Characterization and mechanism of action of suppressor factors derived from human T cell hybridomas

This laboratory developed human T-cell hybridomas which constitutively secrete suppressor factors (SF) capable of inhibiting immune responses (Hybridoma 6:589 (1987). The mechanisms by which human T-cell hybridoma-derived SFs (designated 160 and 169) and Jurkat leukemic T-cell line derived SF inhibit the proliferative response to mitogen by human PBMC were investigated. The Jurkat SF had a pI of 5.2 whereas the 160 and 169 SF had pI of 5.7 and 4.7 (two peaks) and 4.7, respectively. The SF was not transforming growth factor-beta based upon neutralization and iummunoprecipitation experiments with anti-TGF-beta polyclonal antibody. Il-2 production by human PBMC cultured with Con A or OKT3 mAb in the presence of SF was found to be inhibited by greater than 80%. The proliferative responses of SF treated PBMC could not be restored by addition of exogeneous human IL-2. Inhibition of the proliferative responses could not be reversed by addition of exogenous rIL-1, rIL-2 or rIL-4 alone or in paired combinations. The expression of IL-2 receptors (TAC Ag) on Con A activated cultures time points was not affected by treatment with any SFs. Both the 160 and 169 hybridoma-derived SFs were found to arrest PHA induced cell cycle progression in G$\sb0$/G$\sb1$ phase, whereas SF from the Jurkat T-cell line arrested progression in the S phase. Pretreatment of PBMC with SF prior to the addition of mitogen, followed by washing, did not alter the proliferative response of these PBMC nor their cell cycle progression suggesting that cell activation is necessary for these SF to inhibit proliferative responses. Northern blot analysis of total mRNA from mitogen stimulated PBMC in the presence of SF, revealed a time dependent accumulation of an IL-2 specific mRNA of increased size (2.8 kB) in addition to the expected 1.0 kB mature IL-2 message. Interferon-gamma mRNA was of the appropriate size but its half-life was prolonged in SF treated cultures. IL-2 receptor and IL-1 beta mRNA expression was not altered in these cells. ^

A distinct element involved in the lipopolysaccharide activation of the tumor necrosis factor -alpha promoter in a promonocytic cell line, THP-1

TNF-α is a pleiotropic cytokine involved in normal homeostasis and plays a key role in defending the host from infection and malignancy. However when deregulated, TNF-α can lead to various disease states. Therefore, understanding the mechanisms by which TNF-α is regulated may aid in its control. In spite of the knowledge gained regarding the transcriptional regulation of TNF-α further characterization of specific TNF-α promoter elements remains to be elucidated. In particular, the T&barbelow;NF-α A&barbelow;P-1/C&barbelow;RE-like (TAC) element of the TNF-α promoter has been shown to be important in the regulation of TNF-α in lymphocytes. Activating transcription factor-2 (ATF-2) and c-Jun were shown to bind to and transactivate the TAC element However, the role of TAC and transcription factors ATF-2 and c-Jun in the regulation of TNF-α in monocytes is not as well characterized. Lipopolysaccharide (LPS), a potent activator of TNF-α in monocytes, provides a good model to study the involvement of TAC in TNF-α regulation. On the other hand, all-tram retinoic acid (ATRA), a physiological monocyte-differentiation agent, is unable to induce TNF-α protein release. ^ To delineate the functional role of TAC, we transfected the wildtype or the TAC deleted TNF-α promoter-CAT construct into THP-1 promonocytic cells before stimulating them with LPS. CAT activity was induced 17-fold with the wildtype TNF-α promoter, whereas the CAT activity was uninducible when the TAC deletion mutant was used. This daft suggests that TAC is vital for LPS to activate the TNF-α promoter. Electrophoretic mobility shift assays using the TAC element as a probe showed a unique pattern for LPS-activated cells: the disappearance of the upper band of a doublet seen in untreated and ATRA treated cells. Supershift analysis identified c-Jun and ATF-2 as components of the LPS-stimulated binding complex. Transient transfection studies using dominant negative mutants of JNK, c-Jun, or ATF-2 suggest that these proteins we important for LPS to activate the TNF-α promoter. Furthermore, an increase in phosphorylated or activated c-Jun was bound to the TAC element in LPS-stimulated cells. Increased c-Jun activation was correlated with increased activity of Jun N-terminal kinase (JNK), a known upstream stimulator of c-Jun and ATF-2, in LPS-stimulated monocytes. On the other hand, ATRA did not induce TNF-α protein release nor changes in the phosphorylation of c-Jun or JNK activity, suggesting that pathways leading to ATRA differentiation of monocytic cells are independent of TNF-α activation. Together, the induction of TNF-α gene expression seems to require JNK activation, and activated c-Jun binding to the TAC element of the TNF-α promoter in THP-1 promonocytic cells. ^