Immunization with a carbohydrate mimicking peptide augments tumor-specific cellular responses

Abstract

The metastatic potential of some tumor cells is associated with the expression of the neolactoseries antigens sialyl-Lewis x (sLex) and sialyl-Lewis a (sLea) as they are ligands for selectins. We have recently shown that peptide mimetics of these antigens can potentiate IgG2a antibodies, which are associated with a Th1-type cellular response. As L-selectin is preferentially expressed on CD4+ Th1 and CD8+ T cell populations, specific induction of these phenotypes could augment a response to L-selectin ligand-expressing tumor cells. Here we demonstrate that immunization with a multiple antigen peptide (MAP) mimetic of sugar constituents of neolactoseries antigens induces a MHC-dependent peptide-specific cellular response that triggers IFN-gamma production upon peptide stimulation, correlating with IgG2a induction. Surprisingly, T lymphocytes from peptide-immunized animals were activated in vitro by sLex, also triggering IFN-gamma production in a MHC-dependent manner. Stimulation by peptide or carbohydrate resulted in loss of L-selectin on CD4+ T cells confirming a Th1 phenotype. We also observed an enhancement in cytotoxic T lymphocyte (CTL) activity in vitro against sLex-expressing Meth A cells using effector cells from Meth A-primed/peptide-boosted animals. CTL activity was inhibited by both anti-MHC class I and anti-L-selectin antibodies. These results further support a role for L-selectin in tumor rejection along with the engagement by the TCR for most likely processed tumor-associated glycopeptides, focusing on peptide mimetics as a means to induce carbohydrate reactive cellular responses.

Evaluating strategies to enhance the anti-tumor immune response to a carbohydrate mimetic peptide vaccine

“…As in our previous studies (17,32), peptide 106 (sequence, GGIYWRYDIYWRYDIYWRYD) was synthesized as a monomer, a multiple antigen peptide (MAP; Research Genetics, Huntsville, AL) or conjugated to a protein transduction domain, GGAYARAAARQARA (106PTD) (33). Each mouse received 100 μg of 106MAP and 20 μg of QS-21 (Antigenics Inc., Framingham, MA) intraperitoneally (i.p.…”

Deficiency in surface expression of E‐selectin ligand promotes lung colonization in a mouse model of breast cancer

“…Staining, acquisition and analysis were performed as described in a previous study. 30 In brief, cells were harvested using trypsin-free cell dissociation buffer (Invitrogen-GIBCO, Carlsbad, CA); resuspended in buffer containing DPBS, 1% BSA and 0.1% sodium azide (FACS buffer); and then incubated with biotinylated lectins or MAbs (10 lg/ml) for 30 min on ice. Following this, cells were stained with FITC-or PE-conjugated streptavidin (2 lg/ml) or antimouse IgM or IgG (Sigma) for an additional 30 min on ice.…”

Maximizing Immune Response to Carbohydrate Antigens on Breast Tumors

“…It is possible that peptide immunization further expands B and T cells that have been primed via shed E25 -CD4 glycoprotein(s) processing. We propose that peptide 106 immuniza-E tion activated a population ofThl and CTLs with production of IFN-y C 20 (12), and in vivo IL-12 treatment further helps to expand the T-cell population and IFNy production. In previous studies, the failure of E 1S-IL-12 treatment to induce tumor regression was also considered to be Results present the mean value ± SD.…”

“…once daily for 5 days, starting on the day of the last peptide sponses to tumor cells, we have been developing peptide mimics of immunization, Flow Cytometry. Acquisition and analysis were performed as described tumor-associated carbohydrate antigens and have demonstrated t earlier (12). Cells were resuspended in a buffer containing, Dulbecco's PBS, peptides synthesized as multivalent peptides can emulate or mimic the 1% BSA, and 0.1% sodium azide and incubated with biotinylated peanut native clustering or presentation of tumor cell-displayed carbohydrate agglutinin or WGA (10 /rg/ml; Vector laboratories, Burlingame, CA) for 30 antigens (11).…”

“…T-Cell Purification. Splenocytes were harvested from spleens and prepared by lysis of erythrocytes and consequent washing several times with fresh medium (12). Splenocytes were first passed through nylon wool and then,"K." using MiniMACS (Miltenyi Biotec, Auburn, CA), natural killer cells were , depleted using anti-natural killer cell (DX5) microbeads.…”