Expansion of quiescent lung adenocarcinoma CD8+ T cells by MUC1-8-mer peptide-T2 cell-ß2 microglobulin complexes.

Adoptive immunotherapy requires the isolation of CD8+ T cells specific for tumor-associated antigens, their expansion in vitro and their transfusion to the patient to mediate a therapeutic effect. MUC1 is an important adenocarcinoma antigen immunogenic for T cells. The MUC1-derived SAPDTRPA (MUC1-8-mer) peptide is a potent epitope recognized by CD8+ T cells in murine models. Likewise, the T2 cell line has been used as an antigen-presenting cell to activate CD8+ T cells, but so far MUC1 has not been assessed in this context. We evaluated whether the MUC1-8-mer peptide can be presented by T2 cells to expand CD25+CD8+ T cells isolated from HLA-A2+ lung adenocarcinoma patients with stage III or IV tumors. The results showed that MUC1-8-mer peptide-loaded T2 cells activated CD8+ T cells from cancer HLA-A2+ patients when anti-CD2, anti-CD28 antibodies and IL-2 were added. The percentage of CD25+CD8+ T cells was 3-fold higher than those in the non-stimulated cells (P=0.018). HLA-A2+ patient cells showed a significant difference (2.3-fold higher) in activation status than HLA-A2+ healthy control cells (P=0.04). Moreover, 77.6% of MUC1-8-mer peptide-specific CD8+ T cells proliferated following a second stimulation with MUC1-8-mer peptide-loaded T2 cells after 10 days of cell culture. There were significant differences in the percentage of basal CD25+CD8+ T cells in relation to the cancer stage; this difference disappeared after MUC1-8-mer peptide stimulation. In conclusion, expansion of CD25+CD8+ T cells by MUC1-8 peptide-loaded T2 cells plus costimulatory signals via CD2, CD28 and IL-2 can be useful in adoptive immunotherapy.

Impairment of Leishmania mexicana phagocytosis in peritoneal macrophages induced by Amaranthus leucocarpus lectin.

The lectin from Amaranthus leucocarpus (ALL) is specific for N-acetyl-D-galactosamine and inhibits phagocytosis of Leishmania mexicana promastigotes in Balb/c mice peritoneal macrophages by 38%. The lipophosphoglycan (LPG) purified from L. mexicana inhibits penetration of promastigotes into peritoneal macrophages by 31%; interestingly, treatment of macrophages with both, ALL and LPG, inhibits phagocytosis of promastigotes by 72%, confirming that ALL induces modification of the macrophage's phagocytic activity by a different route than mannose or C3b receptors. The Inhibitory effect of ALL was time-dependent. N-acetyl-D-galactosamine (GalNAc) or O-glycosidically linked glycoproteins modified macrophage phagocytosis of Leishmania. These results suggest that macrophage membrane glycoproteins, possessing constitutive GalNAc, can influence the signaling pathways used by this intracellular parasite to infect.

The hydrophobic character of peanut (Arachis hypogaea) isoagglutinins.

Peanut seed lectin (PNA) is widely used to identify tumor-specific antigens on the eukaryotic cell surface. In this work PNA was purified by affinity chromatography, using a column containing glutaraldehyde-treated human erythrocytes, whereas PNA isoforms were purified by hydrophobic interaction chromatography using Phenyl-Sepharose. The affinity-purified PNA and its isoforms consist of four equal subunits of 24.5 kDa each, all of which agglutinated human sialidase-treated erythrocytes equally well; however, differences in their relative thermostabilities and sugar specificities for lactose were observed. Fractions PNA-I and PNA-II possess higher affinity for lactose residues than the more hydrophobic isoforms III and IV. These findings suggest that the differences observed in PNA isoagglutinins are due to hydrophobic regions of the protein that influence the three-dimensional organization of the molecule as well as its thermal stability and sugar specificity.

Isolation of the receptor for Amaranthus leucocarpus lectin from murine peritoneal macrophages.

The receptor for Amaranthus leucocarpus lectin from CD-1 resident macrophages was purified with affinity chromatography with biotin labeled A. leucocarpus lectin and using avidin-agarose as affinity matrix. The receptor is a glycoprotein of 70 kDa that contains 18% of sugar by weight; it is mainly composed of galactose and N-acetyl-D-galactosamine in its saccharidic portion, and lacks sialic acid; the protein is rich in glycine, serine and alanine and lacks cysteine residues. The amino terminus of the receptor is blocked. By ionic strength chromatography on a mono P column in anionic form we purified three isoforms from the affinity purified receptor, each showing quantitative differences in glycosylation. The A. leucocarpus lectin receptor is identified only in resting, not activated, macrophages suggesting that it plays a role in activation mechanisms of macrophages.

Inhibition of phagocytic activity by the N-acetyl-D-galactosamine-specific lectin from Amaranthus leucocarpus.

Amaranthus leucocarpus lectin (ALL), specific for N-acetyl-D-galactosamine, induces inhibition of the erythrophagocytic activity of resident murine peritoneal macrophages and of the macrophage-like cell line J-774. This effect was observed only in macrophages that were Mac-2 (CD11c/CD18 or CR4) negative, indicating that macrophage activation induces important modification to the glycosylation (mainly O-glycosylation) of the membrane. Receptors for IgM and C3b remain unaltered after lectin treatment. Ultrastructural analysis revealed (a) that ALL induced the formation of pinocytic vacuoles, and (b) a regular distribution over the macrophage membrane as well as endosomal vesicles of the gold labeled ALL. Our results suggest that macrophage membrane glycoproteins with constitutive N-acetyl-D-galactosamine residues participate in the regulation of pinocytic-phagocytic vacuole formation.

Purification of the lipophosphoglycan from two Leishmania mexicana strains by lectin affinity chromatography.

Two lipophosphoglycans (LPG) from two Leishmania mexicana (Soberano and Yucatan) strains, isolated in Mexico, were purified by affinity chromatography with the Con A lectin. The LPG from each strain, with a 10 kDa molecular weight, possesses two fractions: one with high mannose concentrations and the other with a more heterogeneous saccharidic composition; the mannose-rich fraction and the heterogeneous fraction from the Yucatan strain show a single band of identity with rabbit IgG against promastigotes from L. mexicana Yucatan. Antigen recognition was abolished by treating the high mannose LPGs with alpha-mannosidase. The presence of non reductor alpha-mannose sequences, as determinant epitopes in L. mexicana Soberano and Yucatan strains, was determined by mass spectrometry analysis and enzymatic cleavage.

Recognition of a CD4+ mouse medullary thymocyte subpopulation by Amaranthus leucocarpus lectin.

We have used the Gal beta(1-->3)GalNAc-specific Amaranthus leucocarpus lectin to isolate a thymus cell subpopulation which is different from that sorted with Arachis hypogaea lectin. The cells recognized by A. leucocarpus lectin were predominantly CD4+, whereas a minor proportion of CD8+ cells (approximately 11%) were also identified. The A. leucocarpus-positive cells were located in the thymus medulla and the cortico-medullary junction. The cortex was negative for A. leucocarpus cells.