Biologically Active Recombinant Rat Granulocyte Macrophage Colony-Stimulating Factor Produced in Escherichia coli

Restoration of Abated T Cell Stimulation Activity of Mature Dendritic Cells

“…Although DC are a minor population (Յ0.1%) of LD-LNC, they account for Ն95% of the T-cell stimulating activity in rat LNC preparations (14, 18). Freshly isolated mature DC rapidly lose their capacity to stimulate T cells (11,18). As shown in Table 1, the T cell stimulation activity of freshly isolated and cultured DC diminishes by 60% within 24 h to less than 20% of their initial activity by 48 h. Our earlier studies and others suggested GM-CSF enhanced the costimulation activity of DC (11,19,20) and that TNF␣ also stimulated the activation of T cells by DC (19,20).…”

“…LNC were obtained by mechanical disruption of mesenteric and cervical lymph nodes from Lewis rats, as described (11,14,15). LNC were subfractionated over bovine plasma albumin (Armour Pharmaceutical Co., Kankakee, IL) density gradients into low density (LD; upper layer and at interface) and high density (HD; sedimented) cells.…”

“…The cDNA encoding mature rat TNF␣ (10) was PCR-amplified with Pfu DNA polymerase (Promega, Madison, WI) (sense primer, 5Ј-TTCTGGATCCCTCAGATCATCT-TCTCAAAACTCGA-3Ј; antisense primer, 5Ј-AGAATTCTAGACT-ATCACAGAGCAATGACTCCAAAGTA-3Ј) and directionaly cloned into the modified maltose binding protein (MBP) expression vector, pMTR-23, as described previously (11,12). Production and purification of the MBP-TNF␣ fusion protein, thrombin proteolysis, and subsequent affinity chromatographic purification of TNF␣ was performed essentially as described for rat GM-CSF (11). Yields of purified TNF␣ were 2.0 -2.5 mg/L of induced cells.…”

“…Responder T cells were purified from HD-LNC by panning to remove MHC class II ϩ cells and B cells (11,16,17). Flow cytometry with pan-T-cell monoclonal antibody W3/13 (anti-CD43) indicated these cells were Ͼ99% T cells (not shown).…”

“…Freshly isolated mature DC rapidly lose their capacity to stimulate T cells (11,18). As shown in Table 1, the T cell stimulation activity of freshly isolated and cultured DC diminishes by 60% within 24 h to less than 20% of their initial activity by 48 h. Our earlier studies and others suggested GM-CSF enhanced the costimulation activity of DC (11,19,20) and that TNF␣ also stimulated the activation of T cells by DC (19,20). We further characterized and extended our studies to include the influence of TNF␣ on cultured DC having diminished T cell stimulation activity, and to characterize the expression of CD80 and CD86 by the lymphokine-treated DC.…”