[Molecular mechanisms regulating trafficking of immune-competent cells: role of cell adhesion molecules].
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12E7 Antigen↗
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Results for “Integrin alpha4”
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[In vitro expansion of cord blood mononuclear cells supported by fetal bone marrow stromal cells and cytokines].
This study was aimed to explore the role of human fetal bone marrow stromal cells (FBMSC) in combination with exogenous cytokines in supporting the in vitro expansion of cord blood mononuclear cells and the expression of CXCR4(+) and CD49d(+) in CD34(+) cells. Mononuclear cells (MNC) separated from cord blood (CB) were cultured in a serum-free support culture system with FBMSC or exogenous cytokines or both of them. On day 0, 6, 10 and 14, total cells were counted, CD34(+), CD34(+)CXCR4(+) and CD34(+)CD49d(+) cells were quantitated by FACS, and hematopoietic progenitor cells were assessed by semisolid culture assay. The results showed that after culturing for 14 days, CD34(+) cells, CD34(+)CXCR4(+) cells, CD34(+) CD49d(+) cells and colony forming unit (CFU) were significantly increased (P < 0.05). Compared with other groups, expansion multiple of CD34(+), CD34(+)CXCR4(+), CD34(+)CD49d(+) cells and CFU were higher than that in FBMSC and cytokine group (P < 0.05). It is concluded that the culture system used in this study can not only support the expansion of CB MNCs but also increase the number of hematopoietic stem and progenitor cells which has chemokine and adhesion capacity. This culture system may be a feasible way for in vitro culture of cord blood cells.
Antigens, CD34↗
Long-term (56-week) oral administration of probiotic Enterococcus faecium M-74 decreases the expression of sICAM-1 and monocyte CD54, and increases that of lymphocyte CD49d in humans.
OBJECTIVES: To study the effects of long-term probiotic [Enterococcus faecium (EF) M-74 strain] application in humans with respect to adhesion molecules, both soluble forms (sICAM-1, sPECAM-1) and their expression on leukocytes. METHODS: Double-blinded randomized and placebo controlled study lasting for 60 weeks. A single capsule containing either 2x10(9) of bacteria EF M-74 with 50 microg of organically bound selenium (E-group) or placebo (P-group) was given to volunteers. Peripheral blood was analyzed for the expression of particular adhesive molecules. RESULTS AND CONCLUSIONS: We observed significant changes in CAMs expression in terms of a decrease in sICAM-1, CD54 on monocytes and CD11b on lymphocytes after one-year administration of Enterococcus faecium M-74 in humans. Anti-adhesion-aimed therapeutic modalities may provide the future approach to prevention and treatment of cardiovascular diseases. Application of probiotics may be part of such strategies. (Tab. 2, Fig. 6, Ref. 41.)
Administration, Oral↗
[Effect of anti-TGF-beta antibody on ex vivo expansion and expression of adhesion molecules of human cord blood CD34+ cells].
TGF-beta, as an inhibitor of hemopoiesis, excreted by hematopoietic stem and progenitor cells, down-regulates the expression of cytokines such as Flt-3 ligand, SCF, IL-3 etc on the stem and progenitor cells. The effect of anti-TGF-beta antibody on ex vivo expansion and expression of adhesive molecules on cord blood CD34(+) cells was studied in this research. The CD34(+) cells from six units of fresh umbilical cord blood were enriched by density gradient sedimentation and purified by miniMACS cell isolation system, and plated them into the SFEM serum free culture system which containing SCF, Flt-3L, TPO and IL-3 in the condition of 37 degrees C, 5% CO2, and saturated moisture. There were three groups in this experiment: (1) blank group: same as the culture system described above; (2) control group: added with normal rabbit IgG into the mentioned culture system; (3) test group: the same culture system with anti-TGF-beta1 antibo-dy. Cultured for 6 days, the number of mononuclear cells (MNC) was counted, the expression of CD34 antigen, CD117 (c-kit) antigen, CD11a antigen, CD49d antigen and CD33 antigen was tested with FCM. Meanwhile, cells of the three groups were plated in the methylcellulose culture system for 14 days, the number of CFU-GEMM, BFU-E, CFU-GM was counted. The results indicated that the expansion multiples of MNC, CD34(+) cells, CD34(+)c-kit(+) cells, CFU-GEMM in the test group (41.82 +/- 13.49, 15.62 +/- 6.95, 13.36 +/- 6.12, 11.07 +/- 4.05) were significantly higher than in the control group (28.86 +/- 9.03, 10.40 +/- 4.98, 9.04 +/- 4.40, 6.36 +/- 2.37) (P = 0.001, 0.002, 0.003, 0.002) respectively. The expansion multiple of more primitive CD34(+)c-kit(-) subpopulation in the test group (69.10 +/- 41.06) was even higher than in the control group (27.29 +/- 10.40) (P = 0.024). Adhesion molecule expression on the CD34(+) cells after short-term expansion: the expression of CD11a on the CD34(+) cells of the original cord blood was (61.73 +/- 4.13)%, and CD49d was (55.12 +/- 5.22)%. After expansion in each group the expression of CD11a on the CD34(+) cells did not change with statistical significance (P > 0.05), the expression of CD49d increased (P < 0.05). Compared with blank group and control group, anti-TGF-beta antibody did not impact on the expression of CD11a and CD49d (P > 0.05). It is concluded that anti-TGF-beta antibody can synergize other cytokines to effectively enhance the proliferation of cord blood NC, CD34(+) cells, progenitor subpopulation of CD34(+)c-kit(-) cells, and increase the output of more primitive progenitor colony, CFU-GEMM and BFU-E. At the same time, anti-TGF-beta antibody did not depresss the expression of adhesion molecules on CD34(+) cells.
Antibodies↗
[Study of estradiol on treatment of preeclampsia in rat model].
OBJECTIVE: To evaluate whether estradiol can inhibit and cure the inflammation of experimental preeclampsia in rats. METHODS: Experimental preeclampsia was induced in 14-day-pregnant rats by infusion of endotoxin (1.0 microg/kg). Rats with normal pregnancy were infused with sodium chloride solution. A group of preeclampsia rats was injected with 17beta-estradiol (17beta-E(2), 1 mg.kg(-1).d(-1)). Blood pressure, albuminuria, inflammation associated adhesion molecule CD(49d) and tumor necrosis factor-alpha (TNF-alpha) were assessed. RESULTS: On pregnant day 19, for normal pregnancy group (group C) the blood pressure was (120.4 +/- 2.0) mm Hg (1 mm Hg = 0.133 kPa), urinary protein (0.47 +/- 0.06) mg/24 hours; for experimental preeclampsia group (group A) blood pressure was (134.2 +/- 2.4) mm Hg, urinary protein (0.79 +/- 0.10) mg/24 hours; for experimental preeclampsia with 17beta-E(2) treatment group (group B) blood pressure was (123.3 +/- 1.7) mm Hg, urinary protein (0.51 +/- 0.08) mg/24 hours. A significant increase of blood pressure and urinary albumin was observed in group A. CD(49d) expression and TNF-alpha concentration were also increased. 17beta-E(2) reduced the expression of CD(49d), concentration of TNF-alpha, blood pressure and albuminuria of experimental preeclampsia. However, the weight of fetuses in 17beta-E(2) treatment group were less than that in other groups. CONCLUSION: 17beta-E(2) can improve the symptoms of experimental preeclampsia, but its effects on fetus need to be further studied.
Animals↗
[Effect of different cytokine combinations on the expression of CD49d and CXCR4 and ex vivo expansion of umbilical cord blood mononuclear cells].
This study was purposed to explore the effect of different cytokine combinations on the expansion of the mononuclear cells drived from umbilical cord blood (CB) ex vivo and expression of CXCR4 and CD49d on CD34+ cells after expansion. Human fresh CB mononuclear cells were cultured in serum-free and stroma-free medium containing different combinations of cytokine for 7 days. At day o and 7, the total cells were counted, CD34+ cells and CD34+CXCR4+, CD34+CD49d+ cells were assayed by flow cytometry, and CFU were determined. According to the different combinations of cytokine, experiments were divided into four groups: control, SF group (SCF + FL), SFT group (SCF + FL + TPO) and SFT6 group (SCF + FL + TPO + IL-6). The results showed that the SF (SF group) combination supported only low expansion of total cells, CD34+ cells and CFU. The addition of TPO in SF group restored UCB stem/progenitors expansion to a higher level than that in SF group, while there was no difference between groups SFT and SFT6 (P > 0.05). The cytokine combinations in groups SF, SFT and SFT6 all could upregulate the expression levels of CD49d and CXCR4 on expanded cord blood CD34+ cells, but there were no significant differences between groups SF, SFT and SFT6 (P > 0.05). It is concluded that SCF + FL has no strong synergistic effects on primitive hematopoietic cells. TPO plays an important role in enhancing expansion of umbilical cord blood hematopoietic cells, while IL-6 only shows a neutral effect on it. SCF + FL + TPO combination not only promotes progenitor cells expansion but also upregulates the expression of CD49d and CXCR4 on CD34+ cells from cord blood.
Antigens, CD34↗
Digestive Disease Week 2006. 21-15 May 2006, Los Angeles, CA, USA.
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Animals↗
Stimulation of human bone marrow stromal cell tyrosine kinases and IL-6 production by contact with B lymphocytes.
The interaction of normal human B cell precursors with the bone marrow (BM) stromal cells that support their growth and maturation is mediated by adhesion of B cell CD49d/CD29 (alpha 4 beta 1 integrin or VLA-4) to stromal cell signal transduction, we generated CD49d-deficient variants of the RAMOS early b Burkitt's lymphoma cell line. RAMOS was chosen due to its lack of expression of CD49e (alpha 5 integrin) and CD44, which could potentially interact with stromal cells or their extracellular matrix. In contrast to the parental RAMOS cell line, CD49d-deficient RAMOS cell lines did not adhere to primary human BM stromal cells. Tyrosine phosphorylation of several stromal cell proteins was stimulated within 5 to 10 min of contact with either CD49d-expressing or CD49d-deficient RAMOS. These proteins included a 110-kDa substrate tentatively identified as pp125FAK. In contrast, mAb cross-linking of stromal cell CD106 had no significant effect on protein tyrosine phosphorylation. Il-6 production by stromal cells was enhanced up to fourfold over 3 days when stromal cells were cultured in contact with RAMOS cells, regardless of CD49d expression. Stromal cell IL-6 production was not significantly enhanced by RAMOS cells cultured above the stromal cells in Transwell inserts. Thus, we have demonstrated through the use of paired CD49d-expressing and CD49d-deficient RAMOS cell lines that protein tyrosine kinase activity and IL-6 production by primary human BM stromal cells can be enhanced in a manner that is dependent on B cell contact but independent of CD49d-mediated adhesion. These results indicate the potential for B cells to interact with cells in their microenvironment and activate feedback mechanisms mediated by stromal cells, which may regulate lymphopoiesis.
Antibodies, Monoclonal↗
Vascular cell adhesion molecule-Ig fusion protein selectively targets activated alpha 4-integrin receptors in vivo. Inhibition of autoimmune diabetes in an adoptive transfer model in nonobese diabetic mice.
The alpha 4 beta 1-integrin (CD49d, CD29) constitutively expressed on leukocytes regulates cell migration to inflammatory sites, cell activation, and development through its interactions with two alternate ligands, vascular cell adhesion molecule-1 (VCAM-1; CD106) expressed on cytokine-activated endothelium, dendritic and stromal cells, and the extracellular matrix protein fibronectin. Another alpha 4-integrin receptor, alpha 4 beta 7, expressed on leukocytes also binds VCAM-1 and fibronectin (FN), and controls homing to mucosal tissues through its interactions with mucosal vascular addressin MAdCAM-1. In vitro studies have shown that alpha 4-dependent cell adhesion is regulated by the activation state of the cell and by divalent cations. However, the existence and role of cells with different alpha 4 activation states in vivo have not been defined. Herein we show that a soluble ligand with the two N-terminal domains of human VCAM-1 fused to a human IgG1 constant region, VCAM-Ig, binds selectively to activated alpha 4-receptors on murine cells, such as those induced by Mn2+ in vitro. To determine whether the cells identified by VCAM-Ig were required under physiologic conditions, we assessed its anti-inflammatory effect. We show that VCAM-Ig is not bound to the majority of murine alpha 4+ cells after in vivo administration, yet it significantly delays the onset of adoptively transferred autoimmune diabetes. Thus, soluble VCAM-Ig can modify alpha 4-dependent disease progression, apparently by its selective action on cells with activated alpha 4-integrin receptors, thereby providing evidence for distinct alpha 4 activation states in vivo.
Animals↗
Variable expression of CD49d antigen in B cell chronic lymphocytic leukemia is related to disease stages.
We have investigated the expression of the dual specific adhesion molecule, VLA-4 (CD49d/CD29) on lymphocytes obtained from 62 patients with B-CLL and compared it with normal controls, patients with other hematological malignancies, and umbilical cord blood. The mean CD49d expression in patients with CLL was lower than the other group of leukemia and the CD19+, CD5+ cells of normal peripheral blood and umbilical cord blood (P < 0.001). The patients in RAI stage 0, I and II (early stage) had even lower CD49d expression, whereas patients in RAI stage III and IV (advanced stage) had relatively higher CD49d levels. In vitro adhesion of lymphocytes to fibronectin, being the extracellular matrix ligand of CD49d, was also investigated. Lymphocytes obtained from B-CLL were found to have lower adhesion to fibronectin than that from controls (P < 0.03). Furthermore, CD49d(low) B-CLL cells had lower adhesion to fibronectin, whereas CD49d(high) B-CLL cells showed normal adhesion ratios (P < 0.002). Further phenotypic analyses revealed the presence of myeloid markers (CD13 and CD33) in most of the advanced stage patients, although these were negative in early stage cases. Expressions of CD11a and sIgM were also low but CD11b was relatively higher in the early stages of the disease. On the basis of these results, we concluded that early stages of CLL are correlated with the expression of CD49d(low), CD11a(low), CD11b(high), CD13-, CD33-, sIgM(low) and also had lower fibronectin adhesion, whereas advanced stages of CLL are associated with CD49d(high), CD11a(high), CD11b(low), CD13+, CD33+, SIgM(high) and show normal fibronectin adhesion.
Antibodies, Monoclonal↗
Homing potentials of circulating lymphocytes in humans depend on the site of activation: oral, but not parenteral, typhoid vaccination induces circulating antibody-secreting cells that all bear homing receptors directing them to the gut.
Specific Ab-secreting cells (ASC) appear in the human blood as a response to oral and parenteral vaccination. The actual contribution of these cells to the defense of the body depends on their final effector site. The homing potentials of mucosally and parenterally induced ASC were compared by examining the homing receptor (HR) expression of circulating specific ASC in the blood of volunteers vaccinated orally or parenterally with the same Ag, Salmonella typhi Ty21a. Circulating lymphocytes were separated into receptor-positive and -negative populations, and the numbers of specific ASC were assayed. The alpha4 beta7 integrin, which acts as a gut HR, was expressed on all (99%) of the mucosally activated ASC, but on only 58% of the parenterally induced ASC or 58% of all Ig-secreting cells of the unvaccinated controls. L-selectin, the peripheral lymph node HR, showed an inverse distribution; it was found on 42% of mucosally activated ASC and on 86% of parenterally induced ASC. These results reveal that all of the circulating ASC after oral vaccination are committed to migrate to the mucosal compartment of the immune system, strongly arguing for a recirculation of activated mucosal cells in humans. By contrast, ASC induced by parenteral vaccination with the same Ag are mostly directed to the systemic compartment, yet a part of them has mucosal homing attitudes as well. These differences indicate that the site of Ag encounter determines the homing potential of the cell.
Administration, Oral↗
Expression of differentiation antigens and adhesion molecules on CD34+ cells in peripheral blood and bone marrow after chemotherapy followed by administration of granulocyte colony stimulating factor.
The expression of differentiation antigens (D/As) and adhesion molecules (A/Ms) was studied on CD34 positive (CD34+) cells in peripheral blood (PB) and bone marrow (BM) of patients treated with myelosuppressive chemotherapy followed by granulocyte colony-stimulating factor (G-CSF) administration (CT/G-CSF). CD33 positive rates of PB and BM CD34+ cells were significantly higher in the patients than those in the normal controls. CD49d positive rate of PB CD34+ was significantly lower than that of BM CD34+ cells in both the patients and the normal controls, and this decreased expression of CD49d on PB CD34+ cells was larger in the patients than in the normal controls. CD117 positive rate was significantly lower in PB CD34+ cells of the patients than that of their BM CD34+ cells and PB CD34+ cells of the normal control. These results suggested that more mature progenitor cells increased in the patients' PB and BM after CT/G-CSF, and that the decreased expression of CD49d played an important role on peripheralisation of CD34+ cells even in steady state, and the decreased expression of CD117 was related with the mobilization of CD34+ cells after CT/G-CSF. CT/G-CSF decreased the expression of CD117 and enhance the decrease of CD49d expression on PB CD34+ cells. These changes appeared to be involved in the mobilization mechanism of hematopoietic progenitor cells from BM into PB after CT/G-CSF.
Adolescent↗
Humanization of a mouse antibody against human alpha-4 integrin: a potential therapeutic for the treatment of multiple sclerosis.
alpha 4 beta 1 integrin (VLA-4) is crucial for the adhesion of leukocytes to human vascular cell adhesion molecule-1 (VCAM-1) on inflamed endothelium. This cell adhesion event is the first step in leukocyte extravasation across the blood-brain barrier in inflammatory diseases of the central nervous system (CNS) such as experimental autoimmune encephalomyelitis (EAE). Prevention of leukocyte infiltration by antibodies against the alpha 4 integrin, which block the alpha 4 beta 1 integrin/VCAM-1 interaction, have been shown to suppress clinical and pathological features of EAE. In this study, two mouse monoclonal antibodies (MAb) directed against human alpha 4 integrin were analyzed in vitro for their ability to block the interaction of leukocytes with VCAM-1 under different assay conditions. The best blocking MAb, AN100226m, was humanized by complementarily-determining region grafting, associated with human C regions and expressed. We found that modification of two structural determinants (H27 and H29) for the heavy chain CDR1 loop in one hand, and modification of framework amino acid H38, H40 and H44 in the other hand, had no effect on antigen binding. In contrast, modification of a structural determinant (H71) for the heavy chain CDR2 loop resulted in loss of binding. The humanized antibody. AN100226, was equivalent to the murine antibody. AN100226m, in binding to alpha 4 beta 1 integrin and in blocking cell adhesion. More importantly, AN100226 was as effective as AN100226m in the reversal of active EAE in guinea pigs and thus may be useful in the treatment of autoimmune diseases such as multiple sclerosis. AN100226 is currently in phase II clinical trials in the UK for the treatment of multiple sclerosis exacerbations.
Amino Acid Sequence↗
P-selectin, L-selectin, and alpha 4 integrin have distinct roles in eosinophil tethering and arrest on vascular endothelial cells under physiological flow conditions.
The adhesive interactions of eosinophils with purified E-, P-, and L-selectins; vascular cell adhesion molecule-1 molecule; and HUVEC were examined in shear flow. Compared with neutrophils, eosinophils showed markedly less binding to E-selectin, but significantly stronger avidity for P-selectin. Both cell types showed a similar level of tethering and rolling on L-selectin. Eosinophils tethered and arrested abruptly on vascular cell adhesion molecule-1. However, some of the tethers were detached within several seconds; this was prevented by stimulation with eotaxin. Eosinophils also showed immediate arrest on HUVEC stimulated with 100 U/ml TNF-alpha for 6 h. Treatment with L-selectin mAb decreased eosinophil accumulation on the HUVEC by abrogating secondary tethers through interactions between flowing and attached eosinophils. mAb to P-selectin but not to E-selectin strongly inhibited primary tethers and accumulation of eosinophils. mAb to the integrin alpha 4 subunit inhibited arrest, induced rolling or detachment of tethered eosinophils, and resulted in partial reduction of eosinophil accumulation. mAb to the integrin beta 2 subunit had only a slight effect, whereas treatment with mAb to the integrin alpha 4 and beta 2 subunits together abolished rolling interactions as well as arrest, and thus almost totally inhibited eosinophil accumulation. Our data indicate that P-selectin, but not E-selectin, is directly involved in eosinophil tethering on inflammatory endothelium while L-selectin mainly mediates intereosinophil interaction. VLA-4 has a crucial role in eosinophil arrest, and arrest is enhanced by exposure to chemoattractants.
Antibodies, Monoclonal↗
IL-4-induced eosinophil accumulation in rat skin is dependent on endogenous TNF-alpha and alpha 4 integrin/VCAM-1 adhesion pathways.
IL-4 has been implicated in the pathogenesis of a number of allergic inflammatory disease states where the accumulation of eosinophils is a prominent feature. The aim of the present study was to use an isotopic in vivo model to investigate the ability of recombinant rat IL-4 in inducing eosinophil accumulation in rat skin. 111In-eosinophil accumulation in response to intradermally injected IL-4 was measured during 0 to 4 h, 24 to 28 h, and 48 to 52 h. Accumulation was detected during the first two periods, but not at the later time point. The accumulation during 24 to 28 h, which was dose dependent, was investigated in detail. Administration i.v. of an anti-rat VCAM-1 mAb, but not an anti-rat ICAM-1 mAb, inhibited the accumulation of 111In-eosinophils induced by IL-4 (maximum inhibition, 80%). Further, when the 111In-eosinophils were pretreated in vitro with an anti-beta 2 integrin mAb, an anti-alpha 4 integrin mAb, or a combination of both mAbs, before their injection into recipient rats, the IL-4-induced cell accumulation was inhibited by 63, 60, and 74%, respectively. Finally, coadministration of IL-4 with the soluble TNF receptor (p55)-IgG fusion protein significantly reduced the 111In-eosinophil accumulation induced by the cytokine, and TNF-alpha was detected in IL-4-injected skin sites by both immunostaining and bioassay. Our results demonstrate that IL-4 is a potent inducer of eosinophil accumulation in vivo, the response being dependent on the endogenous generation of TNF-alpha, beta 2 integrins, and alpha 4 integrin/VCAM-1 interactions.
Animals↗
Contribution of endothelial selectins and alpha 4 integrins to eosinophil trafficking in allergic and nonallergic inflammatory reactions in skin.
The role of endothelial selectins in mediating eosinophil recruitment was assessed using the trafficking of 111In-labeled blood eosinophils in mouse skin. An intradermal injection of chemoattractants (leukotriene B4, macrophage inflammatory protein-l alpha, and eotaxin) resulted in a rapid accumulation of 111In eosinophils that was reduced 49 to 91% by anti-P-selectin mAb. An anti-E-selectin mAb was ineffective, although a combined E- and P-selectin blockade resulted in >95% inhibition of all responses. The accumulation of a pulse of 111In eosinophils at sites of active cutaneous anaphylaxis (ACA) at 4 to 8 h and at 20 to 24 h after Ag challenge was completely dependent upon E- and P-selectin in combination, but not in isolation. In contrast, at 20 to 24 h after Ag challenge in a delayed-type hypersensitivity (DTH) reaction in skin, 111In eosinophil accumulation was largely independent of endothelial selectins, even when L-selectin was also blocked. An anti-alpha 4 integrin mAb significantly reduced 111In eosinophil trafficking in both allergic reactions but was slightly more effective in the DTH reaction compared with the ACA reaction. These results show that P-selectin and to a lesser extent E-selectin mediate eosinophil recruitment in skin in acute inflammatory reactions. In allergic, late-onset inflammatory reactions, neither P- nor E-selectin alone are sufficient to mediate eosinophil accumulation; when combined, they are essential for trafficking in ACA but are less important in the DTH reaction. Whether alpha 4 integrin-based strategies will be more effective than selectin-based strategies at inhibiting eosinophil recruitment in human disease remains to be determined.
Anaphylaxis↗
Eosinophil tethering to interleukin-4-activated endothelial cells requires both P-selectin and vascular cell adhesion molecule-1.
We examined the mechanisms used by eosinophils to tether and accumulate on interleukin-4 (IL-4)-stimulated human umbilical vein endothelial cells (HUVECs) under flow conditions. As previously reported, HUVECs treated for 24 hours with 20 ng/mL IL-4 had increased expression of P-selectin and vascular cell adhesion molecule-1 (VCAM-1) but not E-selectin. We found that eosinophils tethered and rolled on IL-4-stimulated HUVECs at physiologic shear stresses. Eosinophil rolling was quickly followed by firm adhesion. Treatment with either an anti-P-selectin monoclonal antibody (MoAb) or an anti-VCAM-1 MoAb decreased both eosinophil tethering and accumulation at 2 dyn/cm2. VCAM-1 interacts with 4-integrins expressed on eosinophils. We found that an anti-4-integrin MoAb also blocked eosinophil tethering and accumulation at 2 dyn/cm2. None of these MoAbs alone had an impact on eosinophil accumulation at lower shear stresses, but when either an anti-VCAM-1 or an anti-4-integrin MoAb was used in combination with an anti-P-selectin MoAb, all eosinophil tethering and accumulation on IL-4-stimulated HUVECs were blocked. This was true at both high and low shear stresses. These data show that both P-selectin and VCAM-1 are required to tether eosinophils at high shear stresses, but at low shear stresses these adhesion proteins can act independently to recruit eosinophils to IL-4-stimulated HUVECs.
Antibodies, Monoclonal↗
Phenotypic stability of a cytotoxic T-cell line directed against an immunodominant epitope of human carcinoembryonic antigen.
CTL lines have now been generated against defined peptides of a range of human tumor-associated antigens (TAAs). One of the potential uses of these epitope-specific CTLs is in adoptive transfer immunotherapy. This is a modality, however, that will require long-term in vitro culture of CTLs. To date, little has been reported concerning the phenotypic stability of human epitope-specific CTLs as a consequence of long-term in vitro propagation via peptide stimulation. We report here the serial phenotypic characterization of a CTL line directed against an immunodominant epitope (YLSGANLNL, designated CAP-1) of human carcinoembryonic antigen (CEA). This CTL line was derived from peripheral blood mononuclear cells of a patient with metastatic carcinoma who had been treated with a recombinant CEA-vaccinia vaccine in a Phase I trial; the CTLs were analyzed through 20 in vitro cycle passages of stimulation with CAP-1 peptide and interleukin 2 in the presence of autologous antigen-presenting cells. The CTL line was shown to be phenotypically stable in terms of high levels of cytokine (IFN-gamma, tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor) production, expression of homing-adhesion molecules, ability to lyse peptide-pulsed targets, and ability to lyse human carcinoma cells endogenously expressing CEA in a MHC-restricted manner. Vbeta T-cell receptor gene usage was also analyzed. These studies thus present a rationale for the use of long-term cultured epitope-specific human CTLs, directed against a human TAA for potential adoptive transfer immunotherapy protocols.
Antigens, CD↗