IL-17A and IL-17F are related homodimeric proteins of the IL-17 family produced by Th17 cells. In this study, we show that mouse Th17 cells also produce an IL-17F/A heterodimeric protein. Whereas naive CD4+ T cells differentiating toward the Th17 cell lineage expressed IL-17F/A in higher amounts than IL-17A/A homodimer and in lower amounts than IL-17F/F homodimer, differentiated Th17 cells expressed IL-17F/A in higher amounts than either homodimer. In vitro, IL-17F/A was more potent than IL-17F/F and less potent than IL-17A/A in regulating CXCL1 expression. Neutralization of IL-17F/A with an IL-17A-specific Ab, and not with an IL-17F-specific Ab, reduced the majority of IL-17F/A-induced CXCL1 expression. To study these cytokines in vivo, we established a Th17 cell adoptive transfer model characterized by increased neutrophilia in the airways. An IL-17A-specific Ab completely prevented Th17 cell-induced neutrophilia and CXCL5 expression, whereas Abs specific for IL-17F or IL-22, a cytokine also produced by Th17 cells, had no effects. Direct administration of mouse IL-17A/A or IL-17F/A, and not IL-17F/F or IL-22, into the airways significantly increased neutrophil and chemokine expression. Taken together, our data elucidate the regulation of IL-17F/A heterodimer expression by Th17 cells and demonstrate an in vivo function for this cytokine in airway neutrophilia.

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 disulfidelinked 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.Interleukins 17F 3 and 17A (IL-17A) are closely related members of the IL-17 cytokine family, and share 50% amino acid identity. Studies in the mouse have identified Th17 cells as a distinct CD4ϩ T cell lineage that is defined by the production of IL-17F and IL-17A (1-7). IL-6 and transforming growth factor-␤ (TGF-␤) are required for the differentiation of naïve CD4ϩ T cells to Th17 cells (1,8), which are maintained in the presence of IL-23 and IL-1␤. Conversely, IL-4 and interferon-␥ can inhibit the development of Th17 cells (9, 10). Th17 cells have been implicated in the pathology of mouse autoimmune disease models (2).Expression of IL-17F and IL-17A has been detected in activated human peripheral blood lymphocytes. It has been shown by reverse transcriptase-PCR experiments that the cytokines are expressed in activated human CD4ϩ T cells (11,12). Expression of IL-17F and IL-17A has also been observed in tissue samples from various autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, and asthma (2, 3, 13-22).The crystal structure of IL-17F has been solved and shows that the protein forms a disulfide-linked dimeric glycoprotein (23). IL-17A is also a disulfide-linked homodimeric glycoprotein (24), although crystal structure or data defining the precise subunit interactions are lacking. The IL-17F homodimer includes a classical cysteine knot motif, which is found in the TGF-␤, bone morphogenetic protein, and nerve growth factor superfamilies (25, 26). One difference in the cysteine knot motif of IL-17F compared with the other known cysteine knot protein families is that it only utilizes four cysteines instead of the classical six cysteines to form the knot.There have been reports that some of the cysteine knot family members can exist as heterodimers in vivo. TGF-␤1.2 and -␤2.3 were identified in bovine bone extracts, whereas inhibin and activin AB have been found in gonadal fluids (27...

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.

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 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.

Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the ␤1-subunit of L-type voltage-dependent Ca 2؉ channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca 2؉ channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca 2؉ -induced cell death by modulating Ca 2؉ channels and promote neurite outgrowth via FKBP52 binding.immunophilin ͉ L-type voltage-gated calcium channel ͉ natural products ͉ neurodegeneration ͉ stroke

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