Deborah Luxenberg

Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides

Th17 cells are a distinct lineage of effector CD4+ T cells characterized by their production of interleukin (IL)-17. We demonstrate that Th17 cells also expressed IL-22, an IL-10 family member, at substantially higher amounts than T helper (Th)1 or Th2 cells. Similar to IL-17A, IL-22 expression was initiated by transforming growth factor β signaling in the context of IL-6 and other proinflammatory cytokines. The subsequent expansion of IL-22–producing cells was dependent on IL-23. We further demonstrate that IL-22 was coexpressed in vitro and in vivo with both IL-17A and IL-17F. To study a functional relationship among these cytokines, we examined the expression of antimicrobial peptides by primary keratinocytes treated with combinations of IL-22, IL-17A, and IL-17F. IL-22 in conjunction with IL-17A or IL-17F synergistically induced the expression of β-defensin 2 and S100A9 and additively enhanced the expression of S100A7 and S100A8. Collectively, we have identified IL-22 as a new cytokine expressed by Th17 cells that synergizes with IL-17A or IL-17F to regulate genes associated with skin innate immunity.

Engagement of Glucocorticoid-Induced TNFR Family-Related Receptor on Effector T Cells by its Ligand Mediates Resistance to Suppression by CD4+CD25+ T Cells

Nonactivated CD4+CD25+ regulatory T cells constitutively express glucocorticoid-induced TNFR family-related receptor (GITR), a TNFR family member whose engagement was presumed to abrogate regulatory T cell-mediated suppression. Using GITR−/− mice, we report that GITR engagement on CD25−, not CD25+ T cells abrogates T cell-mediated suppression. Mouse APCs constitutively express GITR ligand (GITR-L), which is down-regulated following TLR signaling in vivo. Although GITR−/−CD25− T cells were capable of mounting proliferative responses, they were incapable of proliferation in the presence of physiological numbers of CD25+ T cells. Thus, GITR-L provides an important signal for CD25− T cells, rendering them resistant to CD25+-mediated regulation at the initiation of the immune response. The down-regulation of GITR-L by inflammatory stimuli may enhance the susceptibility of effector T cells to suppressor activity during the course of an infectious insult.

Identification of an Interleukin 17F/17A Heterodimer in Activated Human CD4+ T Cells

IL-17F and IL-17A are members of the IL-17 pro-inflammatory cytokine family. IL-17A has been implicated in the pathogenesis of autoimmune diseases. IL-17F is a disulfide-linked dimer that contains a cysteine-knot motif. We hypothesized that IL-17F and IL-17A could form a heterodimer due to their sequence homology and overlapping pattern of expression. We evaluated the structure of recombinant IL-17F and IL-17A proteins, as well as that of natural IL-17F and IL-17A derived from activated human CD4+ T cells, by enzyme-linked immunosorbent assay, immunoprecipitation followed by Western blotting, and mass spectrometry. We find that both IL-17F and IL-17A can form both homodimeric and heterodimeric proteins when expressed in a recombinant system, and that all forms of the recombinant proteins have in vitro functional activity. Furthermore, we find that in addition to the homodimers of IL-17F and IL-17A, activated human CD4+ T cells also produce the IL-17F/IL-17A heterodimer. These data suggest that the IL-17F/IL-17A heterodimer may contribute to the T cell-mediated immune responses.

Program Death-1 Engagement Upon TCR Activation Has Distinct Effects on Costimulation and Cytokine-Driven Proliferation: Attenuation of ICOS, IL-4, and IL-21, But Not CD28, IL-7, and IL-15 Responses

The program death 1 (PD-1) receptor and its ligands, PD-1 ligand (PD-L)1 and PD-L2, define a novel regulatory pathway with potential inhibitory effects on T, B, and monocyte responses. In the present study, we show that human CD4+ T cells express PD-1, PD-L1, and PD-L2 upon activation, and Abs to the receptor can be agonists or antagonists of the pathway. Under optimal conditions of stimulation, ICOS but not CD28 costimulation can be prevented by PD-1 engagement. IL-2 levels induced by costimulation are critical in determining the outcome of the PD-1 engagement. Thus, low to marginal IL-2 levels produced upon ICOS costimulation account for the greater sensitivity of this pathway to PD-1-mediated inhibition. Interestingly, exogenous IL-2, IL-7, and IL-15 but not IL-4 and IL-21 can rescue PD-1 inhibition, suggesting that among these cytokines only those that activate STAT5 can rescue PD-1 inhibition. As STAT5 has been implicated in the maintenance of IL-2Rα expression, these results suggest that IL-7 and IL-15 restore proliferation under conditions of PD-1 engagement by enhancing high-affinity IL-2R expression and hence, IL-2 responsiveness.

CTLA-4 (CD152) Can Inhibit T cell Activation by Two Different Mechanisms Depending on Its Level of Cell Surface Expression

CTLA-4 (CD152) engagement results in down-regulation of T cell activation. Two mechanisms have been postulated to explain CTLA-4 inhibition of T cell activation: negative signaling and competitive antagonism of CD28:B7-mediated costimulation. We assessed the contributions of these two mechanisms using a panel of T cell lines expressing human CTLA-4 with mutations in the cytoplasmic region. Under conditions of B7-independent costimulation, inhibition of IL-2 production following CTLA-4 engagement required the CTLA-4 cytoplasmic region. In contrast, under B7-dependent costimulation, inhibition of IL-2 production by CTLA-4 engagement was directly proportional to CTLA-4 cell surface levels and did not require its cytoplasmic region. Thus, CTLA-4 down-regulates T cell activation by two different mechanisms—delivery of a negative signal or B7 sequestration—that are operational depending on the levels of CTLA-4 surface expression. These two mechanisms may have distinct functional outcomes: rapid inhibition of T cell activation or induction of T cell anergy.

The Human IL-17F/IL-17A Heterodimeric Cytokine Signals through the IL-17RA/IL-17RC Receptor Complex

IL-17A and IL-17F, produced by the Th17 CD4+ T cell lineage, have been linked to a variety of inflammatory and autoimmune conditions. We recently reported that activated human CD4+ T cells produce not only IL-17A and IL-17F homodimers but also an IL-17F/IL-17A heterodimeric cytokine. All three cytokines can induce chemokine secretion from bronchial epithelial cells, albeit with different potencies. In this study, we used small interfering RNA and Abs to IL-17RA and IL-17RC to demonstrate that heterodimeric IL-17F/IL-17A cytokine activity is dependent on the IL-17RA/IL-17RC receptor complex. Interestingly, surface plasmon resonance studies indicate that the three cytokines bind to IL-17RC with comparable affinities, whereas they bind to IL-17RA with different affinities. Thus, we evaluated the effect of the soluble receptors on cytokine activity and we find that soluble receptors exhibit preferential cytokine blockade. IL-17A activity is inhibited by IL-17RA, IL-17F is inhibited by IL-17RC, and a combination of soluble IL-17RA/IL-17RC receptors is required for inhibition of the IL-17F/IL-17A activity. Altogether, these results indicate that human IL-17F/IL-17A cytokine can bind and signal through the same receptor complex as human IL-17F and IL-17A. However, the distinct affinities of the receptor components for IL-17A, IL-17F, and IL-17F/IL-17A heterodimer can be exploited to differentially affect the activity of these cytokines.

Duplication of primate and rodent B7-H3 immunoglobulin V- and C-like domains: divergent history of functional redundancy and exon loss

B7-H3 is a novel protein structurally related to the B7 family of ligands by the presence of a single set of immunoglobulin-V-like and immunoglobulin-C-like (VC) domains. By multiplex PCR, the dominantly expressed form of human B7-H3 was found to be a splice variant containing tandemly duplicated VC domains (VCVC). In contrast, mouse B7-H3 cDNA contained only one single VC form due to an exon structure corresponding to V-(pseudoexon C)-(pseudoexon V)-C. Comparisons of human, monkey, mouse, and hamster genomic B7-H3 reveal that primates, but not rodents, exhibited a higher degree of intramolecular sequence similarity between VC duplications than between molecules. Both VC and VCVC forms of human B7-H3 inhibited CD4(+) T cell proliferation and downregulated cytokine production upon TCR activation. These results suggest independent, but convergent, paths of B7-H3 active domain duplication followed by divergent histories of exon degeneration in rodents and exon maintenance by humans.

The Inhibitory Function of CTLA-4 Does Not Require Its Tyrosine Phosphorylation

CTLA-4 is a negative regulator of T cell responses. Sequence analysis of this molecule reveals the presence of two cytoplasmic tyrosine residues at positions 165 and 182 that are potential Src homology (SH)-2 domain binding sites. The role of phosphorylation of these residues in CTLA-4-mediated signaling is unknown. Here, we show that sole TCR ligation induces ζ-associated protein (ZAP)-70-dependent tyrosine phosphorylation of CTLA-4 that is important for cell surface retention of this molecule. However, CTLA-4 tyrosine phosphorylation is not required for down-regulation of T cell activation following CD3-CTLA-4 coengagement. Specifically, inhibition of extracellular signal-regulated kinase (ERK) activation and of IL-2 production by CTLA-4-mediated signaling occurs in T cells expressing mutant CTLA-4 molecules lacking the cytoplasmic tyrosine residues, and in lck-deficient or ZAP-70-deficient T cells. Therefore, CTLA-4 function involves interplay between two different levels of regulation: phosphotyrosine-dependent cell surface retention and phosphotyrosine-independent association with signaling molecules.

IL-22R, IL-10R2, and IL-22BP binding sites are topologically juxtaposed on adjacent and overlapping surfaces of IL-22.

Interleukin (IL) 22 is a type II cytokine that is produced by immune cells and acts on nonimmune cells to regulate local tissue inflammation. As a product of the recently identified T helper 17 lineage of CD4(+) effector lymphocytes, IL-22 plays a critical role in mucosal immunity as well as in dysregulated inflammation observed in autoimmune diseases. We used comprehensive mutagenesis combined with mammalian cell expression, ELISA cell-based, and structural methods to evaluate how IL-22 interacts with its cell surface receptor, IL-22R/IL-10R2, and with secreted IL-22 binding protein. This study identifies those amino acid side chains of IL-22 that are individually important for optimal binding to IL-22R, considerably expands the definition of IL-22 surface required for binding to IL-10R2, and demonstrates how IL-22 binding protein prevents IL-22R from binding to IL-22. The IL-22R and IL-10R2 binding sites are juxtaposed on adjacent IL-22 surfaces contributed mostly by helices A, D, and F and loop AB. Our results also provide a model for how IL-19, IL-20, IL-24, and IL-26 which are other IL-10-like cytokines, interact with their respective cell surface receptors.

A simple high-throughput purification method for hit identification in protein screening

Phage and ribosome display technologies have emerged as important tools in the high-throughput screening of protein pharmaceuticals. However, a challenge created by the implementation of such tools is the need to purify large numbers of proteins for screening. While some assays may be compatible with crude bacterial lysates or periplasmic extracts, many functional assays, particularly cell-based assays, require protein of high purity and concentration. Here we evaluate several methods for small-scale, high-throughput protein purification. From our initial assessment we identified the HIS-Select 96-well filter plate system as the method of choice for further evaluation. This method was optimized and used to produce scFvs that were tested in cell-based functional assays. The behavior of HIS-Select purified scFvs in these assays was found to be similar to scFvs purified using a traditional large-scale 2-step purification method. The HIS-Select method allows high-throughput purification of hundreds of scFvs with yields in the 50-100 microg range, and of sufficient purity to allow evaluation in a cell-based proliferation assay. In addition, the use of a similar 96-well-based method facilitates the purification and subsequent screening of large numbers of IgGs and Fc fusion proteins generated through reformatting of scFv fragments.