CD22 associates with protein tyrosine phosphatase 1C, Syk, and phospholipase C-gamma(1) upon B cell activation.

Cross-linking B cell antigen receptor (BCR) elicits early signal transduction events, including activation of protein tyrosine kinases, phosphorylation of receptor components, activation of phospholipase C-gamma (PLC-gamma), and increases in intracellular free Ca2+. In this article, we report that cross-linking the BCR led to a rapid translocation of cytosolic protein tyrosine phosphatase (PTP) 1C to the particulate fraction, where it became associated with a 140-150-kD tyrosyl-phosphorylated protein. Western blotting analysis identified this 140-150-kD protein to be CD22. The association of PTP-1C with CD22 was mediated by the NH2-terminal Src homology 2 (SH2) domain of PTP-1C. Complexes of either CD22/PTP-1C/Syk/PLC-gamma(1) could be isolated from B cells stimulated by BCR engagement or a mixture of hydrogen peroxidase and sodium orthovanadate, respectively. The binding of PLC-gamma(1) and Syk to tyrosyl-phosphorylated CD22 was mediated by the NH2-terminal SH2 domain of PLC-gamma(1) and the COOH-terminal SH2 domain of Syk, respectively. These observations suggest that tyrosyl-phosphorylated CD22 may downmodulate the activity of this complex by dephosphorylation of CD22, Syk, and/or PLC-gamma(1). Transient expression of CD22 and a null mutant of PTP-1C (PTP-1CM) in COS cells resulted in an increase in tyrosyl phosphorylation of CD22 and its interaction with PTP-1CM. By contrast, CD22 was not tyrosyl phosphorylated or associated with PTP-1CM in the presence of wild-type PTP-1C. These results suggest that tyrosyl-phosphorylated CD22 may be a substrate for PTP-1C regulates tyrosyl phosphorylation of CD22.

Differentiation of normal human pre-B cells in vitro.

The differentiation of surface Ig- pre-B cells into surface Ig+ B cells is a critical transition in mammalian B cell ontogeny. Elucidation of the growth factor requirements and differentiative potential of human pre-B cells has been hampered by the absence of a reproducible culture system that supports differentiation. Fluorescence-activated cell sorting and magnetic bead depletion were used to purify fetal bone marrow CD10+/surface mu- cells, which contain 60-70% cytoplasmic mu+ pre-B cells. CD10+/surface mu- cells cultured for 2 d were observed to differentiate into surface mu+ cells. Analysis by Southern blotting provided direct evidence that rearrangement of kappa light chain genes occurs in culture, and flow cytometric analysis revealed the appearance of surface Ig+ B cells expressing mu/kappa or mu/lambda. Unexpectedly, the kappa/lambda ratio in differentiated cells was the inverse of what is normally observed in adult peripheral blood. Differentiation occurs in the absence of exogenous growth factors or cytokines, suggesting that a stimulus-independent differentiative inertia might characterize pre-B cells in vivo. Future use of this model will facilitate our understanding of normal and abnormal human pre-B cell differentiation.

GrpL, a Grb2-related adaptor protein, interacts with SLP-76 to regulate nuclear factor of activated T cell activation.

Propagation of signals from the T cell antigen receptor (TCR) involves a number of adaptor molecules. SH2 domain-containing protein 76 (SLP-76) interacts with the guanine nucleotide exchange factor Vav to activate the nuclear factor of activated cells (NF-AT), and its expression is required for normal T cell development. We report the cloning and characterization of a novel Grb2-like adaptor molecule designated as Grb2-related protein of the lymphoid system (GrpL). Expression of GrpL is restricted to hematopoietic tissues, and it is distinguished from Grb2 by having a proline-rich region. GrpL can be coimmunoprecipitated with SLP-76 but not with Sos1 or Sos2 from Jurkat cell lysates. In contrast, Grb2 can be coimmunoprecipitated with Sos1 and Sos2 but not with SLP-76. Moreover, tyrosine-phosphorylated LAT/pp36/38 in detergent lysates prepared from anti-CD3 stimulated T cells associated with Grb2 but not GrpL. These data reveal the presence of distinct complexes involving GrpL and Grb2 in T cells. A functional role of the GrpL-SLP-76 complex is suggested by the ability of GrpL to act alone or in concert with SLP-76 to augment NF-AT activation in Jurkat T cells.

Targeting pancreatic and ovarian carcinomas using the auristatin-based anti-CD70 antibody-drug conjugate SGN-75.

BACKGROUND: CD70 is an ideal target for antibody-based therapies because of its aberrant high expression in renal carcinomas and non-Hodgkin lymphomas and its highly restricted expression in normal tissues. The expression profiling of CD70 in carcinomas has been limited because of the lack of a CD70-specific reagent that works in formalin-fixed paraffin-embedded (FFPE) tissues. METHODS: We generated murine monoclonal antibodies (mAbs) specific for CD70 and validated their specificity by western blot analysis and developed a protocol for immunohistochemistry on FFPE tissues. CD70+ tumour cell lines were used for testing the anti-tumour activity of the anti-CD70 antibody-drug conjugate, SGN-75. RESULTS: We report novel detection of CD70 expression in multiple cancers including pancreatic (25%), larynx/pharynx (22%), melanoma (16%), ovarian (15%), lung (10%), and colon (9%). Our results show that pancreatic and ovarian tumour cell lines, which express high levels of endogenous or transfected CD70, are sensitive to the anti-tumour activity of SGN-75 in vitro and in vivo. CONCLUSION: Development of murine mAbs for robust and extensive screening of FFPE samples coupled with the detection of anti-tumour activity in novel indications provide rationale for expanding the application of SGN-75 for the treatment of multiple CD70 expressing cancers.

Macrophages and Fc-receptor interactions contribute to the antitumour activities of the anti-CD40 antibody SGN-40.

SGN-40 is a therapeutic antibody targeting CD40, which induces potent anti-lymphoma activities via direct apoptotic signalling cells and by cell-mediated cytotoxicity. Here we show antibody-dependent cellular phagocytosis (ADCP) by macrophages to contribute significantly to the therapeutic activities and that the antitumour effects of SGN-40 depend on Fc interactions.

Cell-cell interactions that regulate the development of B-lineage cells.

Significant progress has been made recently in our understanding of the functions of lymphocyte-associated surface proteins. The latest developments involve the identification of ligands or co-receptors for many of these surface proteins. The signal transduction mechanisms utilized by these molecules are also beginning to be elucidated.

Phospholipase C-gamma1 interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk.

Antigen receptor ligation on lymphocytes activates protein tyrosine kinases and phospholipase C-gamma (PLC-gamma) isoforms. Glutathione S-transferase fusion proteins containing the C-terminal Src-homology 2 [SH2(C)] domain of PLC-gamma1 bound to tyrosyl phosphorylated Syk. Syk isolated from antigen receptor-activated B cells phosphorylated PLC-gamma1 on Tyr-771 and the key regulatory residue Tyr-783 in vitro, whereas Lyn from the same B cells phosphorylated PLC-gamma1 only on Tyr-771. The ability of Syk to phosphorylate PLC-gamma1 required antigen receptor ligation, while Lyn was constitutively active. An mCD8-Syk cDNA construct could be expressed as a tyrosyl-phosphorylated chimeric protein tyrosine kinase in COS cells, was recognized by PLC-gamma1 SH2(C) in vitro, and induced tyrosyl phosphorylation of endogenous PLC-gamma1 in vivo. Substitution of Tyr-525 and Tyr-526 at the autophosphorylation site of Syk in mCD8-Syk substantially reduced the kinase activity and the binding of this variant chimera to PLC-gamma1 SH2(C) in vitro; it also failed to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. In contrast, substitution of Tyr-348 and Tyr-352 in the linker region of Syk in mCD8-Syk did not affect the kinase activity of this variant chimera but almost completely eliminated its binding to PLC-gamma1 SH(C) and completely eliminated its ability to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. Thus, an optimal kinase activity of Syk and an interaction between the linker region of Syk with PLC-gamma1 are required for the tyrosyl phosphorylation of PLC-gamma1.

Signaling via the anti-CD30 mAb SGN-30 sensitizes Hodgkin's disease cells to conventional chemotherapeutics.

SGN-30, a monoclonal antibody with activity against CD30+ malignancies, is currently in phase II clinical evaluation for treatment of Hodgkin's disease (HD) and anaplastic large cell lymphoma. The mechanisms underlying SGN-30's antitumor activity were investigated using cDNA array of L540 cells. SGN-30 treatment activated NF-kappaB and modulation of several messages including the growth regulator p21WAF1/CIP1 (p21) and cellular adhesion marker ICAM-1. p21 protein levels increased coincident with growth arrest and Annexin V/PI staining in treated HD cells. To determine if SGN-30-induced growth arrest would sensitize tumor cells to chemotherapeutics used against HD, L540cy and L428 cells were exposed to SGN-30 in combination with a panel of cytotoxic agents and resultant interactions quantified by the Combination Effects Method. Interactions between SGN-30 and all cytotoxic agents examined were additive or better. These in vitro data translated to increased efficacy of SGN-30 and bleomycin against L540cy tumor xenografts. In addition to direct cell killing, SGN-30 affects growth arrest and drug sensitization through growth regulating and proapoptotic machinery. Importantly, these data suggest that SGN-30 can enhance the efficacy of standard chemotherapies used to treat patients with CD30+ malignancies.

FcgammaRII tyrosine phosphorylation differs between FcgammaRII cross-linking and platelet-activating anti-platelet monoclonal antibodies.

Using glutathione S-transferase Syk fusion proteins, we evaluated the mode of platelet FcgammaRII tyrosine phosphorylation induced by FcgammaRII cross-linking or anti-CD9 monoclonal antibodies (mAb). The N-terminal SH2 domain of Syk (Syk-N-SH2), the C-terminal SH2 domain of Syk (Syk-C-SH2), and the domain having both the N- and C-terminal SH2 of Syk (Syk-NC-SH2) all bound to tyrosine-phosphorylated FcgammaRII with FcgammaRII cross-linking. In the case of anti-CD9 mAb-induced platelet activation, only Syk-C-SH2 and Syk-NC-SH2 bound to tyrosine-phosphorylated FcgammaRII. Since the SH2 domain is specific for a particular structure containing phosphotyrosine, these findings suggest that only one tyrosine residue in the immunoreceptor tyrosine-based activation motif (ITAM) is phosphorylated with anti-CD9 mAb, and that both are phosphorylated with FcgammaRII cross-linking. Synthetic peptides corresponding to the ITAM of human platelet FcgammaRII with the N-terminal tyrosine residue phosphorylated (N-P) or the C-terminal tyrosine residue phosphorylated (C-P), were used. N-P more potently dissociated Syk-C-SH2 from tyrosine-phosphorylated FcgammaRII than C-P, suggesting that the N-terminal tyrosine residue is phosphorylated upon anti-CD9 mAb-induced activation. Furthermore, these findings imply that Syk-N-SH2 binds to the phosphorylated C-terminal tyrosine residue of ITAM, and Syk-C-SH2 to the N-terminal tyrosine. Taken together, our findings suggest that FcgammaRII-dependent platelet activation without FcgammaRII dimerization, such as with anti-CD9 mAb, is distinct from that induced by FcgammaRII cross-linking.

Analysis of expression and function of CD40 on normal and leukemic human B cell precursors.

CD40 was originally identified on circulating and tonsillar human B cells, and anti-CD40 monoclonal antibodies (MoAb) are known to deliver a progression signal to activated B cells. However, the expression and function of CD40 on human B cell precursors (BCP) have not been examined in detail. Two new anti-CD40 MoAb were produced and shown to recognize an epitope indistinguishable from the anti-CD40 antibody G28-5. CD40 was readily detected on normal and leukemic BCP by flow cytometry, and cell surface expression was upregulated by phorbol ester. Despite the ability of normal and leukemic BCP to respond to phorbol ester (PMA) and/or low molecular weight B cell growth factor (L-BCGF), anti-CD40 exerted no stimulatory action and could not enhance the response of these cells to PMA, L-BCGF, or both. Cross-linking anti-CD40 MoAb with rabbit anti-mouse Ig also failed to induce a proliferative response in normal BCP. We conclude that anti-CD40 does not exert demonstrable agonistic effects on normal and leukemic human BCP. Our results suggest that signal delivery through CD40 and/or subsequent intracellular signal processing may require accessory molecules not expressed in BCP, or CD40 may subserve a different function for BCP.

Multiparameter flow cytometric analysis of human fetal bone marrow B cells.

Maturation of adult human bone marrow (BM) B cells is accompanied by the sequential acquisition and loss of characteristic cell surface antigens (Loken et al., Blood 70:1316). Little is known about these changes in fetal BM B cells. In order to compare fetal with adult B cell development, we performed three-color, flow cytometric analyses of cell surface antigens, as well as nuclear TdT staining, on lymphoid cells from fetal BM. Mononuclear cells isolated from fetal BM (18-22 weeks) were stained with combinations of antibodies against CD3, CD10, CD19, CD20, CD21, CD22, CD34, CD45, PCA-1, IgM, and HLA-DR. Analysis of six separate fetal BM specimens indicated that combinations of cell surface antigens were expressed on analogous populations in fetal and adult BM. Consistent with adult BM, greater than 95% of TdT+ cells within the CD10+ population were CD34+, whereas less than 5% were CD34-. This CD10+/CD34+/TdT+ population constituted 30-40% of the total B cell compartment, compared with 10% in adults. Quantitative changes in CD45 expression on fetal BM B cells defined three clear populations, as has been observed in adults. In striking contrast to adult BM, greater than 95% of CD19+ and greater than 95% of surface IgM+ cells were CD10+, indicating that CD10 is a pan-B cell antigen in fetal BM. Virtually no mature B cells expressing CD21, CD22, or PCA-1 were detected in fetal BM. Our results indicate a preponderance of immature phenotypes exist in the fetal BM B cell compartment. These immature cells can be grouped into three distinct populations, and probably correspond to expanded populations found less frequently in adult BM. This striking increase in the earliest identifiable stages of B cell ontogeny is consistent with an active expansion of cells destined to constitute the humoral immune system during fetal development.

Factors associated with the detection of Entamoeba histolytica in homosexual men.

In a study of bowel parasites in 128 Australian homosexual men attending a sexually transmitted diseases (STD) clinic, Entamoeba histolytica was detected in 37%, Giardia intestinalis in 3% and at least one protozoan in 81% of the group. There was no evidence of pathogenicity of E. histolytica, nor was there any association between the detection of E. histolytica and sexual practices, gastrointestinal symptoms, proctitis, human immunodeficiency virus antibody result or T-cell subset values. However it was noted that those subjects with an elevated IgM greater than 4 g/l were more likely to harbour E. histolytica trophozoites than those with a level below 4 g/l (OR 6.54, 95% CI 1.3-32.8). Travel to South-East Asia, India, China, Africa, South America or the Pacific islands in the previous 3 years emerged also as an independent factor (OR 2.70, 95% CI 1.12-6.53) associated with detection of E. histolytica.

Clinical and virological associations between external anogenital warts and cervical HPV infection in an STD clinic population.

To determine the significance of overt anogenital warts as indicators of human papillomavirus (HPV) infection of the cervix, 177 women attending a Sydney STD clinic were screened for evidence of cervical HPV infection using clinical criteria together with cytology and HPV DNA dot hybridization. HPV DNA probing was also performed on biopsies of 50 exophytic warts. A very high prevalence of both anogenital warts (40%), and of cervical HPV infection (58%) was indicated in this group of women. In the exophytic warts, HPV types 6/11 were most commonly detected, whereas the rates of detection of types 6/11 and 16/18 in the cervix were similar. Of the 87 women with evidence of cervical HPV infection, 57 (66%) had a history of either past or current overt exophytic anogenital warts; while the corresponding figure for the 90 women with no evidence of cervical infection was 45 (50%). Cytological evidence of dysplasia (CIN I-III) was detected in 13 (7%) of the cervical smears: of these, 4 were positive for HPV 16/18 only, 2 for 6/11 only and 4 for both 6/11 and 16/18.

Proapoptotic signaling activity of the anti-CD40 monoclonal antibody dacetuzumab circumvents multiple oncogenic transformation events and chemosensitizes NHL cells.

Non-Hodgkin lymphoma (NHL) is a genetically heterogeneous disease with several oncogenic events implicated in the transformation of normal developing B lymphocytes. The objective of this study was to elucidate the signal transduction-based antitumor mechanism(s) of action for the anti-CD40 monoclonal antibody dacetuzumab (SGN-40) in NHL. We report that dacetuzumab activates two distinct proapoptotic signaling pathways, overcoming transformation events key to the pathogenesis of NHL. Dacetuzumab-mediated CD40 signaling constitutively activated the nuclear factor-κB and mitogen-activated protein kinase signaling pathways producing the sustained downregulation of B-cell lymphoma 6 (BCL-6), an oncoprotein implicated in lymphomagenesis. Loss of BCL-6 resulted in c-Myc downregulation and activation of a transcriptional program characteristic of early B-cell maturation, concomitant with reduced proliferation and cell death. In a second mechanism, dacetuzumab signaling induced the expression of the proapoptotic p53 family member TAp63α and downstream proteins associated with the intrinsic and extrinsic apoptotic machinery. Dacetuzumab was synergistic in combination with DNA-damaging chemotherapeutic drugs, correlating with TAp63α upregulation. Furthermore, dacetuzumab augmented the activity of rituximab in combination with multiple chemotherapies in the xenograft models of NHL. The ability of dacetuzumab signaling to circumvent oncogenic events and potentiate the activity of chemotherapy regimens provides a unique therapeutic approach to NHL.

Asparagine transport and the induction of ornithine decarboxylase in H-35 rat hepatoma cells.

A number of amino acids, most noticeably asparagine, were capable of inducing ornithine decarboxylase in H-35 rat hepatoma cells. The effective amino acids were all neutral and were substrates of the Na+-dependent transport systems A, ASC and N. Transport inhibitor studies indicated that there was an excellent correlation between the level of enzyme activity induced and the initial rate of asparagine transport into the cells by the A and the N systems. It is proposed that the activation of the Na+-dependent, pH-sensitive amino acid transport systems and the subsequent intracellular pH and ionic perturbation constitute part of the initiation signals for cell activation.

The induction of ornithine decarboxylase by L-asparagine and intracellular alkalinization.

The uptake of transport systems A and N amino acids, most noticeably L-asparagine, is essential for the induction of ornithine decarboxylase (L-ornithine carboxylase, EC 4.1.1.17) in cultured cells and we have proposed that the uptake-associated pH and ionic changes might constitute part of the cell activation signal (1). In the present study, it was shown that extracellular L-asparagine caused an immediate and transient increase in intracellular pH which was continuously monitored by the fluorescence probe BCECF (2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein). NH4Cl and NH4OH which caused intracellular alkalinization also caused ornithine decarboxylase activity to increase.

Possible role of the membrane Na+/H+ antiport in ornithine decarboxylase induction by L-asparagine.

Amino acids of transport systems A and N play certain important role in cell activation. For example, the presence of these amino acids is essential in the induction of ornithine decarboxylase by growth factors and hormones. At mM concentrations, each of these amino acids, particularly L-asparagine, can also induce the enzyme without being further metabolized or incorporated into proteins. We have reported that the addition of 10 mM L-asparagine to quiescent Reuber's H-35 rat hepatoma cells caused an immediate and transient increase in intracellular pH. Here we report that concomitant with the intracellular alkalinization was an increase in H+ extrusion which was amiloride-sensitive and Na+-dependent. The induction of ornithine decarboxylase by L-asparagine was also amiloride-sensitive.

Rectal spirochaetosis in homosexual men: the association with sexual practices, HIV infection and enteric flora.

OBJECTIVE: To determine the prevalence of rectal spirochaetosis in homosexual men attending a sexually transmissible diseases clinic and investigate the association between their presence and sexual practices, HIV infection and enteric flora. DESIGN: The study included 144 male homosexual subjects who each completed a questionnaire, underwent physical examination, proctoscopy and investigations for STD and HIV screening, rectal biopsies and collection of faecal samples. SETTING: The Sexual Health Centre, Sydney Hospital, Sydney, Australia. RESULTS: Spirochaetes were detected in 39% of the rectal biopsies, using histological criteria. Logistic regression analysis showed that rectal spirochaetosis was significantly associated with: oral-anal contact. (P < 0.05, OR 3.45, 95% CI 1.48-8.05); detection of 3-5 different non-pathogenic protozoa in faeces (P < 0.01, OR 11.68, 95% C.I. 2.33-58) and a positive HIV antibody test (P < 0.01) OR 4.48, 95% C.I. 1.28-15.72). CONCLUSIONS: These findings indicate that rectal spirochaetosis is relatively common in homosexual men. The association with non-pathogenic protozoa is most likely attributable to the common mode of transmission viz oral-anal contact. However it is difficult to determine whether the association with HIV infection is cause or effect because of the limitations in the study design. Further information is required to determine the clinical significance of infection with these organisms.

Effects of polyamines on the uptake of neurotransmitters by rat brain synaptosomes.

The influence of putrescine, spermidine, spermine, and some aliphatic alpha, w-diamines on the uptake spermine, and some aliphatic alpha, w-diamines on the uptake of neurotransmitters by rat forebrain synaptosomes was investigated. Choline uptake was most effectively inhibited by spermine (IC50 = 0.22 mM), less so by spermidine (IC50 = 4.0 mM), but not by putrescine (IC50 greater than 100 mM). At 10 mM, 1,3-diaminopropane, cadaverine, and 1,8-diaminooctane all inhibited choline uptake by 50% or more. Spermine and spermidine inhibited the uptake of dopamine with IC50 values of 2.7 and 2.2 mM, respectively. Putrescine was only slightly inhibitory (IC50 = 17.3 mM) and the other diamines were inactive. The uptake of gamma-aminobutyrate (GABA) was only slightly inhibited (15-40%) by the polyamines at 10 mM. With the exception of inhibition of glycine uptake by 1,8-diaminooctane (60%) and of glutamate uptake by cadaverine (35%) none of the polyamines, tested at 10 mM, affected the uptake of adenosine, glutamate, and glycine significantly. A possible modulatory role for polyamines in synaptic transmission through interaction by negatively charged groups of the synaptic membrane with the polycationic compounds is discussed.