Biomedical subjects

Leonid S Metelitsa

Publications and source records attributed to Leonid S Metelitsa.

6 recordsLinked to original sources

PubMed2006

Synthesis and evaluation of an alpha-C-galactosylceramide analogue that induces Th1-biased responses in human natural killer T cells.

An alpha-galactosylceramide (alphaGalCer, 1) and its isosteric C-glycoside analogue (2) were found to possess promising immunostimulatory activity because of their ability to activate natural killer T (NKT) cells. We report the synthesis of compound 3, a truncated nonisosteric C-alphaGalCer analogue, that like 2 is not enzymatically labile at the glycosidic linkage, but has the anomeric carbon directly bonded to the C1 of the phytoceramide backbone. We compared the biological activity of the three ligands using an in vitro system with human dendritic cells as the antigen-presenting cells and human NKT cells as the responding cells. Although 3 was a less potent agonist for NKT cells than 1 and 2, it induced cytokine production with the highest IFN-gamma:IL-4 and IFN-gamma:IL-13 ratios. Therefore, our data suggest that the new mimetic of alphaGalCer might preferentially promote Th1-immune responses and serve as a potent adjuvant in the immunotherapy of cancer and infectious diseases.

Cells, Cultured↗

PubMed2004

Distinct homeostatic requirements of CD4+ and CD4- subsets of Valpha24-invariant natural killer T cells in humans.

CD1d-restricted Valpha24-invariant natural killer T cells (iNKTs) are important in immunoregulation. CD4(+) and CD4(-) iNKTs develop with similar frequencies in murine thymus and depend on interleukin-15 (IL-15) in periphery. However, homeostatic requirements of iNKTs have not been analyzed in humans. We evaluated thymic production, peripheral dynamics, and functional maturation of human iNKTs. CD4(+) subset comprises 90% of iNKTs in mature thymocytes and cord blood (CB) but only 40% in adult blood. Using T-cell receptor excision circle (TREC) analysis, we directly measured in vivo replicative history of CD4(+) and CD4(-) iNKT cells. Compared to CD4(+), CD4(-) iNKTs contain fewer TRECs, express higher levels of IL-2Rbeta, and proliferate with higher rate in response to IL-15. In contrast, CD4(+) cells express higher levels of IL-7Ralpha and better respond to IL-7. Neither thymic nor CB iNKTs are able to produce cytokines unless they are induced to proliferate. Therefore, unlike in the mouse, human CD4(+) iNKTs are mainly supported by thymic output and limited peripheral expansion, whereas CD4(-) cells undergo extensive peripheral expansion, and both subsets develop their functions in periphery. These findings reveal important differences in homeostatic requirements and functional maturation between murine and human iNKTs that are to be considered for clinical purposes.

Adult↗

PubMed2004

Natural killer T cells infiltrate neuroblastomas expressing the chemokine CCL2.

CD1d-restricted Valpha24-Jalpha18-invariant natural killer T cells (iNKTs) are potentially important in tumor immunity. However, little is known about their localization to tumors. We analyzed 98 untreated primary neuroblastomas from patients with metastatic disease (stage 4) for tumor-infiltrating iNKTs using TaqMan((R)) reverse transcription polymerase chain reaction and immunofluorescent microscopy. 52 tumors (53%) contained iNKTs, and oligonucleotide microarray analysis of the iNKT(+) and iNKT(-) tumors revealed that the former expressed higher levels of CCL2/MCP-1, CXCL12/SDF-1, CCL5/RANTES, and CCL21/SLC. Eight tested neuroblastoma cell lines secreted a range of CCL2 (0-21.6 ng/ml), little CXCL12 (</=0.1 ng/ml), and no detectable CCL5 or CCL21. CCR2, the receptor for CCL2, was more frequently expressed by iNKT compared with natural killer and T cells from blood (P < 0.001). Supernatants of neuroblastoma cell lines that produced CCL2 induced in vitro migration of iNKTs from blood of patients and normal adults; this was abrogated by an anti-CCL2 monoclonal antibody. CCL2 expression by tumors was found to inversely correlate with MYCN proto-oncogene amplification and expression (r = 0.5, P < 0.001), and MYCN-high/CCL2-low expression accurately predicted the absence of iNKTs (P < 0.001). In summary, iNKTs migrate toward neuroblastoma cells in a CCL2-dependent manner, preferentially infiltrating MYCN nonamplified tumors that express CCL2.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2004

Flow cytometry for natural killer T cells: multi-parameter methods for multifunctional cells.

Valpha24-Jalpha18 invariant natural killer T cells (iNKT) are an evolutionary conserved sub-lineage of T cells with effector-memory phenotype that often express an NK cell surface antigen CD161 and is characterized by reactivity to self-glycolipids and alpha-galactosylceramide (alphaGalCer) that are presented by monomorphic HLA class-I-like molecule CD1d. Upon antigen recognition, iNKT cells can rapidly produce multiple cytokines and chemokines and regulate development of Th-2 and Th-1 immune responses. Potential importance of iNKT cells has been demonstrated in several animal models of infection diseases, autoimmunity, and cancer. Multi-parameter flow cytometry has been the main tool to study human and murine iNKT cells. Analysis of human iNKT cells is particularly demanding since their frequency among peripheral blood T cells is relatively low ranging from less than 0.01% to 1%, with a mean of about 0.1%. Herein, we discuss flow cytometry applications that are utilized for iNKT cell identification and enumeration, subset characterization, detection of intracellular cytokines, quantitative analysis of multiple secreted molecules, cell-mediated cytotoxicity, and migration.

Animals↗

PubMed2002

Antidisialoganglioside/granulocyte macrophage-colony-stimulating factor fusion protein facilitates neutrophil antibody-dependent cellular cytotoxicity and depends on FcgammaRII (CD32) and Mac-1 (CD11b/CD18) for enhanced effector cell adhesion and azurophil granule exocytosis.

Polymorphonuclear leukocytes (PMNs) mediate antibody-dependent cellular cytotoxicity (ADCC), which is increased by the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF). We sought to determine whether PMN ADCC also would be increased by the addition of an antibody/GM-CSF fusion protein and whether this would be associated with the up-regulation and activation of Mac-1 (CD11b/CD18) and with azurophil granule exocytosis. ADCC against LA-N-1 human neuroblastoma cells was evaluated with 4-hour calcein acetoxymethyl ester (calcein-AM) microcytotoxicity assay, electron microscopy, and multi-parameter flow cytometry. With the calcein-AM assay, LA-N-1 cell survival was 10%, 55%, and 75% when PMN ADCC was mediated by the antidisialoganglioside (anti-GD2) immunocytokine hu14.18/GM-CSF, by monoclonal antibody (mAb) hu14.18 mixed with GM-CSF, and by hu14.18 alone. Function-blocking mAbs demonstrated that FcgammaRII and FcgammaRIII were required for ADCC with hu14.18 alone or mixed with GM-CSF, but that only FcgammaRII was required for ADCC with hu14.18/GM-CSF. ADCC mediated by hu14.18 and hu14.18/GM-CSF was Mac-1 dependent. Electron microscopy demonstrated the greatest PMN adhesion, spreading, and lysis of targets with hu14.18/GM-CSF. Monoclonal antibodies blocking Mac-1 function allowed the tethering of PMN to targets with hu14.18/GM-CSF but prevented adhesion, spreading, and cytolysis. Flow cytometry showed that hu14.18 with or without GM-CSF and hu14.18/GM-CSF all mediated Mac-1-dependent PMN-target cell conjugate formation but that GM-CSF was required for the highest expression and activation of Mac-1, as evidenced by the mAb24-defined beta(2)-integrin activation epitope. Hu14.18/GM-CSF induced the highest sustained azurophil granule exocytosis, almost exclusively in PMNs with activated Mac-1. Thus, hu14.18/GM-CSF facilitates PMN ADCC against neuroblastoma cells associated with FcgammaRII and Mac-1-dependent enhanced adhesion and degranulation.

Antibodies↗

PubMed2002

NKT and T cells: coordinate regulation of NK-like phenotype and cytokine production.

A maturation-dependent change in phenotype and cytokine production from relatively immature CD161(-) or CD161(+), IL-13(+)IL-4(+), IFN-gamma(-), to mature CD161(+)CD56(+) IFN-gamma(+) cells occurs in primary human alpha-galactosyl ceramide-reactive CD1d-restricted natural killer T (NKT) cells under the control of IL-12 and other monokines. Modulation of this process upon alpha-galactosyl ceramide stimulation explains the opposite roles of NKT cells to drive type 1 and type 2 immune responses. Because the same developmental changes occurred and were similarly regulated in T cells, the data establish that NKT cells should no longer be considered a functionally unique regulatory subset. However, the results of their analysis can be taken as a model for immunotherapeutic approaches with T cells for which a nominal or surrogate antigen is defined.

Adult↗