Functional sialylated O-glycan to platelet aggregation on Aggrus (T1alpha/Podoplanin) molecules expressed in Chinese hamster ovary cells

Aggrus, also called T1alpha and podoplanin, is a novel platelet aggregation-inducing factor that is expressed in various carcinoma cells. Aggrus/T1alpha/podoplanin is known to be expressed in lung type I alveolar cells or lymphatic endothelial cells. However, its physiological role has not been clarified. To assess the attribution of glycosylation to Aggrus platelet aggregation activity, recombinant molecules were stably expressed in a series of Chinese hamster ovary (CHO) cell mutants, N-glycan-deficient Lec1, CMP-sialic acid transporter-deficient Lec2, and UDP-galactose transporter-deficient Lec8. A new anti-human Aggrus monoclonal antibody, YM-1, was established to detect the expression of human Aggrus on these CHO cell mutants. Aggrus on Lec1 cells induced platelet aggregation, but those on Lec2 and Lec8 cells did not. Further, the glycans on Aggrus were analyzed by lectin blotting. Aggrus expressed in CHO and Lec1 cells showed Wheat-germ agglutinin, Jacalin, and Vicia villosa lectin bindings. Lectin blotting results indicated that sialylated core 1 structures, sialic acid plus Galbeta1,3GalNAc-Ser/Thr, were critical for the platelet aggregation activity. This oligosaccharide structure is known as tumor-associated antigen, which is potentially related to the metastasis process of cancer cells.     

Platelets Promote Tumor Growth and Metastasis via Direct Interaction between Aggrus/Podoplanin and CLEC-2

The platelet aggregation-inducing factor Aggrus, also known as podoplanin, is frequently upregulated in several types of tumors and enhances hematogenous metastasis by interacting with and activating the platelet receptor CLEC-2. Thus, Aggrus–CLEC-2 binding could be a therapeutic molecular mechanism for cancer therapy. We generated a new anti-human Aggrus monoclonal antibody, MS-1, that suppressed Aggrus–CLEC-2 binding, Aggrus-induced platelet aggregation, and Aggrus-mediated tumor metastasis. Interestingly, the MS-1 monoclonal antibody attenuated the growth of Aggrus-positive tumors in vivo. Moreover, the humanized chimeric MS-1 antibody, ChMS-1, also exhibited strong antitumor activity against Aggrus-positive lung squamous cell carcinoma xenografted into NOD-SCID mice compromising antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities. Because Aggrus knockdown suppressed platelet-induced proliferation in vitro and tumor growth of the lung squamous cell carcinoma in vivo, Aggrus may be involved in not only tumor metastasis but also tumor growth by promoting platelet-tumor interaction, platelet activation, and secretion of platelet-derived factors in vivo. Our results indicate that molecular target drugs inhibiting specific platelet–tumor interactions can be developed as antitumor drugs that suppress both metastasis and proliferation of tumors such as lung squamous cell carcinoma.     

Functional glycosylation of human podoplanin: glycan structure of platelet aggregation-inducing factor

Podoplanin (Aggrus) is a mucin-type sialoglycoprotein that plays a key role in tumor cell-induced platelet aggregation. Podoplanin possesses a platelet aggregation-stimulating (PLAG) domain, and Thr52 in the PLAG domain of human podoplanin is important for its activity. Endogenous or recombinant human podoplanin were purified, and total glycosylation profiles were surveyed by lectin microarray. Analyses of glycopeptides produced by Edman degradation and mass spectrometry revealed that the disialyl-corel (NeuAc alpha2-3Gal beta l-3(NeuAc alpha2-6)GalNAc alpha l-O-Thr) structure was primarily attached to a glycosylation site at residue Thr52. Sialic acid-deficient podoplanin recovered its activity after additional sialylation. These results indicated that the sialylated Corel at Thr52 is critical for podoplanin-induced platelet aggregation.     

Inhibition of tumor cell-induced platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating domain

The mucin-type sialoglycoprotein, podoplanin (aggrus), is a platelet-aggregating factor on cancer cells. We previously described up-regulated expression of podoplanin in malignant astrocytic tumors including glioblastoma. Its expression was associated with tumor malignancy. In the present study, we investigated podoplanin expression and platelet-aggregating activities of glioblastoma cell lines. First, we established a highly reactive anti-podoplanin antibody, NZ-1, which inhibits podoplanin-induced platelet aggregation completely. Of 15 glioblastoma cell lines, LN319 highly expressed podoplanin and induced platelet aggregation. Glycan profiling using a lectin microarray showed that podoplanin on LN319 possesses sialic acid, which is important in podoplanin-induced platelet aggregation. Interestingly, NZ-1 neutralized platelet aggregation by LN319. These results suggest that podoplanin is a main reason for platelet aggregation induced by LN319. We infer that NZ-1 is useful to determine whether platelet aggregation is podoplanin-specific or not. Furthermore, podoplanin might become a therapeutic target of glioblastoma for antibody-based therapy.     

Aggretin, a C-type lectin protein, induces platelet aggregation via integrin alpha(2)beta(1) and GPIb in a phosphatidylinositol 3-kinase independent pathway.

Aggretin purified from Calloselasma rhodostoma venom was previously identified as alpha(2)beta(1) agonist in triggering platelet aggregation, and exists as a heterodimer sharing a great homologous sequence to GPIb binding proteins. We show here that binding to GPIb is also required in aggregation-inducing activity of aggretin. A2-IIE10, an anti-integrin alpha(2) monoclonal antibody, delayed platelet aggregation while agkistin, a GPIb antagonist, only slightly inhibited platelet aggregation caused by aggretin. However, the aggretin-induced platelet aggregation was completely abolished by a combination of A2-IIE10 and agkistin. Either A2-IIE10 or agkistin significantly inhibited the binding of FITC-aggretin toward fixed platelets. Aggretin and collagen induced a similar signal transduction in platelets involving a time-dependent tyrosine phosphorylation of p125(FAK) and PLCgamma2, but aggretin caused a much-delayed tyrosine-phosphorylation of PI 3-kinase compared with collagen. LY294002, a PI 3-kinase inhibitor, showed a significant inhibitory effect on collagen, but not aggretin-stimulated platelet aggregation. These findings indicate aggretin induces platelet aggregation via binding of alpha(2)beta(1) and GPIb, causing phosphorylation of p125(FAK) and PLCgamma2 leading to platelet activation without the involvement of PI 3-kinase activation.     

Integrin alpha3beta1 expressed by human colon cancer cells is a major carrier of oncodevelopmental carbohydrate epitopes

Oncodevelopmental carbohydrate epitopes are a common feature of human colorectal carcinoma, yet their biological significance remains unclear. We have shown previously that monoclonal antibody (MAb) 3A7, which recognizes a determinant on type 2 chain blood group A and B oligosaccharides, detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and some human colon cancer cell lines. (Laferté S et al. [19951 J. Cell. Biochem. 57:101-119). In this study, we set out to purify gp140, the major glycoprotein carrier of the 3A7 epitope expressed by human colon cancer cells, as a first step towards elucidating the contribution of the 3A7 epitope and its major glycoprotein carrier to colon cancer progression. To this end, gp140 was purified from HT29 cells and used for the preparation of polypeptide-specific monoclonal antibodies. Five monoclonal antibodies (7A8, 7B11, 8C7, 8H7, and 11D4) immunoprecipitated a 3A7-immunoreactive glycoprotein complex of 140 kDa which was subsequently identified by partial protein sequencing as alpha3beta1 integrin. Flow cytometric analysis of Chinese hamster ovary (CHO) cells expressing different human integrin chains revealed that MAbs 7A8 and 7B11 detect the alpha3 integrin subunit whereas MAbs 8C7 and 8H7 detect the beta1 integrin subunit. MAb 11D4, which did not bind to any of the CHO transfectants, detected type 2 chain blood group A determinant. Flow cytometric analysis of a panel of human colon carcinoma cell lines obtained from blood group A, AB, or B individuals revealed a direct correlation between cell-surface expression of the 3A7 epitope and alpha3 integrin subunit, suggesting that alpha3beta1 integrin is a preferred target of the 3A7 epitope in colon cancer cells. Using lectins and glycosidases to examine further the carbohydrate structure of alpha3beta1 integrin, we demonstrated that it is a sialoglycoprotein containing both N- and O-linked oligosaccharides. In addition, both alpha3 and beta1 integrin subunits express beta1-6 branched Asn-linked oligosaccharides and short poly-N-acetyllactosamine units (Galbeta1-4GlcNAc-R; n < or = 3), glycans previously implicated in cancer metastasis.Thus, alpha3beta1 integrin expressed by human colon carcinoma cells is a major carrier of oncodevelopmental carbohydrate epitopes whose presence may modulate tumor cell adhesion, migration, and/or invasion.     

Lack of effector cell function and altered tetramer binding of tumor-infiltrating lymphocytes

Tumor-specific CD8 T cell responses to MCA102 fibrosarcoma cells expressing the cytotoxic T cell epitope gp33 from lymphocytic choriomeningitis virus were studied. MCA102(gp33) tumors grew progressively in C57BL/6 mice, despite induction of peripheral gp33-tetramer(+) T cells that were capable of mediating antiviral protection, specific cell rejection, and concomitant tumor immunity. MCA102(gp33) tumors were infiltrated with a high number ( approximately 20%) of CD11b(+)CD11c(-) macrophage-phenotype cells that were able to cross-present the gp33 epitope to T cells. Tumor-infiltrating CD8 T cells exhibited a highly activated phenotype but lacked effector cell function. Strikingly, a significant portion of tumor-infiltrating lymphocytes expressed TCRs specific for gp33 but bound MHC tetramers only after cell purification and a 24-h resting period in vitro. The phenomenon of "tetramer-negative T cells" was not restricted to tumor-infiltrating lymphocytes from MCA102(gp33) tumors, but was also observed when Ag-specific T cells derived from an environment with high Ag load were analyzed ex vivo. Thus, using a novel tumor model, allowing us to trace tumor-specific T cells at the single cell level in vivo, we demonstrate that the tumor microenvironment is able to alter the functional activity of T cells infiltrating the tumor mass.     

The F2-isoprostane, 8-epi-prostaglandin F2 alpha, a potent agonist of the vascular thromboxane/endoperoxide receptor, is a platelet thromboxane/endoperoxide receptor antagonist.

F2-isoprostanes are a recently discovered series of prostaglandin (PG)F2-like compounds that are produced in vivo in humans by nonenzymatic free radical catalyzed peroxidation of arachidonic acid. One of the compounds that can be produced in abundance by this mechanism is 8-epi-PGF2 alpha. 8-epi-PGF2 alpha is a potent vasoconstrictor in the rat, an effect that has been shown to be mediated via interaction with vascular thromboxane (TxA2)/endoperoxide (PGH2) receptors. In an effort to further understand the biological properties of this prostanoid in relation to its ability to interact with TxA2/PGH2 receptors, we examined its effects on human and rat platelets. At concentrations of 10(-6) M and 10(-5) M, 8-epi-PGF2 alpha induced only a shape change in human platelets and at higher concentrations (10(-4) M) induced reversible but not irreversible aggregation. Both the shape change and reversible aggregation were unaffected by indomethacin but were inhibited by the TxA2/PGH2 receptor antagonist SQ29548. Conversely, 8-epi-PGF2 alpha inhibited platelet aggregation induced by the TxA2/PGH2 receptor agonists U46619 (10(-6) M) and IBOP (3.3 x 10(-7) M) with an IC50 of 1.6 x 10(-6) M and 1.8 x 10(-6) M, respectively. 8-epi-PGF2 alpha also inhibited platelet aggregation induced by arachidonic acid. Similarly, in rat platelets, 8-epi-PGF2 alpha alone induced only modest reversible aggregation but completely inhibited U46619-induced aggregation.     

Alpha IIb beta 3 integrin expression and function in subpopulations of murine tumors.

Subpopulations of B16 amelanotic melanoma (B16a) cells, isolated by centrifugal elutriation from enzymatically dispersed solid tumors, demonstrated different abilities to form lung colonies when injected intravenously. In contrast, no differences in experimental metastasis were observed among subpopulations obtained from Lewis lung (3LL) tumors. Lung colonization by B16a and 3LL subpopulations correlated positively with observed differences (B16a) or lack of differences (3LL) in tumor cell ability to induce aggregation of homologous platelets, to adhere to subendothelial matrix or fibronectin, and with the percentage of cells in the G2/M phase of the cell cycle. Both B16a and 3LL cells express alpha IIb beta 3 integrin receptors; however, differences in the receptor expression level were found only among B16a subpopulations. Comparison of the amount of alpha IIb beta 3 receptor expressed on cell surface with tumor cell ability to induce platelet aggregation (TCIPA) and to adhere to fibronectin or subendothelial matrix revealed a positive correlation. Pretreatment of tumor cells with alpha IIb beta 3-specific antibodies inhibited tumor cell matrix adhesion, TCIPA, and lung colony formation. We propose that alpha IIb beta 3 integrin receptor expression, tumor cell matrix adhesion, and tumor cell-induced platelet aggregation can be important parameters to indicate the metastatic potential of some tumor cells and that the alpha IIb beta 3 is a multifunctional receptor involved in both tumor cell-matrix and tumor cell-platelet interactions. Further, the correlation among cell cycle phase, metastatic ability, and receptor expression suggests that metastatic propensity may be transiently expressed and/or increased in some tumor cell subpopulations.     

Involvement of the snake toxin receptor CLEC-2, in podoplanin-mediated platelet activation, by cancer cells

Podoplanin (aggrus), a transmembrane sialoglycoprotein, is involved in tumor cell-induced platelet aggregation, tumor metastasis, and lymphatic vessel formation. However, the mechanism by which podoplanin induces these cellular processes including its receptor has not been elucidated to date. Podoplanin induced platelet aggregation with a long lag phase, which is dependent upon Src and phospholipase Cgamma2 activation. However, it does not bind to glycoprotein VI. This mode of platelet activation was reminiscent of the snake toxin rhodocytin, the receptor of which has been identified by us as a novel platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2) (Suzuki-Inoue, K., Fuller, G. L., Garcia, A., Eble, J. A., Pohlmann, S., Inoue, O., Gartner, T. K., Hughan, S. C., Pearce, A. C., Laing, G. D., Theakston, R. D., Schweighoffer, E., Zitzmann, N., Morita, T., Tybulewicz, V. L., Ozaki, Y., and Watson, S. P. (2006) Blood 107, 542-549). Therefore, we sought to evaluate whether CLEC-2 serves as a physiological counterpart for podoplanin. Association between CLEC-2 and podoplanin was confirmed by flow cytometry. Furthermore, their association was dependent on sialic acid on O-glycans of podoplanin. Recombinant CLEC-2 inhibited platelet aggregation induced by podoplanin-expressing tumor cells or lymphatic endothelial cells, suggesting that CLEC-2 is responsible for platelet aggregation induced by endogenously expressed podoplanin on the cell surfaces. These findings suggest that CLEC-2 is a physiological target protein of podoplanin and imply that it is involved in podoplanin-induced platelet aggregation, tumor metastasis, and other cellular responses related to podoplanin.     

Immunohistochemical detection of a cross-reacting virus antigen in mouse mammary tumors and human breast carcinomas.

An indirect immunoperoxidase method was first used to localize mouse mammary tumor virus (MMTV) antigens in paraffin sections of mammary tumors of Paris RIII and CD8F1 mice. By using the same method, an antigen with cross-reactivity to a group-specific antigen (gp52, a 52,000 dalton glycoprotein) of MMTV was detected in paraffin sections of human breast carcinomas. The specificity of this reaction with antibody against MMTV was examined by absorption of the IgG with: a) purified gp52; b) several relevant and irrelevant viral preparations; c) normal human plasma, leukocytes, breast tissue, milk, actin, collagen, and hyaluronic acid; d) sheep erythrocytes, bovine mucin and fetal calf serum. Only MMTV and prufied gp52 eliminated the immunohistochemical reaction in human breast tumors. Positive reactions were seen in 73 of 191 (38%) breast carcinomas of various histopathologic types, while negative reactions were obtained in all 137 normal and benign cases tested. A positive reaction of uncertain specificity was observed in foci of apocrine metaplasia. With one exception, 99 carcinomas from 13 organs other than breast and eight cystosarcomas were negative.     

CTL-dependent and -independent antitumor immunity is determined by the tumor not the vaccine

Previously, we compared the efficiency of direct injection with an adenovirus (Ad) expressing human gp100 (hgp100) to immunization with dendritic cells (DC) loaded with the same vector ex vivo. The DC vaccine provided the greatest protection against challenge with B16F10 melanoma, and antitumor immunity was found to be CD8(+) T cell-independent. In the current study, we sought to determine whether lack of CD8(+) T cell-mediated antitumor immunity was a function of the vaccine platform or the tumor line. Both Ad and DC/Ad vaccines elicited CD8(+) CTL reactive against hgp100 and provided protection against B16F10 engineered to express hgp100 demonstrating that both vaccination platforms can effectively generate protective CD8(+) T cell-mediated immunity. The hgp100-induced CTL cross-reacted with murine gp100 (mgp100) and lysed B16F10 cells pulsed with mgp100 peptide indicating that the resistance of B16F10 cells to CTL elicited by hgp100 vaccination may be due to a defect in processing of the endogenous mgp100. Indeed, introduction of the TAP-1 cDNA into B16F10 rendered the cells sensitive to lysis by gp100-specific CTL. Furthermore, gp100-immunized mice were protected from challenge with B16F10-TAP1 cells through a mechanism dependent upon CD8(+) T cells. These results demonstrate that tumor phenotype, not the vaccination platform, ultimately determines CD8(+) or CD4(+) T cell-mediated tumor clearance.     

Expression of the full-length form of gp2 of equine herpesvirus 1 (EHV-1) completely restores respiratory virulence to the attenuated EHV-1 strain KyA in CBA mice

Wild-type equine herpesvirus 1 (EHV-1) strains express a large (250-kDa) glycoprotein, gp2, that is encoded by EUs4 (gene 71) located within the unique short region of the genome. DNA sequence analysis revealed that EUs4 of the pathogenic EHV-1 strain RacL11 is an open reading frame of 2,376 bp that encodes a protein of 791 amino acids. The attenuated EHV-1 vaccine strain KyA harbors an in-frame deletion of 1,242 bp from bp 222 to 1461 and expresses a truncated gp2 of 383 amino acids. To determine the relative contribution of gp2 to EHV-1 pathogenesis, we compared the course of respiratory infection of CBA mice infected with either wild-type RacL11, attenuated KyA, or a recombinant KyA that expresses the full-length gp2 protein (KyARgp2F). Mice infected with KyA lost a negligible amount of body weight (0.18% total weight loss) on day 1 postinfection and regained weight thereafter, whereas mice infected with KyARgp2F or RacL11 steadily lost weight beginning on day 1 and experienced a 20 and 18% loss in body weight, respectively, by day 3. Immunohistochemical and flow cytometric analyses revealed higher numbers of T and B lymphocytes and an extensive consolidation consisting of large numbers of Mac-1-positive cells in the lungs of animals infected with KyARgp2F compared to animals infected with KyA. RNase protection analyses revealed increased expression of numerous cytokines and chemokines, including interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor alpha, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, MIP-2, interferon gamma-inducible protein, monocyte chemotactic protein 1, and T-cell activation gene 3 at 12 h postinfection with KyARgp2F. Three independent DNA array experiments confirmed these results and showed a 2- to 13-fold increase in the expression of 31 inflammatory genes at 8 and 12 h postinfection with KyARgp2F compared to infection with KyA. Taken together, the results indicate that expression of full-length gp2 is sufficient to restore full respiratory virulence to the attenuated KyA strain and raise caution concerning the inclusion of full-length gp2 in the development of EHV-1 vaccines.     

Cytokine-enhanced expression of glycoprotein Ib alpha in human endothelium

Platelet glycoprotein Ib is a major platelet membrane protein composed of two disulfide-linked chains, termed the alpha and beta chains. The larger alpha chain (GpIb alpha), a platelet receptor for von Willebrand factor, plays a major role in mediating platelet adhesion to the subendothelium. Our laboratories have previously reported synthesis of a protein in human endothelial cells that is immunoprecipitated with polyclonal and monoclonal antibodies to platelet GpIb alpha. Lopez et al. (Lopez, J. A., Chung, D. W., Fujikawa, K., Hagan, F. S., Papayannopoulou, T., and Roth, G. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5615-5619) have reported the cloning of GpIb alpha from a human erythroleukemia (HEL) cell cDNA library. Using this clone as probe, we have isolated two partial GpIb alpha clones from a human umbilical vein endothelial cell lambda gt11 cDNA library. These clones were localized within HEL-derived GpIb alpha cDNA by sequence and restriction enzyme analysis. Additionally, they detected the same message species in HEL and tonsilar RNA that was detected with the HEL GpIb alpha cDNA. Low level GpIb alpha mRNA expression was detected in cultured human umbilical vein endothelial cells, which was increased by treatment of the cells with tumor necrosis factor-alpha. This effect was enhanced by pretreatment with interferon-gamma. Additionally, localization of GpIb alpha in endothelium of fresh tonsilar tissue was demonstrated by immunohistochemistry and in situ hybridization. GpIb alpha may play a role in mediating platelet or other effector cell adhesion to activated endothelium.     

Induction of antigen-specific immune responses against malignant brain tumors by intramuscular injection of sindbis DNA encoding gp100 and IL-18

We constructed pSin-SV40-HDV-SV40pA, an improved Sindbis DNA expression vector, and evaluated the potential of this vector system for brain tumor therapy. We investigated whether immunizing mice with xenogeneic DNA encoding human gp100 and mouse IL-18 would enhance the antitumor responses. To study the immune mechanisms involved in tumor regression, we examined tumor growth in B16-gp100-implanted brain tumor models using T-cell subset-depleted and IFN-gamma-neutralized mice. Hugp100/mIL-18 vaccination was also investigated for its antitumor effects against the wild-type murine B16 tumor, which expresses the murine gp100 molecule. Genetic immunization using plasmid pSin 9001 DNA codelivery of human gp100 and mouse IL-18 resulted in enhanced protective and therapeutic effects on the malignant brain tumors. The antitumor and protective effects were mediated by both CD4(+)/CD8(+) T cells and IFN-gamma. Vaccination with hugp100/mIL-18 conferred a significant survival merit to wild-type B16 tumor-harboring mice. Immunogene therapy with the improved Sindbis virus vector expressing xenogeneic gp100 and syngeneic IL-18 may be an excellent approach for developing a new treatment protocol. Thus, the Sindbis DNA system may represent a novel approach for the treatment of malignant brain tumors.     

Inhibition of human tumor cell induced platelet aggregation by antibodies to platelet glycoproteins Ib and IIb/IIIa

Tumor cell induced platelet aggregation was shown to be inhibited in a dose dependent manner by preincubation of human platelets with antibodies to platelet glycoprotein Ib and the IIb/IIIa complex. Combination of antibody to Ib and antibody to the IIb/IIIa complex at concentrations which produced half maximal inhibition of platelet aggregation alone caused complete inhibition of tumor cell induced platelet aggregation. Antibodies to platelet glycoproteins Ib and the IIb/IIIa complex also inhibited platelet synthesis of thromboxane A2, but not synthesis of 12-hydroxyeicosatrienoic acid. Inhibition of tumor cell induced platelet aggregation with antibodies against platelet glycoproteins suggests a role for these glycoproteins in tumor cell-platelet interactions and possibly platelet facilitated tumor cell metastasis.     

Induction of podoplanin by transforming growth factor-beta in human fibrosarcoma

Podoplanin/aggrus is increased in tumors and its expression was associated with tumor malignancy. Podoplanin on cancer cells serves as a platelet-aggregating factor, which is associated with the metastatic potential. However, regulators of podoplanin remain to be determined. Transforming growth factor-beta (TGF-beta) regulates many physiological events, including tumorigenesis. Here, we found that TGF-beta induced podoplanin in human fibrosarcoma HT1080 cells and enhanced the platelet-aggregating-ability of HT1080. TGF-beta type I receptor inhibitor (SB431542) and short hairpin RNAs for Smad4 inhibited the podoplanin induction by TGF-beta. These results suggest that TGF-beta is a physiological regulator of podoplanin in tumor cells.     

Modulation of CD103 expression on human colon carcinoma-specific CTL

Recent results have shown a correlation between survival and frequency of tumor-infiltrating T cells in colorectal cancer patients. However, the mechanisms controlling the ability of human T lymphocytes to infiltrate colon carcinoma remain unclear. Although, it is known that expression of the integrin CD103alpha(E)/beta(7) by intraepithelial lymphocytes controls the retention of lymphocytes in epithelial layers, very little is known about the expression of intestinal homing receptors in human T lymphocytes. In particular, it remains unknown whether expression of CD103/beta(7) by human colon cancer-specific T lymphocytes is controlled by recognition of tumor Ags and is imprinted during T cell priming, facilitating its expression during memory T cell activation. In this study, we demonstrate that expression of CD103/beta(7) in human colon carcinoma-specific CTL is synergistically enhanced by the simultaneous TGF-beta1 stimulation and Ag recognition. These results were confirmed by using a panel of human CTL clones. Finally, we show that priming of naive CD8(+) T cells in the presence of TGF-beta1 ensures up-regulation of CD103/beta(7) in recall responses, at concentrations of TGF-beta1 significantly lower than those required by memory T cells primed in the absence of TGF-beta1. These results indicate a role of TGF-beta1 during T cell priming in modulating expression of CD103/beta(7) and controlling retention of human memory CD8(+) T cells into tumor epithelium.     

N-terminal sequence of human leukocyte glycoprotein Mo1: conservation across species and homology to platelet IIb/IIIa

Mo1 and gp160-gp93 are two surface membrane glycoprotein heterodimers present on granulocytes and monocytes derived from humans and guinea pigs, respectively. We purified both antigens and found that their alpha subunits had identical N-termini which were significantly homologous to the alpha subunit of the human adhesion platelet glycoprotein IIb/IIIa.     

Tumor-specific idiotype vaccines. I. Generation and characterization of internal image tumor antigen

The concept of idiotype vaccines against tumor-associated antigens (TAA) was tested in the DBA/2 L1210 lymphoma subline, L1210/GZL. Monoclonal antibodies against a TAA that cross-reacts with the envelope glycoprotein gp52 of the mammary tumor virus were used to make hybridoma anti-idiotype antibodies (Ab2). In this report we describe the characterization of monoclonal anti-idiotypic antibodies against the combining site of 11C1 (Ab1), which recognizes a shared determinant of gp52 of mouse mammary tumor virus (MMTV) and the TAA of L1210/GZL. Hybridomas expressing the internal image of gp52 were screened by an idiotype inhibition assay. Mice sensitized with radiated L1210/GZL cells produced specific delayed type hypersensitivity (DTH) against the Ab2 hybridoma. Five Ab2 hybridomas were selected and were used to immunize DBA/2 mice. Such immunized animals showed specific DTH reaction against a challenge with the L1210/GZL tumor cells. Similar results were obtained in mice immunized with purified Ab2. Fluorescence-activated cell sorter analysis demonstrated that fluorescence staining of L1210/GZL cells by 11C1 can be completely inhibited with preabsorption on Ab2 hybridoma cells. Mice immunized with 2F10 and 3A4 coupled to keyhole limpet hemocyanin (KLH) contained antibodies binding to MMTV. But only in mice immunized with 2F10-KLH was significant inhibition of L1210/GZL tumor growth observed. Collectively, these results indicate that certain anti-idiotypic antibodies can mimic the MMTV gp52 antigen, as well as the gp52-like epitope expressed on the L1210/GZL tumor cells. These properties of anti-idiotypic antibodies mimicking TAA could be exploited for making idiotype vaccines against tumors.