Monocytes are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs.
Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs within certain flanking base pairs are recognized as a danger signal by the innate immune system of vertebrates. Using lymphocyte proliferative response (LPR) and IFN-gamma secretion assays, a panel of 38 ODN was screened for immunostimulatory activity on bovine peripheral blood mononuclear cells. ODN composed of a nuclease resistant phosphorothioate backbone and a leading 5'-TCGTCGTT-3' motif with two 5'-GTCGTT-3' motifs were highly stimulatory in both assays. Flow cytometric analysis and cell-specific surface marker labeling determined that B-cells (surface IgM(+)) were the primary cell population responding in the LPR assay. Depletion of T cells (CD3(+)) from the PBMC population did not affect IFN-gamma secretion or B-cell proliferation when cultured with CpG-ODN. However, depletion of monocytes (DH59B(+)) completely abrogated the ability of CpG-ODN to stimulate IFN-gamma secretion, and significantly reduced the B-cell proliferative response. These data establish the identity of an optimal immunostimulatory CpG motif for cattle and demonstrate that monocytes play a pivotal role in the ability of cell populations to respond to CpG-ODN. These data provide insight for future studies investigating the mechanism of CpG-ODN bioactivity and its application in novel vaccine formulations and immunotherapy.
Pontarollo
R A
RA
Veterinary Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Sask., S7N 5E3, Saskatoon, Canada.
Rankin
R
R
Babiuk
L A
LA
Godson
D L
DL
Griebel
P J
PJ
Hecker
R
R
Krieg
A M
AM
van Drunen Littel-van den Hurk
S
S
D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D005260:Female; D016130:Immunophenotyping; D007371:Interferon-gamma / Q000096:biosynthesis; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000276:immunology; D008213:Lymphocyte Activation / Q000187:drug effects; D008297:Male; D009000:Monocytes / Q000502:physiology*; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000502:physiology
D000276:Adjuvants, Immunologic; C408982:CPG-oligonucleotide; D009838:Oligodeoxyribonucleotides; D007371:Interferon-gamma