thy-1⁺ Ly-1⁺2⁺ T lymphocytes were required for transfer. Treatment with anti-Ia serum neither suppressed nor enhanced adoptive transfer. Thus Ia⁺ B lymphocytes were not required, and Ia⁺ suppressor T cells were not active in the response. Three injections per week of anti-Ly-1 monoclonal antibody beginning 5 days before infection led to a 10-fold increase in bacterial numbers 25 days after infection when acquired immunity was well established in untreated mice. The delayed hypersensitivity response was unaffected. In addition cells from these in vivo treated mice were unable to transfer resistance. Beginning the treatment on the day of infection abolished the IgG antibody response without affecting bacterial numbers. The studies emphasize the unique role of Ly-1⁺2⁺ T cells in immunity to Brucella and indicate the usefulness of these techniques in dissecting out those components of the immune response which contribute to recovery from infection.     

Isolated soluble fractions from the murine B16 melanoma induce primary in vitro syngeneic antitumor responses

Summary This paper extends our previous studies, which documented our ability to isolate immunogenic entities from nonimmunogenic or weakly immunogenic tumors.B16 melanoma cells failed, in our in vitro experimental system, to induce anti-B16 cytotoxic responses in spleen cells derived from normal syngeneic C57BL/6 mice. The B16 melanoma cellular homogenate was fractionated on an Ultrogel AcA 34 column, and the various fractions were tested for their ability to induce anti-B16 cytotoxic responses under the same conditions as those used for intact B16, the nonimmungenic tumor cells. Certain fractions, some of them with relatively low protein concentrations, induced anti-B16 cytotoxic responses in spleen cells of normal C57BL/6 mice, whereas others, some of them with relatively high protein concentrations, failed to induce such responses. One fraction (Fr.), designated Fr. 5/6, was examined in detail. It was found that in normal syngeneic spleen cells this fraction induced effector cells that efficiently killed (at various E : T ratios) the relevant B16 target cells and RBL5 syngeneic tumor cells, but not the YAC allogeneic tumor cells or C57BL/6 lymphoblasts. Furthermore, an excess of unlabeled B16 cells most efficiently blocked the ability of these anti-B16 effector cells to kill radiolabeled B16 target cells. RBL5 tumor cells, YAC tumor cells, or C57BL/6 lymphoblasts failed to block these effector cells efficiently. A significant fraction of the effector cells induced with Fr. 5/6 was characterized as thymus-derived cells (Thy-1⁺, Thy-2⁺3⁺ cells). It was suggested that another fraction of the cellular population was natural killer cells, which cytolyzed the RBL5 target cells. Various theoretical and practical aspects of these findings are discussed.     

Cellular interactions in the generation and expression of histamine-induced suppressor activity

The tissue, anatomic, and developmental distribution of Maclura pomifera (MP) lectin binding to rat lymphoid cells was examined. Analysis was performed by immunofluorescence microscopy and by the fluorescence-activated cell sorter. Comparison with anti-rat Ig to detect B cells and the monoclonal antibodies W3/13, W3/25, and OX 8 to detect T cells revealed that the MP lectin reacted with all T cells and not B cells in spleen and lymph node of young adult rats. The lectin also bound selectively to the thymus-dependent areas in frozen sections of spleen and lymph node. Using the MP lectin in conjunction with anti-Thy1 antibody and the monoclonal antibodies, W3/25 and OX 8, four T-cell subpopulations in spleen and lymphnode were identified on the basis of their cell surface antigenic phenotype. The T-cell developmental distribution of MP binding revealed that 100% of normal and neoplastic thymocytes bound the lectin whereas approximately 25% of TdT⁺ bone marrow cells, putative thymocyte progenitors, were MP⁺. Thus, the MP lectin is a nonimmunoglobulin reagent which binds to prethymic, thymic, and post-thymic cells of the T-lymphocyte lineage. Affinity chromatography experiments indicated that the MP lectin binds, at least in part, to the major thymocyte cell surface glycoprotein which is recognized by the W3/13 monoclonal antibody.     

The B cell surface molecule B1 is functionally linked with B cell activation and differentiation

The B1 molecule is a 32,000 m.w. phosphorylated cell surface protein expressed exclusively by B cells from the mid pre-B until the plasma cell stage of differentiation. Two monoclonal antibodies (gamma 2a and mu) reactive with this molecule were used to assess the role of B1 in B cell activation, proliferation, and differentiation. The anti-B1 antibodies at concentrations ranging from 0.1 to 100 micrograms/ml significantly inhibited B cell proliferation induced by anti-mu antibodies, Staphylococcus aureus Cowan strain 1, activated T cells, and Epstein Barr virus. Although capable of inhibiting proliferation, anti-B1 antibody in soluble form or coupled to beads did not activate B cells or induce proliferation. Antibodies of comparable isotypes or against other B cell-restricted antigens, including B2, B4, B5, and HB-5, did not inhibit activation. Pretreatment of B cells with anti-B1 antibody did not inhibit activation, indicating that B cells had to be cultured with anti-B1 antibody for anti-B1-mediated inhibition to occur. Maximum inhibition was obtained when anti-B1 antibody was added at the initiation of culture. In agreement with this, growth factor-dependent proliferation of preactivated B cells was not inhibited by anti-B1 antibodies. Comparable inhibition of B cell activation was noted with antibodies reactive with class II antigens of the major histocompatibility complex with the exception that anti-B1 antibody inhibited immunoglobulin secretion in pokeweed mitogen assays, whereas anti-DR antibody did not. These results suggest that the B1 molecule may serve a central role in the regulation of B cell activation and differentiation.     

Systemic perfusion: a method of enhancing relative tumor uptake of radiolabeled monoclonal antibodies

We evaluated the feasibility of systemic vascular perfusion with saline (mimicking plasmapheresis) as a method to enhance tumor-specific monoclonal antibody (MoAb) tumor/background ratios. Initially, groups of rats were injected intravenously (i.v.) with ¹³¹I-5G6.4 MoAb (murine IgG2aK reactive with ovarian carcinoma). These animal's radioactivity levels were determined by dose calibrator and they were imaged before and after perfusion which was conducted at 4 or 24 h post-antibody injection. Animals were sacrificed after perfusion, as were controls, and normal organ radioactivity levels determined. In addition, nude mice bearing HTB77 ovarian cancers subcutaneously were injected i.v. with ¹³¹I-5G6.4 MoAb and were imaged before and after systemic perfusion with saline 24 h post-5G6.4 injection. Perfusion in rats dropped whole-body 5G6.4 levels significantly at both perfusion times (P < 0.0005). The drop in whole-body radioactivity with perfusion was significantly greater for the animals perfused at 4 h post i.v. 5G6.4 antibody injection (48.3 ± 5.1%) than for those perfused at 24 h post i.v. antibody injection (32.9 ± 2.9%) (P < 0.025). In the nude mice with ovarian cancer xenografts, γ camera images of tumors were visually and quantitatively (by computer image analysis) enhanced by perfusion, with a 2.33-fold greater decline in whole body uptake than in the tumor (P < 0.05). These studies show that (1) much background antibody radioactivity can be removed using whole-body perfusion with saline, (2) that the decline in whole body activity is larger with 4 than 24 h perfusion and (3) tumor imaging can be enhanced by this approach. This and similar approaches that increase relative tumor antibody uptake such as plasmapheresis may be useful in imaging and therapy with radiolabeled antibodies.     

99mTc-CD19 monoclonal antibody is not useful for imaging of B cell non-Hodgkin’s lymphoma

 In this study we investigated the applicability of ^99mTc-labeled CD19 monoclonal antibody (mAb) for tumor imaging in patients with B cell non-Hodgkin’s lymphoma. A 1-mg sample of murine CD19 mAb was labeled with approximately 550 MBq [^99mTc]pertechnetate. The labeled mAb was administered i. v. to seven patients, four without and three with pretreatment with 10 mg unlabeled CD19 mAb. The number of circulating B cells was decreased by 44±5% 1 h after injection of the radiolabeled mAb. Peripheral B cells were coated with CD19, resulting in partial modulation of CD19, most pronounced in the three pretreated patients. Whole-body images were obtained with a gamma camera and compared with results obtained by conventional imaging techniques. Initially, blood-pool activity dominated, whereas 24 h after injection the radioactivity was mainly located in the spleen, kidneys and liver. In two patients, a lesion in the spleen appeared as an unlabeled spot. In one patient, a lesion in the femur, which was detected by computed tomography (CT) and gallium-67 scans, was also seen on the CD19 scan from 1 h after administration of the radioimmunoconjugate onwards. Good imaging of bone marrow infiltration was observed in one of three patients. Lymph node involvement was not observed in any of the patients in whom affected lymph nodes were detected by CT or gallium-67 scan. In conclusion, in the present study radioimmunodetection with ^99mTc-labeled CD19 mAb was found to be inferior to CT and gallium-67 scanning in the diagnosis of patients with B cell non-Hodgkin’s lymphoma. Received: 14 March 1996 / Accepted: 14 May 1996     

Studies of the mouse Ly-6 alloantigen system

Three monoclonal antibodies were produced by fusing mouse myeloma cell line NS-1 with spleen cells from C3H/An mice hyperimmunized with B6-H-2^k spleen cells. These antibodies recognized an alloantigen displaying a similar strain distribution pattern to the Ly-6.2 and Ala-1.2 alloantigens. Analysis of C×B and B×H recombinant inbred mice revealed close linkage of genes controlling Ly-m6 and Ly-6. The monoclonal antibodies lysed 70 percent of cells in lymph nodes and 60 percent in spleen in direct cytotoxicity assays, but did not lyse significant numbers of cells of thymus and bone marrow. Separated T and B cells were reactive with the antibodies, but T cells were more sensitive to the antibody and complement than B cells. Virtually all cells in cultures of cells activated in the mixed lymphocyte reaction or by Concanavalin A were reactive with the monoclonal antibodies. Direct plaque-forming cells were completely eliminated by the monoclonal antibody and complement. By absorption tests, cells from all organs tested so far (thymus, lymph node, spleen, bone marrow, brain, kidney and liver) were shown to express the Ly-m6 determinant. Tumor cell lines with T, B or stem cell characteristics were reactive with the monoclonal antibody by direct cytotoxicity and absorption assays.     

Development of T lymphocytes in Xenopus laevis: appearance of the antigen recognized by an anti-thymocyte mouse monoclonal antibody

Development of T lymphocytes in Xenopus laevis was studied using a mouse monoclonal antibody (mAb), XT-1, that was produced against surface determinants on thymocytes of J strain frog. Ontogenic studies, employing immunofluorescence, showed that cells positive for the determinant recognized by XT-1 mAb (XT-1⁺ cells) were first detected in the thymus of J strain Xenopus by Nieuwkoop and Faber stage 48 (7 days postfertilization) and then in the spleen, liver and kidney by stage 52 (20 days postfertilization). Percentages of XT-1⁺ cells in the thymus increased rapidly by stage 49 (10 days postfertilization) and reached adult levels by stage 52, and those in the spleen, liver, and kidney reached adult levels by stage 56 (40 days postfertilization). Electron microscopic immunohistochemistry revealed that most XT-1⁺ cells in thymuses from stage 56 larvae were typical small lymphocytes (4–7 μm in diameter). In contrast, many XT-1⁺ cells in larval thymuses at stage 49 are large (8–10 μm in diameter) lymphoblastoid cells. Thymectomy at stage 46 (5 days postfertilization) depleted XT-1⁺ cells in larval and adult lymphoid organs to background levels. These results suggest that the XT-1⁺ cells are differentiated from the lymphoid precursor cells in the thymus before the appearance of small lymphocytes and migrate into peripheral lymphoid organs. The cell surface determinant recognized by the XT-1 mAb may provide an important marker for the differentiation of T lymphocytes in Xenopus.     

Production and characterization of monoclonal antibodies against the two subunits proteins B1 and B2 of Escherichia coli ribonucleotide reductase

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, named protein B1 (170 000) and protein B2 (87 000). We purified and characterized five monoclonal antibodies against B1 and three against B2 from hybridomas obtained by fusion of spleen cells from immunized mice and the myeloma cell line P3-X63Ag8. All are of the IgG1 class with a high affinity for the antigen with dissociation constants in the nanomolar range. Four of the anti-B1 monoclonals and all three anti-B2 monoclonals neutralize reductase activity while one anti-B1 monoclonal binds tightly to B1 without affecting its activity. Fab fragments prepared from three anti-B1 monoclonals had similar dissociation constants. The anti-B1 monoclonals interacted with separate epitopes while two of the anti-B2 monoclonals appeared to react with the same epitope. In the case of B1, various allosteric states of the protein induced by binding of effectors had no apparent effect on the interaction with monoclonals, nor did their binding prevent subsequent binding of effectors. With B2, binding of monoclonals did not affect the typical electron paramagnetic resonance spectrum of the protein and thus did not involve either the tyrosyl free radical or the iron center of B2. All neutralizing antibodies interfered with the interaction between the two subunits, explaining their effect on enzyme activity, since active ribonucleotide reductase consists of a B1-B2 complex.     

BAFF Receptor mAb Treatment Ameliorates Development and Progression of Atherosclerosis in Hyperlipidemic ApoE?/? Mice

Aims

Option to attenuate atherosclerosis by depleting B2 cells is currently limited to anti-CD20 antibodies which deplete all B-cell subtypes. In the present study we evaluated the capacity of a monoclonal antibody to B cell activating factor-receptor (BAFFR) to selectively deplete atherogenic B2 cells to prevent both development and progression of atherosclerosis in the ApoE^−/− mouse.

Methods and Results

To determine whether the BAFFR antibody prevents atherosclerosis development, we treated ApoE^−/− mice with the antibody while feeding them a high fat diet (HFD) for 8 weeks. Mature CD93⁻ CD19⁺ B2 cells were reduced by treatment, spleen B-cell zones disrupted and spleen CD20 mRNA expression decreased while B1a cells and non-B cells were spared. Atherosclerosis was ameliorated in the hyperlipidemic mice and CD19⁺ B cells, CD4⁺ and CD8⁺ T cells were reduced in atherosclerotic lesions. Expressions of proinflammatory cytokines, IL1β, TNFα, and IFNγ in the lesions were also reduced, while MCP1, MIF and VCAM-1 expressions were unaffected. Plasma immunoglobulins were reduced, but MDA-oxLDL specific antibodies were unaffected. To determine whether anti-BAFFR antibody ameliorates progression of atherosclerosis, we first fed ApoE^−/− mice a HFD for 6 weeks, and then instigated anti-BAFFR antibody treatment for a further 6 week-HFD. CD93⁻ CD19⁺ B2 cells were selectively decreased and atherosclerotic lesions were reduced by this treatment.

Conclusion

Anti-BAFFR monoclonal antibody selectively depletes mature B2 cells while sparing B1a cells, disrupts spleen B-cell zones and ameliorates atherosclerosis development and progression in hyperlipidemic ApoE^−/− mice. Our findings have potential for clinical translation to manage atherosclerosis-based cardiovascular diseases.     

Regulation of natural killer cell activity by anti-I-region monoclonal antibodies

The effects of a monoclonal antibody directed against immune response gene products on mouse NK activity were examined. In vivo administration of an anti-I-A^k antibody to C3H/He (H-2^k) mice modulated their peritoneal cell (PC) and spleen cell (SC) natural killer (NK) activity against YAC-1 lymphoma target cells in vitro. No such effect was observed when BALB/c (H-2^d) mice were treated with this antibody. Administration of anti-I-A^k antibody to mice before and after infection with Toxoplasma or treatment with poly(I:C) leads to suppression of NK activity in comparison to NK activity of mice infected with Toxoplasma or injected with poly(I:C) alone. A similar treatment regimen with M5/114 antibody which reacts with I-A^b, I-A^d, I-E^d, and I-E^k molecules resulted in decreased NK activity in B10.D2 (H-2^d) but not in B10.BR (H-2^k) mice. Serum and cell culture supernatant interferon (IFN) concentrations were not altered as a result of anti-I-A^k treatment. Removal of adherent cells did not restore NK activity of anti-I-A^k-treated Toxoplasma-infected mice to levels obtained with mice infected with Toxoplasma. In contrast, depletion of Ly 2.1⁺ cells from nylon-wool nonadherent SC of mice treated with anti-I-A^k antibody, before and after infection with Toxoplasma, resulted in restoration of NK activity to the same level as that observed in Toxoptasma-infected mice.     

Primary in vitro antibody response of rabbit lymphoid cells and T-B cell collaboration in the absence of detectable mitogens

To investigate whether the antibody response and T-B-cell collaboration in vitro can be obtained in the absence of mitogens, a method of obtaining an in vitro primary anti-sheep red blood cell antibody response by rabbit spleen and lymph node cells was developed. We used Marbrook culture vessels and a specially prepared medium containing 10% autologous serum and maintained at pH 7.4–7.6. The system was shown to be devoid of any polyclonal mitogens as assessed by [³H]thymidine incorporation and by direct examination for blast cells in stained smears. The primary response increased continuously over the 5-day cultivation period and only IgM but not IgG plaque-forming cells (PFC) were detected. In over 20 experiments, the response ranged from 357 ± 17 to 4425 ± 110 PFC/10⁷ cultured cells with a median stimulation index of 52. The spleen cells required less antigen than the lymph node cells and 2-mercaptoethanol inhibited the response of the spleen cells but not that of the lymph node cells. Lymphocytes were separated into highly pure T- and B-cell populations by negative selection using antibody-coated human erythrocytes to rosette either T or B cells and Ficoll-Hypaque centrifugation to remove rosetted cells. Upon cultivation, B cells alone gave a low IgM response, whereas B cells reconstituted with T cells gave a response similar to that obtained with unseparated lymphoid cells. We concluded that: (a) optimal conditions for obtaining primary in vitro antibody responses using rabbit spleen and lymph node cells were established, (b) T-B-cell collaboration was demonstrated in the rabbit primary antibody response to sheep erythrocytes, and (c) the primary antibody response in vitro and T-B-cell collaboration may occur in the absence of detectable polyclonal mitogens.     

Immunological properties of Fc receptor on lymphocytes. 6. Characterization of suppressive B-cell factor (SBF) released from Fc receptor-bearing B cells.

EA, i.e., antigen-antibody complexes are able to induce an antigen-nonspecific suppressive factor(s) from FcR⁺ B cells by binding on FcR. This factor, termed “suppressive B-cell factor (SBF)” was only effective on H-2 compatible, but not on H-2 incompatible spleen cells in an adoptive cell transfer system. Furthermore, SBF, prepared from B10.A (H-2^a) splenic FcR⁺ B cells, suppressed the adoptive primary response of B10.D2 mice (H-2^d), in addition to A/J mice (H-2^a) against DNP-DE, by the pretreatment of cells with SBF in vitro. Absorption with affinity columns demonstrated that active components) of SBF from C3H/He mice (H-2^k) was eliminated by both B6 anti-CBA (H-2^b anti-H-2^k) and B10.D2 anti-B10.BR (H-2^d anti-H-2^k), but not B10 anti-B10.A (H-2^b anti-H-2^a). In contrast, the suppressive activity of SBF was eliminated neither by anti-mouse Ig nor by a heat-aggregated human γ-globulin column. These results indicate that SBF contains a product coded by the right-hand side of H-2 gene complex, but does not contain Ig determinants nor FcR. Thus, it is conceivable that a compatibility of the right-hand side of H-2 gene complex is required for inducing effective suppression of spleen cells by SBF. SBF was considered to be a trypsin-resistant and heat-labile substance with a molecular weight of 30,000–63,000. The target cells for SBF were FcR^? B precursors, but not helper T cells.     

An improved method of direct labeling monoclonal antibodies with 99mTc

An improved method of direct labeling MAbs with ^99mTc is described. Two murine monoclonal antibodies, designated Lym-1 and B72.3, have been successfully labeled with ^99mTc in 0.1 M borate buffer at pH 9.3. The choice of buffer and pH was essential for obtaining a radiolabeling yield ⩾98%. In vitro studies demonstrated that the radiolabeled antibodies were stable and retained their immunoreactivity. Imaging and biodistribution studies using Raji and LS174T human tumor-bearing nude mice demonstrated a significant tumor uptake at 24-h post-injection of ^99mTc-labeled MAbs. This improved labeling method showed better stability than those of previously published methods and resulted in significant improvement in the uptake of antibody in tumor. External images at 24 h post-injection revealed clearly visible tumors demonstrating the benefit of this method for tumor immunoscintigraphy.     

Nature of neonatal splenic suppressor cells in the mouse

Due to the controversy in the recent literature concerning the T-cell nature of the neonatal mouse splenic suppressor cell, we have reexamined the nature of these cells at different stages after birth. With the use of monoclonal anti-Thy antibody we can detect T-cell products on spleen suppressor cells from BDF₁ mice at 3, 4, 5, 6, and 7 days of age. The suppressor cells are assayed by their ability to inhibit mixed-lymphocyte reactivity, which is an in vitro example of cell-mediated immunity. The neonatal spleen suppressor cells also carry products of the Ly 1 and I-J locus. Neonatal thymectomy results in a premature decrease of suppressor cell activity. These data suggest that the mouse neonatal splenic suppressor cell is a short-lived Ly 1⁺, I-J⁺ T cell.     

Th17-Related Cytokines in Systemic Lupus Erythematosus Patients with Dilated Cardiomyopathies: A Possible Linkage to Parvovirus B19 Infection

Dilated cardiomyopathies (DCM) are a major cause of mortality in patients with systemic lupus erythematosus (SLE). Immune responses induced by human parvovirus B19 (B19) are considered an important pathogenic mechanism in myocarditis or DCM. However, little is known about Th17-related cytokines in SLE patients with DCM about the linkage with B19 infection. IgM and IgG against B19 viral protein, and serum levels of Th17-related cytokines were determined using ELISA in eight SLE patients with DCM and six patients with valvular heart disease (VHD). Humoral responses of anti-B19-VP1u and anti-B19-NS1 antibody were assessed using Western blot and B19 DNA was detected by nested Polymerase Chain Reaction (PCR). Levels of interleukin (IL)-17, IL-6, IL-1β, and tumor necrosis factor (TNF)-α were significantly higher in SLE patients with DCM (mean ± SEM, 390.99±125.48 pg/ml, 370.24±114.09 pg/ml, 36.01±16.90 pg/ml, and 183.84±82.94 pg/ml, respectively) compared to healthy controls (51.32±3.04 pg/ml, p<0.001; 36.88±6.64 pg/ml, p<0.001; 5.39±0.62 pg/ml, p<0.005; and 82.13±2.42 pg/ml, p<0.005, respectively). Levels of IL-17 and IL-6 were higher in SLE patients with DCM versus those with VHD (both p<0.01). Five (62.5%) of DCM patients had detectable anti-B19-NS1 IgG and four (50.0%) of them had anti-B19-VP1u IgG, whereas only one (16.7%) of VHD patients had detectable anti-B19-NS1 IgG and anti-B19-VP1u IgG. Serum levels of IL-17, IL-6 and IL-1β were markedly higher in SLE patients with anti-B19-VP1u IgG and anti-B19-NS1 IgG compared to those without anti-B19-VP1u IgG or anti-B19-NS1 IgG, respectively. These suggest a potential association of B19 with DCM and Th17-related cytokines implicated in the pathogenesis of DCM in SLE patients.     

Imaging Axl expression in pancreatic and prostate cancer xenografts

The receptor tyrosine kinase Axl is overexpressed in and leads to patient morbidity and mortality in a variety of cancers. Axl–Gas6 interactions are critical for tumor growth, angiogenesis and metastasis. The goal of this study was to investigate the feasibility of imaging graded levels of Axl expression in tumors using a radiolabeled antibody. We radiolabeled anti-human Axl (Axl mAb) and control IgG1 antibodies with ¹²⁵I with high specific radioactivity and radiochemical purity, resulting in an immunoreactive fraction suitable for in vivo studies. Radiolabeled antibodies were investigated in severe combined immunodeficient mice harboring subcutaneous CFPAC (Axl^high) and Panc1 (Axl^low) pancreatic cancer xenografts by ex vivo biodistribution and imaging. Based on these results, the specificity of [¹²⁵I]Axl mAb was also validated in mice harboring orthotopic Panc1 or CFPAC tumors and in mice harboring subcutaneous 22Rv1 (Axl^low) or DU145 (Axl^high) prostate tumors by ex vivo biodistribution and imaging studies at 72 h post-injection of the antibody. Both imaging and biodistribution studies demonstrated specific and persistent accumulation of [¹²⁵I]Axl mAb in Axl^high (CFPAC and DU145) expression tumors compared to the Axl^low (Panc1 and 22Rv1) expression tumors. Axl expression in these tumors was further confirmed by immunohistochemical studies. No difference in the uptake of radioactivity was observed between the control [¹²⁵I]IgG1 antibody in the Axl^high and Axl^low expression tumors. These data demonstrate the feasibility of imaging Axl expression in pancreatic and prostate tumor xenografts.