Chia-Chun J. Chang

Evolution of a cytokine using DNA family shuffling

DNA shuffling of a family of over 20 human interferon-α (Hu-IFN-α) genes was used to derive variants with increased antiviral and antiproliferation activities in murine cells. A clone with 135,000-fold improved specific activity over Hu-IFN-α2a was obtained in the first cycle of shuffling. After a second cycle of selective shuffling, the most active clone was improved 285,000-fold relative to Hu-IFN-α2a and 185-fold relative to Hu-IFN-α1. Remarkably, the three most active clones were more active than the native murine IFN-αs. These chimeras are derived from up to five parental genes but contained no random point mutations. These results demonstrate that diverse cytokine gene families can be used as starting material to rapidly evolve cytokines that are more active, or have superior selectivity profiles, than native cytokine genes.

Monocyte-derived CD1a+ and CD1a- dendritic cell subsets differ in their cytokine production profiles, susceptibilities to transfection, and capacities to direct Th cell differentiation.

We describe a phenotypically and functionally novel monocyte-derived dendritic cell (DC) subset, designated mDC2, that lacks IL-12 synthesis, produces high levels of IL-10, and directs differentiation of Th0/Th2 cells. Like conventional monocyte-derived DC, designated mDC1, mDC2 expressed high levels of CD11c, CD40, CD80, CD86, and MHC class II molecules. However, in contrast to mDC1, mDC2 lacked expression of CD1a, suggesting an association between cytokine production profile and CD1a expression in DC. mDC2 could be matured into CD83+ DC cells in the presence of anti-CD40 mAbs and LPS plus IFN-γ, but they remained CD1a− and lacked IL-12 production even upon maturation. The lack of IL-12 and CD1a expression by mDC2 did not affect their APC capacity, because mDC2 stimulated MLR to a similar degree as mDC1. However, while mDC1 strongly favored Th1 differentiation, mDC2 directed differentiation of Th0/Th2 cells when cocultured with purified human peripheral blood T cells, further indicating functional differences between mDC1 and mDC2. Interestingly, the transfection efficiency of mDC2 with plasmid DNA vectors was significantly higher than that of mDC1, and therefore mDC2 may provide improved means to manipulate Ag-specific T cell responses after transfection ex vivo. Taken together, these data indicate that peripheral blood monocytes have the capacity to differentiate into DC subsets with different cytokine production profiles, which is associated with altered capacity to direct Th cell differentiation.

SLAM and its role in T cell activation and Th cell responses.

Following the initial events of T cell activation, triggered by binding of specific peptide‐MHC complex to the TCR for antigen and engagement of costimulatory molecules, a number of activation molecules are expressed on the cell surface. Many of these molecules regulate T cell function. T‐T cell interactions and the interaction of T cells with other cells. One such molecule is SLAM, a multifunctional 70 kDa glycoprotein member of the Ig superfamily with multiple isoforms. SLAM is rapidly induced on natve T cells and B cells following activation. Engagement of SLAM by a specific antibody (mAb A12) results in IL‐2‐independent T cell expansion and induction/up‐regulation of IFN‐γ by activated T cells, including Th2 cells. SLAM was found to be a high‐affinity self‐ligand mediating molecular and cellular homophilic interactions. In this review we discuss SLAM as a receptor involved in T cell expansion and in directing immune responses to a Th0‐Th 1 pathway.

Brequinar Sodium, Mycophenolic Acid, and Cyclosporin A Inhibit Different Stages of IL-4- or IL-13-Induced Human IgG4 and IgE Production In Vitro

We investigated the effect of cyclosporin A (CsA), mycophenolic acid (MPA), and brequinar sodium (BQ) on human IgG4 and IgE synthesis induced by IL-4 or IL-13. BQ inhibited IL-4 and IL-13-induced IgG4 and IgE synthesis in cultures of peripheral blood mononuclear cells (PBMC) or highly purified B cells costimulated by anti-CD40 mAbs in a dose-dependent fashion. CsA and MPA had either suppressive or enhancing effects depending on the concentrations tested. Interestingly, BQ inhibited IgG4 and IgE synthesis at concentrations of 10(-6)-10(-8) M, which did not affect T or B cell proliferation, indicating that the inhibitory effects of BQ on Ig production were not directly related to inhibition of T or B cell proliferation. In contrast, the inhibitory effects of MPA on Ig production were directly associated with inhibitory effects on T and B cell proliferation. CsA blocked T and B cell proliferation at the same concentration (10(-7) M) which enhanced IgG4 and IgE synthesis, indicating that reduction in T or B cell proliferation correlated with enhanced IgE production. CsA also inhibited CD40 ligand expression and IL-2, IL-4, IL-5, IFN-gamma, and GM-CSF production by activated CD4+ T cell clones, whereas MPA and BQ were ineffective, indicating that these compounds do not inhibit early events in T cell activation. Collectively, our data indicate that BQ, MPA, and CsA block different stages of the IgG4 and IgE production process. In addition, we observed that CsA and MPA, in contrast to BQ, at lower concentrations can also have potentiating effects on the production of these Ig isotypes.

Expression of soluble human signaling lymphocytic activation molecule in vivo

BACKGROUND Signaling lymphocytic activation molecule (SLAM) is a novel glycoprotein expressed on activated T and B cells. Ligation of cell surface SLAM, either by anti-SLAM mAbs or the recombinant soluble form of SLAM (sSLAM), enhanced the proliferation of T and B cells in vitro. In addition, the engagement of SLAM on T cells preferentially induced IFN-gamma production even by allergen-specific TH2 clones. OBJECTIVE In this study we investigated the expression of sSLAM in vivo in healthy individuals and in disease conditions that are associated with increased TH1 - or TH2 -cell responses. METHODS The expression of mRNA encoding sSLAM in peripheral blood and synovial fluid (SF) lymphocytes was studied by using reverse transcriptase-PCR, and the presence of sSLAM protein in serum and SF samples was investigated by using a specific ELISA. RESULTS Lymphocytes from patients with rheumatoid arthritis (RA) and healthy individuals consistently expressed mRNA encoding sSLAM. In addition, sSLAM protein was present in 38% of serum and 54% of SF samples from patients with RA and in 47% of serum samples from healthy individuals. The levels of sSLAM in positive serum and SF samples from patients with RA and in positive serum samples from healthy individuals were not significantly different. In contrast, the levels of sSLAM were significantly lower in patients with reactive arthritis or in patients with elevated IgE levels than in patients with RA. Similarly, the frequency of positive SF samples was significantly lower in reactive arthritis (28%) than in RA (54%). CONCLUSION These results indicate that sSLAM is present in serum and SF, further suggesting that sSLAM regulates T- and B-cell function in vivo. Moreover, these data suggest an association between low sSLAM production and the occurrence of TH2 responses in vivo.